Antibodies and Their Related Molecules and Uses

Abstract

The present invention relates to isolated antibodies that selectively bind to CLEC14A, wherein the antibody (a) comprises at least one heavy chain variable region comprising 3 CDRs and at least one light chain variable region comprising 3 CDRs, wherein the heavy chain variable region comprises: (i) variable heavy (VH) CDR1 having the amino acid sequence SEQ ID NO.105, preferably SEQ ID NO:2 or 42; (ii) VH CDR2 having the amino acid sequence SEQ ID NO.106, preferably SEQ ID NO:3 or 43; and / or (iii) VH CDR3 having the amino acid sequence SEQ ID NO.107, preferably SEQ ID NO:4 or 44; and / or wherein the light chain variable region comprises: (iv) variable light (VL) CDR1 having the amino acid sequence SEQ ID NO.108, preferably SEQ ID NO:6 or 46; (v) VL CDR2 having the amino acid sequence SEQ ID NO.109, preferably SEQ ID NO:7 or 47; and / or (vi) VL CDR3 having the amino acid sequence SEQ ID NO.110, preferably SEQ ID NO:8 or 48; or (b) comprises at least one heavy chain variable region comprising 3 CDRs and at least one light chain variable region comprising 3 CDRs, wherein the heavy chain variable region comprises: (i) variable heavy (VH) CDR1 having the amino acid sequence SEQ ID NO:22; (ii) VH CDR2 having the amino acid sequence SEQ ID NO:23; and / or (iii) VH CDR3 having the amino acid sequence SEQ ID NO:24; and / or wherein the light chain variable region comprises: (iv) variable light (VL) CDR1 having the amino acid sequence SEQ ID NO:26; (v) VL CDR2 having the amino acid sequence SEQ ID NO:27; and / or (vi) VL CDR3 having the amino acid sequence SEQ ID NO:28; or (c) is an antibody that can compete with antibody (a) or (b) for binding to CLEC14A. The present invention also provides chimeric antigen receptors, nucleic acid molecules encoding the antibodies or chimeric antigen receptors of the present invention, vectors, cells and methods / uses of the antibodies and chimeric antigen receptors.

Description

[0001] The present invention generally relates to antibodies, CLEC14A biology and related therapies, such as in the field of chimeric antigen receptors (CARs). More specifically, it provides antibodies that bind to CLEC14A and CARs directed against the antigen CLEC14A, which are expressed in immune effector cells to target target cells expressing CLEC14A. Such anti-CLEC14A antibodies and immune effector cells have therapeutic uses in diseases and conditions associated with CLEC14A. For example, the present invention provides products for inhibiting angiogenesis and combating diseases associated with unwanted angiogenesis, such as cancer. The antibodies of the present invention can also have diagnostic utility in monitoring or predicting tumor growth and progression, for example, by imaging the vascular system. The antibody-based compositions and methods of the present invention also extend to the use of immunoconjugates and other therapeutic combinations, kits and methods. The present invention also provides nucleic acid molecules encoding such antibodies and CARs and vectors containing them, which can be used to modify host cells, such as immune effector cells, to express the antibody or CAR. In particular, the CARs of the present invention comprise antigen-binding domains derived from the antibodies of the present invention.

[0002] CLEC14A (also known as epidermal growth factor receptor 5 (EGFR5)) is a single-pass transmembrane glycoprotein that belongs to the vascular-restricted C-type lectin family 14, and its other members include CD248 / TEM1 / endosialin, thrombomodulin, and CD93. It is a single-pass type I transmembrane protein that is 490 amino acids (aa) in length and contains a signal peptide (aa 1-21), an extracellular region (aa 22-398), a transmembrane domain (aa 399-421), and a cytoplasmic domain (aa 422-490). The extracellular region has a C-type lectin-like domain (aa 22-173) and an epidermal growth factor-like region (aa 245-287). The human and mouse CLEC14A proteins show 67% amino acid sequence identity, with even greater sequence conservation in the C-type lectin and epidermal growth factor-like domains. Available data on CLEC14A suggest that manipulating CLEC14A levels or function (e.g., using blocking antibodies) can regulate endothelial migration (WO2011 / 027132).

[0003] Endothelial cells form a single cell layer that serves as the lining of all blood vessels and regulates the exchange between blood flow and the surrounding tissues. During a process called angiogenesis, new blood vessels develop from the walls of existing small blood vessels through the outgrowth of endothelial cells. When isolated in culture, endothelial cells even have the ability to form hollow capillaries. Once the vascular system is fully developed, the endothelial cells of blood vessels generally remain quiescent, with no new blood vessel formation, except during natural wound healing. However, some tumors attract new blood supplies by secreting factors that stimulate nearby endothelial cells to build new capillary sprouts. Angiogenesis plays a major role in the progression of solid tumors and is widely regarded as a rate-limiting process in solid tumor growth. The growth of tumors that fail to attract a blood supply is severely restricted. Therefore, the ability to inhibit inappropriate, unwanted, or unnecessary angiogenesis can be used to treat solid tumors.

[0004] The development of new blood vessels is essential for both local tumor progression and the development of distant metastases. In fact, the growth and survival of tumors depend on their ability to obtain a blood supply, and it has been shown that damage imposed on tumor endothelium effectively eradicates tumors. Tumor angiogenesis involves the degradation of the basement membrane by activated tissue or circulating endothelial precursors, the proliferation and migration of endothelial cells, interaction with the extracellular matrix, morphological differentiation, cell adhesion, and vessel formation. Therefore, inhibiting tumor angiogenesis is a goal of anti-tumor therapy, either using angiogenesis inhibitors alone or in combination with standard cancer treatments. However, targeting anti-tumor agents to the sites of angiogenesis depends on the identification of specific markers of tumor angiogenesis. Endothelium plays an important role in many physiological and pathological processes and is known to be a particularly active transcriptional site. Approximately 1,000 different genes are expressed in endothelial cells, although many of them are not endothelial cell-specific.

[0005] CLEC14A has been identified as a tumor endothelial marker (WO2011 / 027132). CLEC14A is highly expressed on the cell surface of cells that line the vascular systems of many common human cancers, including breast cancer, liver cancer, prostate cancer, pancreatic cancer, bladder cancer, and ovarian cancer, but is expressed low or undetectable in the vascular systems of healthy tissues. It is induced under conditions of low shear stress, such as occurs in the poorly formed blood vessels of tumor tissues, and interacts with MMRN2 within the extracellular matrix. It mediates filipodia formation and endothelial migration and plays a role in sprouting angiogenesis and promoting tumor growth in mice. Therefore, blocking or inhibiting the function or activity of CLEC14A is expected to result in the inhibition of angiogenesis, particularly tumor angiogenesis. Therefore, reagents that inhibit or block the function or activity of CLEC14A and reagents that target cells expressing CLEC14A, particularly tumor cells (such as the tumor vascular system), are needed.

[0006] In recent years, immunotherapies using antibodies, particularly monoclonal antibodies, have emerged as a safe and selective method for treating various diseases, including cancer. Antibodies have also been found to be effective in various diagnostic and prognostic analyses in vitro, in vivo, and ex vivo. However, not all antibodies capable of binding to a specific antigen are effective in therapy. Accordingly, there is a need and a desire for antibodies that specifically bind to CLEC14A, particularly antibodies that bind to different epitopes than previously characterized CLEC14A antibodies and that may also be therapeutically effective and / or capable of blocking or inhibiting the function or activity of CLEC14A, e.g., for inhibiting angiogenesis, particularly tumor angiogenesis, e.g., for treating cancer.

[0007] Therapies involving the expression of chimeric antigen receptors (CARs) in T cells or other immune effector cells, such as NK cells, have also been developed in recent years. CARs, which are now widely known and described in the art, are fusion proteins that contain an antigen-binding domain linked to a signaling domain (or equivalent) of the T cell receptor (TcR) complex, typically but not always derived from an antibody, and which can be used to direct T cells or other immune effector cells against tumors if an appropriate antigen-binding domain or antibody is selected.

[0008] CAR constructs typically contain an antigen-binding domain, optionally a hinge domain (which functions as a spacer to extend the antigen-binding domain away from the plasma membrane of the immune effector cell in which it is expressed), a transmembrane domain, an intracellular signaling domain (e.g., the signaling domain of the zeta chain (CD3ζ) of the CD3 molecule of the TcR complex, or an equivalent), and optionally one or more co-stimulatory domains, which can assist in the signaling or functionality of the cell expressing the CAR. The different domains can be directly linked or linked via a linker. There are a variety of options for these different domains and linkers. Accordingly, there is a need for CARs that can target cells expressing CLEC14A, particularly tumor cells, e.g., for inhibiting angiogenesis, particularly tumor angiogenesis, e.g., for treating cancer.

[0009] The present inventors have generated antibodies that specifically and potently bind CLEC14A (referred to herein as CRT-2 and CRT-3). In addition, as discussed in more detail in the Examples, the present inventors have determined that the antibodies can have useful therapeutic properties alone and in combination with cytotoxic drugs (e.g., as antibody-drug conjugates (also referred to as immunoconjugates)). Notably, the inventors have characterized the antigen-binding domains of the antibodies and determined that the domains, more specifically, the variable regions (VL and VH chains) of the domains, can provide effective CARs for adoptive cell transfer therapy against cells expressing CLEC14A. In more specific embodiments, as will be described in more detail below, the CAR can comprise an antigen-binding domain based on or comprising the VL and VH chains of the antibody of the present invention, particularly based on its hypervariable regions or CDRs (complementary determining regions), in combination with a "signal transduction tail" that includes a combination of hinge, transmembrane, co-stimulatory, and intracellular signal transduction domains.

[0010] Accordingly, in one aspect, the present invention provides antibodies, particularly isolated antibodies, that selectively bind CLEC14A, wherein the antibody:

[0011] (a) comprises at least one heavy chain variable region comprising 3 CDRs and at least one light chain variable region comprising 3 CDRs,

[0012] wherein the heavy chain variable region comprises:

[0013] (i) variable heavy (VH) CDR1, which has the amino acid sequence SEQ ID NO.105, preferably SEQ ID NO:2 or 42, or a sequence substantially homologous to any of the foregoing SEQ ID NOs with 1, 2, 3, or 4 amino acid substitutions, additions, and / or deletions;

[0014] (ii) VH CDR2, which has the amino acid sequence SEQ ID NO.106, preferably SEQ ID NO:3 or 43, or a sequence substantially homologous to any of the foregoing SEQ ID NOs with 1, 2, 3, or 4 amino acid substitutions, additions, and / or deletions; and / or

[0015] (iii) VH CDR3, which has the amino acid sequence SEQ ID NO.107, preferably SEQ ID NO:4 or 44, or a sequence substantially homologous to any of the foregoing SEQ ID NOs with 1, 2, 3, or 4 amino acid substitutions, additions, and / or deletions; and / or

[0016] wherein the light chain variable region comprises:

[0017] (iv) Variable light (VL) CDR1, which has the amino acid sequence SEQ ID NO.108, preferably SEQ ID NO:6 or 46, or a sequence substantially homologous to any of the foregoing SEQ ID NOs with 1, 2, 3 or 4 amino acid substitutions, additions and / or deletions;

[0018] (v) VL CDR2, which has the amino acid sequence SEQ ID NO.109, preferably SEQ ID NO:7 or 47, or a sequence substantially homologous to any of the foregoing SEQ ID NOs with 1, 2, 3 or 4 amino acid substitutions, additions and / or deletions; and / or

[0019] (vi) VL CDR3, which has the amino acid sequence SEQ ID NO.110, preferably SEQ ID NO:8 or 48, or a sequence substantially homologous to any of the foregoing SEQ ID NOs with 1, 2, 3 or 4 amino acid substitutions, additions and / or deletions; or

[0020] (b) Comprising at least one heavy chain variable region containing 3 CDRs and at least one light chain variable region containing 3 CDRs,

[0021] wherein the heavy chain variable region comprises:

[0022] (i) Variable heavy (VH) CDR1, which has the amino acid sequence SEQ ID NO:22 or a sequence substantially homologous to it with 1, 2, 3 or 4 amino acid substitutions, additions and / or deletions;

[0023] (ii) VH CDR2, which has the amino acid sequence SEQ ID NO:23 or a sequence substantially homologous to it with 1, 2, 3 or 4 amino acid substitutions, additions and / or deletions; and / or

[0024] (iii) VH CDR3, which has the amino acid sequence SEQ ID NO:24 or a sequence substantially homologous to it with 1, 2, 3 or 4 amino acid substitutions, additions and / or deletions; and / or

[0025] wherein the light chain variable region comprises:

[0026] (iv) Variable light (VL) CDR1, which has the amino acid sequence SEQ ID NO:26 or a sequence substantially homologous to it with 1, 2, 3 or 4 amino acid substitutions, additions and / or deletions;

[0027] (v) VL CDR2, which has the amino acid sequence SEQ ID NO:27 or a sequence substantially homologous to it with 1, 2, 3 or 4 amino acid substitutions, additions and / or deletions; and / or

[0028] (vi) A VL CDR3 having the amino acid sequence SEQ ID NO:28 or a sequence substantially homologous thereto with 1, 2, 3 or 4 amino acid substitutions, additions and / or deletions; or

[0029] (c) is an antibody that can compete with antibody (a) or (b) for binding to CLEC14A.

[0030] In some embodiments, the present invention provides an antibody, particularly an isolated antibody, that selectively binds to CLEC14A, wherein the antibody:

[0031] (a) comprises at least one heavy chain variable region containing 3 CDRs and at least one light chain variable region containing 3 CDRs,

[0032] wherein the heavy chain variable region comprises:

[0033] (i) A variable heavy (VH) CDR1 having the amino acid sequence SEQ ID NO.105, particularly SEQ ID NO:2 or 42;

[0034] (ii) A VH CDR2 having the amino acid sequence SEQ ID NO.106, particularly SEQ ID NO:3 or 43; and / or

[0035] (iii) A VH CDR3 having the amino acid sequence SEQ ID NO.107, particularly SEQ ID NO:4 or 44; and / or

[0036] wherein the light chain variable region comprises:

[0037] (iv) A variable light (VL) CDR1 having the amino acid sequence SEQ ID NO.108, particularly SEQ ID NO:6 or 46;

[0038] (v) A VL CDR2 having the amino acid sequence SEQ ID NO.109, particularly SEQ ID NO:7 or 47; and / or

[0039] (vi) A VL CDR3 having the amino acid sequence SEQ ID NO.110, particularly SEQ ID NO:8 or 48; or

[0040] (b) is an antibody that can compete with antibody (a) for binding to CLEC14A.

[0041] Thus, in some embodiments, the antibody (a) has a VH domain of SEQ ID NO: 1 or 41 and / or a VL domain of SEQ ID NO: 5 or 45; or (b) is an antibody that can compete with antibody (a) for binding to CLEC14A. For example, the antibody can comprise at least one heavy chain variable region comprising 3 CDRs and at least one light chain variable region comprising 3 CDRs,

[0042] wherein the heavy chain variable region comprises

[0043] (i) VH CDR1, which has the amino acid sequence SEQ ID NO. 105, particularly SEQ ID NO: 2 or 42 or a sequence substantially homologous thereto;

[0044] (ii) VH CDR2, which has the amino acid sequence SEQ ID NO. 106, particularly SEQ ID NO: 3 or 43 or a sequence substantially homologous thereto; and / or

[0045] (iii) VH CDR3, which has the amino acid sequence SEQ ID NO. 107, particularly SEQ ID NO: 4 or 44 or a sequence substantially homologous thereto; and / or

[0046] wherein the light chain variable region comprises:

[0047] (iv) VL CDR1, which has the amino acid sequence SEQ ID NO. 108, particularly SEQ ID NO: 6 or 46 or a sequence substantially homologous thereto;

[0048] (v) VL CDR2, which has the amino acid sequence SEQ ID NO. 109, particularly SEQ ID NO: 7 or 47 or a sequence substantially homologous thereto; and / or

[0049] (vi) VL CDR3, which has the amino acid sequence SEQ ID NO. 110, particularly SEQ ID NO: 8 or 48 or a sequence substantially homologous thereto.

[0050] Thus, in some embodiments, the antibodies of the invention comprise one or more CDRs selected from the group consisting of SEQ ID NO: 2, 3, 4, 6, 7, 8, 42, 43, 44, 46, 47, and 48 or sequences substantially homologous to any of the foregoing SEQ ID NOs.

[0051] Thus, in some preferred embodiments, the antibody comprises two or more heavy chain CDRs from SEQ ID NO:2, 3, and 4 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs. In particularly preferred embodiments, the antibody comprises three heavy chain CDRs from SEQ ID NO:2, 3, and 4 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs.

[0052] Thus, in some preferred embodiments, the antibody comprises two or more heavy chain CDRs from SEQ ID NO:42, 43, and 44 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs. In particularly preferred embodiments, the antibody comprises three heavy chain CDRs from SEQ ID NO:42, 43, and 44 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs.

[0053] In additional or alternative embodiments, the antibody comprises two or more light chain CDRs from SEQ ID NO:6, 7, and 8 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs. In particularly preferred embodiments, the antibody comprises three light chain CDRs from SEQ ID NO:6, 7, and 8 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs.

[0054] In additional or alternative embodiments, the antibody comprises two or more light chain CDRs from SEQ ID NO:46, 47, and 48 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs. In particularly preferred embodiments, the antibody comprises three light chain CDRs from SEQ ID NO:46, 47, and 48 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs.

[0055] Thus, in some embodiments, the antibody comprises three heavy chain CDRs from SEQ ID NO:2, 3, and 4 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs and three light chain CDRs from SEQ ID NO:6, 7, and 8 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs.

[0056] In some embodiments, the antibody comprises three heavy chain CDRs from SEQ ID NO:42, 43, and 44 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs and three light chain CDRs from SEQ ID NO:6, 7, and 8 or sequences that are substantially homologous to any one of the foregoing SEQ ID NOs.

[0057] In some embodiments, the antibody comprises three heavy chain CDRs in SEQ ID NO:2, 3, and 4 or a sequence substantially homologous to any of the foregoing SEQ ID NOs, and three light chain CDRs in SEQ ID NO:46, 47, and 48 or a sequence substantially homologous to any of the foregoing SEQ ID NOs.

[0058] In some embodiments, the antibody comprises three heavy chain CDRs in SEQ ID NO:42, 43, and 44 or a sequence substantially homologous to any of the foregoing SEQ ID NOs, and three light chain CDRs in SEQ ID NO:46, 47, and 48 or a sequence substantially homologous to any of the foregoing SEQ ID NOs.

[0059] In some embodiments, the antibody comprises a heavy chain and / or a light chain, wherein the heavy chain comprises the amino acid sequence SEQ ID NO:1 or 41 or a sequence substantially homologous thereto, and the light chain comprises the amino acid sequence SEQ ID NO:5 or 45 or a sequence substantially homologous thereto.

[0060] In some embodiments, the antibody is a single-chain fragment variable (scFv). Thus, in some aspects, the antibody comprises the amino acid sequence SEQ ID NO:9, 49, 50, or 51 or a sequence substantially homologous thereto.

[0061] Without wishing to be bound by theory, it is hypothesized that the antibody defined in part (a) above is capable of binding to an epitope within the C-type lectin domain of CLEC14A (residues 22 - 173 of CLEC14A). (It should be understood that different alignment programs may predict alternative localizations of the domain within CLEC14A. Thus, the C-type lectin domain may more particularly be located at residues 22 - 175 of CLEC14A, 23 - 173 or 23 - 175 of CLEC14A, or 32 - 173 or 32 - 175 of CLEC14A, depending on the alignment used). In particular, the antibody is thought to bind an epitope within residues 22 - 96 of CLEC14A, such as 32 - 96 and / or residues 109 - 175. Thus, in some embodiments, the antibody does not bind to residues 97 - 108 of CLEC14A. Whether the antibody binds to any of these epitopes or regions can be evaluated using standard techniques in the art, including the binding assays described herein, such as ELISA.

[0062] In a preferred embodiment, an antibody that can compete with the antibody defined in part (a) above can bind to substantially the same epitope as the antibody defined in part (a) above. Thus, in some embodiments, an antibody that can compete with the antibody defined in part (a) above can bind to an epitope within residues 22-96 of CLEC14A, such as 32-96 and / or residues 109-175, such as 109-173. It should be noted that unless otherwise specified, the amino acid numbering discussed herein refers to the amino acid sequence of human CLEC14A as shown in SEQ ID NO.52.

[0063] In an alternative embodiment, the present invention provides antibodies, particularly isolated antibodies, that selectively bind to CLEC14A, wherein the antibody:

[0064] (a’) comprises at least one heavy chain variable region comprising 3 CDRs and at least one light chain variable region comprising 3 CDRs,

[0065] wherein the heavy chain variable region comprises:

[0066] (i) variable heavy (VH) CDR1, which has the amino acid sequence SEQ ID NO:22;

[0067] (ii) VH CDR2, which has the amino acid sequence SEQ ID NO:23; and / or

[0068] (iii) VH CDR3, which has the amino acid sequence SEQ ID NO:24; and / or

[0069] wherein the light chain variable region comprises:

[0070] (iv) variable light (VL) CDR1, which has the amino acid sequence SEQ ID NO:26;

[0071] (v) VL CDR2, which has the amino acid sequence SEQ ID NO:27; and / or

[0072] (vi) VL CDR3, which has the amino acid sequence SEQ ID NO:28; or

[0073] (b’) is an antibody capable of competing with antibody (a’) for binding to CLEC14A.

[0074] Thus, in some embodiments, the antibody (a') has a VH domain of SEQ ID NO:21 and / or a VL domain of SEQ ID NO:25; or (b') is an antibody that can compete with the antibody (a') for binding to CLEC14A. For example, the antibody can comprise at least one heavy chain variable region comprising three CDRs and at least one light chain variable region comprising three CDRs,

[0075] wherein the heavy chain variable region comprises

[0076] (i) VH CDR1, which has an amino acid sequence of SEQ ID NO:22 or a sequence substantially homologous thereto;

[0077] (ii) VH CDR2, which has an amino acid sequence of SEQ ID NO:23 or a sequence substantially homologous thereto; and / or

[0078] (iii) VH CDR3, which has an amino acid sequence of SEQ ID NO:24 or a sequence substantially homologous thereto; and / or

[0079] wherein the light chain variable region comprises:

[0080] (iv) VL CDR1, which has an amino acid sequence of SEQ ID NO:26 or a sequence substantially homologous thereto;

[0081] (v) VL CDR2, which has an amino acid sequence of SEQ ID NO:27 or a sequence substantially homologous thereto; and / or

[0082] (vi) VL CDR3, which has an amino acid sequence of SEQ ID NO:28 or a sequence substantially homologous thereto.

[0083] In some embodiments, the antibody of the invention comprises one or more CDRs selected from SEQ ID NO:22, 23, 24, 26, 27, and 28 or sequences substantially homologous to any one of the foregoing SEQ ID NOs.

[0084] In some preferred embodiments, the antibody comprises two or more heavy chain CDRs of SEQ ID NO:22, 23, and 24 or sequences substantially homologous to any one of the foregoing SEQ ID NOs. In particularly preferred embodiments, the antibody comprises three heavy chain CDRs of SEQ ID NO:22, 23, and 24 or sequences substantially homologous to any one of the foregoing SEQ ID NOs.

[0085] In additional or alternative embodiments, the antibody comprises two or more light chain CDRs from SEQ ID NO: 26, 27, and 28 or sequences that are substantially homologous to any of the foregoing SEQ ID NOs. In particularly preferred embodiments, the antibody comprises three light chain CDRs from SEQ ID NO: 26, 27, and 28 or sequences that are substantially homologous to any of the foregoing SEQ ID NOs.

[0086] Thus, in some embodiments, the antibody comprises three heavy chain CDRs from SEQ ID NO: 22, 23, and 24 or sequences that are substantially homologous to any of the foregoing SEQ ID NOs and three light chain CDRs from SEQ ID NO: 26, 27, and 28 or sequences that are substantially homologous to any of the foregoing SEQ ID NOs.

[0087] In some embodiments, the antibody comprises a heavy chain and / or a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 21 or a sequence that is substantially homologous thereto, and the light chain comprises SEQ ID NO: 25 or a sequence that is substantially homologous thereto.

[0088] In some embodiments, the antibody is a single-chain fragment variable (scFv). Thus, in some aspects, the antibody comprises the amino acid sequence SEQ ID NO: 29 or a sequence that is substantially homologous thereto.

[0089] Without wishing to be bound by theory, it is hypothesized that the antibody defined in part (a') above does not bind to an epitope in the C-type lectin domain of CLEC14A (e.g., residues 22-173 of CLEC14A). In particular, it is believed that the antibody may bind to an epitope within the sushi-like region of CLEC14A (residues 174-244), which is proximal to the C-type lectin, e.g., within residues 174-210, 174-200, 174-190.

[0090] In preferred embodiments, an antibody that can compete with the antibody defined in part (a') above can bind to substantially the same epitope as the antibody defined in part (a') above. Thus, in some embodiments, an antibody that can compete with the antibody defined in part (a') above can bind to an epitope within the region between the C-type lectin domain and the sushi-like domain of CLEC14A. Standard techniques in the art, including the binding assays described herein such as ELISA, can be used to evaluate whether an antibody binds to any of these epitopes or regions.

[0091] As used herein, the term "competitive antibody" refers to an antibody that binds to substantially the same, essentially or substantially the same, or even the same epitope as a "reference antibody". "Competitive antibodies" include antibodies having overlapping epitope specificities. Thus, a competitive antibody is capable of effectively competing with a reference antibody for binding to CLEC14A. Preferably, a competitive antibody binds to the same epitope as the reference antibody. Alternatively, a competitive antibody preferably has the same epitope specificity as the reference antibody.

[0092] As used herein, a "reference antibody" is an antibody that can bind to an epitope of CLEC14A (e.g., the extracellular domain of CLEC14A, preferably human CLEC14A), and has one or more CDR sequences as defined herein, preferably VH and VL domains as defined herein.

[0093] Given the provision of the antibodies of the invention, i.e., the reference antibodies (CRT-2 and CRT-3), the identification of one or more competitive antibodies is a straightforward technical matter. Since the identification of a competitive antibody is determined by comparison with a reference antibody, it should be understood that it is not necessary in any way to actually determine the epitope(s) bound by either or both of the antibodies to identify a competitive antibody. However, if desired, epitope mapping can be performed using standard techniques.

[0094] By way of example, the following methods for identifying and defining epitopes are mentioned herein. The amino acid sequence of CLEC14A is known, and thus synthetic peptides can be used for epitope mapping, e.g., using a Pepscan assay. Site-directed mutagenesis is also a powerful tool in epitope mapping and can be used to evaluate the role of individual amino acids in immune complex formation. Protein footprinting relies on the fact that an epitope is protected from cleavage when bound as an antibody-antigen complex. Enzyme-linked immunosorbent assay (ELISA) and hemagglutination and slot blot assays can also be used for epitope mapping. Crystallization of an antigen with an antibody can be used to map non-linear epitopes. Protocols for performing such methods are widely available, and those skilled in the art will be aware of suitable alternative methods for epitope mapping.

[0095] The identification of competitive antibodies can be readily determined using any of a variety of immunological screening assays in which antibody competition can be evaluated. All such assays are conventional in the art, and each of U.S. Patent Nos. 6,342,219, 6,524,583, 7,056,509, 6,887,468, 6,342,221, 6,676,941, 6,703,020, and 6,416,758 is hereby expressly incorporated by reference herein for the purpose of supplementing this teaching on how to identify competitive antibodies.

[0096] For example, in cases where the test antibody to be assayed is obtained from animals of different sources or is even of different isotypes, a simple competitive assay can be used, in which the reference and test antibodies are mixed (or pre-adsorbed) and applied to a composition containing CLEC14A, preferably cells expressing CLEC14A, phages displaying CLEC14A, or a biochip containing immobilized CLEC14A. ELISA-based protocols are particularly suitable for such simple competitive studies.

[0097] In certain embodiments, a reference antibody (such as CRT2 or CRT3 as defined herein) can be pre-mixed with different amounts of the test antibody (e.g., 1:10, 1:100, or 1:1000) for a period of time and then applied to the antigen composition. In other embodiments, the reference and different amounts of the test antibody can simply be mixed during exposure to the antigen composition. In any case, by using a species or isotype secondary antibody, only the bound reference antibody will be able to be detected, and its binding will be reduced by the presence of the "competitive" bound test antibody.

[0098] In conducting antibody competition studies between a reference antibody and any test antibody (regardless of species or isotype), the reference (e.g., CRT-2 or CRT-3) can first be labeled with a detectable label (such as biotin or an enzyme or a radioactive label) for subsequent identification. In these cases, the labeled reference antibody can be pre-mixed or incubated with the test antibody to be assayed at different ratios (e.g., 1:10, 1:100, or 1:1000), and (optionally after a suitable period of time), then the reactivity of the labeled reference antibody is assayed and compared to a control value in which the potential competitive test antibody is not included in the incubation.

[0099] The assay can be any of a series of immunoassays based on antibody binding, and the reference antibody can be detected by detecting its label, e.g., using streptavidin in the case of a biotinylated antibody or by using a chromogenic substrate that binds to an enzyme label (such as 3,3'5,5'-tetramethylbenzidine (TMB) substrate with peroxidase) or simply detecting a radioactive label. Antibodies that compete with the reference antibody for binding to CLEC14A will be able to effectively or significantly reduce the binding of the reference antibody to CLEC14A, as demonstrated by a decrease in the binding of the label.

[0100] The reactivity of the (labeled) reference antibody in the presence of a completely irrelevant antibody will be a control high value. When competition occurs, a control low value is obtained by incubating the labeled reference (e.g., CRT-2 or CRT-3) antibody with an unlabeled antibody of the exact same type and reducing the binding of the labeled antibody. In a test assay, a significant decrease in the reactivity of the labeled antibody in the presence of the test antibody indicates that the test antibody "competes" with the labeled antibody for binding to CLEC14A.

[0101] The significant decrease is "reproducible", i.e., a consistently observed reduction in binding. For the purposes of this application, a "significant decrease" is defined as a reproducible decrease of at least about 20%, more preferably at least about 25, 30, 35, 40, 45, 50, 55, 60 or 65%, even more preferably at least about 70%, about 75% or about 80% of the binding of the reference antibody to CLEC14A in ELISA at any ratio between about 1:10 and about 1:100. Antibodies with even more stringent competitive activity will exhibit a reproducible decrease of at least about 82%, about 85%, about 88%, about 90%, about 92% or about 95% etc. of the binding of the reference antibody to CLEC14A in ELISA or other suitable assays at any ratio between about 1:10 and about 1:100. Of course, complete or near-complete competition is not excluded, e.g., exhibiting a reproducible decrease in the binding of the reference antibody to CLEC14A of about 99%, about 98%, about 97% or about 96% etc., although the practice of the present invention does not require this at all.

[0102] The above method is only one example of a suitable competition assay. Those skilled in the art will be aware of other suitable methods and variations. Another competition assay is described below.

[0103] Prior to performing an alternative competition assay using flow cytometry, a quantity of the test antibody should be labeled, e.g., by biotinylation. Determine the functionality of the biotinylated product (retaining cell-binding properties) and the minimum concentration of the biotinylated antibody (Ab1) of the present invention that gives sub-maximal binding to a fixed number of CLEC14A+ cells. Harvest a total of 10 6 cells from an exponentially growing culture and incubate them with various antibody concentrations for a suitable time at a suitable temperature, e.g., for 1 hour at 4°C. Wash the cells and incubate them with a suitable detection antibody for a suitable period of time at a suitable temperature, e.g., for another 1 hour at 4°C. After washing, analyze the cells by flow cytometry. For each test antibody, generate a saturation curve from the data by plotting the median fluorescence intensity (MFI) against the antibody concentration.

[0104] For alternative competitive assays, CLEC14A+ cells can be prepared as described above and treated in duplicate with a fixed concentration of labeled (biotinylated) antibody (bio-Ab1) and increasing concentrations of unlabeled competing antibody. The fixed concentration is the minimum concentration of antibody that produces a reasonable fluorescent signal against a fixed number of tumor cells determined as above. Ideally, this fixed concentration in nM should be lower than the affinity (Kd) of the antibody being treated at equilibrium. In such cases, the method described can be used to estimate the affinity of the competing antibody. The antibody mixture is incubated with the target cells for a suitable period of time at a suitable temperature, e.g., 1 hour at 4°C. The cells are washed and the cell binding of the biotinylated antibody is revealed by incubation with FITC-labeled streptavidin. After subtracting the background fluorescence (PBS - 5% FCS) from the median fluorescence reading of each test sample (bio-Ab1+Ab2), the percent inhibition for each Ab2 concentration “c” is calculated according to the following formula:

[0105] % Inhibition = (1 - MFI bio-Ab1+Ab2”c” / MFI bio-Ab1) x 100

[0106] As described above, the competing antibody can comprise one or more CDRs that are substantially homologous to the CDR amino acid sequences disclosed herein. With respect to CDR sequences, the term “substantially homologous” means a sequence having 1, 2, 3, or 4 substitutions, deletions, or additions relative to the CDR sequences disclosed herein, preferably 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the substitutions can be conservative or non-conservative amino acid substitutions, or mixtures thereof.

[0107] Thus, in some embodiments, a substantially homologous sequence can be a sequence that contains up to 1, 2, or 3, preferably up to 1 or 2 altered amino acids in one or more of the CDR regions disclosed herein. In some preferred embodiments, the amino acid substitutions are conservative amino acid substitutions.

[0108] In all embodiments, an antibody containing a substantially homologous sequence retains the ability to bind CLEC14A.

[0109] Preferred antibodies of the invention comprise at least one heavy chain variable region containing three CDRs and at least one light chain variable region containing three CDRs. Exemplary and preferred sequences of these CDRs are described herein.

[0110] As used herein, unless otherwise specifically stated or clear from scientific context, the shorthand term “CLEC14A” refers to C-type lectin domain family member 14A (also known as epidermal growth factor receptor 5 (EGFR5)).

[0111] CLEC14A can be free CLEC14A, such as recombinant or purified CLEC14A, but preferably it is present in its native form, such as on the cell surface.

[0112] The antibodies of the present invention can also bind to fragments of CLEC14A, particularly fragments comprising or consisting of the extracellular domain, or can bind to entities comprising CLEC14A or CLEC14A fragments. In fact, as described above, the epitope of the antibodies of the present invention is believed to be located in the extracellular region of CLEC14A.

[0113] "CLEC14A" can also refer to any form of CLEC14A, particularly since CLEC14A is conserved among mammalian species. Thus, the antibodies or antibody fragments of the present invention can bind to human, monkey (e.g., cynomolgus monkey), cow (bovine), mouse, rat, hamster, ferret, guinea pig, and / or rabbit CLEC14A. Preferably, the antibodies or antibody fragments of the present invention bind at least to human CLEC14A. Thus, unless otherwise indicated, any reference to "CLEC14A" herein can be construed to refer to "human CLEC14A". In certain preferred embodiments, the antibodies or antibody fragments of the present invention will bind at least to human and monkey (e.g., cynomolgus monkey) CLEC14A. In other preferred embodiments, the antibodies or antibody fragments of the present invention will bind at least to human and mouse CLEC14A. In other preferred embodiments, the antibodies or antibody fragments of the present invention will bind at least to human, monkey, and mouse CLEC14A. In other preferred embodiments, the antibodies or antibody fragments of the present invention bind at least to human, monkey, guinea pig, and mouse CLEC14A.

[0114] As used herein, in the context of the antibodies or antibody fragments of the present invention, the terms "bind to CLEC14A / binding to CLEC14A" or "anti-CLEC14A" refer to an antibody or antibody fragment capable of having one or more of the following; preferably, more than one of the following; most preferably, meeting all of the following:

[0115] (a) Bind to CLEC14A expressed on the cell surface, e.g., as assessed by flow cytometry or immunohistochemistry;

[0116] (b) Bind to a conformation-dependent (e.g., non-linear) CLEC14A epitope, e.g., as assessed by binding to CLEC14A in a Western blot under non-reducing conditions;

[0117] (c) Bind to free CLEC14A on a solid support; e.g., recombinantly expressed CLEC14A, e.g., as assessed by ELISA assay or BIAcore assay; and / or

[0118] (d) binds at least to human CLEC14A, more preferably to human and monkey CLEC14A or human and mouse CLEC14A, and most preferably to human, monkey and mouse CLEC14A.

[0119] The preferred antibodies or antibody fragments of the present invention may also be able to localize to tumors after administration to animals suffering from tumors.

[0120] In the context of binding to CLEC14A+ cells, it should be understood that the antibodies of the present invention bind to CLEC14A+ cells and do not significantly bind to CLEC14A- cells.

[0121] It should be understood that the term "does not significantly bind to CLEC14A- cells" means that any binding of the antibody to CLEC14A- cells does not preclude the use of the antibody for therapeutic or diagnostic purposes. Thus, "insignificant" binding to CLEC14A- cells means that the binding of the antibody to CLEC14A- cells is weaker than its binding to one or more CLEC14A+ cells. Thus, some cross-reactivity with normal cells may occur, but such a level of binding may be considered "background" binding. For therapeutic or diagnostic purposes, the main consideration is that the antibody must bind more strongly to one or more types of CLEC14A+ cells than to any CLEC14A- cells that may come into contact with the antibody during therapeutic or diagnostic applications.

[0122] The antibodies of the present invention may be referred to as "CLEC14A-specific". The term "CLEC14A-specific" should be interpreted such that the binding of the antibody to cells expressing CLEC14A is specific enough to allow the use of the antibody for therapeutic or diagnostic purposes. By comparing the binding strength to target CLEC14A+ cells with the binding strength to one or more types of CLEC14A- cells, such as wild-type (i.e., not transformed with CLEC14A) HUVEC cells or HEK293T cells, one skilled in the art can readily determine whether any given antibody is CLEC14A-specific.

[0123] One skilled in the art will appreciate that the binding to CLEC14A+ cells compared to CLEC14A- cells can be evaluated by any suitable means known in the art, such as using flow cytometry. Similarly, species cross-reactivity can be determined using known methods.

[0124] Other preferred properties include the lack of significant toxicity in vivo and the lack of significant other side effects in vivo when the antibodies of the present invention are administered.

[0125] "CLEC14A+ cells" refers to cells that express CLEC14A on their surface, preferably at least substantially in its wild-type conformation. CLEC14A+ cells can be naturally positive for CLEC14A, or they can be transformants that express recombinant CLEC14A.

[0126] As used herein, the terms "antibody" and "immunoglobulin" broadly refer to any immunoconjugate or molecule that contains an antigen-binding domain, including polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, intact antibodies are assigned to one of five main classes: IgA, IgD, IgE, IgG, and IgM, and the antibodies of the present invention can be any of these classes. Several of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0127] Generally, when intact antibodies rather than antigen-binding regions (i.e., antibody fragments) are used in the present invention, IgG and / or IgM are preferred because they are the most common antibodies in physiological situations and because they are the easiest to prepare in a laboratory setting. IgG1 antibodies are particularly preferred.

[0128] The "light chains" of mammalian antibodies are assigned to one of two distinct types based on the amino acid sequence of their constant domains and some of the amino acids in the framework regions of their variable domains: kappa (κ) and lambda (λ). There is no preference for using κ or λ light-chain constant regions in the antibodies of the present invention.

[0129] As will be understood by those skilled in the art, the immunoconjugate reagents encompassed by the term "antibody" extend to all antibodies and their antigen-binding fragments, including intact antibodies, dimeric, trimeric, and multimeric antibodies; bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies and their fragments.

[0130] Accordingly, the term "antibody" is used to refer to any antibody-like molecule having an antigen-binding region, and the term includes antibody fragments containing an antigen-binding domain, such as Fab', Fab, F(ab')2, single-domain antibodies (DABs), TandAbs dimers, Fv, scFv (single-chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibodies, tribodies (scFv-Fab fusions, bispecific or trispecific respectively); sc-diabodies; kappa(lambda) bodies (scFv-CL fusions); bispecific T cell engagers (BiTE) (scFv-scFv tandems to recruit T cells); dual variable domain (DVD)-Ig (bispecific form); small immunoproteins (SIPs) (minibody class); SMIPs ("small modular immunopharmaceuticals" scFv-Fc dimers; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics containing one or more CDRs, etc.

[0131] Techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 647-669, 1991, which is hereby incorporated by reference in its entirety). In particular, diabodies are further described in WO93 / 11161; and linear antibodies are further described in Zapata et al. (Protein Eng., 8(10):1057-1062, 1995).

[0132] Antibody fragments can be generated using conventional techniques. For example, F(ab')2 fragments can be produced by treating an antibody with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bonds to produce Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, Fab', and F(ab')2, scFv, Fv, dsFv, Fd, dAb, TandAb, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for generating antibody fragments are well known in the art and have been described.

[0133] Antibodies or antibody fragments can be produced naturally or can be produced synthetically in whole or in part. Thus, antibodies can be from any suitable source, such as recombinant sources and / or produced in transgenic animals or transgenic plants, or produced in eggs using IgY technology. Thus, antibody molecules can be produced in vitro or in vivo.

[0134] Preferably, the antibody or antibody fragment comprises an antibody light chain variable region (VL) containing three CDR domains and an antibody heavy chain variable region (VH) containing three CDR domains. The VL and VH typically form the antigen binding site.

[0135] The "Fv" fragment is the smallest antibody fragment containing the complete antigen recognition and binding site. This region has a dimer of one heavy chain and one light chain held together by non-covalent bonds. In this construct, the three hypervariable regions (CDRs) of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. The six hypervariable regions (CDRs) together confer antigen binding specificity to the antibody.

[0136] However, it is well documented in the art that the presence of three CDRs from the light chain variable region and three CDRs from the heavy chain variable region of an antibody is not necessary for antigen binding. Thus, constructs smaller than the above-described classical antibody fragments are known to be effective.

[0137] For example, camelid antibodies have a broad antigen binding repertoire but lack a light chain. In addition, results with single domain antibodies containing either a single VH domain or a single VL domain show that these domains can bind antigen with acceptable high affinity. Thus, three CDRs can effectively bind antigen. In addition, it is also known that a single CDR or two CDRs can effectively bind antigen.

[0138] Notably, it is known that two CDRs can effectively bind antigen and even confer properties superior to those of the parental antibody. For example, it has been shown that two CDRs (VH CDR1 and VL CDR3 regions) from a parental antibody can retain the antigen recognition properties of the parental molecule but have excellent tumor penetrating ability. Connecting these CDR domains with a suitable linker sequence (e.g., from VH FR2) and orienting the CDRs in a manner similar to the native parental antibody results in even better antigen recognition. Thus, it is known in the art that antigen binding antibody mimics can be constructed that comprise two CDR domains (preferably one from the VH domain and one from the VL domain, and more preferably, one of the two CDR domains is the CDR3 domain), which are oriented by an appropriate framework region to maintain the conformation found in the parental antibody.

[0139] Thus, although the preferred antibodies of the invention may comprise six CDR regions (three from the light chain and three from the heavy chain), the invention includes antibodies having fewer than six CDR regions and as few as one or two CDR regions. In addition, antibodies having CDRs only from the heavy chain or the light chain are also encompassed. In this regard, the invention further provides antibodies that selectively bind CLEC14A and comprise at least one of the following:

[0140] (i) VH CDR1 having the amino acid sequence SEQ ID NO:2 or 42 or a sequence substantially homologous thereto;

[0141] (ii) VH CDR2 having the amino acid sequence SEQ ID NO:3 or 43 or a sequence substantially homologous thereto; and / or

[0142] (iii) VH CDR3 having the amino acid sequence SEQ ID NO:4 or 44 or a sequence substantially homologous thereto; and / or

[0143] (iv) VL CDR1 having the amino acid sequence SEQ ID NO:6 or 46 or a sequence substantially homologous thereto;

[0144] (v) VL CDR2 having the amino acid sequence SEQ ID NO:7 or 47 or a sequence substantially homologous thereto; and / or

[0145] (vi) VL CDR3 having the amino acid sequence SEQ ID NO:8 or 48 or a sequence substantially homologous thereto.

[0146] Antibodies are further provided that selectively bind CLEC14A and comprise at least one of the following:

[0147] (i) VH CDR1 having the amino acid sequence SEQ ID NO:22 or a sequence substantially homologous thereto;

[0148] (ii) VH CDR2 having the amino acid sequence SEQ ID NO:23 or a sequence substantially homologous thereto; and / or

[0149] (iii) VH CDR3 having the amino acid sequence SEQ ID NO:24 or a sequence substantially homologous thereto; and / or

[0150] (iv) VL CDR1 having the amino acid sequence SEQ ID NO:26 or a sequence substantially homologous thereto;

[0151] (v) A VL CDR2 having the amino acid sequence SEQ ID NO:27 or a sequence substantially homologous thereto; and / or

[0152] (vi) A VL CDR3 having the amino acid sequence SEQ ID NO:28 or a sequence substantially homologous thereto.

[0153] Preferred antibodies of the invention that bind CLEC14A comprise at least one heavy chain variable region comprising three CDRs and at least one light chain variable region comprising three CDRs, wherein the heavy chain variable region comprises:

[0154] (a) A variable heavy (VH) CDR1 having the amino acid sequence SEQ ID NO:2 or 42 or a sequence substantially homologous thereto,

[0155] (b) A VH CDR2 having the amino acid sequence SEQ ID NO:3 or 43 or a sequence substantially homologous thereto, and

[0156] (c) A VH CDR3 having the amino acid sequence SEQ ID NO:4 or 44 or a sequence substantially homologous thereto; or

[0157] (d) A variable heavy (VH) CDR1 having the amino acid sequence SEQ ID NO:22 or a sequence substantially homologous thereto,

[0158] (e) A VH CDR2 having the amino acid sequence SEQ ID NO:23 or a sequence substantially homologous thereto, and

[0159] (f) A VH CDR3 having the amino acid sequence SEQ ID NO:24 or a sequence substantially homologous thereto.

[0160] Preferred light chain CDR regions for use in combination with the specified heavy chain CDR regions are described elsewhere herein. However, other light chain variable regions comprising three CDRs are also contemplated for use in combination with the heavy chain variable regions of the invention. Those skilled in the art can readily identify suitable light chain variable regions that can be used in combination with the heavy chain variable regions of the invention and produce antibodies that bind CLEC14A.

[0161] For example, the heavy chain variable regions of the invention can be combined with a single light chain variable region or a repertoire of light chain variable regions, and the resulting antibodies tested for binding to CLEC14A. It is expected that a reasonable number of such combinations of the heavy chain variable regions of the invention with different light chain variable regions will retain the ability to bind CLEC14A.

[0162] A similar approach can be used to identify alternative heavy chain variable regions for use in combination with the preferred light chain variable regions of the invention.

[0163] In certain embodiments, the antibody or antibody fragment comprises all or part of a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgG1 heavy chain constant region or a portion thereof. In addition, the antibody or antibody fragment can comprise the whole or part of a kappa light chain constant region or a lambda light chain constant region or a portion thereof. Such whole or partial constant regions can occur naturally or can be wholly or partially synthetic. Suitable sequences for such constant regions are well known in the art and are described in the art. When the complete complement of constant regions from the heavy and light chains is included in the antibodies of the invention, such antibodies are generally referred to herein as "full-length" antibodies or "complete" antibodies.

[0164] Antibodies containing an Fc region are preferred for certain uses, particularly for in vivo therapeutic uses.

[0165] In a preferred embodiment, the antibodies of the invention are monoclonal antibodies, which can be humanized or human monoclonal antibodies. In this regard, human or humanized antibodies generally have at least three potential advantages for human therapy. First, the human immune system should not recognize the antibody as foreign. Second, the half-life in the human circulation is similar to that of naturally occurring human antibodies, allowing for smaller and less frequent doses to be administered. Third, since the effector portion is human, it will interact better with other parts of the human immune system.

[0166] Thus, the specific antibodies disclosed in the examples can be "humanized" in a known manner, for example by inserting the CDR regions of the murine antibody into the framework of a human antibody. The techniques and methods described in Verhoeyen et al (1988) Science, 239, 1534-1536 and Kettleborough et al, (1991) Protein Engineering, l4(7), 773-783 can be used to prepare humanized antibodies. In some cases, Fv framework residues of the human immunoglobulin are replaced with the corresponding non-human residues. Generally, a humanized antibody will contain a variable region in which all or most of the CDR regions correspond to the CDR regions of a non-human immunoglobulin, and the framework regions are substantially or entirely those of human immunoglobulin consensus sequences.

[0167] Fully human antibodies can be generated using recombinant techniques. Large libraries containing billions of different antibodies are typically used. Contrary to prior art that employs, for example, chimerization or humanization of murine antibodies, this technique does not rely on immunization of animals to generate specific antibodies. Instead, the recombinant library contains a large number of pre-prepared antibody variants, where the library may have at least one antibody specific to any antigen. Thus, in the context of the present invention, such libraries can be used to identify competitive antibodies with desired binding characteristics. To find good binders in the library in an efficient manner, various systems have been designed where the phenotype, i.e., the antibody or antibody fragment, is linked to its genotype, i.e., the encoding gene. The most commonly used such system is the so-called phage display system, where antibody fragments are expressed and displayed on the surface of filamentous phage particles as fusions with phage coat proteins, while carrying the genetic information encoding the displayed molecule (McCafferty et al, 1990, Nature 348:552 - 554). Phages displaying antibody fragments specific to a particular antigen can be selected by binding to the antigen under discussion. The isolated phages can then be amplified, and optionally, the genes encoding the selected antibody variants can be transferred to other antibody forms, e.g., full-length immunoglobulins, and expressed in large quantities using suitable vectors and host cells well known in the art. Alternatively, "human" antibodies can be prepared by immunizing transgenic mice that are substantially human immunoglobulin gene-containing (Vaughan et al (1998) Nature Biotechnol. 16, 535 - 539).

[0168] "Human" and "humanized" antibodies of the present invention may include amino acid residues not encoded by human sequences, such as mutations introduced by in vitro random or site-directed mutagenesis, e.g., mutations introduced by in vitro cloning or PCR. Specific examples of such mutations are mutations involving conservative substitutions or other mutations (non-conservative substitutions, additions, and / or deletions) in a small number of residues of the antibody, e.g., in up to 5, 4, 3, 2, or 1 residue of the antibody, preferably, e.g., in up to 5, 4, 3, 2, or 1 residue(s) constituting one or more CDRs of the antibody. Certain examples of such "human" and "humanized" antibodies include antibodies and variable regions that have undergone standard modification techniques to reduce the amount of potential immunogenic sites.

[0169] Thus, "human" and "humanized" antibodies of the present invention include sequences derived from and related to those found in humans, but that may not naturally occur in the human antibody germline repertoire in vivo. In addition, the human and humanized antibodies of the present invention include proteins containing human consensus sequences identified from human sequences, or sequences that are substantially homologous to human sequences.

[0170] In addition, the human and humanized antibodies of the present invention are not limited to the combinations of VH, VL, CDR or FR regions that are found in combination in human antibody molecules per se. Thus, the human and humanized antibodies of the present invention can include or correspond to combinations of such regions that do not necessarily occur naturally in humans.

[0171] In some embodiments, the human antibody can be a fully human antibody. As used herein, a "fully human" antibody is an antibody that comprises a "human" variable region and / or CDR as defined above and has no substantial non-human antibody sequence or no non-human antibody sequence at all. For example, an antibody that comprises a human variable region and / or CDR and "has no substantial non-human antibody sequence" is an antibody, domain and / or CDR in which only up to 5, 4, 3, 2 or 1 amino acid is an amino acid not encoded by a human antibody sequence. Thus, a "fully human" antibody can be distinguished from a "humanized" antibody, which is based on a substantially non-human variable region, such as a murine variable region, in which certain amino acids have been altered to better correspond to the amino acids that are typically present in human antibodies.

[0172] The "fully human" antibody of the present invention can be a human variable region and / or CDR with no other substantial antibody sequence, such as a single-chain antibody. Alternatively, the "fully human" antibody of the present invention can be a human variable region and / or CDR that is integrated or operably linked to one or more human antibody constant regions. Certain preferred fully human antibodies are IgG antibodies that have a complete complement of IgG constant regions.

[0173] In other embodiments, the "human" antibody of the present invention is a partially human chimeric antibody. As used herein, a "partially human chimeric" antibody is an antibody that comprises a "human" variable region and / or CDR that is operably linked or grafted onto a constant region of a non-human species (such as a rat or a mouse). Such partially human chimeric antibodies can be used, for example, in preclinical studies, where the constant region is preferably of the same animal species as that used in preclinical testing. These partially human chimeric antibodies can also be used, for example, in in vitro diagnostics, where the constant region of the non-human species can provide additional options for antibody detection.

[0174] As used herein, the term "fragment" refers to a biologically relevant fragment, e.g., one that contributes to antigen binding, such as forming part of an antigen-binding site, and / or one that contributes to inhibiting or reducing the function or activity of the CLEC14A antigen. Certain preferred fragments comprise the heavy-chain variable region (VH domain) and / or the light-chain variable region (VL domain) of the antibody of the present invention. Other preferred fragments comprise one or more heavy-chain CDRs of the antibody of the present invention (or the VH domain of the present invention), or one or more light-chain CDRs of the antibody of the present invention (or the VL domain of the present invention). Certain preferred fragments are at least 5 amino acids in length and comprise at least one CDR region, preferably the CDR3 region, more preferably the heavy-chain CDR3 region.

[0175] In embodiments where the antibody of the invention comprises fragments of any defined sequence (such as fragments comprising SEQ ID NO: 1, 5, 21, 25, 41 or 45), for example an antibody comprising the VH and / or VL domains of the invention, or an antibody comprising one or more CDRs of the invention, these regions / domains are generally separated within the antibody such that each region / domain can perform its biological function and thus the contribution to antigen binding is retained. Thus, the VH and VL domains are preferably separated by a suitable scaffolding sequence / linker sequence, and the CDRs are preferably separated by a suitable framework region, such as the framework regions found in naturally occurring antibodies and / or effective engineered antibodies. Thus, the VH, VL and individual CDR sequences of the invention are preferably provided or incorporated within a suitable framework or scaffold to effect antigen binding. Such framework sequences or regions may correspond to the naturally occurring framework regions FR1, FR2, FR3 and / or FR4, suitably forming a suitable scaffold, or may correspond to consensus framework regions, such as identified by comparing various naturally occurring framework regions. Alternatively, non-antibody scaffolds or frameworks, such as T cell receptor frameworks, may be used.

[0176] Suitable sequences that can be used for the framework regions are well known and described in the art, and any of these sequences may be used. Preferred sequences for the framework regions are one or more (i.e., one, two, three or four) framework regions that constitute the VH and / or VL domains of the invention, i.e., one or more framework regions found within SEQ ID NO: 1, 5, 21, 25, 41 or 45 or framework regions that are substantially homologous thereto, particularly framework regions that allow the maintenance of antigen specificity, such as framework regions that result in a substantially identical or identical 3D structure of the antibody.

[0177] In the context of the framework regions of the antibody of the invention, the term "substantially homologous" includes sequences having at least 60%, 65%, 70% or 75%, preferably at least 80%, even more preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequences disclosed herein. Thus, the substantially homologous sequences of the invention include single or multiple base or amino acid alterations (additions, substitutions, insertions or deletions) to the sequences of the invention. At the amino acid level, preferred substantially homologous sequences contain only up to 1, 2, 3, 4 or 5, preferably up to 1, 2 or 3, more preferably up to 1 or 2 altered amino acids in one or more framework regions and / or one or more CDRs that constitute the sequences of the invention. The alterations may be with conservative or non-conservative amino acids, or mixtures thereof. Preferably, the alterations are conservative amino acid substitutions.

[0178] Thus, for example, an antibody of the present invention may comprise an antigen-binding domain comprising a VH sequence having the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 60% sequence identity thereto and a VL sequence having the amino acid sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 60% sequence identity thereto, preferably with the proviso that the CDR sequences of SEQ ID NOs. 2, 3, 4, 6, 7 and 8 are retained (i.e., not modified or altered).

[0179] In some embodiments, an antibody of the present invention may comprise an antigen-binding domain comprising a VH sequence having the amino acid sequence shown in SEQ ID NO.41 or an amino acid sequence having at least 60% sequence identity thereto and a VL sequence having the amino acid sequence shown in SEQ ID NO.45 or an amino acid sequence having at least 60% sequence identity thereto, preferably with the proviso that the CDR sequences of SEQ ID NOs. 42, 43, 44, 46, 47 and 48 are retained (i.e., not modified or altered).

[0180] In other embodiments, an antibody of the present invention may comprise an antigen-binding domain comprising a VH sequence having the amino acid sequence shown in SEQ ID NO.21 or an amino acid sequence having at least 60% sequence identity thereto and a VL sequence having the amino acid sequence shown in SEQ ID NO.25 or an amino acid sequence having at least 60% sequence identity thereto, preferably with the proviso that the CDR sequences of SEQ ID NOs. 22, 23, 24, 26, 27 and 28 are retained (i.e., not modified or altered).

[0181] In still other embodiments, an antibody of the present invention may be a scFv comprising the amino acid sequence shown in SEQ ID NO.9 or an amino acid sequence having at least 60% sequence identity thereto, preferably with the proviso that the CDR sequences of SEQ ID NOs. 2, 3, 4, 6, 7 and 8 are retained (i.e., not modified or altered).

[0182] In some embodiments, an antibody of the invention can be an scFv that comprises an amino acid sequence shown in SEQ ID NO. 49 or an amino acid sequence having at least 60% sequence identity thereto, preferably provided that the CDR sequences of SEQ ID NOs. 42, 43, 44, 6, 7, and 8 are retained (i.e., not modified or altered). In some embodiments, an antibody of the invention can be an scFv that comprises an amino acid sequence shown in SEQ ID NO. 50 or an amino acid sequence having at least 60% sequence identity thereto, preferably provided that the CDR sequences of SEQ ID NOs. 2, 3, 4, 46, 47, and 48 are retained (i.e., not modified or altered).

[0183] In some embodiments, an antibody of the invention can be an scFv that comprises an amino acid sequence shown in SEQ ID NO. 51 or an amino acid sequence having at least 60% sequence identity thereto, preferably provided that the CDR sequences of SEQ ID NOs. 42, 43, 44, 46, 47, and 48 are retained (i.e., not modified or altered).

[0184] In other embodiments, an antibody of the invention can be an scFv that comprises an amino acid sequence shown in SEQ ID NO. 29 or an amino acid sequence having at least 60% sequence identity thereto, preferably provided that the CDR sequences of SEQ ID NOs. 22, 23, 24, 26, 27, and 28 are retained (i.e., not modified or altered).

[0185] Thus, it should be understood that in such embodiments, the CDR sequences of the antibody are retained or substantially retained (i.e., they can optionally be modified within the limitations set forth above, such as 1 to 4 amino acid substitutions, additions, or deletions, such that the binding specificity of the antibody is retained (e.g., not changed)).

[0186] The term "substantially homologous" also includes modifications or chemical equivalents of the amino acids of the present invention that perform substantially the same function with the proteins or nucleic acid molecules of the present invention in substantially the same manner. For example, any substantially homologous antibody (or substantially homologous nucleic acid encoding it) should retain the ability to bind CLEC14A as discussed above. Preferably, any substantially homologous antibody should retain the functions of an antibody, such as an antibody as defined elsewhere herein. Preferably, any substantially homologous antibody should retain the ability to specifically bind the same epitope of CLEC14A recognized by the antibody as discussed, such as the same epitope recognized by the CDR domain of the present invention or the VH and VL domains of the present invention as described herein. Binding to the same epitope / antigen can be easily tested by methods well known and described in the art, such as using binding assays, such as competition assays. Retention of other functional properties can also be easily tested by methods well known and described in the art.

[0187] Thus, those skilled in the art will understand that binding assays can be used to test whether "substantially homologous" antibodies have the same binding specificity as the antibodies and antibody fragments of the present invention. For example, binding assays, such as ELISA assays or BIAcore, can be easily used to establish whether such "substantially homologous" antibodies can bind CLEC14A. As outlined above, competition binding assays can be used to test whether "substantially homologous" antibodies retain the ability to specifically bind substantially the same epitope of CLEC14A recognized by the antibodies of the present invention.

[0188] Substantially homologous sequences of the proteins of the present invention include, but are not limited to, conservative amino acid substitutions, or alterations that, for example, do not affect the VH, VL, or CDR domains of the antibody, such as scFv antibodies using different linker sequences or antibodies with tag sequences or other components added that do not contribute to antigen binding, or alterations that convert one type or form of antibody molecule or fragment to another type or form of antibody molecule or fragment (e.g., conversion from Fab to scFv or vice versa), or conversion of an antibody molecule to a specific class or subclass of antibody molecule (e.g., conversion of an antibody molecule to IgG or its subclass, such as IgG1 or IgG3).

[0189] As used herein, "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0190] Homology can be assessed by any convenient method. However, to determine the degree of homology between sequences, computer programs for performing multiple sequence alignments are useful, such as Clustal W. If desired, the Clustal W algorithm can be used with a BLOSUM 62 scoring matrix and a gap opening penalty of 10 and a gap extension penalty of 0.1 to obtain a highest order match between two sequences, where at least 50% of the total length of one of the sequences is involved in the alignment. Other methods for calculating the percent identity between two amino acid sequences are generally recognized in the art and include, for example, those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects.

[0191] Generally, computer programs can be employed for such calculations. Programs for comparing and aligning pairs of sequences, such as ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG, can also be used for this purpose. In addition, the Dali server at the European Bioinformatics Institute provides structure-based protein sequence alignments.

[0192] By providing a reference point, sequences according to the invention having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology, sequence identity, etc. can be determined using the ALIGN program with default parameters (e.g., available on the GENESTREAM web server on the Internet, IGH, Montpellier, France).

[0193] In addition, although the preferred antibodies of the invention are composed of the VH, VL or CDRs of the invention, it should be noted that the antibodies of the invention also encompass combinations of one or more of the VH, VL or CDRs of the invention with other VH, VL or CDRs that are not of the invention, provided that the CLEC14A binding characteristics or anti-CLEC14A characteristics of the antibodies of the invention as outlined herein still exist.

[0194] As used herein, the term "heavy chain complementarity determining region" ("heavy chain CDR") refers to the hypervariable regions within the variable region of the heavy chain (VH domain) of an antibody molecule. The variable region of the heavy chain has three CDRs, which are designated heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 from the amino terminus to the carboxyl terminus. The variable region of the heavy chain also has four framework regions (FR1, FR2, FR3 and FR4 from the amino terminus to the carboxyl terminus). These framework regions separate the CDRs.

[0195] As used herein, the term "variable region of the heavy chain" (VH domain) refers to the variable region of the heavy chain of an antibody molecule.

[0196] As used herein, the term "light chain complementarity determining region" ("light chain CDR") refers to the hypervariable regions within the variable region of the light chain (VL domain) of an antibody molecule. The variable region of the light chain has three CDRs, designated light chain CDR1, light chain CDR2 and light chain CDR3 from the amino terminus to the carboxyl terminus. The variable region of the light chain also has four framework regions (FR1, FR2, FR3 and FR4 from the amino terminus to the carboxyl terminus). These framework regions separate the CDRs.

[0197] As used herein, the term "variable region of the light chain" (VL domain) refers to the variable region of the light chain of an antibody molecule.

[0198] It should be noted that Kabat nomenclature is followed herein when necessary to define the positioning of the CDRs (Kabat et al., 1991 (supra), which is hereby incorporated by reference in particular).

[0199] In some embodiments, the antibodies of the invention (e.g., in IgG form) can have high binding affinity for CLEC14A, i.e., K d at 1x10 -8 M or 1x10 -9in the range of M or lower. Importantly, antibodies with such affinities are within the established range that has been shown to be useful for therapy. Preferably, the antibodies of the present invention (e.g., in IgG form) have a binding affinity for CLEC14A that corresponds to less than 30 nM, 20 nM, 15 nM or 10 nM, more preferably less than 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or 1 nM, and most preferably less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 nM d.

[0200] Any suitable method for determining K d can be used. However, preferably, K d is determined by testing various concentrations of the test antibody against various concentrations of the antigen (CLEC14A) in vitro to establish a saturation curve, for example using the Lineweaver - Burk method, or by using commercially available binding model software, such as the 1:1 binding model in BIAcore 1000 evaluation software.

[0201] Regarding the determination of K d values, those skilled in the art will understand that the apparent K d values obtained from binding experiments using cells expressing the target (e.g., CLEC14A) cannot be considered an absolute indication of affinity because the experimental conditions will affect the apparent binding affinity. For example, the expression level of CLEC14A can vary depending on the conditions of culturing the cells, as well as differences between different cell types. Therefore, it is best to compare the apparent K d values obtained in a set of experiments, and it may not always be appropriate to compare the K d values obtained in one set of experiments with the K d values obtained in different experimental groups, especially if the experimental conditions vary significantly.

[0202] Alternatively, the dissociation rate and antibody half - life on CLEC14A - positive cell surfaces can be determined by performing a cell - surface retention assay (Adams et al., 1998, Br J Cancer 77:1405 - 12; Le Gall et al., 1999, FEBS Lett 453:164 - 8). The Le Gall method allows for a more appropriate simulation of the real situation in human patients under therapeutic conditions.

[0203] In some embodiments, the antibodies of the invention may bind to both human CLEC14A and monkey CLEC14A. Such cross-reactivity between species, particularly between humans and species commonly used as preclinical animal models, can be advantageous because it allows for more efficient translation from preclinical studies to clinical use. For example, the use of antibodies that cross-react with the native CLEC14A present in the particular animal model used means that the results in the model are more likely to reflect the situation in human patients, thereby allowing for more accurate assessments of, for example, the administration to be performed and the increased likelihood of identifying any potentially relevant or problematic side effects.

[0204] For example, the ability of the antibodies of the invention to bind to both human and monkey CLEC14A means that such antibodies can be tested in preclinical toxicity studies to assess adverse side effects of treatment and to find an appropriately tolerated dose.

[0205] Furthermore, the ability to bind both human CLEC14A and mouse CLEC14A means that the results shown by such antibodies of the invention in a mouse model, for example, a mouse syngeneic model using immunocompetent mice, are more likely to represent the activity of the antibodies in human subjects. The reason for this is that an antibody that can bind to human CLEC14A but not mouse CLEC14A will bind to CLEC14A expressed by human tumor cells in a mouse model, but will not be able to bind to endogenous murine CLEC14A. This is of course different from the situation in a human patient, where both CLEC14A expressed by the tumor and endogenous CLEC14A will be present.

[0206] In preferred embodiments, the antibodies of the invention bind with similar affinities, e.g., 10 nM or less or 5 nM or less, more preferably 3 nM or less or 2 nM or less, most preferably 1 nM or less. d Binds to human and monkey CLEC14A and / or binds to human and mouse CLEC14A.

[0207] "Similar affinity" also means that the binding affinity of the antibody to human CLEC14A and one or more other species of interest (e.g., monkey or mouse) is comparable, for example, no more than 20-fold different. More preferably, the difference between the binding affinities is less than 15-fold, more preferably less than 10-fold, most preferably less than 5-fold, 4-fold, 3-fold or 2-fold.

[0208] However, in other embodiments, the antibodies of the invention may not bind to monkey CLEC14A and / or they may not bind to mouse CLEC14A.

[0209] Thus, according to the present invention, a series of anti-CLEC14A antibodies can be prepared and used in a variety of embodiments, including treating any disease discussed elsewhere herein, particularly diseases or conditions involving unwanted or undesired angiogenesis, particularly tumor angiogenesis, such as for treating cancer.

[0210] Those skilled in the art will understand that the proteins and polypeptides of the present invention, such as light and heavy CDRs, light and heavy chain variable regions, antibodies, antibody fragments and immunoconjugates (described in more detail below), can be prepared by any of several methods known and described in the art, but are most preferably prepared using recombinant methods.

[0211] Thus, in another embodiment, the present invention provides nucleic acid molecules encoding the antibodies or portions or fragments thereof of the present invention as described herein, or nucleic acid molecules that are substantially homologous thereto. Thus, in some embodiments, the present invention provides nucleic acid molecules comprising one or more sequences selected from SEQ ID NOs. 12, 13, 14, 16, 17 and 18, one or more sequences selected from SEQ ID NOs. 32, 33, 34, 36, 37 and 38 or one or more sequences selected from SEQ ID NOs. 55, 56, 57, 59, 60 and 61 (i.e., encoding the CDRs described above). In some embodiments, the present invention provides nucleic acid molecules comprising a sequence selected from SEQ ID NOs. 11 and 15 or a sequence selected from SEQ ID NOs: 31 and 35 or a sequence selected from SEQ ID NOs: 54 and 58 (i.e., encoding the variable chains described above). In some embodiments, the present invention provides nucleic acid molecules comprising a sequence selected from SEQ ID NOs: 19, 39, 62, 63 and 64 (i.e., encoding the scFv polypeptides described above).

[0212] In some embodiments, the nucleic acid molecules encoding the antibodies of the present invention can be substantially homologous to the nucleic acid molecules exemplified herein. Thus, in some embodiments, the nucleic acid molecules can encode polypeptides comprising an antigen-binding domain, wherein the antigen-binding domain comprises a VH sequence as described herein, wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO. 11, 31 or 54 or a nucleotide sequence having at least 60% sequence identity thereto, preferably provided that the nucleotide sequences encoding the CDR sequences of SEQ ID NOs. 2, 3 and 4 or 22, 23 and 24 or 42, 43 and 44 are retained, for example, the nucleotide sequence comprises SEQ ID NO: 12, 13 and 14 or 32, 33 and 34 or 55, 56 and 57.

[0213] Thus, in some embodiments, a nucleic acid molecule can encode a polypeptide comprising an antigen-binding domain, wherein the antigen-binding domain comprises a VL sequence as described herein, and wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.15, 35, or 58 or a nucleotide sequence having at least 60% sequence identity thereto, preferably with the proviso that the nucleotide sequences encoding the CDR sequences of SEQ ID NO.6, 7, and 8 or 26, 27, and 28 or 46, 47, and 48 are retained. For example, the nucleotide sequence comprises SEQ ID NO:16, 17, and 18 or 36, 37, and 38 or 59, 60, and 61.

[0214] In a further embodiment, a nucleic acid molecule can encode a polypeptide comprising an scFv as described herein, wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.SEQ ID NO.19, 39, 49, 50, or 51 or a nucleotide sequence having at least 60% sequence identity thereto, preferably with the proviso that the nucleotide sequences encoding the CDR sequences of SEQ ID NO.2, 3, 4, 6, 7, and 8 or 22, 23, 24, 26, 27, and 28, or 42, 43, 44, 6, 7, and 8 or 2, 3, 4, 46, 47, and 48 or 42, 43, 44, 46, 47, and 48 are retained. For example, the nucleotide sequence comprises SEQ ID NO:12, 13, 14, 16, 17, and 18 or 32, 33, 34, 36, 37, and 38 or 55, 56, 57, 16, 17, and 18 or 12, 13, 14, 59, 60, and 61 or 55, 56, 57, 59, 60, and 61.

[0215] Thus, fragments of the antibodies of the invention as defined herein, or sequences that are substantially homologous thereto, or nucleic acid molecules comprising sequences encoding such fragments form a further aspect of the invention.

[0216] Nucleic acid fragments encoding the light and heavy chain variable regions of the antibodies of the invention can be derived or produced by any suitable method, such as by cloning or synthesis. For example, such sequences can be prepared by cloning suitable sequences from, for example, human germline genes and then making any necessary modifications to the germline sequences using methods well known and described in the art to obtain the sequences of the invention. An alternative and more efficient method is to synthesize appropriate light or heavy chain variable region sequences as overlapping primers and use primer extension to obtain the complete sequences. The complete sequences can then be amplified by PCR using primers containing appropriate restriction sites for further cloning and manipulation, such as cloning into a suitable expression vector. Five to seven overlapping primers for each variable region are generally sufficient, making the technique very efficient and precise.

[0217] Once nucleic acid fragments encoding the light and heavy chain variable regions of the antibodies of the invention are obtained, these fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region fragments into full-length antibody molecules having appropriate constant regions, or specific forms of antibody fragments discussed elsewhere herein, such as Fab fragments, scFv fragments, etc. Typically, or as part of this further manipulation procedure, the nucleic acid fragments encoding the antibody molecules of the invention are incorporated into a suitable expression vector to facilitate the production of the antibodies of the invention.

[0218] As used herein, the term "nucleic acid sequence" or "nucleic acid molecule" refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes sequences that contain modified or substituted monomers or portions thereof that are not naturally occurring. The nucleic acid sequences of the invention can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA), and can include naturally occurring bases, including adenine, guanine, cytosine, thymidine and uracil. The sequences can also contain modified bases. Examples of such modified bases include azido and deazaadenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid molecule can be double-stranded or single-stranded. The nucleic acid molecule can be wholly or partially synthetic or recombinant.

[0219] As used herein in connection with nucleic acid sequences, the term "substantially homologous" includes sequences having at least 60%, 65%, 70% or 75%, preferably at least 80%, even more preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the disclosed nucleic acid sequences. Thus, substantially homologous sequences of the invention include single-base or multi-base changes (additions, substitutions, insertions or deletions) to the sequences of the invention.

[0220] Substantially homologous nucleic acid sequences also include nucleotide sequences that hybridize to the disclosed nucleic acid sequences (or their complementary sequences) under at least moderately stringent hybridization conditions, e.g., nucleotide sequences that hybridize (or their complementary sequences) to the nucleotide sequences encoding one or more light or heavy chain CDRs of the invention, the light or heavy chain variable regions of the invention, or the antibodies of the invention.

[0221] "At least moderately stringent hybridization conditions" refers to conditions selected to promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization can be performed on all or part of a nucleic acid sequence molecule. The length of the hybridizing portion is typically at least 15 (e.g., 20, 25, 30, 40 or 50) nucleotides. Those skilled in the art will recognize that the stability of a nucleic acid duplex or hybrid is determined by Tm, which is a function of sodium ion concentration and temperature in a sodium-containing buffer (Tm = 81.5°C - 16.6(Log10[Na+]) + 0.41(%(G+C) - 600 / l) or a similar equation). Thus, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. To identify molecules that are similar but not identical to a known nucleic acid molecule, it is assumed that a 1% mismatch results in a decrease in Tm of approximately 1°C. For example, if a nucleic acid molecule with >95% identity is sought, the final wash temperature will be decreased by approximately 5°C. Based on these considerations, those skilled in the art will be able to readily select appropriate hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. By way of example, the following conditions can be employed to achieve stringent hybridization: based on the above equation, hybridize at Tm - 5°C in 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt's solution / 1.0% SDS, then wash at 60°C in 0.2x SSC / 0.1% SDS. Moderately stringent hybridization conditions include a wash step in 3x SSC at 42°C. As another example, "hybridizing" sequences are those that bind (hybridize) under non-stringent conditions (e.g., 6x SSC, 50% formamide at room temperature) and are washed under low stringency conditions (e.g., 2x SSC, room temperature, more preferably 2x SSC, 42°C) or higher stringency conditions (e.g., 2x SSC, 65°C) (where SSC = 0.15M NaCl, 0.015M sodium citrate, pH 7.2).

[0222] However, it should be understood that equivalent stringency can be achieved using alternative buffers, salts and temperatures. Additional guidance on hybridization conditions can be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1 - 6.3.6 and Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 3.

[0223] In general, sequences that hybridize under high stringency conditions are preferred, but so are sequences that will hybridize under high stringency conditions due to coding degeneracy.

[0224] The present invention also encompasses nucleotide sequences that are degenerate variants of the nucleotide sequences defined herein. It should be understood that due to the degeneracy of the genetic code, multiple nucleotide sequences can encode a single amino acid sequence. In this regard, such degenerate sequences form part of the present invention. In this context, it may be desirable to codon-optimize the nucleotide sequences of the present invention for expression in a particular host organism or cell (e.g., for expression in humans or mice). This can involve modifying the nucleotide sequences of the present invention in order to select codons (from among the codons encoding a particular amino acid) that are preferentially expressed in a particular host cell / organism. Such procedures are well known in the art and generally do not alter the encoded amino acid sequence. Accordingly, the present invention further encompasses codon-optimized forms of the nucleotide sequences of the present invention. In particular, the present invention encompasses polynucleotide sequences comprising codon-optimized sequences of SEQ ID NOs 101 - 104. Also encompassed are variants of SEQ ID NOs: 101 - 104, wherein said variants have at least 60% identity to the sequences of SEQ ID NOs 101 - 104 and encode an antibody or a portion thereof that is capable of binding CLEC14A. In this regard, SEQ ID NOs 101 and 103 are codon-optimized sequences for expression in a human host or cell, and SEQ ID NOs 102 and 104 are codon-optimized sequences for expression in a murine host or cell, wherein SEQ ID NOs 101 - 104 encode the scFv of the present invention. In particular, SEQ ID NOs: 101 and 102 represent the human and murine expression codon-optimized sequences of SEQ ID NO.19, respectively, and SEQ ID NOs 103 and 104 represent the human and murine expression codon-optimized sequences of SEQ ID NO.39, respectively.

[0225] The antibody and nucleic acid molecules of the present invention are generally "isolated" or "purified" molecules insofar as they are distinguishable from any such components that may be present in situ within the body of a human or animal or within a tissue sample derived from the body of a human or animal. However, the sequences may correspond to or be substantially homologous to sequences found within the body of a human or animal. Thus, as used herein with respect to nucleic acid molecules or sequences and proteins or polypeptides (e.g., antibodies), the terms "isolated" or "purified" refer to such molecules when separated, purified, or substantially removed from their natural environment, e.g., isolated or purified from the body of a human or animal (if they are indeed naturally occurring), or to such molecules when produced by technical means, i.e., including recombinantly and synthetically produced molecules.

[0226] Thus, when used in conjunction with nucleic acid molecules, such terms can refer to nucleic acids that are substantially free of substances naturally associated therewith, such as other nucleic acids / genes or polypeptides. These terms can also refer to nucleic acids that are substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Isolated or purified nucleic acids can also be substantially free of sequences that flank the nucleic acid in nature (i.e., sequences located at the 5' and 3' ends of the nucleic acid), or sequences that have been prepared, for example, by genetic engineering to flank the nucleic acid (e.g., tag sequences or other sequences that have no therapeutic value).

[0227] Thus, when used in conjunction with protein or polypeptide molecules, such as light chain CDR1, 2, and 3, heavy chain CDR1, 2, and 3, light chain variable region, heavy chain variable region, and the antibodies of the present invention (including full-length antibodies), the terms "isolated" or "purified" generally refer to proteins that are substantially free of cellular material or other proteins from their source of derivation. In some embodiments, particularly in the case of administering the protein to a human or animal, such isolated or purified proteins are substantially free of culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Such isolated or purified proteins can also be free of flanking sequences, such as those described above for isolated nucleic acid molecules.

[0228] As mentioned above, the inventors have determined that the antigen-binding domains of the antibodies of the present invention, more particularly the variable regions (VL and VH chains) of said domains, can provide effective CARs for adoptive cell transfer therapy against cells expressing CLEC14A, particularly tumor cells (e.g., tumor vasculature).

[0229] Accordingly, the present invention further provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR) against the antigen CLEC14A, wherein the CAR is capable of binding to the antigen CLEC14A expressed on the surface of a target cell when expressed on the surface of an immune effector cell, and comprises an antigen-binding domain, the antigen-binding domain comprising:

[0230] (1)(a) VH CDR sequences, which comprise:

[0231] (i) VH CDR1, which has the amino acid sequence SEQ ID NO:2 or 42; and / or

[0232] (ii) VH CDR2, which has the amino acid sequence SEQ ID NO:3 or 43; and / or

[0233] (iii) VH CDR3, which has the amino acid sequence SEQ ID NO:4 or 44; and / or

[0234] (b) VL CDR sequences, comprising:

[0235] (i) VL CDR1, having the amino acid sequence SEQ ID NO: 6 or 46; and / or

[0236] (ii) VL CDR2, having the amino acid sequence SEQ ID NO: 7 or 47; and / or

[0237] (vi) VL CDR3, having the amino acid sequence SEQ ID NO: 8 or 48; and / or

[0238] One or more sequences that are substantially homologous to the SEQ ID NOs shown in (a) or (b); or

[0239] (2) (a) VH CDR sequences, comprising:

[0240] (i) VH CDR1, having the amino acid sequence SEQ ID NO: 22; and / or

[0241] (ii) VH CDR2, having the amino acid sequence SEQ ID NO: 23; and / or

[0242] (iii) VH CDR3, having the amino acid sequence SEQ ID NO: 24; and / or

[0243] (b) VL CDR sequences, comprising:

[0244] (i) VL CDR1, having the amino acid sequence SEQ ID NO: 26; and / or

[0245] (ii) VL CDR2, having the amino acid sequence SEQ ID NO: 27; and / or

[0246] (iii) VL CDR3, having the amino acid sequence SEQ ID NO: 28; and / or

[0247] One or more sequences that are substantially homologous to the SEQ ID NOs shown in (a) or (b).

[0248] In some embodiments, the CAR comprises more than one CDR, e.g., 2, 3, 4, 5, or 6 CDRs. Thus, in some embodiments, the CAR can comprise a VH sequence containing one, two, or three CDRs and / or a VL sequence containing one, two, or three CDRs. Accordingly, in some embodiments, the present invention provides a nucleic acid molecule encoding a CAR against the antigen CLEC14A, wherein the CAR, when expressed on the surface of an immune effector cell, is capable of binding to the antigen CLEC14A expressed on the surface of a target cell and comprises an antigen-binding domain, the antigen-binding domain comprising a VH sequence and a VL sequence each comprising three CDR sequences, wherein:

[0249] (1) The VH sequence comprises:

[0250] (i) VH CDR1, which has the amino acid sequence SEQ ID NO:2 or 42;

[0251] (ii) VH CDR2, which has the amino acid sequence SEQ ID NO:3 or 43; and / or

[0252] (iii) VH CDR3, which has the amino acid sequence SEQ ID NO:4 or 44; and / or

[0253] wherein the VL sequence comprises:

[0254] (iv) VL CDR1, which has the amino acid sequence SEQ ID NO:6 or 46;

[0255] (v) VL CDR2, which has the amino acid sequence SEQ ID NO:7 or 47; and / or

[0256] (vi) VL CDR3, which has the amino acid sequence SEQ ID NO:8 or 48; or

[0257] One or more sequences substantially homologous to the SEQ ID NOs shown in (1)(i)-(1)(vi); or

[0258] (2) The VH sequence comprises:

[0259] (i) VH CDR1, which has the amino acid sequence SEQ ID NO:22;

[0260] (ii) VH CDR2, which has the amino acid sequence SEQ ID NO:23; and / or

[0261] (iii) VH CDR3, which has the amino acid sequence SEQ ID NO:24; and / or

[0262] Wherein said VL sequence comprises:

[0263] (iv) VL CDR1, which has the amino acid sequence SEQ ID NO: 26;

[0264] (v) VL CDR2, which has the amino acid sequence SEQ ID NO: 27; and / or

[0265] (vi) VL CDR3, which has the amino acid sequence SEQ ID NO: 28; or

[0266] One or more sequences that are substantially homologous to the SEQ ID NOs shown in (2)(i)-(2)(vi).

[0267] In a preferred embodiment, the substantially homologous sequences have 1, 2, or 3 amino acid substitutions, additions, or deletions in said CDRs. Alternatively, one or more of said CDR sequences may optionally be modified by substitution, addition, or deletion of 1 to 3 amino acids. In a particularly preferred embodiment, the amino acid substitutions are conservative substitutions.

[0268] The CDR sequences in (1) are or correspond to the CDR sequences contained in the VH sequences of SEQ ID NO. 1 and 41 and the VL sequences of SEQ ID NO. 5 and SEQ ID NO. 45. The CDR sequences in (2) are or correspond to the CDR sequences contained in the VH sequence of SEQ ID NO. 21 and the VL sequence of SEQ ID NO. 25. SEQ ID NO. 1, 41, 5, and 45 and SEQ ID NO. 21 and 25 represent the amino acid sequences of the VH and VL regions of the antibodies described above (SEQ ID NO. 11, 54, 15, and 58 and SEQ ID NO: 31 and 35 represent the nucleotide sequences encoding said amino acid sequences).

[0269] In a preferred embodiment, the CAR comprises at least the corresponding VH and VL sequences, such as the CDR3s of SEQ ID NO: 4 and 8, SEQ ID NO: 44 and 48 or SEQ ID NO: 24 and 28 (preferably the unmodified sequences). In a particularly preferred embodiment, the CAR comprises at least two CDRs of the corresponding VH and VL sequences, such as at least two of SEQ ID NO: 2, 3, and 4 (i.e., at least 2 and 3, 2 and 4, or 3 and 4) or at least two of SEQ ID NO: 42, 43, and 44 (i.e., at least 42 and 43, 42 and 44, or 43 and 44) and at least two of SEQ ID NO: 6, 7, and 8 (i.e., at least 6 and 7, 6 and 8, or 7 and 8) or at least two of SEQ ID NO: 46, 47, and 48 (i.e., at least 46 and 47, 46 and 48, or 47 and 48), or a combination thereof, or at least two of SEQ ID NO: 22, 23, and 24 (i.e., at least 22 and 23, 22 and 24, or 23 and 24) and at least two of SEQ ID NO: 26, 27, and 28 (i.e., at least 26 and 27, 26 and 28, or 27 and 28). Preferably, the at least two CDRs are unmodified. In a particularly preferred embodiment, the CAR comprises all of the CDRs of the corresponding VH and VL sequences, i.e., SEQ ID NO: 2-4 and 6-8, or 42-44 and 46-48, or SEQ ID NO: 22-24 and 26-28, preferably wherein the CDRs are unmodified.

[0270] More specifically, when expressed on the surface of an immune effector cell, the CAR is capable of directing the immune effector cell against a target cell expressing CLEC14A. In other words, the immune cell can direct its action or function, e.g., its cytotoxic activity against the target cell, particularly a target cancer cell, e.g., an endothelial cell in a blood vessel, particularly an endothelial cell in a tumor blood vessel.

[0271] As is known in the art and described elsewhere herein, each of the VL and VH chains of an antibody comprises 3 CDRs separated by frameworks, which framework regions serve as scaffolds for the CDRs. Thus, the VL and VH sequences of the CARs of the present invention comprise the CDR sequences of the VL and VH sequences of the antibodies of the present invention separated by frameworks. The framework regions can be the framework regions of the VL and VH chains of the antibodies of the present invention, but need not be so. Thus, the framework regions of the VL and VH chains of the antibody can be modified, which includes that they can be replaced (and thus the amino acid sequence of the framework region can be modified and / or replaced), e.g., they can be humanized, as described elsewhere herein.

[0272] In a specific embodiment, the present invention provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR) against the antigen CLEC14A, wherein the CAR, when expressed on the surface of an immune effector cell, is capable of binding to the antigen CLEC14A expressed on the surface of a target cell and comprises an antigen-binding domain that comprises a VH sequence of SEQ ID NO.1 or 41 or an amino acid sequence having at least 95% sequence identity therewith, and / or a VL sequence of SEQ ID NO.5 or 45 or an amino acid sequence having at least 95% sequence identity therewith.

[0273] In another embodiment, the present invention provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR) against the antigen CLEC14A, wherein the CAR, when expressed on the surface of an immune effector cell, is capable of binding to the antigen CLEC14A expressed on the surface of a target cell and comprises an antigen-binding domain that comprises a VH sequence of SEQ ID NO.21 or an amino acid sequence having at least 95% sequence identity therewith and a VL sequence of SEQ ID NO.25 or an amino acid sequence having at least 95% sequence identity therewith.

[0274] In other embodiments, the framework regions of the VL and VH sequences are modified, and the CAR may comprise an antigen-binding domain that comprises a VH sequence having an amino acid sequence as shown in SEQ ID NO.1 or 41 or an amino acid sequence having at least 60% sequence identity therewith and / or a VL sequence having an amino acid sequence as shown in SEQ ID NO.5 or 45 or an amino acid sequence having at least 60% sequence identity therewith, preferably provided that the CDR sequences of SEQ ID NOs.2, 3, 4, 6, 7, and 8 and / or SEQ ID NOs.42, 43, 44, 46, 47, and 48 are retained (i.e., not modified or altered).

[0275] In other embodiments, the framework regions of the VL and VH sequences are modified, and the CAR may comprise an antigen-binding domain that comprises a VH sequence having an amino acid sequence as shown in SEQ ID NO.21 or an amino acid sequence having at least 60% sequence identity therewith and / or a VL sequence having an amino acid sequence as shown in SEQ ID NO.25 or an amino acid sequence having at least 60% sequence identity therewith, preferably provided that the CDR sequences of SEQ ID NOs.22, 23, 24, 26, 27, and 28 are retained (i.e., not modified or altered).

[0276] Accordingly, it should be understood that in such embodiments, the CDR sequences of the antibody are retained or substantially retained (i.e., they may optionally be modified within the limitations set forth above, such as substitutions, additions or deletions of 1 to 3 amino acids, such that the binding specificity of the antibody is retained (e.g., unchanged)).

[0277] The antigen-binding domain is extracellular (i.e., when the CAR is expressed on an immune effector cell). Thus, the CAR comprises an extracellular domain containing the antigen-binding domain, which antigen-binding domain comprises antibody-based VL and VH sequences as defined above. As will be described in more detail below, the extracellular domain may also comprise a signal sequence, more particularly a plasma membrane targeting sequence, especially a plasma membrane targeting sequence based on the VL chain.

[0278] The nucleic acid molecules of the invention can be used to prepare immune effector cells (more particularly, modified immune effector cells) directed against cells expressing CLEC14A. Such (modified) immune effector cells express the CAR on their cell surface and are capable of recognizing or binding to target cells expressing CLEC14A, such as endothelial cells in blood vessels, particularly endothelial cells in tumor blood vessels. Thus, the nucleic acid molecules enable immune effector cells expressing the CAR (i.e., the CAR encoded by the nucleic acid molecules) to have effector activity (such as cytotoxic activity) against (e.g., killing) target cells expressing CLEC14A. Accordingly, the modified immune effector cells are genetically modified or engineered immune effector cells, or immune effector cells that have been transduced with the nucleic acid molecules of the invention.

[0279] In a method of generating CLEC14A-specific immune effector cells, the immune effector cells modified by introduction of the nucleic acid molecules of the invention can be obtained from a subject to be treated (such as a subject suffering from a tumor). After modification of the immune effector cells and optionally in vitro expansion, the subject can be re-administered (i.e., administered) the modified immune effector cells expressing the CAR. Accordingly, autologous immune effector cells can be used in the therapeutic compositions, methods and uses of the invention discussed further below. Alternatively, heterologous (i.e., donor or allogeneic, or syngeneic or xenogeneic) immune effector cells can be used.

[0280] The immune effector cells can be any immune cells capable of an immune response against target cells expressing CLEC14A. More specifically, the immune effector cells are capable of eliminating, destroying or deleting the target cells, i.e., reducing or inhibiting the viability of the target cells, preferably killing the target cells (in other words, making the target cells fewer or non-viable). Accordingly, the immune effector cells are preferably cytotoxic immune effector cells.

[0281] The term "cytotoxic" is synonymous with "cytolytic" and is used herein to refer to cells capable of inducing cell death by lysis or apoptosis in target cells.

[0282] As used herein, the term "immune effector cell" includes not only mature or fully differentiated immune effector cells, but also their precursor cells (or progenitor cells), including stem cells (more particularly hematopoietic stem cells, HSCs) or cells derived from HSCs. Thus, immune effector cells can be T cells, NK cells, NKT cells, neutrophils, macrophages, or cells derived from hematopoietic tissue, such as CD34+ cell populations derived from HSCs contained in bone marrow, umbilical cord blood, or blood, such as mobilized peripheral blood, which differentiate into mature immune effector cells after administration to a subject. As will be described in more detail below, in a preferred embodiment, the immune effector cells are T cells or NK cells. Primary cells can be used, such as cells isolated from a subject to be treated or from a donor subject, optionally in the presence of an intervening cell culture step (such as to expand the cells) or other cultured cells or cell lines (such as an NK cell line, such as the NK92 cell line).

[0283] The term "directed against antigen CLEC14A" is synonymous with "specific for CLEC14A" or "anti-CLEC14A", i.e., it merely means that the CAR is capable of specifically binding CLEC14A. In particular, the antigen-binding domain of the CAR is capable of specifically binding CLEC14A (more particularly when the CAR is expressed on the surface of an immune effector cell). Specific binding can be distinguished from non-specific binding to non-target antigens (in this case antigens other than CLEC14A). Thus, immune effector cells expressing a CAR according to the invention are redirected to specifically bind to target cells expressing CLEC14A and exhibit cytotoxicity (e.g., killing) against target cells expressing CLEC14A. Alternatively, immune effector cells are modified to redirect cytotoxicity to target cells expressing CLEC14A.

[0284] In one embodiment, specific binding to CLEC14A can mean that the antigen-binding domain (or CAR comprising the antigen-binding domain) binds or associates with CLEC14A (or more particularly a target cell expressing CLEC14A on its cell surface) with an affinity or Ka (i.e., equilibrium binding constant) of greater than or equal to about 10 5 M -1 , for example at least 10 6 M -1 , 10 7 M -1 , or 10 8 M -1 .

[0285] Binding of the antigen - binding domain of a CAR to its target antigen on the surface of a target cell delivers an activating stimulus to the CAR - containing cell, resulting in induction of effector cell signaling pathways. Thus, binding to the target antigen can trigger proliferation, cytokine production, phagocytosis, lytic activity, and / or the production of molecules that can mediate the death of the target cell in an MHC - independent manner. Although CARs containing an intracellular domain with a signaling domain consisting of only CD3ζ or FcRγ can deliver potent signals for immune cell activation and effector function, they may not be sufficient to initiate signals that promote the survival and expansion of immune effector cells in the absence of an accompanying co - stimulatory signal. Thus, it can be preferred that the CAR contains one or more co - stimulatory signaling domains.

[0286] Thus, the CARs of the present invention generally comprise 3, 4, or preferably 5 domains as follows:

[0287] (1) An antigen - binding domain capable of specifically binding CLEC14A, which comprises VH and VL sequences based on or derived from SEQ ID NO.1 and 5 or 41 and 45 or 21 and 25 as defined above;

[0288] (2) Optionally, a hinge domain that extends the antigen - binding domain away from the surface of the immune effector cell;

[0289] (3) A transmembrane domain that anchors the CAR to the effector cell and connects the extracellular domain containing the antigen - binding domain to the intracellular signaling domain;

[0290] (4) An intracellular domain containing a signaling domain; and optionally or preferably;

[0291] (5) One or more co - stimulatory signaling domains.

[0292] The CAR can also comprise (6) a signal sequence (i.e., a targeting domain), and in particular a sequence that targets the CAR to the plasma membrane of the immune effector cell. This is generally located adjacent to or near the antigen - binding domain at the end of the CAR molecule / construct, typically upstream of the antigen - binding domain.

[0293] Thus, it can be seen that a CAR can comprise an extracellular domain containing an antigen-binding domain and a signal sequence (if present), connected via an optional hinge domain and a transmembrane domain to an intracellular domain containing one or more signaling domains. In one aspect, the intracellular domain or the optional hinge, transmembrane, and intracellular domains can be regarded as the "signaling tail" in the CAR construct. Thus, the order of domains in the CAR construct is N-terminus to C-terminus: extracellular domain - optional hinge domain - transmembrane domain - intracellular domain. Within the extracellular and intracellular domains, the separate domains can be arranged in any order. However, preferably, the order is signal sequence - antigen-binding domain in the extracellular domain. In one embodiment, within the intracellular domain, the order can be co-stimulatory domain - intracellular signaling domain. In another embodiment, the order can be intracellular signaling domain - co-stimulatory domain.

[0294] In the CAR of the present invention, the "antigen-binding domain" comprising the variable region sequence derived from the antibody of the present invention can be provided in various forms, as long as it contains the VL and VH sequences as defined above. Thus, it can be or can correspond to a natural or synthetic antibody sequence. Thus, the nucleotide sequence encoding the antigen-binding domain in the nucleic acid molecule of the present invention can be derived from or can correspond to a natural sequence or can encode a genetically engineered product. Thus, the antigen-binding domain can be (or more precisely can correspond to) a fragment of the antibody of the present invention comprising variable regions (antibody light and heavy chain variable regions; VL and VH regions), such as Fv or Fab or Fab2 or the light and heavy chain variable regions can be joined together in a single chain and in either orientation (e.g., VL-VH or VH-VL). As discussed above, the VL and / or VH sequences can be modified. In particular, the framework regions can be modified (e.g., substitutions, such as humanizing the antigen-binding domain).

[0295] In a preferred embodiment, the binding domain is a single-chain antibody (scFv) derived from the antibody of the present invention, such as based on or derived from SEQ ID NO.9, 29, 49, 50, or 51.

[0296] In a preferred embodiment, the VL and VH are joined together by a linker sequence. More precisely, this can be referred to as a "variable region linker sequence", which is an amino acid sequence that joins the heavy chain variable region to the light chain variable region and provides a spacer function compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains the specific binding affinity for the same target molecule as the antibody comprising the same light and heavy chain variable regions. The linker sequence can be used to provide appropriate spacing and conformation of the molecule.

[0297] Thus, in one embodiment, the scFv comprises a VH sequence of SEQ ID NO.1 or 41 or a sequence having at least 95% sequence identity thereto, linked to a VL sequence of SEQ ID NO.5 or 45 or a sequence having at least 95% sequence identity thereto, preferably linked in the order of VL-VH.

[0298] In a further embodiment, the scFv comprises a VH sequence of SEQ ID NO.21 or a sequence having at least 95% sequence identity thereto, linked to a VL sequence of SEQ ID NO.25 or a sequence having at least 95% sequence identity thereto, preferably linked in the order of VL-VH.

[0299] In another embodiment, the scFv comprises a VH sequence of SEQ ID NO.1 or 41 or a sequence having at least 60% sequence identity thereto, linked to a VL sequence of SEQ ID NO.5 or 45 or a sequence having at least 60% sequence identity thereto, preferably linked in the order of VL-VH. As described above, this is subject to the condition that the CDR sequences as defined above are retained, and preferably the condition that the CDR sequences are unchanged.

[0300] In a further embodiment, the scFv comprises a VH sequence of SEQ ID NO.21 or a sequence having at least 60% sequence identity thereto, linked to a VL sequence of SEQ ID NO.25 or a sequence having at least 60% sequence identity thereto, preferably linked in the order of VL-VH. As described above, this is subject to the condition that the CDR sequences as defined above are retained, and preferably the condition that the CDR sequences are unchanged.

[0301] More preferably, the VL sequence is linked to the VH via a linker sequence. The linker sequence can be 1-30, more preferably 1-25, 1-22 or 1-20 amino acids in length. The linker can be a flexible linker. Suitable linkers can be readily selected and can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids or longer.

[0302] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers, where n is an integer of at least 1, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can thus serve as neutral tethers between domains of fusion proteins such as CARs as described herein. In a representative embodiment, the linker sequence can be (G4S)3 (SEQ ID NO. 65).

[0303] Accordingly, in a representative embodiment, a nucleic acid molecule of the invention can comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 9 or a sequence having at least 95% sequence identity thereto, the amino acid sequence sequentially comprising the VH of SEQ ID NO. 1, the linker of SEQ ID NO. 65, and the VL of SEQ ID NO. 5.

[0304] In another representative embodiment, a nucleic acid molecule of the invention can comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 49 or a sequence having at least 95% sequence identity thereto, the amino acid sequence sequentially comprising the VH of SEQ ID NO. 41, the linker of SEQ ID NO. 65, and the VL of SEQ ID NO. 5.

[0305] In another representative embodiment, a nucleic acid molecule of the invention can comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 50 or a sequence having at least 95% sequence identity thereto, the amino acid sequence sequentially comprising the VH of SEQ ID NO: 1, the linker of SEQ ID NO. 65, and the VL of SEQ ID NO. 45.

[0306] In another representative embodiment, a nucleic acid molecule of the invention can comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 51 or a sequence having at least 95% sequence identity thereto, the amino acid sequence sequentially comprising the VH of SEQ ID NO. 41, the linker of SEQ ID NO. 65, and the VL of SEQ ID NO. 45.

[0307] In a representative embodiment, a nucleic acid molecule of the invention can comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 10 or a sequence having at least 95% sequence identity thereto, the amino acid sequence comprising the VH of SEQ IDNO. 1 and the VL of SEQ ID NO. 5. Accordingly, a nucleic acid molecule of the invention can comprise a nucleotide sequence containing SEQ ID NO. 20 or a sequence having at least 95% sequence identity thereto.

[0308] In another representative embodiment, a nucleic acid molecule of the invention can comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 30 or a sequence having at least 95% sequence identity thereto. The amino acid sequence comprises the VH of SEQ ID NO. 21 and the VL of SEQ ID NO. 25. Accordingly, a nucleic acid molecule of the invention can comprise a nucleotide sequence comprising SEQ ID NO. 40 or a sequence having at least 95% sequence identity thereto.

[0309] In some embodiments, the VH and VL regions can be encoded by nucleotide sequences comprising the nucleotide sequences of SEQ ID NOs. 11 and 15, respectively, or nucleotide sequences having at least 95% nucleotide sequence identity thereto.

[0310] In some embodiments, the VH and VL regions can be encoded by nucleotide sequences comprising the nucleotide sequences of SEQ ID NOs. 54 and 58, respectively, or nucleotide sequences having at least 95% nucleotide sequence identity thereto.

[0311] In some embodiments, the VH and VL regions can be encoded by nucleotide sequences comprising the nucleotide sequences of SEQ ID NOs. 31 and 35, respectively, or nucleotide sequences having at least 95% nucleotide sequence identity thereto.

[0312] In another embodiment, the VH and VL regions can be encoded by nucleotide sequences comprising the nucleotide sequences of SEQ ID NOs. 11 and 15 or SEQ ID NOs. 54 and 58, respectively, or nucleotide sequences having at least 60% nucleotide sequence identity thereto. As above, this is subject to the condition that the CDR sequences encoded by the nucleotide sequences as defined above are retained, and preferably the condition that the CDR sequences are unchanged.

[0313] In another embodiment, the VH and VL regions can be encoded by nucleotide sequences comprising the nucleotide sequences of SEQ ID NOs. 31 and 35, respectively, or nucleotide sequences having at least 60% nucleotide sequence identity thereto. As above, this is subject to the condition that the CDR sequences encoded by the nucleotide sequences as defined above are retained, and preferably the condition that the CDR sequences are unchanged.

[0314] If desired, the VL and VH sequences can be humanized by modifying one or more framework regions to correspond to at least one human framework region. A "human framework region" refers to the wild-type (i.e., naturally occurring) framework region of a human immunoglobulin variable region, a modified framework region of a human immunoglobulin variable region in which less than about 50% (e.g., preferably less than about 45%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%) of the amino acids in the region are deleted or replaced (e.g., replaced with one or more amino acid residues of a non-human immunoglobulin framework region at the corresponding position), or a modified framework region of a non-human immunoglobulin variable region in which less than about 50% (e.g., less than about 45%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%) of the amino acids in the region are deleted or replaced (e.g., at the position of exposed residues and / or replaced with one or more amino acid residues of a human immunoglobulin framework region at the corresponding position), thereby reducing immunogenicity in one aspect.

[0315] Thus, in one specific embodiment, the framework regions of the VH sequences of SEQ ID NOs. 1, 21 and 41 and the VL sequences of SEQ ID NOs. 5, 25 and 45 can be modified (more specifically, the amino acid sequences of the framework regions can be modified), while retaining or substantially retaining the amino acid sequences of the CDRs.

[0316] Thus, in another embodiment, the framework region of the VH sequence in the CAR has at least 60% amino acid sequence identity with the framework regions of SEQ ID NOs. 1, 21 and 41 and / or the framework region of the VL sequence in the CAR has at least 60% amino acid sequence identity with the framework regions of SEQ ID NOs. 5, 25 and 45.

[0317] As described above, the CAR, more particularly its extracellular region, can also comprise a signal sequence (or targeting domain). Such sequences are typically provided at the N-terminus of the molecule (construct) and can function to direct the trafficking of the molecule in a co-translational or post-translational manner. In particular, the signal sequence can be a sequence that targets the CAR to the plasma membrane of an immune effector cell. This can be linked directly or indirectly (e.g., through a linker sequence) to the antigen-binding domain, typically upstream of the antigen-binding domain, at the N-terminus of the CAR molecule / construct. The linker sequence can be the linker as described above in connection with the variable region linker. In one embodiment, the signal sequence is linked directly to the N-terminus of the antigen-binding domain, e.g., to the N-terminus of the VL sequence.

[0318] The antigen-binding domain of the CAR is optionally followed by a hinge domain. The hinge region in the CAR is typically located between the transmembrane domain and the antigen-binding domain. In certain embodiments, the hinge region is an immunoglobulin hinge region and can be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region, such as a truncated hinge region. Other exemplary hinge regions that can be used include hinge regions derived from the extracellular regions of type I membrane proteins such as CD8α, CD4, CD28, and CD7, which can be wild-type hinge regions from these molecules or can be altered. Preferably, the hinge region is or is derived from the hinge region of human CD8α, CD4, CD28, or CD7. Alternatively (and interchangeably), the hinge region is referred to as a spacer or spacer region.

[0319] "Altered wild-type hinge region" or "altered hinge region" or "altered spacer" means (a) a wild-type hinge region having up to 30% amino acid variation (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid variation, such as substitutions or deletions), (b) a portion of a wild-type hinge region that is at least 10 amino acids in length (e.g., at least 12, 13, 14, or 15 amino acids) and has up to 30% amino acid variation (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid variation, such as substitutions or deletions), or (c) a portion of a wild-type hinge region that includes the core hinge region (the length of which can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). When the altered wild-type hinge region is inserted between the CLEC14A-specific binding domain and another region (such as the transmembrane domain) in the chimeric antigen receptor described herein and connects them, it allows the chimeric fusion protein to maintain specific binding to CLEC14A.

[0320] In certain embodiments, one or more cysteine residues in the wild-type immunoglobulin hinge region can be replaced by one or more other amino acid residues (e.g., one or more serine residues). Alternatively / additionally, the altered immunoglobulin hinge region can have a proline residue of the wild-type immunoglobulin hinge region replaced by another amino acid residue (e.g., a serine residue).

[0321] Hinge regions containing CH2 and CH3 constant regions have been described in the art for use in CARs (e.g., the CH2CH3 hinge, referred to as the "Fc hinge" or "IgG hinge", as shown in SEQ ID NO. 72). However, preferably, when the hinge domain is based on or derived from an immunoglobulin, it does not contain the CH3 domain, e.g., it can contain the CH2 domain or a fragment or portion thereof or consist of the CH2 domain or a fragment or portion thereof, excluding the CH3.

[0322] In a preferred embodiment, the hinge domain has or comprises the amino acid sequence of SEQ ID NO.66 (which represents the hinge domain of CD8α) or an amino acid sequence having at least 95% sequence identity therewith.

[0323] In another preferred embodiment, the hinge domain has or comprises the amino acid sequence of SEQ ID NO.67 (which represents a shortened IgG hinge) or an amino acid sequence having at least 95% sequence identity therewith.

[0324] The hinge domain may be attached to the transmembrane domain via a linker sequence, which may be the linker sequence as defined above. An exemplary linker sequence is KDPK (SEQ ID NO.68). A shortened IgG hinge with a linker sequence is shown in SEQ ID NO.69. Such a sequence or a sequence having at least 95% sequence identity therewith may be included in the CARs of the present invention. More particularly, such a sequence may be included between the extracellular domain (such as the scFv portion) and the transmembrane domain.

[0325] The transmembrane domain may be based on or derived from the transmembrane domain of any transmembrane protein. Generally, it may be or may be derived from the transmembrane domains of CD8α, CD28, CD4, CD3ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154, preferably of the human origin. In one embodiment, the transmembrane domain may be or may be derived from the transmembrane domain of CD8α, CD28, CD4 or CD3ζ, preferably from the transmembrane domains of human CD28, CD4 or CD3ζ. In another embodiment, the transmembrane domain may be synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine.

[0326] In a preferred embodiment, the transmembrane domain is the CD8α transmembrane domain having the amino acid sequence of SEQ ID NO.70 or an amino acid sequence having at least 95% sequence identity therewith. This transmembrane sequence may be further attached to the hinge domain from CD8α as shown in SEQ ID NO.66 or an amino acid sequence having at least 95% sequence identity therewith.

[0327] In another embodiment, the transmembrane domain may be the transmembrane domain of human CD28 having the amino acid sequence of SEQ ID NO.71 or an amino acid sequence having at least 95% sequence identity therewith.

[0328] "Intracellular signaling domain" refers to a portion of the CAR protein that is involved in transducing information about the effective binding of the CAR to the target antigen into the interior of the immune effector cell to initiate effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to the target cells bound by the CAR, or other cellular responses induced by the binding of the antigen to the extracellular CAR domain. The term "effector function" refers to the specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or help or activity including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs the cell to perform specialized functions. While the entire intracellular signaling domain can be used, in many cases, it is not necessary to use the entire domain. In terms of using truncated portions of the intracellular signaling domain, such truncated portions can be used to replace the entire domain as long as they transduce effector function signals. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce effector function signals. The intracellular signaling domain is also referred to as the "signal transduction domain" and typically derives from portions of human CD3ζ or FcRy chains.

[0329] In addition, to permit or enhance the full activation of immune effector cells, a secondary or co-stimulatory domain can be provided to the CAR. Thus, the intracellular signaling domain can initiate antigen-dependent primary activation (i.e., can be a primary cytoplasmic signaling sequence), and the co-stimulatory domain can act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequence (S)). The primary cytoplasmic signaling sequence can regulate primary activation, including in an inhibitory manner. A primary cytoplasmic signaling sequence that acts in a co-stimulatory manner can contain signaling motifs, which are called immunoreceptor tyrosine-based activation motifs or ITAMs.

[0330] Examples of ITAMs containing primary cytoplasmic signaling sequences that can be used in the present invention include those derived from TCRζ, FcRy, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In certain specific embodiments, the intracellular signaling domain is derived from CD3ζ or FcRγ, preferably human CD3ζ or FcRγ.

[0331] In a preferred representative embodiment, the intracellular signaling domain is preferably the human CD3ζ domain, more preferably the human CD3ζ domain having the amino acid sequence of SEQ ID NO.73 or an amino acid sequence having at least 95% sequence identity thereto.

[0332] The term "costimulatory signaling domain" or "costimulatory domain" refers to the CAR portion that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a second signal, which is generally required for the effective activation and function of an immune effector cell (e.g., a T cell) upon binding of an antigen. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H2, and the ligand that specifically binds CD83, more particularly the intracellular domain of such molecules. However, in some embodiments, a costimulatory molecule provides a second signal that blocks the stimulation of an immune effector cell (e.g., a T cell) upon binding of a ligand. For example, and as discussed in more detail below, it may be useful to provide an immune effector cell that contains more than one CAR, wherein the second CAR is capable of binding a ligand presented on a non-target cell, such as a non-tumor cell, such that if the second (or additional) CAR binds its ligand, it provides a negative signal that blocks the stimulation of the immune effector cell. Examples of such costimulatory molecules include PD-1 and CTLA4. Preferably, the molecule is human. Thus, although exemplary or preferred costimulatory domains are derived from 4-1BB, CD28, or OX40 (CD134), other costimulatory domains for use with the CARs described herein are encompassed. Costimulatory domains may be used alone or in combination (i.e., may include one or more costimulatory domains). Incorporating one or more costimulatory signals can enhance the efficacy and expansion of immune effector cells expressing a CAR.

[0333] The intracellular signaling and costimulatory signaling domains can be tandemly linked to the carboxyl terminus of the transmembrane domain in any order.

[0334] In a preferred embodiment, the costimulatory domain is the intracellular domain of 4-1BB having the amino acid sequence of SEQ ID NO. 74 or an amino acid sequence having at least 95% sequence identity thereto.

[0335] In another embodiment, the costimulatory domain can be or can comprise the intracellular domain of human CD28 having the amino acid sequence of SEQ ID NO. 75 or an amino acid sequence having at least 95% sequence identity thereto and / or the OX40 (CD134) costimulatory domain having the amino acid sequence of SEQ ID NO. 76 or an amino acid sequence having at least 95% sequence identity thereto.

[0336] In a preferred embodiment of the present invention, the CAR (or more specifically, its "signaling tail") comprises an optional hinge domain from CD8α or a truncated IgG hinge domain lacking the CH3 domain, a CD8α transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain.

[0337] In other embodiments, the CAR (or its "signaling tail") comprises an optional hinge domain from CD8α or a truncated IgG hinge domain lacking the CH3 domain, a CD28 transmembrane domain, a CD28 intracellular domain, and / or an OX40 co-stimulatory domain, and a CD3ζ intracellular signaling domain.

[0338] Furthermore, the polynucleotides of the present invention can encode a CAR that comprises 1) a transmembrane and co-stimulatory domain from CD28 and an intracellular signaling domain from CD3ζ; 2) a transmembrane domain from CD8α, a co-stimulatory domain from 4-1BB, and an intracellular signaling domain from CD3ζ; 3) a transmembrane domain from CD8α, a co-stimulatory domain from OX40, and an intracellular signaling domain from CD3ζ; 4) a transmembrane domain from CD28, co-stimulatory domains from CD28 and 4-1BB, and an intracellular signaling domain from CD3ζ; 5) a transmembrane domain from CD28, co-stimulatory domains from CD28 and OX40, and an intracellular signaling domain from CD3ζ; 6) a transmembrane domain from CD8α, co-stimulatory domains from 4-1BB and OX40, and an intracellular signaling domain from CD3ζ; 7) a transmembrane domain from CD8α, a co-stimulatory domain from CD28, and an intracellular signaling domain from CD3ζ; 8) a transmembrane domain from CD8α, co-stimulatory domains from CD28 and 4-1BB, and an intracellular signaling domain from CD3ζ; or 9) a transmembrane domain from CD8α, co-stimulatory domains from CD28 and OX40, and an intracellular signaling domain from CD3ζ. In particular, any construct comprising a transmembrane domain from CD8α can further comprise a hinge or spacer that also derives from CD8α.

[0339] Such CARs according to the present invention can include an scFv antigen-binding domain as defined above and can also comprise a plasma membrane targeting sequence located upstream of the scFv.

[0340] Thus, in certain representative embodiments, the CARs of the present invention comprise an extracellular domain having the sequence of SEQ ID NO. 9, 19, 49, 50, or 51 or a sequence having at least 95% sequence identity thereto, in addition to the signaling tail as defined above.

[0341] Thus, representative CARs according to the present invention can have or comprise the amino acid sequence of SEQ ID NO. 10 or 30 or an amino acid having at least 95% sequence identity thereto.

[0342] The nucleic acid molecule of the present invention may comprise the nucleotide sequence of SEQ ID NO.20 or SEQ ID NO:40, or a nucleotide sequence having at least 95% sequence identity thereto.

[0343] The present disclosure provides CAR polypeptides and fragments thereof. In particular, the present invention provides a CAR encoded by a nucleic acid molecule of the present invention as previously defined. The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acids, not limited to any particular length. The term does not exclude modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and addition or deletion of signal sequences. The term "polypeptide" or "protein" or "peptide" refers to one or more chains of amino acids, where each chain contains amino acids covalently linked by peptide bonds, and where the polypeptide or protein may comprise multiple chains non-covalently and / or covalently linked together by peptide bonds, having the sequence of a natural protein (i.e., a protein produced by a naturally occurring and in particular non-recombinant cell, or a genetically engineered or recombinant cell), and comprising a molecule having the amino acid sequence of a natural protein, or a molecule having a deletion, addition, and / or substitution of one or more amino acids of the natural sequence. The terms "polypeptide" and "protein" specifically encompass the CARs of the present disclosure, or sequences having a deletion, addition, and / or substitution of one or more amino acids of a CAR as disclosed herein.

[0344] As is clear from the foregoing, the various domains of a CAR may comprise one or more amino acid sequence modifications relative to the natural sequence of the molecule from which they are derived. For example, it may be desirable to improve the binding affinity and / or other biological properties of the CAR. For example, amino acid sequence variants of a CAR, or a binding domain, or its stimulatory signaling domain, can be prepared by introducing appropriate nucleotide changes into the polynucleotide encoding the CAR or its domain. Such modifications include, for example, deletions of residues within the amino acid sequence of the CAR, and / or insertions and / or substitutions. For example, any combination of deletions, insertions, and substitutions can be made to obtain the final CAR, provided that the final construct possesses the desired characteristics, such as specific binding of the binding domain to CLEC14A, or increased signaling achieved through the intracellular signaling domain and / or co-stimulatory domain. Amino acid changes can also alter the post-translational processes of the CAR, such as changing the number or location of glycosylation sites. Any of the foregoing changes and modifications can be included in the CARs of the present invention.

[0345] In a specific embodiment, the various domains of the CAR (except for the VL and VH sequences) can have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% or 99% identical to the native sequence of the domain of the protein from which they are derived. Thus, in certain embodiments, the domain can have an amino acid sequence having at least 80, 85, 90, 95, 98 or 99% sequence identity to any of SEQ ID NOs. 66 - 76.

[0346] Those of ordinary skill in the art will understand that due to the degeneracy of the genetic code, there are many nucleotide sequences that can encode the CAR as described herein.

[0347] The nucleic acid molecule can be introduced into a host cell, particularly an immune effector cell, as mRNA or DNA for expression in the cell. A vector can be used to transfer the nucleic acid molecule into the cell or to generate the nucleic acid for transfer (e.g., to generate mRNA for transfer, or to generate a nucleic acid molecule for preparing an expression vector for transfer into the cell).

[0348] Thus, another aspect of the invention provides a vector comprising the nucleic acid molecule of the invention as defined herein.

[0349] The vector can be, for example, an mRNA expression vector, a cloning vector or an expression vector for transfer into immune cells, such as a viral vector.

[0350] Thus, another aspect of the invention provides a virus comprising the nucleic acid molecule or vector of the invention as defined herein.

[0351] Another aspect of the invention provides a host cell, particularly an immune effector cell, comprising the nucleic acid molecule or vector (or CAR) of the invention as defined herein.

[0352] In a preferred embodiment, the immune effector cell can be a T cell or an NK cell.

[0353] Also provided is a method of generating a host cell, particularly a CLEC14A - specific immune effector cell, the method comprising introducing the nucleic acid molecule or vector of the invention as defined herein into a host cell, particularly an immune effector cell.

[0354] Such methods can include stimulating the cells and inducing their proliferation before and / or after introducing the nucleic acid molecule or vector.

[0355] The nucleic acid molecules and vectors of the present invention can be introduced into host cells to produce the antibodies or CARs of the present invention. Accordingly, another aspect of the present invention is a method for producing the antibodies or CARs of the present invention, which includes culturing host cells containing the nucleic acid molecules or vectors of the present invention under conditions for expressing the antibodies, and recovering the molecules so produced.

[0356] The vector or construct (nucleic acid molecule) can be introduced into the cells of the present invention by a variety of means, including chemical transfection agents (such as calcium phosphate, branched organic compounds, liposomes or cationic polymers), electroporation, cell squeezing, sonoporation, optical transfection, hydrodynamic delivery or viral transduction. In a preferred embodiment, the vector or construct is introduced by viral transduction. This can allow for more persistent expression of the CAR. However, in certain cases, such as in clinical trials, or in certain clinical situations, it may be desirable to have a more transient CAR protein expression period. In such cases, it may be desirable to deliver the nucleic acid molecule as mRNA to immune effector cells. The mRNA expression vectors for producing mRNA can be prepared according to methods known in the art (such as using Gateway Technology) and are known in the art (such as pClpA102, -Larssen et al, 2002, J. Immunol. Methods 259, p 191-203 and pCIpA120-G, et al, 2011, PLoS ONE 6(11)e27930).

[0357] mRNA can be produced in vitro, for example, by in vitro transcription. Then, the mRNA can be introduced into immune effector cells, for example, as naked mRNA, for example, by electroporation (such as as described in Almasbak et al., Cytotherapy 2011, 13, 629-640, Rabinovich et al., Hum. Gene Ther., 2009, 20, 51-60 and Beatty et al., Cancer Immunol. Res. 2014, 2, 112-120). Alternatively, the mRNA can be introduced by other means, such as by liposomes or cationic molecules, etc. The heterologous nucleic acid molecule introduced into the cell can be expressed in an episomal form or can be integrated into the genome of the cell at a suitable locus.

[0358] Gene segments that render the immune effector cells (e.g., T cells) of the present invention susceptible to negative selection in vivo are within the scope of the present invention. "Negative selection" refers to the elimination of the infused cells that can be caused by a change in the in vivo condition of an individual. A negative selection phenotype can be caused by the insertion of a gene (e.g., a so-called suicide gene) that confers sensitivity to a reagent (e.g., a compound) administered. Negative selection genes are known in the art and particularly include the following: Herpes simplex virus type I thymidine kinase (HSV-I TK) gene that confers sensitivity to ganciclovir (Wigler et al., Cell 11(1):223-232, 1977); cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33-37(1992)).

[0359] In some embodiments, it may be useful to include a positive marker in the genetically modified immune effector cells (e.g., T cells) that can select for cells with a negatively selectable phenotype in vitro. A positive selection marker can be a gene that, when introduced into a host cell, expresses a dominant phenotype that allows for the positive selection of cells carrying the gene. Genes of this type are known in the art and particularly include the hygromycin-B phosphotransferase gene (hph) that confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene (neo or aph) from Tn5 that encodes resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multidrug resistance (MDR) gene. Other positive selection markers can include genes that encode proteins expressed in or on the cell membrane that allow for the sorting of transduced cells, e.g., genes that encode fluorescent proteins (e.g., GFP), and the fluorescent proteins enable the selection of transduced cells using fluorescence-activated cell sorting (FACS). Any suitable positive selection marker can be used in the present invention.

[0360] Preferably, a positive selection marker and a negative selectable element are linked such that loss of the negative selectable element is necessarily accompanied by loss of the positive selection marker. Even more preferably, the positive and negative selection markers are fused such that loss of one necessarily results in loss of the other. An example of a fusion polynucleotide that produces a polypeptide as an expression product that confers both the desired positive and negative selection characteristics is the hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene produces a polypeptide that confers hygromycin B resistance for positive selection in vitro and ganciclovir sensitivity for negative selection in vivo. See Lupton S.D., et al, Mol. and Cell. Biology 11:3374-3378, 1991.

[0361] In some embodiments, it may be desirable to express a polypeptide from the CAR expression vector of the invention to permit detection of CAR expression in immune effector cells. Thus, it may be possible to identify successful transduction of immune effector cells with the vector and successful expression of the CAR molecule by detecting the expression of another polypeptide directed under the control of the same (or a different promoter) as the nucleotide sequence encoding the CAR. In particular, the CAR molecule of the invention may additionally comprise a CD34 molecule or a modified CD34 molecule, such as a truncated CD34 molecule, wherein such molecule comprises an extracellular portion that permits detection by known techniques, e.g., immunofluorescent detection using a suitable antibody and label. In one specific embodiment, the vector of the invention may additionally comprise the nucleotide sequence of SEQ ID NO.77 or a nucleotide sequence having at least 80% sequence identity thereto. Alternatively, the vector may additionally encode the amino acid sequence of SEQ ID NO.78 or a sequence having at least 80% sequence identity thereto.

[0362] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC). Thus, representative immune effector cells include T lymphocytes, particularly cytotoxic T cells (CTL; CD8+ T cells) and helper T cells (HTL; CD4+ T cells). Other T cell populations may also be used herein, such as naive T cells and memory T cells. Other immune effector cells include NK cells, NKT cells, neutrophils, and macrophages. As described above, immune effector cells also include progenitors of effector cells, where such progenitors can be induced to differentiate into immune effector cells in vivo or in vitro.

[0363] T cells, particularly CD8+ T cells and NK cells represent preferred immune effector cells according to the invention.

[0364] The term "NK cell" refers to large granular lymphocytes, which are cytotoxic lymphocytes derived from common lymphoid progenitors and do not naturally contain antigen-specific receptors (such as T cell receptors or B cell receptors). NK cells can be phenotypically distinguished by their CD3 - , CD56 + . Thus, as used herein, the term includes any known NK cell or any NK-like cell or any cell having NK cell characteristics. Thus, primary NK cells can be used, or in alternative embodiments, NK cells that have been previously isolated and cultured and are known in the art can be used. Thus, NK cell lines can be used. Many different NK cells are known and reported in the literature, and any of these cells can be used, or cell lines can be prepared from primary NK cells, for example, by viral transformation (Vogel et al. 2014, Leukaemia 28:192-195). In addition to NK-92, suitable NK cells include (but are by no means limited to) NK-YS, NK-YT, MOTN-1, NKL, KHYG-1, HANK-1, or NKG cell lines. In a preferred embodiment, the cells are NK-92 cells (Gong et al. 1994, Leukaemia 8:652-658) or variants thereof. Many different variants of the original NK-92 cells have been prepared and described or are available, including non-immunogenic NK-92 variants. Any such variant can be used and is included in the term "NK-92". Variants of other cell lines can also be used.

[0365] When the immune effector cell is a non-autologous cell (i.e., a donor cell) for therapeutic use, preferably it is non-immunogenic so that when administered to a subject, it does not produce an effect, interfere with, or prevent the immune response in which the cell is used in the treatment.

[0366] NK cells can be naturally non-immunogenic, but NK cells or other immune effector cells can be modified to be non-immunogenic. Naturally non-immunogenic NK cells do not express MHC molecules or express MHC molecules only weakly, or can express non-functional MHC molecules that do not stimulate an immune response. Immune effector cells that are immunogenic can be modified to eliminate the expression of MHC molecules or to express MHC molecules only weakly on their surface. Alternatively, such cells can be modified to express non-functional MHC molecules.

[0367] Any means of disrupting the expression of functional MHC molecules is encompassed. Thus, this can include knocking out or knocking down the molecules of the MHC complex, and / or it can include modifications that prevent proper trafficking to the cell surface and / or proper expression of MHC molecules or the entire complex at the cell surface.

[0368] In particular, the expression of one or more functional MHC class I proteins on the surface of the cells of the present invention can be disrupted. In one embodiment, the cells can be HLA-negative human cells and thus the expression of one or more HLA molecules, such as the molecules of the HLA-MHC class I complex, is disrupted (e.g., knocked out).

[0369] In a preferred embodiment, disruption of MHC class I can be carried out by knocking out the gene encoding β2-microglobulin (β2m) (a component of the mature MHC class I complex). The expression of β2m can be eliminated by targeted disruption of the β2m gene, for example, by site-directed mutagenesis of the β2m promoter (to inactivate the promoter), or by introducing an inactivating mutation within the gene encoding the β2m protein that prevents the expression of the β2m protein, such as by introducing a frameshift mutation or a premature "stop" codon within the gene. Alternatively, site-directed mutagenesis can be used to generate a non-functional β2m protein that cannot form an active MHC protein at the cell surface. In this way, the β2m protein or MHC can be retained intracellularly, or can be present at the cell surface but be non-functional.

[0370] Alternatively, immune effector cells can be irradiated prior to administration to a subject. Without wishing to be bound by theory, it is believed that irradiation of the cells results in cells that are only transiently present in the subject, thus reducing the time available for the subject's immune system to mount an immune response against the cells. Although such cells can express functional MHC molecules on their cell surface, they can also be considered non-immunogenic. The radiation can be from any source of α, β or γ radiation, or can be X-ray radiation or ultraviolet light. A radiation dose of 5-10 Gy can be sufficient to eliminate proliferation, but other suitable radiation doses can be 1-10, 2-10, 3-10, 4-10, 6-10, 7-10, 8-10 or 9-10 Gy, or higher doses, such as 11, 12, 13, 14, 15 or 20 Gy. Alternatively, the cells can be modified to express a "suicide gene" that allows the cells to be induced to die or prevents replication in response to an external stimulant.

[0371] Thus, the immune effector cells according to the present invention can be modified to be non-immunogenic, which is achieved by reducing their proliferative ability or capacity, i.e., by reducing their proliferative ability.

[0372] The modified immune effector cells of the present invention can also be modified in other ways, such as altering or modifying other aspects of the cell function or behavior, and / or expressing other proteins. For example, the cells can be modified to express homing receptors or localization receptors that act to target or improve the localization of the cells to a specific tissue or location within the body.

[0373] For example, it may be desirable to further modify immune effector cells transduced with the vectors or nucleic acids of the present invention or transduced therewith. In particular, it may be desirable to modify immune cells that prolong or enhance their response to CLEC14A. For example, TGFβ is known to be secreted by tumors and this can inhibit the induction of T cells. In this regard, it may be desirable that the modified immune effector cells of the present invention, such as T cells (i.e., those transduced with the nucleic acids or vectors of the present invention), are capable of neutralizing the effects of TGFβ, for example, by expressing a dominant negative TGFβ receptor II. Additionally / or, the immune effector cells of the present invention can be transduced with nucleic acids encoding cytokines such as IL-15 or IL-2, IL-7, IL-12, etc., which can enhance the effector function of the cells. Any additional nucleic acid sequences can be expressed from the same or different vectors as the CAR molecule.

[0374] It should also be understood that the immune effector cells of the present invention can comprise more than one nucleic acid or vector of the present invention. In particular, the immune effector cells of the present invention can comprise 2, 3, 4 or 5 or more nucleic acids or vectors of the present invention, each expressing a different CAR molecule. Thus, the immune effector cells of the present invention can comprise different CAR molecules that are capable of binding CLEC14A, for example, at the same or different positions on CLEC14A.

[0375] Thus, in some embodiments, the immune effector cells of the present invention comprise more than one nucleic acid molecule as described below, for example, 2, 3 or 4 nucleic acid molecules as described below, such as a nucleic acid molecule encoding an antigen-binding domain comprising (1) and a nucleic acid molecule encoding an antigen-binding domain comprising (2), wherein each said nucleic acid molecule encodes a CAR against the antigen CLEC14A, wherein the CAR is capable of binding the antigen CLEC14A expressed on the surface of a target cell when expressed on the surface of the immune effector cell, and comprises an antigen-binding domain that comprises:

[0376] (1)(a)VH CDR sequences, which comprise:

[0377] (i)VH CDR1, which has the amino acid sequence SEQ ID NO:2 or 42; and / or

[0378] (ii)VH CDR2, which has the amino acid sequence SEQ ID NO:3 or 43; and / or

[0379] (iii)VH CDR3, which has the amino acid sequence SEQ ID NO:4 or 44; and / or

[0380] (b)VL CDR sequences, which comprise:

[0381] (i) A VL CDR1 having the amino acid sequence SEQ ID NO: 6 or 46; and / or

[0382] (ii) A VL CDR2 having the amino acid sequence SEQ ID NO: 7 or 47; and / or

[0383] (vi) A VL CDR3 having the amino acid sequence SEQ ID NO: 8 or 48; and / or

[0384] One or more sequences that are substantially homologous to the SEQ ID NO shown in (a) or (b); or

[0385] (2)(a) VH CDR sequences, comprising:

[0386] (i) A VH CDR1 having the amino acid sequence SEQ ID NO: 22; and / or

[0387] (ii) A VH CDR2 having the amino acid sequence SEQ ID NO: 23; and / or

[0388] (iii) A VH CDR3 having the amino acid sequence SEQ ID NO: 24; and / or

[0389] (b) VL CDR sequences, comprising:

[0390] (i) A VL CDR1 having the amino acid sequence SEQ ID NO: 26; and / or

[0391] (ii) A VL CDR2 having the amino acid sequence SEQ ID NO: 27; and / or

[0392] (iii) A VL CDR3 having the amino acid sequence SEQ ID NO: 28; and / or

[0393] One or more sequences that are substantially homologous to the SEQ ID NO shown in (a) or (b). In some embodiments, in addition to the CAR of the invention that is expressed, the immune effector cells of the invention can comprise at least one other receptor, particularly a foreign receptor (e.g., multiple receptors), which can be used in combination with the CAR to bind to target cells (e.g., tumor cells expressing CLEC14A, such as the tumor vasculature) in a combinatorial approach. Thus, in such an approach, binding of the CAR and at least one other receptor to the target cell may be required to stimulate an immune response against the target cell (e.g., each CAR / receptor may only provide a partial signal for immune cell stimulation, which alone may not be sufficient to effect immune cell stimulation, but together allow immune effector cell stimulation). In the case where the immune effector cells of the invention are T cells, for T cell stimulation, binding of the CAR to CLEC14A and binding of at least one other receptor to its ligand on CLEC14A-expressing cells are necessary. The at least one other receptor can be an additional CAR molecule.

[0394] In a variant of this embodiment, expression of the CAR within the immune effector cells of the invention can be induced. In particular, in this embodiment, binding of at least one other receptor expressed on the immune effector cell to its target can permit or control expression of the CAR molecule. Thus, in such a case, binding of at least one other receptor to its ligand is required before CAR expression occurs, and thus immune effector cell stimulation requires binding of at least one other receptor to its ligand and subsequent binding of the CAR to the target cell. Such a particular system can include additional expression of a SynNotch receptor engineered to have an extracellular ligand-binding domain for an antigen of interest, such as CD19, and an orthogonal transcription factor (e.g., TetR or Gal4). After binding to the antigen of interest, the orthogonal transcription factor is cleaved from the tail of the SynNotch receptor and activates expression of the CAR. Thus, the immune effector cells of the invention can further comprise a nucleic acid or vector encoding a receptor that binds an antigen other than CLEC14A, particularly a tumor-associated antigen other than CLEC14A.

[0395] Alternatively, a combinatorial approach can also be used, where an additional receptor other than the CAR of the invention is expressed on the immune effector cells of the invention, and the additional receptor is capable of binding to off-target cells or tissues (e.g., non-tumor cells). In such a case, if the additional receptor binds its ligand, a negative signal is generated that blocks immune cell stimulation (e.g., T cell stimulation).

[0396] Further combinatorial approaches can use additional receptors in combination with the CARs of the invention, where the two receptors bind different targets and induce different effects to treat cancer. Thus, the two anti-cancer effects can be completely independent of each other, but together can present an effective therapy against cancer. In this regard, the CARs of the invention can be used in combination with TCR therapy, where immune cells can be transduced with one or more nucleic acid molecules encoding the CARs and TCRs of the invention, the TCRs being capable of binding specific MHC / peptide combinations that can be found on cancer cells (e.g., on a specific type of cancer cell or on any cancer cell). Alternatively, immune cells transduced with nucleic acid encoding a CAR and a separate population of immune cells transduced with nucleic acid encoding a TCR can be provided separately, sequentially, or simultaneously. Also contemplated is gene therapy using one or more nucleic acids encoding the CARs of the invention and TCRs that recognize cancer MHC / peptide combinations.

[0397] The invention provides methods for preparing immune effector cells that express CARs as described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from a subject such that the immune effector cells express one or more CARs as described herein. In certain embodiments, immune effector cells are isolated from a subject and modified by introduction of a nucleic acid molecule without further manipulation in vitro. Such cells can then be re-administered directly to the subject. In a further embodiment, the immune effector cells are first activated and stimulated to proliferate in vitro and then modified to express a CAR. In this regard, the immune effector cells can be cultured before or after genetic modification (i.e., transduction or transfection to express a CAR as described herein).

[0398] T cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymic issues, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, a variety of techniques known to those of skill in the art such as FICOLL can be used TMIsolate T cells from a blood unit collected from a subject. In one embodiment, cells of the circulating blood from a subject are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and the cells can be placed in a suitable buffer or medium for subsequent processing. In one embodiment of the present invention, the cells are washed with PBS. In an alternative embodiment, the washing solution lacks calcium and / or magnesium, or may lack many (if not all) divalent cations. As will be understood by those of ordinary skill in the art, the washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automatic flow-through centrifuge. For example, the Cobe 2991 cell processor, Baxter CytoMate, etc. After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions with or without buffer. In certain embodiments, the unwanted components of the apheresis sample can be removed in the medium in which the cells are directly resuspended.

[0399] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes (e.g., by centrifugation through a PERCOLL TM gradient). Specific T cell subsets, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be achieved by an antibody combination directed against surface markers specific for the negatively selected cells. One method used herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a monoclonal antibody mixture directed against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically contains antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate the cell population of interest for the present invention.

[0400] PBMCs can be directly genetically modified using the methods described herein. In certain embodiments, after isolating PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets before and / or after genetic modification and / or expansion. CD8+ cells can be obtained by using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of those types of CD8+ cells. In embodiments, memory T cells are present in both the CD62L+ and CD62L subsets of CD8+ peripheral blood lymphocytes. After staining with anti-CD8 and anti-CD62L antibodies, PBMCs are sorted into CD62L-CD8+ and CD62L+CD8+ fractions. In some embodiments, the expression of phenotypic markers of central memory TCM includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and are positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0401] Immune effector cells, such as T cells, can be modified after isolation, or immune effector cells can be activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to modification. In another embodiment, immune effector cells, such as T cells, are modified by introducing a nucleic acid molecule and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in US6905874; US6867041; US6797514; WO2012079000. Generally, such methods include contacting PBMCs or isolated T cells with stimulants and co-stimulants, such as anti-CD3 and anti-CD28 antibodies, which are typically attached to beads or other surfaces, in a medium having appropriate cytokines such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same bead act as "surrogate" antigen-presenting cells (APCs). In other embodiments, methods such as those described in US6040177; US5827642; and WO2012129514 can be used to activate and stimulate T cells to proliferate in the presence of feeder cells and appropriate antibodies and cytokines.

[0402] In one embodiment, CD34+ cells are transduced or transfected with a CAR-encoding nucleic acid molecule according to the present invention. In certain embodiments, the modified (e.g., transfected or transduced) CD34+ cells differentiate in vivo into mature immune effector cells after being administered to a subject, typically the subject from whom the cells were initially isolated. In another embodiment, CD34+ cells can be stimulated in vitro with one or more of the following cytokines before or after introduction of the nucleic acid molecule according to methods known in the art: Flt-3 ligand (FL), stem cell factor (SF), thrombopoietin (TPO), IL-3, and IL-6.

[0403] The present invention provides modified immune effector cells for use in the therapies described in more detail below, modified immune effector cells expressing a CAR as disclosed herein. For example, the modified immune effector cells can be prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient diagnosed with cancer, particularly a solid tumor.

[0404] Standard procedures can be used to store (e.g., cryopreserve) the modified immune effector cells and / or formulate them for use in humans or other subjects.

[0405] Immune effector cells expressing a CAR can be used in adoptive immunotherapy methods and compositions according to known techniques. In some embodiments, the cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating the cells in a therapeutically effective amount in a medium and container system suitable for administration ("pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or Ringer's lactate can also be used. The infusion medium can be supplemented with human serum albumin. A therapeutically effective amount of cells in the composition is at least 2 cells (e.g., at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell subset) or more typically greater than 10 2 cells, and up to 10 6 and including 10 8 or 10 9 cells, and can exceed 10 10 cells. The number of cells will depend on the intended end use of the composition and the cell types included therein. For the uses provided herein, the cells typically have a volume of 1 liter or less, 500 ml or less, even 250 ml or 100 ml or less. Thus, the density of the desired cells is typically greater than 10 6 cells / ml, typically greater than 10 7cells / ml, typically 10 cells / ml or greater. Clinically relevant numbers of immune cells can be allotted to multiple infusions that cumulatively equal or exceed 10 5 , 10 6 ,10 7 ,10 8 ,10 9 ,10 10 ,10 11 , or 10 12 cells in multiple infusions. For example, 2, 3, 4, 5, 6 or more separate infusions can be administered to a patient at intervals of 24 or 48 hours, or every 3, 4, 5, 6 or 7 days. Infusions can also be separated by weekly, bi-weekly or monthly intervals, or intervals of 6 weeks or 2, 3, 4, 5 or 6 months. Annual infusions can also be administered. In some aspects of the invention, due to redirecting all infused cells to a specific target antigen (i.e., CLEC14A), lower numbers of cells can be used, in the range of 10 6 / kg (10 6 -10 9 per patient). The cell compositions can be administered multiple times at doses within these ranges. If desired, the treatment can also include administering a mitogen (e.g., PHA) or lymphokines, cytokines and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, etc.) to enhance the induction of an immune response.

[0406] The CAR-expressing immune effector cells of the invention can be administered alone, or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the invention can comprise a population of CAR-expressing immune effector cells, such as the T cells described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the invention are preferably formulated for intravenous administration and are further described below.

[0407] As noted elsewhere regarding in vivo selection markers for vectors encoding CAR, adverse events can be minimized by transducing immune effector cells containing CAR with a suicide gene (e.g., inducible caspase 9 or thymidine kinase) before, after or simultaneously with modifying the cells with the nucleic acid molecules of the invention.

[0408] The immune responses induced in a subject by administering the CAR-expressing immune effector cells described herein can include cell-mediated immune responses mediated by cytotoxic T cells capable of killing infected cells, regulatory T cells, and helper T cell responses. Humoral immune responses mediated primarily by helper T cells capable of activating B cells resulting in antibody production can also be induced.

[0409] When “effective amount” is indicated, the precise amount of the composition to be administered can be determined by a physician taking into account individual differences in age, weight, malignancy, and the general condition of the patient (subject). By monitoring the signs of the disease in the subject and adjusting the treatment accordingly, one of ordinary skill in the medical arts can readily determine the optimal dosage and treatment regimen for a particular patient.

[0410] The term “target cell” refers to any cell that is killed or eliminated by the modified immune effector cells of the present invention. As described above, it is typically an endothelial cell expressing CLEC14A (e.g., endothelial cells within a tumor, preferably a solid tumor, such as cancer cells).

[0411] Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), provided that the vector is compatible with the host cell used. The expression vector “is suitable for transforming a host cell,” which means that the expression vector contains the nucleic acid molecule of the present invention and regulatory sequences selected based on the host cell to be used for expression, which are operably linked to the nucleic acid molecule. Operably linked means that the nucleic acid is linked to the regulatory sequences in a manner that permits expression of the nucleic acid.

[0412] Accordingly, the present invention encompasses recombinant expression vectors that contain the nucleic acid molecule of the present invention or a fragment thereof, as well as regulatory sequences necessary for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the present invention.

[0413] Suitable regulatory sequences can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The selection of suitable regulatory sequences depends on the host cell selected as discussed below and can be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: transcriptional promoters and enhancers or RNA polymerase binding sequences, ribosome binding sequences, including translation initiation signals. Additionally, depending on the host cell selected and the vector used, other sequences, such as origins of replication, additional DNA restriction sites, enhancers, and sequences conferring the ability to induce transcription, can be incorporated into the expression vector.

[0414] The recombinant expression vector of the present invention may also contain a selectable marker gene, which helps to select host cells transformed or transfected with the recombinant molecule of the present invention. Examples of selectable marker genes are genes encoding the following: proteins conferring resistance to certain drugs such as neomycin and hygromycin, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or parts thereof, such as the Fc part of an immunoglobulin, preferably IgG. The transcription of the selectable marker gene is monitored by changes in the concentration of a selectable marker protein such as β-galactosidase, chloramphenicol acetyltransferase or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance, such as neomycin resistance, G418 can be used to select transformed cells. Cells incorporating the selectable marker gene will survive, while other cells will die. This allows visualization and determination of the expression of the recombinant expression vector of the present invention, particularly to determine the effect of mutations on expression and phenotype. It should be understood that the selectable marker can be introduced on a vector separate from the nucleic acid of interest.

[0415] The recombinant expression vector may also contain a gene encoding a fusion moiety that provides increased expression of the recombinant protein; increases the solubility of the recombinant protein; and aids in the purification of the target recombinant protein by acting as a ligand in affinity purification (e.g., appropriate "tags" may be present to enable purification and / or identification, such as His tag or myc tag). For example, a proteolytic cleavage site can be added to the target recombinant protein to allow separation of the recombinant protein from the fusion protein after purification of the fusion protein. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A to the recombinant protein, respectively.

[0416] A recombinant expression vector can be introduced into a host cell to produce a transduced host cell. The terms "transduce with", "transform with", "transfect with", "transform" and "transfect" are intended to encompass the introduction of a nucleic acid (such as a vector) into a cell by one of many possible techniques known in the art. As used herein, the term "transformed host cell" is also intended to include cells capable of glycosylation that have been transformed with the recombinant expression vectors of the present invention. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride-mediated transformation. For example, nucleic acids can be introduced into mammalian cells by conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al., 1989 (supra) and other laboratory textbooks.

[0417] Suitable host cells include an extremely wide variety of eukaryotic and prokaryotic host cells. For example, the proteins (such as antibodies) of the present invention can be expressed in yeast cells or mammalian cells. In addition, the proteins of the present invention can be expressed in prokaryotic cells, such as Escherichia coli.

[0418] Yeast and fungal host cells suitable for practicing the present invention include, but are not limited to, Saccharomyces cerevisiae, Pichia, Kluyveromyces, and various species of Aspergillus. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, CA). Protocols for transforming yeast and fungi are well known to those of ordinary skill in the art.

[0419] Mammalian cells suitable for practicing the present invention particularly include: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL2), 293 (ATCC No. 1573), NS-1 cells, NS0 (ATCC CRL-11177), and (Crucell, Leiden, Netherlands). Suitable expression vectors for directing expression in mammalian cells typically include a promoter (e.g., derived from viral material such as polyoma, adenovirus 2, cytomegalovirus, and simian virus 40), as well as other transcriptional and translational control sequences. Examples of mammalian expression vectors include pCDM8 and pMT2PC.

[0420] In view of the teachings provided herein, promoters, terminators, and methods for introducing suitable types of expression vectors into plant, avian, and insect cells can also be readily achieved. For example, in one embodiment, the protein of the present invention can be expressed from plant cells.

[0421] Insect cells suitable for practicing the present invention include cells and cell lines from the species Bombyx, Trichoplusia, or Spodotera. Baculovirus vectors useful for expressing proteins in cultured insect cells (SF 9 cells) include the pAc series and the pVL series.

[0422] Alternatively, the protein of the present invention can also be expressed in non-human transgenic animals such as rats, rabbits, sheep, and pigs (U.S. Patent No. 4,736,866).

[0423] The protein of the present invention can also be prepared by chemical synthesis using techniques well known in protein chemistry, such as solid-phase synthesis or synthesis in homogeneous solution.

[0424] Antibodies of the present invention and N-terminal or C-terminal fusion proteins of the protein conjugated with other molecules (e.g., immunoconjugates) (e.g., proteins) can be prepared by fusion via recombinant techniques. The resulting fusion proteins contain the antibody or protein of the present invention fused with a selected protein or marker protein, or a tag protein as described herein. The antibodies and proteins of the present invention can also be conjugated with other proteins by known techniques. For example, heterobifunctional thiol-containing linkers, N-succinimidyl-3-(2-pyridyldithio)-propionate, or N-succinimidyl-5-thioacetate as described in WO 90 / 10457 can be used to couple proteins. Examples of proteins useful for preparing fusion proteins or conjugates include cell-binding proteins such as immunoglobulins, hormones, growth factors, lectins, insulin, low-density lipoprotein, glucagon, endorphin, transferrin, bombesin, asialo-glycoprotein, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0425] As discussed in more detail above and in the following examples, the inventors have determined that the antibodies of the invention have an effect on angiogenesis. Accordingly, the antibodies of the invention (including their fusion proteins and their conjugates, such as immunoconjugates, etc.), CARs, nucleic acid molecules (including vectors, particularly expression vectors comprising said nucleic acid molecules), and immune effector cells are useful in therapy. Accordingly, other aspects of the invention include:

[0426] Compositions, particularly therapeutic or pharmaceutical compositions, comprising an antibody of the invention, a CAR (i.e., a CAR polypeptide), a nucleic acid molecule (e.g., encoding a CAR, such as an expression vector), or an immune effector cell as defined herein and at least one physiologically acceptable carrier or excipient;

[0427] An antibody of the invention, a CAR (i.e., a CAR polypeptide), a nucleic acid molecule (e.g., encoding a CAR, such as an expression vector), or an immune effector cell or composition as defined herein for use in therapy, particularly adoptive cell transfer therapy;

[0428] An antibody of the invention, a CAR (i.e., a CAR polypeptide), a nucleic acid molecule (e.g., encoding a CAR, such as an expression vector), or an immune effector cell or composition as defined herein for use against a disease or condition associated with CLEC14A expression, such as for inhibiting angiogenesis, particularly tumor angiogenesis, such as for treating cancer;

[0429] A method of treating a disease or condition associated with CLEC14A expression, such as a method of inhibiting angiogenesis, particularly tumor angiogenesis, such as a method of treating cancer, the method comprising administering to a subject in need thereof an antibody of the invention, a CAR (i.e., a CAR polypeptide), a nucleic acid molecule (e.g., encoding a CAR, such as an expression vector), an immune effector cell or composition as defined herein, particularly an effective amount of said antibody, CAR, nucleic acid molecule, cell or composition; and

[0430] Use of an antibody of the invention, a CAR (i.e., a CAR polypeptide), a nucleic acid molecule (e.g., encoding a CAR, such as an expression vector), or an immune effector cell as defined herein for the manufacture of a medicament (or composition) for use against a disease or condition associated with CLEC14A expression, such as for inhibiting angiogenesis, particularly tumor angiogenesis, such as for treating cancer.

[0431] To avoid doubt, it should also be understood that the present invention also includes in vitro or ex vivo methods of inhibiting angiogenesis (e.g., tumor angiogenesis), including in vitro or ex vivo administration of an antibody as defined herein, a CAR (i.e., a CAR polypeptide), a nucleic acid molecule (e.g., encoding a CAR, e.g., an expression vector), an immune effector cell or a composition to a tissue or a cell. The cell can be an established cell line or a cell that has been removed from an individual. The tissue or cell is preferably a mammalian tissue or cell (e.g., endothelial tissue or cell), most preferably a human tissue or cell. When the method is an ex vivo method, the reagent can be administered ex vivo to an angiogenesis model. Suitable angiogenesis assays include assays of endothelial cell proliferation, migration and invasion, sponge assays and aortic ring assays. Further angiogenesis assays are described below and in the examples.

[0432] By "counteracting", we include the meaning that the method can be used to alleviate the symptoms of a disorder (i.e., palliative use method), or to treat a disorder or to prevent and augment (i.e., prophylactic use of the method).

[0433] By a disease or condition associated with CLEC14A expression, we include any disease or condition associated with cells expressing CLEC14A. For example, the cell can be an unwanted cell. An unwanted cell can be any cell that is not desired to be present in a host. Thus, a disease or condition associated with CLEC14A expression can be any condition characterized by the presence of cells expressing CLEC14A and that are unwanted, e.g., any biological or medical condition or disorder in which at least part of the pathology is mediated by the presence of such unwanted cells expressing CLEC14A. The condition can be caused by the presence of the unwanted cells, or the presence of the unwanted cells can be an effect of the condition.

[0434] By expression of CLEC14A, we include the meaning that the CLEC14A protein can be detected on or in a cell or in an extract prepared from a cell, or the expression of the polypeptide can be inferred by detecting CLEC14A mRNA. To confirm the expression of CLEC14A in a cell, a variety of assays can be performed. Such assays include, for example, biochemical assays well known to those skilled in the art, such as by immunological means (ELISA and Western blotting), or molecular biology assays well known to those skilled in the art, such as Northern blotting, RT-PCR and PCR for detecting the presence of CLEC14A mRNA to detect the presence of a specific protein (i.e., CLEC14A).

[0435] As mentioned above, the inventors have found that CLEC14A is an endothelial cell marker, and thus it is understood that the antibodies, CARs, nucleic acid molecules, expression vectors, immune effector cells and compositions of the present invention are particularly useful in combating any disease or condition involving unwanted, undesired or inappropriate angiogenesis. Such conditions include tumors / cancers, psoriasis, menorrhagia, endometriosis, arthritis (including both inflammatory and rheumatoid arthritis), macular degeneration, Paget's disease, retinopathy and its vascular complications (including proliferative and pre - term and diabetic retinopathy), benign vascular proliferation, fibrosis, obesity and inflammation.

[0436] The term "inhibiting angiogenesis" is intended to mean reducing the rate or level of angiogenesis. The reduction can be a low - level reduction of about 10%, or about 20%, or about 30%, or about 40% of the angiogenesis rate or level. Preferably, the reduction is a moderate - level reduction of about 50%, or about 60%, or about 70%, or about 80% of the angiogenesis rate or level. More preferably, the reduction is a high - level reduction of about 90%, or about 95%, or about 99%, or about 99.9% of the angiogenesis rate or level. Most preferably, inhibition can also include eliminating angiogenesis or reducing it to an undetectable level. Methods and assays for determining the rate or level of angiogenesis, and thus for determining whether and to what extent an antibody, CAR, nucleic acid molecule, expression vector, immune effector cell or composition inhibits angiogenesis, are known in the art and are described in more detail herein, including in the Examples.

[0437] Typically, the angiogenesis that is inhibited is tumor angiogenesis. Thus, an individual can have a solid tumor, which can be treated by inhibiting tumor angiogenesis, i.e., the solid tumor is associated with neovascularization. The term "tumor" should be understood to refer to all forms of neoplastic cell growth, including but not limited to, breast, ovarian, liver, bladder, prostate, kidney, pancreas, stomach, esophageal, lung and thyroid tumors.

[0438] Typically, tumors are associated with unwanted neovascularization. Reduction of unwanted neovascularization can halt tumor progression and can result in a clinically useful reduction in tumor size and growth. Thus, inhibiting tumor angiogenesis can be used to treat tumors, e.g., to prevent (further) tumor growth, prevent tumor spread (metastasis), or reduce the size of the tumor.

[0439] Preferably, the antibodies, CARs, nucleic acid molecules, expression vectors, immune effector cells, methods and compositions of the present invention are used for treating humans, in which case the antibody, CAR or immune effector cell is capable of binding to human CLEC14A or a nucleic acid molecule or expression vector encoding an antibody or CAR capable of binding to human CLEC14A. However, it should be understood that when the antibodies, CARs, nucleic acid molecules, expression vectors, immune effector cells, methods and compositions of the present invention are used for treating non-human mammals, preferably the antibody, CAR or immune effector cell is capable of binding to CLEC14A from other species or a nucleic acid molecule or expression vector encoding an antibody or CAR capable of binding to CLEC14A from other species.

[0440] As discussed above, CLEC14A is expressed on certain tumor cells (such as endothelial cells), and the antibodies of the present invention are localized to CLEC14A+ cells. Thus, the antibodies of the present invention can target body parts (such as tumors) where CLEC14A+ cells are present, and thus the antibodies can act on the target site. In particular, the ability of the antibodies to localize to CLEC14A+ endothelial cells in tumor blood vessels means that the antibodies of the present invention can target body parts where CLEC14A+ tumor cells are present, and thus the antibodies can act on the target site.

[0441] As shown in the examples, the antibodies of the present invention can themselves have an anti-CLEC14A+ cell effect (such as an inhibitory effect on angiogenesis or an anti-cancer effect), i.e., as naked antibodies, for example, by inhibiting, reducing or blocking the function or activity of CLEC14A. This ability to act as a naked antibody is advantageous, and thus in some embodiments, the compositions, uses and methods of the present invention use the antibodies of the present invention that are not conjugated to any other active agent such as a therapeutic active agent.

[0442] It is known that when administered in combination with standard chemotherapy, the angiogenesis inhibitor anti-VEGF monoclonal antibody bevacizumab improves the clinical outcomes of many solid tumors. Combinations that have been used include bevacizumab combined with irinotecan, fluorouracil and leucovorin; bevacizumab combined with FOLFOX4 (a regimen of oxaliplatin, 5-fluorouracil and leucovorin); bevacizumab combined with paclitaxel; and bevacizumab combined with paclitaxel and carboplatin.

[0443] Thus, those skilled in the art will understand that although the antibodies, CARs, nucleic acid molecules, expression vectors, compositions and immune effector cells of the present invention can be clinically effective without any other therapeutic agent (such as an anti-cancer and / or anti-angiogenic compound / reagent), it can be advantageous to use the antibodies, CARs, nucleic acid molecules, expression vectors, compositions and immune effector cells of the present invention in combination with other therapeutic agents (such as anti-cancer and / or anti-angiogenic).

[0444] Thus, in another embodiment of the invention, the method may further comprise administering to the individual at least one additional or other therapeutic agent (e.g., an anti-cancer and / or anti-angiogenic compound / reagent). The method may comprise administering to the individual a pharmaceutical composition comprising an antibody, CAR, nucleic acid molecule, expression vector or immune effector cell and an additional therapeutic agent (e.g., an anti-cancer and / or anti-angiogenic compound / reagent). However, it should be understood that the antibody, CAR, nucleic acid molecule, expression vector, composition or immune effector cell of the invention and the other therapeutic agent (e.g., an anti-cancer and / or anti-angiogenic compound / reagent) may be administered separately, e.g., by separate routes of administration. Additionally, the antibody, CAR, nucleic acid molecule, expression vector, composition or immune effector cell of the invention and at least one other therapeutic agent (e.g., an anti-cancer and / or anti-angiogenic compound / reagent) may be administered sequentially or (substantially) simultaneously. They may be administered in the same pharmaceutical formulation or drug, or they may be formulated and administered separately.

[0445] In a specific embodiment, the invention provides a method for combating a disease or condition associated with CLEC14A expression, e.g., a method for inhibiting angiogenesis, particularly tumor angiogenesis, e.g., a method for treating cancer, the method comprising administering an antibody, CAR, nucleic acid molecule, expression vector, immune effector cell or composition of the invention as defined herein, particularly an effective amount of said antibody, cell or composition, and administering one or more additional active (e.g., therapeutic) agents (e.g., anti-cancer and / or anti-angiogenic compounds / reagents) to a subject in need thereof, either separately, simultaneously or sequentially.

[0446] Alternatively, there is provided an antibody, CAR, nucleic acid molecule, expression vector, immune effector cell or composition of the invention as defined herein, which is used in combination with one or more additional active (e.g., therapeutic) agents (e.g., anti-cancer and / or anti-angiogenic compounds / reagents) for combating a disease or condition associated with CLEC14A expression, e.g., for inhibiting angiogenesis, particularly tumor angiogenesis, e.g., for treating cancer.

[0447] Thus, there is provided the use of an antibody, CAR, nucleic acid molecule, expression vector or immune effector cell of the invention as defined herein in the manufacture of a medicament which is used in combination with one or more additional active agents (e.g., therapeutic agents) (e.g., anti-cancer and / or anti-angiogenic compounds / reagents) for combating a disease or condition associated with CLEC14A expression, e.g., for inhibiting angiogenesis, particularly tumor angiogenesis, e.g., for treating cancer.

[0448] Thus, in one embodiment, the medicament may further comprise one or more additional active (e.g., therapeutic) agents (e.g., anti-cancer and / or anti-angiogenic compounds / reagents).

[0449] The medicament may be in the form of a single composition that contains both the antibody, CAR, nucleic acid molecule, expression vector, or immune effector cell of the invention as defined herein and one or more additional active agents (e.g., therapeutic agents) (e.g., anti-cancer and / or anti-angiogenic compounds / reagents), or it may be in the form of a kit or product containing them for separate (e.g., simultaneous or sequential) administration.

[0450] In some embodiments, the additional therapeutic agent is an anti-cancer agent. The additional anti-cancer agents may be selected from alkylating agents, including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-phenylalanine mustard), and chlorambucil; ethyleneimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozocin (streptozotocin); and triazines such as dacarbazine (DTIC; dimethyltriazenoimidazole-carboxamide); antimetabolites, including folic acid analogs such as methotrexate; pyrimidine analogs such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR), and cytarabine (cytosine arabinoside); and purine analogs and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin); natural products, including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes; miscellaneous agents, including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenediones such as mitoxantrone and anthracyclines; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); and adrenocortical inhibitors such as mitotane (o,p'-DDD) and aminoglutethimide; paclitaxel and analogs / derivatives; cell cycle inhibitors; proteasome inhibitors such as bortezomib Signal transduction enzyme (e.g., tyrosine kinase) inhibitors such as imatinib COX-2 inhibitors and hormone agonists / antagonists such as flutamide and tamoxifen.

[0451] Anticancer agents used clinically are usually grouped by mechanism of action: alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA / DNA antimetabolites, DNA antimetabolites, and antimitotic agents. The National Institutes of Health / National Cancer Institute website lists 122 compounds (http: / / dtp.nci.nih.gov / docs / cancer / searches / standard_mechanism.html), all of which can be used in conjunction with the antibodies, compositions, or immune effector cells of the present invention. They include alkylating agents, including Asaley, AZQ, BCNU, busulfan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorthiazide, cisplatin, clomesone, cyanomorpholino-doxorubicin, cyclodisone, dianhydrogalactitol, fluorodopan, hepsulfam, heinothione, melphalan, methyl CCNU, mitomycin C, mitozolamide, mechlorethamine, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, tilorone, tetraplatin, picoplatin (SP-4-3) (cis-amminedichloro(2-methylpyridine)) (Pt-II)), thiotepa, trimetrexate, uracil mustard, Yoshi-864; antimitotic agents, including allocolchicine, Halichondrin B, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel, paclitaxel derivatives, thiocolchicine, tritylcysteine, vinblastine sulfate, vincristine sulfate; topoisomerase I inhibitors, including camptothecin, camptothecin, sodium salt, aminocamptothecin, 20 camptothecin derivatives, morpholinodoxorubicin; topoisomerase II inhibitors, including doxorubicin, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, mitoxantrone, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26, VP-16;RNA / DNA antimetabolites, including L-alanosine, 5-azacytidine, 5-fluorouracil, asivicin, 3-aminopterin derivatives, antifolates, Baker's soluble antifolates, dichlorallyl lawsone, brequinar, tegafur (prodrug), 5,6-dihydro-5-azacytidine, methotrexate, methotrexate derivatives, N-(phosphonacetyl)-L-aspartate (PALA), pyrazomycin, trimetrexate; DNA antimetabolites, including 3-HP, 2'-deoxy-5-fluorouridine, 5-HP, α-TGDR, aphidicolin glycinate, ara-C, 5-aza-2'-deoxycytidine, β-TGDR, cyclocytidine, guanazole, hydroxyurea, inosine glycodialdehyde, macbecin II, pyrazoloimidazole, thioguanine, and thiopurine.

[0452] In some preferred embodiments, at least one other anti-cancer agent is selected from cisplatin; carboplatin; picoplatin; 5-fluorouracil; paclitaxel; mitomycin C; doxorubicin; gemcitabine; topotecan; pemetrexed; methotrexate; irinotecan, fluorouracil, and leucovorin; oxaliplatin, 5-fluorouracil, and folinic acid; and paclitaxel and carboplatin.

[0453] When an additional anti-cancer agent has been shown to be particularly effective against a specific tumor type, the antibodies, CARs, nucleic acid molecules, expression vectors, compositions, or immune effector cells of the present invention can preferably be used in combination with the additional anti-cancer agent to treat the specific tumor type.

[0454] In some embodiments, the anti-angiogenic compound can be selected from any of the following: bevacizumab itraconazole; carboxyamidotriazole; TNP-470 (fumagillin analogue); CM101; IFN-α; IL-12; platelet factor-4; suramin; SU5416; thrombospondin; VEGFR antagonists; angiostatic steroids + heparin; cartilage-derived angiogenesis inhibitor; matrix metalloproteinase inhibitor; angiostatin; endostatin; 2-methoxyestradiol; tecogalan; tetrathiomolybdate; thalidomide; prolactin; αVβ3 inhibitor; linomide; tasquinimod; ranibizumab; sorafenib; sunitinib Pazopanib and everolimus

[0455] In some embodiments, the additional therapeutic agent can be an immune checkpoint inhibitor, particularly for use in combination with the immune effector cells of the present invention. Such inhibitors generally act by blocking the interaction between immune cells and target cells (such as tumor cells), which interaction prevents or downregulates the stimulation of immune cells. In particular, checkpoint inhibitors prevent or reduce the interaction between proteins expressed on T cells and proteins expressed on tumor cells, which interaction would prevent or reduce the stimulation of T cells. Checkpoint inhibitors can, for example, prevent the interaction between PD1 and PDL1, and in particular can be reagents that bind to PD1. Alternatively, checkpoint inhibitors can bind to CTLA-4. Such checkpoint inhibitors are well known in the art and include monoclonal antibodies such as Pembrolizumab, Nivolumab, or Ipilimumab.

[0456] The additional active agent can also be a sphingosine-1-phosphate agonist, such as FTY720, which is capable of sequestering lymphocytes in lymphoid organs by blocking signals from sphingosine-1-phosphate receptors. In this way, such compounds can limit the competition for cytokines such as IL-7 and IL-15, and can therefore increase the proliferation of administered immune effector cells. In particular, such compounds can be administered prior to the nucleic acids, expression vectors, CARs, or immune effector cells of the present invention, for example, at least 12 hours, 24 hours, 36 hours, or 48 hours prior.

[0457] Alternatively or additionally, the antibodies of the present invention can have anti-CLEC14A+ cell effects (such as an inhibitory effect on angiogenesis or an anti-cancer effect) by conjugating with additional therapeutic molecules as described herein, such as toxins or other anti-cancer molecules or anti-angiogenic agents.

[0458] In this regard, as shown in the examples, some of the antibodies of the present invention are capable of internalizing into the cells to which they bind. Thus, in some embodiments of the present invention, the antibodies are capable of being internalized. This property can be particularly advantageous for use in immunoconjugates, since any other reagent attached to the antibody molecule should be internalized together with the antibody molecule. In other embodiments, no significant internalization is seen.

[0459] Accordingly, in another embodiment, the present invention provides a series of conjugated antibodies and fragments thereof (as defined herein), wherein the antibody or fragment is operably attached to at least one other therapeutic or diagnostic agent. The term "immunoconjugate" is widely used to define the operative association of an antibody (including fragments thereof) with another active agent and is not intended to be limited to any particular type of operative association and is particularly not limited to chemical "conjugation". Recombinant fusion proteins are specifically encompassed. As long as the delivery or targeting agent is capable of binding to the target and the therapeutic or diagnostic agent has sufficient functionality upon delivery, the mode of attachment will be suitable.

[0460] Accordingly, in some embodiments, the antibodies of the present invention can be immunoconjugates and / or the antibodies of the present invention for the methods, compositions, and uses defined herein are immunoconjugates, i.e., antibodies conjugated (i.e., coupled, attached, or linked) to additional therapeutic molecules such as toxins or other anti-cancer molecules or anti-angiogenic agents or diagnostic molecules.

[0461] Carter & Senter (2008), Cancer J. 14(3):154 - 69 and Chari et al (2014) Angewandte Chemie International Edition 53:3751 (which are incorporated herein by reference) reviewed antibody - drug conjugates (ADCs), e.g., for cancer therapy, and it should be understood that the immunoconjugates of the present invention can be considered to include such antibody - drug conjugates (see also US 5,773,001; US 5,767,285; US 5,739,116; US 5,693,762; US 5,585,089; US 2006 / 0088522; US 2011 / 0008840; US 7,659,241; Hughes (2010) Nat Drug Discov 9:665, Lash (2010); In vivo: The Business & Medicine Report 32 - 38; Mahato et al (2011) Adv Drug Deliv Rev 63:659; Jeffrey et al (2006) BMCL16:358; Drugs R D 11(1):85 - 95). ADCs typically comprise a monoclonal antibody directed against a target present on tumor cells, a cytotoxic drug, and a linker that attaches the antibody to the drug.

[0462] Accordingly, in another aspect, the present invention provides immunoconjugates (such as antibody - drug conjugates) that comprise an antibody of the present invention and a cytotoxic moiety.

[0463] The cytotoxic moiety can be directly or indirectly toxic to cells in the new vascular system or to cells that are very close to and associated with the new vascular system. By "direct cytotoxicity", we include the meaning that the moiety is a moiety that is itself cytotoxic. By "indirect cytotoxicity", we include the meaning that the moiety, although not itself cytotoxic, can induce cytotoxicity, for example, by its action on another molecule or by further action on it. For example, an indirectly cytotoxic moiety can act to recruit immune cells (such as cytotoxic immune cells, such as cytotoxic T cells), thereby indirectly inducing a cytotoxic effect.

[0464] Typically, the cytotoxic moiety is selected from direct cytotoxic chemotherapeutic agents, direct cytotoxic polypeptides, moieties capable of converting a prodrug into a cytotoxic drug, radiosensitizers, direct cytotoxic nucleic acids, nucleic acid molecules encoding direct or indirect cytotoxic polypeptides, or radioactive atoms. Examples of such cytotoxic moieties and methods for preparing conjugates comprising an antibody and a cytotoxic moiety are provided in WO 02 / 36771, WO 2004 / 046191, and WO 2011 / 027132, which are incorporated herein by reference.

[0465] In some embodiments, the cytotoxic moiety is a cytotoxic chemotherapeutic agent. Cytotoxic chemotherapeutic agents such as anticancer agents are well known in the art and include those described above.

[0466] The various cytotoxic moieties mentioned above, such as cytotoxic chemotherapeutic agents, have previously been attached to antibodies and other targeting agents, and thus those skilled in the art can readily prepare the immunoconjugates of the present invention comprising these agents. For example, carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159) can be used to couple a variety of agents (including doxorubicin) to an antibody. Other methods for conjugating a cytotoxic moiety to an antibody can also be used. For example, sodium periodate oxidation followed by reductive alkylation with an appropriate reactant can be used, and glutaraldehyde crosslinking can also be used. Methods for crosslinking polypeptides are known in the art and are described in WO 2004 / 046191. However, it should be recognized that, regardless of the method chosen to produce the compounds of the present invention, it must be determined that the antibody retains its targeting ability and that the attached moiety retains its relevant function.

[0467] In some embodiments of the present invention, the cytotoxic moiety can be a cytotoxic peptide or polypeptide moiety, and by said cytotoxic peptide or polypeptide moiety, we include any moiety that causes cell death. Cytotoxic peptides and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor, etc. Methods of conjugating them to targeting moieties such as antibodies are also known in the art and include, for example, conventional methods of cross-linking polypeptides and using recombinant DNA technology to fuse polypeptides to produce compounds. The use of ricin as a cytotoxic agent is described in Burrows & Thorpe (1993) Proc. Natl. Acad. Sci. USA 90, 8996-9000, and the use of tissue factor (which causes local blood coagulation and tumor infarction) has been described by Ran et al (1998) Cancer Res. 58, 4646-4653 and Huang et al (1997) Science 275, 547-550. The conjugation of abrin A chain to monoclonal antibodies is described in Tsai et al (1995) Dis. Colon Rectum 38, 1067-1074. Other ribosome-inactivating proteins are described as cytotoxic agents in WO 96 / 06641. Pseudomonas exotoxin can also be used as a cytotoxic polypeptide moiety (Aiello et al (1995) Proc. Natl. Acad. Sci. USA 92, 10457-10461).

[0468] Certain cytokines, such as TNFα, INFγ, and IL-2, can also be used as cytotoxic agents.

[0469] If delivered in sufficient doses, certain radioactive atoms can also be cytotoxic. Thus, in some embodiments, the cytotoxic moiety can comprise a radioactive atom that delivers a sufficient amount of radioactivity to the target site in use to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-111, rhenium-186, rhenium-188, or yttrium-90, or any other isotope that emits sufficient energy to disrupt adjacent cells, organelles, or nucleic acids. Preferably, the isotope and density of the radioactive atom in the compounds of the present invention are such that a dose of more than 4000 cGy (preferably at least 6000, 8000, or 10000 cGy) is delivered to the target site, and preferably to the cells at the target site and their organelles, particularly the nucleus.

[0470] Radioactive atoms can be attached to antibodies in a known manner. For example, EDTA or another chelating agent can be attached to the antibody and used to attach 111 In or 90 Y. Tyrosine residues can be used with 125I or 131 I label.

[0471] In some embodiments, the cytotoxic moiety can be a radiosensitizer. Radiosensitizers include fluoropyrimidines, thymidine analogs, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3-aminophenyl diamide (3-aminobenzodiamide), etanixadole, pimonidazole, and misonidazole (see, e.g., McGinn et al (1996) J. Natl. Cancer Inst. 88, 1193-11203; Shewach & Lawrence (1996) Invest. New Drugs 14, 257-263; Horsman (1995) Acta Oncol. 34, 571-587; Shenoy & Singh (1992) Clin. Invest. 10, 533-551; Mitchell et al (1989) Int. J. Radiat. Biol. 56, 827-836; Iliakis & Kurtzman (1989) Int. J. Radiat. Oncol. Biol. Phys. 16, 1235-1241; Brown (1989) Int. J. Radiat. Oncol. Biol. Phys. 16, 987-993; Brown (1985) Cancer 55, 2222-2228).

[0472] In some embodiments, the cytotoxic moiety can be a procoagulant factor, such as the extracellular domain of tissue factor (Rippmann et al (2000) “Fusion of the tissue factor extracellular domain to a tumour stroma specific single-chain fragment variable antibody results in an antigen-specific coagulation-promoting molecule.” Biochem J. 349:805-12; Huang et al (1997) “Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature.” Science. 275(5299):547–550.

[0473] In some embodiments, the cytotoxic moiety can be an indirect cytotoxic polypeptide. In particularly preferred embodiments, the indirect cytotoxic polypeptide is a polypeptide having enzymatic activity and capable of converting a relatively non-toxic prodrug into a cytotoxic drug. When the targeting moiety is an antibody, this type of system is commonly referred to as ADEPT (antibody-directed enzyme prodrug therapy). This system requires the targeting moiety to localize the enzymatic moiety to a desired site within the patient's body (e.g., the site of neovascular tissue associated with a tumor), and after a time allowing the enzyme to localize at that site, a prodrug that is a substrate for the enzyme is administered, and the catalytic end product is a cytotoxic compound. The aim of this method is to maximize the concentration of the drug at the desired site and minimize the concentration of the drug in normal tissues ((Senter et al (1988) “Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate” Proc. Natl. Acad. Sci. USA 85, 4842-4846; Bagshawe (1987) Br. J. Cancer 56, 531-2; and Bagshawe, et al (1988) “A cytotoxic agent can be generated selectively at cancer sites” Br. J. Cancer. 58, 700-703); Bagshawe (1995) Drug Dev. Res. 34, 220-230 and WO 2004 / 046191 describe various enzyme / prodrug combinations, which can be applicable to the context of the present invention.

[0474] Typically, a prodrug is relatively non-toxic compared to a cytotoxic drug. Generally, it has less than 10% toxicity, preferably less than 1% toxicity, as measured in a suitable in vitro cytotoxicity test.

[0475] As used in this application, the term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that is less active than the parent drug and is capable of being enzymatically activated or converted into a more active parent form (see, for example, D.E.V. Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions 14, 375 - 382 (615th Meeting, Belfast 1986) and V. J. Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery", Directed Drug Delivery, R. Borchardt et al. (eds.), pp. 247 - 267 (Humana Press 1985)).

[0476] It is possible that the moiety capable of converting the prodrug into a cytotoxic drug may be active when separated from the remainder of the compound, but it is necessary that it become active only when (a) it is combined with the remainder of the compound and (b) the compound is attached to, adjacent to, or internalized in the target cell.

[0477] The cytotoxic moiety can be a moiety that becomes cytotoxic or releases a cytotoxic moiety upon irradiation. For example, upon appropriate irradiation, the boron - 10 isotope releases cytotoxic alpha particles (US 4,348,376; Primus et al (1996) Bioconjug. Chem. 7:532 - 535).

[0478] Similarly, the cytotoxic moiety can be a cytotoxic moiety useful for photodynamic therapy, such as a photosensitizer (see, for example, Dougherty et al (1998) J. Natl. Cancer Inst. 90, 889 - 905).

[0479] In some embodiments, the cytotoxic moiety is an antibody, such as an antibody that specifically binds to immune cells, such as cytotoxic immune cells (e.g., T cells). Thus, in such cases, the compounds of the present invention can be asymmetric IgG-like antibodies (e.g., triomab / quadroma, Trion Pharma / Fresenius Biotech; knobs-into-holes, Genentech; Cross MAbs, Roche; electrostatically matched antibodies, AMGEN; LUZ-Y, Genentech; strand-exchange engineered domain (SEED) bodies, EMD Serono; biolonic, erus; and Fab-exchange antibodies, Genmab), symmetric IgG-like antibodies (e.g., dual-targeting (DT)-Ig, GSK / Domantis; two-in-one antibody, Genentech; cross-linked MAb, karmanos cancer center; mAb<2>, F-star; and Cov X-body, Cov X / Pfizer), IgG fusions (e.g., dual-variant domain (DVD)-lg, Abbott; IgG-like bispecific antibodies, Eli Lilly; Ts2Ab, Medimmune / AZ; BsAb, ZymoGenetics; HERCULES, Biogen Idee; TvAb, Roche) Fc fusions (e.g., ScFv / Fc fusions, Academic Institution; SCORPION, Emergent BioSolutions / Trubion, ZymoGenetics / BMS; bispecific affinity retargeting technology (Fc-DART), MacroGenics; dual (ScFv)2-Fab, National Research Center for Antibody Medicine) Fab fusions (e.g., F(ab)2, Medarex / AMGEN; bifunctional or bisFab, Genentech; Dock-and-Lock (DNL), ImmunoMedics; bivalent bispecific, Biotechnol; and Fab-Fv, UCB-Celltech), ScFv- and diabody-based antibodies (e.g., bispecific T cell engager (BiTE), Micromet; tandem diabody (Tandab), Affimed; DART, MacroGenics; single-chain diabody, Academic; TCR-like antibodies, AIT, Receptor Logics;Human serum albumin ScFv fusions, Merrimack; and COMBODIES, Epigen Biotech), IgG / non-IgG fusions (e.g., immunocytokines, EMD Serono, Philogen, ImmunGene, ImmunoMedics; superantigen fusion proteins, Active Biotech; and immune mobilizing mTCRs for cancer, ImmTAC) and oligoclonal antibodies (such as Symphogen and Merus).;

[0480] In some embodiments, the cytotoxic moiety is a pyrrolobenzodiazepine (PBD) dimer. PBDs are potent anti-cancer agents that have shown broad-spectrum anti-tumor activity in vivo. These drugs exert their activity by binding to the minor groove of DNA and cross-linking two DNA strands in a manner that is difficult for cells to recognize and repair. Thus, in some embodiments, the immunoconjugates of the invention can be antibodies of the invention that comprise a PBD. Further information on PBDs can be found in Hartley et al, 2012 (Invest New Drugs 30:950-958).

[0481] As discussed above, in some embodiments, the immunoconjugates of the invention can be fusion proteins / polypeptides that comprise an antibody of the invention and a cytotoxic polypeptide. Thus, in another embodiment, the invention provides a nucleic acid molecule encoding an immunoconjugate of the invention, such as a fusion protein / polypeptide, that comprises an antibody of the invention and a cytotoxic polypeptide.

[0482] Thus, alternatively, the invention can also be regarded as providing a method of targeting a cytotoxic agent to the neovasculature in a subject's body, the method comprising administering to the subject an immunoconjugate of the invention (such as an antibody of the invention conjugated to a cytotoxic agent). Preferably, the neovasculature is tumor neovasculature.

[0483] Thus, another aspect of the invention includes an immunoconjugate of the invention (such as an antibody of the invention conjugated to a cytotoxic agent) for targeting a cytotoxic agent to the neovasculature in a subject's body (such as tumor neovasculature).

[0484] Thus, another aspect of the invention includes the use of an immunoconjugate of the invention (such as an antibody of the invention conjugated to a cytotoxic agent) in the preparation or manufacture of a medicament for targeting a cytotoxic agent to the neovasculature in a subject's body (such as tumor neovasculature).

[0485] Those skilled in the art will understand that targeting cytotoxic agents to the new vascular system will serve to inhibit angiogenesis. Thus, it is evident that the immunoconjugates of the present invention can be used in the treatment methods and uses described above. It will also be understood that although the immunoconjugates of the present invention may be clinically effective in the absence of any other therapeutic agents (e.g., anti-cancer and / or anti-angiogenic compounds / reagents), it may still be advantageous to administer the immunoconjugates in combination (separately, sequentially or subsequently) with additional anti-cancer and / or anti-angiogenic compounds / reagents as described above.

[0486] The antibody of the present invention binds to CLEC14A. Thus, the antibody of the present invention can be used to detect CLEC14A in vivo or in vitro, particularly to detect CLEC14A+ cells. For example, when CLEC14A is expressed on certain tumor cells, the antibody of the present invention can be used to detect tumor cells in vivo or in vitro, particularly when conjugated (coupled, attached or linked) to a detectable moiety.

[0487] Thus, in another embodiment, the present invention provides an immunoconjugate comprising the antibody of the present invention and a detectable moiety. Such compounds can be used in conjunction with appropriate detection methods to detect the location of CLEC14A, particularly CLEC14A+ cells, in a subject, thereby identifying the sites and extent of angiogenesis (e.g., tumor angiogenesis) in the subject, as well as the inhibition of angiogenesis (e.g., tumor angiogenesis) in the subject.

[0488] Alternatively, the present invention provides a diagnostic or imaging agent comprising the antibody of the present invention linked to a label (detectable moiety) that directly or indirectly produces a detectable signal.

[0489] In some embodiments, the immunoconjugates of the present invention can be fusion proteins / polypeptides comprising the antibody of the present invention and a detectable moiety comprising a polypeptide (e.g., a polypeptide that directly gives a signal or can be used to generate or produce a detectable signal). Thus, in another embodiment, the present invention provides a nucleic acid molecule encoding the immunoconjugates of the present invention, such as a fusion protein / polypeptide, comprising the antibody of the present invention and a detectable moiety comprising a polypeptide.

[0490] By "detectable moiety", we include the meaning that this moiety is a moiety that can generally be detected non-invasively from outside the body and at the site of the targeted object after administering the compound of the present invention to a patient. Thus, the immunoconjugates of the present invention can also be used for imaging and diagnosis, particularly in the imaging and diagnosis of the new vascular system of solid tumors, as further described below.

[0491] In some embodiments, the detectable moiety is or comprises magnetic nanoparticles, radionuclides or fluorophores.

[0492] Thus, in some embodiments, the detectable moiety can be a radioisotope atom useful for imaging. Suitable radioisotope atoms include technetium 99m or iodine 123 for scintigraphy studies. Others can be selected from: iodine 124 ; iodine 125 ; iodine 126 ; iodine- 131 ; iodine 133; indium -111 ; indium -113m , fluorine 18 ; fluorine 19 ; carbon 11 ; carbon -13 ; copper 64 ; copper 67 ; nitrogen 13 ; nitrogen 15 ; oxygen 15 ; oxygen 17 ; arsenic 72 ; gadolinium; manganese; iron; deuterium; tritium; yttrium 86 ; zirconium 89 ; bromine 77 , gallium 67 ; gallium 68 , ruthenium 95 ; ruthenium 97 ; ruthenium 103 ; ruthenium 105 ; mercury 107 ; rhenium 99m ; rhenium 101 ; rhenium 105 ; scandium 47 . As known in the art, such radioisotopes can be conjugated to the antibody using suitable methods - directly or through chelating agents such as EDTA or DTPA.

[0493] In some embodiments, the detectable moiety includes X-ray detectable compounds such as bismuth(III), gold(III), lanthanum(III) or lead(II); radioactive ions such as gallium 67 ; gallium 68 ; mercury 177 ; mercury 203 ; rhenium 186 ; rhenium 188 ; rubidium 97 ; rubidium 103 ; or yttrium 90 ; nuclear magnetic resonance spin isotopes such as cobalt(II), copper(II), chromium(III), dysprosium(III), erbium(III), gadolinium(III), holmium(III), iron(II), iron(III), manganese(II), neodymium(III), nickel(II), samarium(III), terbium(III), vanadium(II) or ytterbium(III); or rhodamine or fluorescein.

[0494] Other readily detectable moieties that can be used include, for example, spin labels for magnetic resonance imaging (MRI), such as iodine 123 , iodine 131 , indium 111 , fluorine 19 , carbon 13 , nitrogen 15 , oxygen 17 , gadolinium, manganese or iron.

[0495] Radioactive labels or other labels can be incorporated into the compound in a known manner. For example, if the antibody can be biosynthesized or synthesized by chemical amino acid synthesis using suitable amino acid precursors, said amino acid precursors including, for example, fluorine 19 in place of hydrogen. Labels such as 99mTc, 123I, 186Rh, 188Rh and 111In can be linked via cysteine residues in the antibody. Yttrium 90 can be attached via lysine residues. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Comm. 80, 49 - 57) can be used to incorporate iodine 123 . Other methods are described in detail in the reference (“Monoclonal Antibodies in Immunoscintigraphy”, J.F. Chatal, CRC Press, 1989).

[0496] Many suitable fluorophores and detection methods are well known in the art and are described, for example, by Stefan Andersson - Engels et al (1997) “In vivo fluorescence imaging for tissue diagnostics. Phys. Med. Biol. 42:815 - 824; et al (2008) “Near-Infrared Emitting Fluorophore-Doped Calcium Phosphate Nanoparticles for In Vivo Imaging of Human Breast Cancer” ACS Nano 2(10):2075-84; and Chin et al (2009) “In-vivo optical detection of cancer using chlorin e6–polyvinylpyrrolidone induced fluorescence imaging and spectroscopy” BMC Medical Imaging 9:1 (doi:10.1186 / 1471-2342-9-1). Examples include fluorescein and its derivatives, fluorescent dyes, rhodamine and its derivatives, green fluorescent protein (GFP), dansyl, umbelliferone, etc. In such conjugates, the antibody or its functional fragment of the present invention can be prepared by methods known to those skilled in the art.

[0497] The detectable moiety can include a detectable enzyme, such as peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, or glucose-6-phosphate dehydrogenase.

[0498] The detectable moiety can comprise a molecule, such as biotin, digoxygenin, or 5-bromodeoxyuridine.

[0499] The detectable moiety can comprise a chemiluminescent label, such as luminol and dioxetanes, or a bioluminescent label, such as luciferase and luciferin.

[0500] Accordingly, in another embodiment, the present invention provides a method for imaging a neovascular system in a subject, the method comprising:

[0501] administering to the subject an imaging agent of the present invention (e.g., an immunoconjugate comprising an antibody of the present invention and a detectable moiety), and

[0502] imaging the detectable moiety in vivo. Preferably, the neovascular system is a tumor neovascular system.

[0503] In some embodiments, a subject can have a solid tumor, preferably such solid tumors as described above, and image the new blood vessel system of the tumor. Thus, the localization of an imaging agent (such as an immunoconjugate) at a particular organ in the body indicates that the individual may have a solid tumor at that organ or may be forming a solid tumor. The method can be used, for example, to determine the size of a previously diagnosed solid tumor, determine the effectiveness of a therapy for a solid tumor, or determine the extent of tumor metastasis. Methods for imaging detectable portions within the body are well known in the art and include PET (positron emission tomography).

[0504] Thus, it can also be seen that the present invention provides a method for detecting, diagnosing, and prognosing solid tumors in a subject, the method comprising: administering an imaging agent (such as an immunoconjugate) of the present invention to the subject and detecting the presence and / or location of a detectable portion within the body.

[0505] It is apparent from the above discussion that the present invention provides various compositions, such as medicaments, therapies, diagnostics, imaging, comprising the antibodies (including the immunoconjugates described above) of the present invention and / or the immune effector cells of the present invention, and pharmaceutically acceptable diluents, carriers, or excipients. In this regard, it should be understood that the reagents of the present invention (i.e., antibodies, immune effector cells, carriers, viruses, etc.) can generally be formulated into pharmaceutical compositions for administration to an individual (i.e., a subject), i.e., together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0506] "Pharmaceutically acceptable" includes that the formulation is sterile and pyrogen-free. Suitable pharmaceutical carriers, diluents, and excipients are well known in the pharmaceutical art. The carrier must be "acceptable" in the sense of being compatible with the medicament and not harmful to its recipient. Generally, the carrier will be water or saline, which will be sterile and pyrogen-free; however, other acceptable carriers can be used.

[0507] In some embodiments, the pharmaceutical composition or formulation of the present invention is for parenteral administration, more particularly for intravenous administration. In a preferred embodiment, the pharmaceutical composition is suitable for intravenous administration to a patient, for example, by injection.

[0508] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents.

[0509] Liquid pharmaceutical compositions, whether they are solutions, suspensions or other similar forms, may include one or more of the following: sterile diluents such as water for injection, saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic monoglycerides or diglycerides which may act as solvents or suspending media, polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetates, citrates or phosphates and agents for regulating osmotic pressure such as sodium chloride or dextrose. Parenteral formulations may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0510] The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0511] Preferably, the formulation is a unit dose containing the active ingredient, a daily sub-dose or an appropriate fraction thereof.

[0512] The reagent or composition of the present invention may be administered orally or by any parenteral route in the form of a pharmaceutical preparation in a pharmaceutically acceptable dosage form, which contains the active ingredient, optionally in the form of a non-toxic organic or inorganic acid or base addition salt. Depending on the condition and the patient to be treated, as well as the route of administration, the composition may be administered in different dosages.

[0513] In human therapy, the reagent or composition of the present invention is usually administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected according to the intended route of administration and standard pharmaceutical practice.

[0514] For example, the reagent or composition of the present invention may be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate, delayed or controlled release applications. The reagent or composition of the present invention may also be administered by intracavernosal injection.

[0515] Suitable tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, and glycine, disintegrants such as starch (preferably corn, potato, or tapioca starch), sodium starch glycolate, croscarmellose sodium, and certain complex silicates, and granulating binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricants such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be included.

[0516] Solid compositions of a similar type can also be used as fillers in gelatin capsules. In this regard, preferred excipients include lactose, starch, cellulose, lactose, or high molecular weight polyethylene glycol. For aqueous suspensions and / or elixirs, the compounds of the present invention can be combined with various sweetening or flavoring agents, coloring agents or dyes, emulsifying and / or suspending agents, and diluents such as water, ethanol, propylene glycol, and glycerol and combinations thereof.

[0517] The reagents or compositions of the present invention can also be administered parenterally, such as intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternal, intracranially, intramuscularly, or subcutaneously, or they can be administered by infusion techniques. They are preferably used in the form of a sterile aqueous solution, which may contain other substances, such as sufficient salts or glucose to render the solution isotonic with blood. If necessary, the aqueous solution should be appropriately buffered (preferably pH 3 to 9). Preparation of suitable parenteral formulations under sterile conditions can be readily accomplished by standard pharmaceutical techniques known to those skilled in the art.

[0518] The formulations can be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a lyophilized (freeze-dried) state, requiring only the immediate addition of a sterile liquid carrier, such as water for injection, before use. Temporary injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the aforementioned types.

[0519] For oral and parenteral administration to human patients, the daily dosage level of the reagents or compositions of the present invention is generally from 1 to 1,000 mg per adult (i.e., about 0.015 to 15 mg / kg), administered as a single dose or divided doses.

[0520] Thus, for example, tablets or capsules of the reagents or compositions of the present invention can contain from 1 mg to 1,000 mg of the active agent, for single or two or more administrations at a time, as appropriate. In any case, the physician will determine the actual dose most suitable for any individual patient, and it will vary with the age, weight, and response of the particular patient. The above dosages are exemplary of the general case. Of course, there can be individual cases where higher or lower dosage ranges are advantageous, and these are all within the scope of the present invention.

[0521] The reagent or composition of the present invention can also be administered intranasally or by inhalation, and is conveniently delivered from a pressurized container, pump, nebulizer or atomizer in the form of a dry powder inhaler or aerosol spray using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkanes such as 1,1,1,2-tetrafluoroethane (HFA134A) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide or other suitable gases. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. The pressurized container, pump, nebulizer or atomizer can contain a solution or suspension of the active compound, for example using a mixture of ethanol and a propellant as a solvent, which can additionally contain a lubricant, such as sorbitan trioleate. Capsules and cartridges (e.g., made of gelatin) for inhalers or insufflators can be formulated to contain a powder mixture of the antibody and a suitable powder matrix such as lactose or starch. Such formulations can be particularly useful for treating solid tumors of the lung, such as, for example, small cell lung cancer, non-small cell lung cancer, pleuropulmonary blastoma or carcinoid tumors.

[0522] The aerosol or dry powder formulation is preferably arranged such that each metered dose or "puff" contains at least 1 mg of the inhibitor for delivery to the patient. It should be understood that the total daily dose with the aerosol will vary from patient to patient and can be administered as a single dose, or more commonly, in divided doses throughout the day.

[0523] In some embodiments, the reagent or composition of the present invention can be administered in the form of a suppository or pessary, particularly for treating or targeting tumors of the colon, rectum or prostate.

[0524] In some embodiments, the reagent or composition of the present invention can be administered by the ocular route. For ocular use, the reagent or composition of the present invention can be formulated, for example, as a micronized suspension in isotonic pH-adjusted sterile saline, or preferably, as a solution in isotonic pH-adjusted sterile saline, optionally in combination with a preservative such as benzalkonium chloride. Alternatively, the reagent or composition of the present invention can be formulated as an ointment, such as petrolatum. Such formulations can be particularly useful for treating solid tumors of the eye, such as retinoblastoma, medulloepithelioma, uveal melanoma, rhabdomyosarcoma, intraocular lymphoma or orbital lymphoma.

[0525] In some embodiments, the reagents or compositions of the present invention may be suitable for topical administration to a patient. The reagents or compositions of the present invention may be topically administered in the form of a lotion, solution, cream, ointment or powder, or may be administered transdermally, for example, by using a skin patch. For topical administration to the skin, the reagents or compositions of the present invention may be formulated into a suitable ointment containing the active compound suspended or dissolved in a mixture with, for example, one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compounds, emulsifying wax and water. Alternatively, they may be formulated into a suitable emulsion or cream, suspended or dissolved in a mixture with, for example, one or more of the following: mineral oil, sorbitan stearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Such formulations may be particularly useful for treating solid skin tumors, such as basal cell carcinoma, squamous cell carcinoma or melanoma.

[0526] For skin cancer, the reagents or compositions of the present invention may also be delivered by electroincorporation (EI). EI occurs when small particles up to 30 microns in diameter on the skin surface are subjected to electrical pulses identical or similar to those used in electroporation. In EI, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles may be loaded or coated with an inhibitor, or may simply act as "bullets" that create pores in the skin through which the reagents or compositions of the present invention may enter.

[0527] Preparations suitable for topical administration in the mouth include lozenges, which contain a flavoring base, usually sucrose, and the active ingredient in gum arabic or tragacanth; troches, which contain an inert matrix such as gelatin and glycerin or sucrose and the active ingredient in gum arabic; and mouthwashes, which contain the active ingredient in a suitable liquid carrier. Such preparations may be particularly useful for treating solid tumors of the mouth and pharynx.

[0528] In some embodiments, injectable sustained release drug delivery systems may be used to deliver the reagents or compositions of the present invention. These are specifically designed to reduce the frequency of injections. An example of such a system is Nutropin Depot, which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that slowly release rhGH over a sustained period of time once injected.

[0529] The reagents or compositions of the present invention may be administered by a surgical implant device that releases the drug directly into the desired site, for example, into the eye to treat eye tumors. Such direct application to the diseased site enables effective treatment without significant systemic side effects.

[0530] An alternative method for delivering the reagent or composition of the present invention is the thermosensitive ReGel injectable system. Below body temperature, ReGel is an injectable liquid that immediately forms a gel depot at body temperature. The gel depot slowly erodes and dissolves into known, safe, and biodegradable polymers. As the biopolymer dissolves, the active drug is delivered over time.

[0531] The reagent or composition of the present invention can also be delivered orally. This process uses the natural process of oral uptake of vitamin B12 in the body to co-deliver proteins and peptides. By utilizing the vitamin B12 uptake system, the protein or peptide can cross the intestinal wall. A complex is synthesized between a vitamin B12 analogue and a drug, which retains both a significant affinity for the intrinsic factor (IF) in the vitamin B12 moiety of the complex and a significant biological activity of the drug moiety of the complex.

[0532] The nucleic acid molecule reagents of the present invention (such as nucleic acid molecules, vectors, etc.) can be administered as suitable genetic constructs as described below and delivered to a patient in the case of expressing it. Generally, the nucleic acid in the genetic construct is operably linked to a promoter that can express the compound in the cell. The genetic constructs of the present invention can be prepared using methods well known in the art, such as the methods in Sambrook et al. (2001).

[0533] Although the genetic constructs for delivering polynucleotides can be DNA or RNA, preferably they are DNA.

[0534] Preferably, the genetic construct is suitable for delivery to human cells. For example, a transposon system (such as sleeping beauty, piggyBac) can be used to integrate the target DNA into the host cell genome. Means and methods for introducing the genetic construct into cells are known in the art, including the use of immunoliposomes, liposomes, viral vectors (including vaccinia, modified vaccinia, lentivirus, parvovirus, retrovirus, adenovirus, and adeno-associated virus (AAV) vectors), and by direct DNA delivery, such as using gene guns and electroporation. In addition, methods for delivering polynucleotides to target tissues of a patient for treatment are also well known in the art. In another method, a highly efficient nucleic acid delivery system that utilizes receptor-mediated endocytosis to carry large DNA molecules into cells is used. This is achieved by conjugating the iron-transporting protein transferrin to a polycation that binds nucleic acids. Delivery of the DNA construct of the present invention or other genetic constructs using highly efficient receptor-mediated endocytosis is carried out using the endosome-disrupting activity of defective or chemically inactivated adenovirus particles generated by the method of Cotten et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 6094-6098. It should be understood that "naked DNA" and DNA complexed with cationic and neutral lipids can also be used to introduce the DNA of the present invention into the cells of an individual to be treated. Non-viral methods of gene therapy are described in Ledley (1995, Human Gene Therapy 6, 1129-1144).

[0535] Although it may be useful to use a tissue-specific promoter in a vector encoding a polynucleotide inhibitor for a particular tissue cancer / tumor, this is not necessary because the risk of expression of the nucleic acid molecule agent in the body at locations other than the cancer / tumor is expected to be tolerable compared to the therapeutic benefit for a patient with cancer / tumor. It may be desirable to be able to temporarily regulate the expression of the polynucleotide inhibitor in cells, although this is also not necessary.

[0536] The reagents and compositions of the present invention can be lyophilized for storage and reconstituted in a suitable vehicle prior to use. Any suitable lyophilization method (such as spray drying, cake drying) and / or reconstitution technique can be used. Those skilled in the art will understand that lyophilization and reconstitution can result in varying degrees of loss of antibody activity (e.g., for conventional immunoglobulins, IgM antibodies tend to have a greater loss of activity than IgG antibodies) and the use level may have to be adjusted upward to compensate. In one embodiment, when rehydrated, the lyophilized (freeze-dried) antibody loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (before lyophilization).

[0537] The reagents of the present invention for administration (such as antibodies, immune effector cells, nucleic acid molecules, vectors, etc.) can be appropriately modified for use in pharmaceutical compositions. For example, the reagents can be stabilized against degradation in the compositions of the present invention, for example, by using appropriate additives such as salts or non-electrolytes, acetates, EDTA, citrates, Tris, phosphates or acetate buffers, mannitol, glycine, HSA (human serum albumin) or Polysorbate. Many stabilizers are known in the art.

[0538] As defined herein, the antibodies of the present invention can also be used as molecular tools for in vitro or in vivo applications and assays. Since antibodies have antigen-binding sites, these can act as members of specific binding pairs, and these molecules can be used in any assay that requires a member of a specific binding pair.

[0539] Accordingly, other aspects of the present invention provide reagents comprising the antibodies of the present invention as defined herein and the use of such antibodies as molecular tools, for example, in in vitro or in vivo assays.

[0540] The present invention further includes kits comprising one or more of the following: the antibodies of the present invention, CARs, immunoconjugates or compositions, or one or more nucleic acid molecules encoding the antibodies or CARs of the present invention, or one or more recombinant expression vectors comprising the nucleic acid sequences of the present invention, or one or more host cells (including immune effector cells) or viruses comprising the recombinant expression vectors or nucleic acid sequences of the present invention. Preferably, the kits are for the methods and uses as described herein, such as the therapeutic, diagnostic or imaging methods described herein, or for in vitro assays or methods as described herein. The antibodies in such kits can preferably be antibody conjugates (immunoconjugates) as described elsewhere herein, for example, which can be conjugated to a detectable moiety or a therapeutic or cytotoxic agent. Preferably, the kits include instructions for using the kit components, for example, for diagnosis. Preferably, the kits are for diagnosing or treating diseases as described elsewhere herein and optionally include instructions for using the kit components to diagnose or treat such diseases.

[0541] Cancer treatment can also be carried out by:

[0542] (a) forming a tumor image by administering to an animal or patient (i.e., subject) having a tumor a diagnostic amount of at least a first detectable-labeled anti-CLEC14A antibody of the present invention (such as an imaging agent of the present invention), thereby forming a detectable image of the tumor; and

[0543] (b) Subsequently administer a therapeutically optimized amount of at least a first therapeutic agent of the present invention (e.g., an antibody, immune effector cell or composition) to the same animal or patient, thereby eliciting an anti-tumor effect.

[0544] Any reference herein to "tumor" also refers to "cancer" or "neoplasm". Metastatic cancer can also be treated, which can reduce the metastasis of the primary tumor. The so-called minimal residual disease (MRD) remaining in postoperative patients can be suitable for immunotherapy with the anti-CLEC14A antibody and other agents of the present invention, such as immune effector cells or compositions.

[0545] The subject to be treated using the methods and cells of the present invention can be any mammalian species. For example, the subject can be any type of domestic pet, such as a mouse, rat, gerbil, rabbit, guinea pig, hamster, cat or dog, or livestock, such as a goat, sheep, pig, cow or horse. In another preferred embodiment of the present invention, the subject can be a primate, such as a monkey, gibbon, gorilla, orang-utang, chimpanzee or bonobo. However, in a preferred embodiment of the present invention, the subject is a human.

[0546] As used throughout the application, the terms "a" and "an" are used in the sense that they mean "at least one / species", "at least first", "one / species or more than one / species" or "more than one / species" of the recited component or step, except in cases where an upper limit is specifically stated thereafter. Thus, as used herein, "antibody" means "at least a first antibody". Based on the present disclosure, one of ordinary skill in the art will know the operable limitations and parameters of the combination, as well as the amount of any single reagent.

[0547] The present invention will now be described in more detail in the following non-limiting examples with reference to the accompanying drawings, wherein:

[0548] Figure 1 A graph showing the relative expression of CLEC14A in HUVEC and other primary cells. CLEC14A is specifically expressed in endothelial cells (HUVEC), but not in human aortic smooth muscle cells (HASMC), human lung fibroblasts (MRC5), human bronchial epithelial cells (HBE), hepatocytes or peripheral blood mononuclear cells (PBMC).

[0549] Figure 2 It is shown that [A] siRNA duplex targeting CLEC14A can effectively knockdown CLEC14A mRNA expression in HUVECs, as determined by qPCR. Relative expression was determined by normalizing expression to flotilin2. [B] Knockdown of CLEC14A at the protein level was determined by Western blot analysis. Tubulin was used as a loading control. [C] Representative images of sprout outgrowth from control or clec14a-targeting siRNA-treated HUVECs after 16 h. [D] Quantification of sprouts from 27 spheroids (9 spheroids from 3 umbilical cords) for control and CLEC14A knockdown HUVECs; Mann-Whitney statistical test p<0.001. [E] Representative images of sprout outgrowth after 24 h for mixed control (green) and clec14a-targeting siRNA-treated HUVECs (red). [F] Quantification of the percentage of tip and stalk cells from control (CON) and CLEC14A knockdown (KD) HUVECs; two-way ANOVA statistical test using Bonferroni post-test *** = p<0.001, ns = not significant.

[0550] Figure 3 Schematic representation of the clec14a gene in C57BL / 6 (clec14a+ / +) or C57BL / 6 (Cle14atm1(KOMP)Vlcg) (clec14a- / -) mice. [B] Quantitative PCR analysis of the 5' untranslated region (UTR), coding sequence (CDS), and 3' UTR of clec14a on cDNA generated from three clec14a+ / + mice (white bars) and three clec14a- / - mice (black bars). Relative expression was determined by normalizing expression to flotilin2. [C] Western blot analysis of CLEC14A protein expression in lung lysates from clec14a+ / + and clec14a- / - mice using a polyclonal antiserum against murine CLEC14A. Tubulin was used as a loading control. [D] Representative images of aortic ring sprouting assays from clec14a+ / + and clec14a- / - mice. Quantification of the number of tubes formed per ring [E], and quantification of the maximum distance migrated by endothelial tubes from aortic rings [F], data from 48 rings per genotype, 6 mice per genotype; Mann-Whitney statistical test p<0.001. [G] Representative images of hematoxylin and eosin stained sections of sponge implants from clec14a+ / + and clec14a- / - mice, sections analyzed at the center of the sponge. [H] Quantification of cell invasion of the sponge implants shown in G; Mann-Whitney statistical test p<0.05. [I] Quantification of vascular density; Mann-Whitney statistical test p<0.001. [J] Hematoxylin and eosin counterstained sections of x-gal stained liver and sponge tissues from clec14a- / - mice.

[0551] Figure 4 Shown are [A] Lewis lung carcinoma (LLC) tumor growth in clec14a+ / + (black dotted line) and clec14a− / − (black square line) mice; two-way ANOVA statistical analysis, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. [B] Representative images of LLC tumors. [C] Endpoint tumor weights of 7 clec14a + / + (dots) and 7 clec14a− / − (squares) mice; Mann-Whitney statistical test p < 0.001. [D] Representative images of immunofluorescent staining of LLC tumor sections against murine CD31. Quantification of [E] vascular density and [F] percentage endothelial coverage from clec14a+ / + and clec14a− / − mice; Mann-Whitney statistical test p < 0.0001. [G] Liver and LLC tumor tissue sections from clec14a− / − mice stained with x-gal and counterstained with hematoxylin and eosin.

[0552] Figure 5 Optical microscopy images showing the results of (A) HUVEC scratch wound healing assays, where the anti-CLEC14A monoclonal antibody CRT-3 showed a delay in wound healing. (B) Graphic representation of the results in (A).

[0553] Figure 6 Shown is the analysis of tube formation assays of HUVECs treated with CLEC14A antibodies. HUVECs were treated with 20 μg / ml CRT2, 3 or 4 or murine IgG isotype control. Tube images were taken at 16 h and total tube length, number of junctions, number of branches, branch length, number of meshes and total mesh area were analyzed. Data shown represent three experiments, each analyzing five data points. Error bars show SEM. *P, 0.05. **P < 0.01..

[0554] Figure 7 Flow cytometry analysis plots showing the binding of CRT-2 and CRT-3 to [A] HEK293T transfected with CLEC14A and (B) HEK293T transfected with thrombomodulin.

[0555] Figure 8 Flow cytometry analysis plots showing the association of CRT-2 and CRT-3 with the following: (A) HEK293T transfected with a chimera containing the CTLD of thrombomodulin and the remainder of CLEC14A, (B) HEK293T transfected with a chimera containing the sushi-like domain of thrombomodulin and the remainder of CLEC14A, and (C) HEK293T transfected with a chimera containing the loop residues 97-108 of thrombomodulin and the remainder of CLEC14A.

[0556] Figure 9 Alignment of CLEC14A region 1-42 of CD141; CLEC14A region 97-108 of CD141; and CLEC14A region 122-142 of CD141 is shown.

[0557] Figure 10 Retroviral CAR vectors (based on pMP71) co-expressing a truncated CD34 marker gene and a scFv fragment / CD3ζ chain chimeric receptor are shown. Expression is driven by an LTR promoter, and the 2A peptide linker ensures equimolar expression of both CD34 and CAR. The second-generation CAR construct includes a CD28 co-stimulatory domain. B shows CD34 staining by flow cytometry analysis, demonstrating successful transduction of T cells using a retroviral construct co-expressing a CLEC14A-specific CAR. The first-generation CARs based on antibody CRT3 are designated CRT3.z. The second-generation CARs based on antibody CRT3 are designated CRT3.28z. (C) shows cells by flow cytometry analysis, directly stained for CAR expression using CLEC14A-Fc (% values show specific binding of CLEC14A-Fc, which has had background staining with Fc alone subtracted).

[0558] Figure 11 CAR-transduced T cells are shown to respond to CLEC14A in vitro. T cells transduced to express a first- or second-generation CAR based on antibody CRT3 or mock-transduced (control) T cells are tested for their ability to respond to CLEC14A, which is expressed as (A) a plate-bound recombinant Fc fusion protein, (B) expressed on engineered CHO cells, or (C) expressed on human umbilical vein endothelial cells (HUVECs) that naturally express CLEC14A when cultured in static culture. T cell responses are measured using ELISA for interferon γ production. The data shown represent data obtained from 3-7 replicate experiments. T cells are adjusted to equalize the frequency of transgenic-expressing cells. All histograms show mean responses + SD.

[0559] Figure 12 Shows further in vitro functional testing of CLEC14A - specific CAR - transduced T cells. T cells transduced to express first - or second - generation CARs based on the antibody CRT3 or mock - transduced (control) T cells were tested for their ability to respond to CLEC14A in the following functional assays: (A) Cytotoxicity, using CHO cells engineered to express human CLEC14A (subtracting the background level of lysis of CHO alone (control cells)). Data shown represent 5 replicate experiments. (B) Proliferation, using CFSE - labeled CAR - transduced T cells. When co - cultured with HUVECs for 4 days, we measured the proliferation of CAR+(CD34+) and CAR-(CD34-) cell subsets. Data shown represent 2 replicate experiments. (C) Response of CLEC14A - specific CAR - transduced T cells to both human and mouse CLEC14A was evaluated using interferon - γ release. T cells were adjusted to equalize the frequency of transgenic - expressing cells. Data shown represent 6 replicate experiments. All histograms show mean response + SD.

[0560] Figure 13 Shows histological pictures of tissue samples after in vivo toxicity testing in healthy C57 / BL6 mice injected with CLEC14A - specific CAR - transduced murine T cells.

[0561] Figure 14 Shows a graph of Lewis lung tumor volume from mice treated with T cells transduced to express the second - generation CAR based on the antibody CRT - 3 or mock - transduced (control) T cells. Mice received a total of 20 million T cells (CD8:CD4 = 5:2), with CRT3.28z expressed in 2.2 million of these cells. Tumor growth was then monitored using (A) bioluminescence or (B) calipers.

[0562] Figure 15 Shows bar graphs depicting tumor weight (A), percentage of tumor tissue area covered by blood vessels (B), and percentage of tumor tissue stained with fibrinogen (C) in Lewis lung tumors from mice injected with T cells transduced to express the second - generation CAR based on the antibody CRT - 3 or mock - transduced (control) T cells.

[0563] Figure 16 Shows images of HUVECs treated with CRT - 3 at (i) 0 minutes and (ii) 90 minutes and demonstrates antibody internalization; (B) shows a graph of the cell viability of HUVECs treated with a CRT - 3 - antibody drug conjugate (immunoconjugate).

[0564] Figure 17 Photographs of mouse lung tissues are shown (A) 24 hours after treatment with control ADC (B12-ADC) and (B) 24 hours after treatment with CRT-3-ADC.

[0565] Figure 18 CRT1, 3, and 5 CAR (with CD28 co-stimulatory domain) T cell responses to titrated concentrations of human and mouse recombinant CLEC14A are shown.

[0566] Figure 19 Designs of constructs of CARs with different co-stimulatory domains are shown; 1) tCD34-F2A-scFv-CD28TM-CD28 signal-CD3zeta, 2) tCD34-F2A-scFv-CD8TM-4-1BB signal-CD3zeta, 3) tCD34-F2A-scFv-CD8TM-OX40 signal-CD3zeta, 4) tCD34-F2A-scFv-CD28TM-CD28 signal-4-1BB signal–CD3zeta, 5) tCD34-F2A-scFv-CD28TM-CD28 signal-OX40 signal-CD3zeta, 6) tCD34-F2A-scFv-CD8TM-4-1BB signal–OX40 signal-CD3zeta. tCD34 is included to identify successfully transduced cells, so this and F2A can be excluded from the construct. A hinge or spacer region, e.g., one from CD8a, can be included additionally.

[0567] Figure 20 Results of cytotoxicity assays of CRT1, 3, and 5 CARs against mouse endothelial cells expressing CLEC14A are shown ( Figure 20 A). Results of proliferation assays of CRT 1, 3, and 5 CARs are shown in Figure 20 B.

[0568] Figure 21 Functional tests of CRT3 CAR T cells containing different co-stimulatory domains are shown and IFNγ production in response to titrated numbers of CHO cells expressing human CLEC14A is shown.

[0569] Figure 22 IFNγ release of CRT1, 3, and 5 CAR (CD28 co-stimulatory domain) T cells is shown after incubation with 293 or SEND cells engineered to express CLEC14A chimeras (A1 - human CLEC14A with mouse intracellular domain, B1 - human CLEC14A with mouse transmembrane and intracellular domains, huCLEC - human CLEC14A). After incubation with SEND cells, cytotoxicity data of CAR T cells are shown in Figure 22 B.

[0570] Figure 23 Shows a schematic diagram of a suitable vector for generating RNA for electroporation by in vitro transcription.

[0571] Figure 24 Shows a construct encoding a CAR that can be used to transduce murine T cells. The construct contains transmembrane, co-stimulatory, and intracellular signaling sequences from murine proteins (see SEQ ID NOs 116 - 121). The construct may further contain a hinge or spacer domain from murine CD8α.

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590] * Refers to variant sequences

[0591] X refers to the absence of an amino acid Examples

[0592] Analysis of CLEC14A Expression in Example 1

[0593] Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords donated by the UK National Health Service after donor informed consent. The umbilical cords were dissected from the placenta and the veins were washed in sterile PBS to remove blood. 1 mg / ml collagenase diluted in M199 medium (Sigma) was injected into the veins and then incubated at 37 °C for 20 minutes to isolate the endothelial cells. The HUVECs were collected by washing in M199 complete medium containing 10% FCS, 10% large vessel endothelial cell growth supplement (TCS Cell Works) and 4 mM L-glutamine, and plated on 0.1% type I gelatin from pig skin (Sigma)-coated dishes.

[0594] Primary cell source

[0595] Human aortic smooth muscle cells (HASMCs) and human bronchial epithelial cells (HBECs) were purchased from TCS Cell Works. Human lung fibroblasts (MRC5) were obtained from Cancer Research UK Central Services. Human peripheral blood mononuclear cells (PBMCs) were obtained from the Institute of Cancer at the University of Birmingham. Hepatocytes were a gift from Professor David Adams of the School of Immunology and Infection at the University of Birmingham.

[0596] RNA extraction and real-time PCR

[0597] Total RNA was isolated from cultured primary cells using TRI reagent (Sigma), and then cDNA synthesis was performed using the High-Capacity cDNA Archive kit (Applied Biosystems) with the provided random primers. The ProbeLibrary real-time PCR assay system (Exiqon) was used in the screening of primary cells for CLEC14A expression. Flotillin 2 was selected as the housekeeping gene and the expression of CLEC14A was normalized relative to it. The Primer and probe sets for CLEC14A and Flotillin 2 were designed by ProbeFinder software (Roche). For CLEC14A, the primer and probe set was:

[0598] 5'-CTGGGACCGAGGTGAGTG-3' (SEQ ID NO:79), and

[0599] 5'-CGCGATGCAAGTAACTGAGA-3' (SEQ ID NO:80), probe number 24.

[0600] For Flotillin 2, the primer and probe sets were:

[0601] 5'-TGTTGTGGTTCCGACTATAAACAG-3' (SEQ ID NO:81) and

[0602] 5'-GGGCTGCAACGTCATAATCT-3' (SEQ ID NO:82), probe number 28. Quantitative PCR reactions were performed on a Rotor-Gene RG3000 thermocycler (Corbett Research). For each primary cell type, reaction mixtures were prepared in triplicate and 5 ng of cDNA was applied in each reaction. Fold changes were calculated using the ΔΔCt method.

[0603] HUVEC Immunofluorescence

[0604] HUVEC were cultured in glass micropore chambers (Nunc) fixed in ice-cold methanol, washed with PBST, and blocked in 10% FCS 3% BSA in PBST. Cells were then stained with the CLEC14A antibody or co-stained with a 5 μg / ml mouse monoclonal IgG antibody against human VE-cadherin (kind gift from Professor Maria Grazia Lampugnani, Fire Institute for Molecular Oncology, Milan) following the same protocol used for paraffin-embedded sections. Sections were analyzed for staining with a 510 laser scanning confocal microscope (Carl Zeiss).

[0605] Results

[0606] Figure 1 is a graph showing the relative expression of CLEC14A in HUVEC and other primary cells. CLEC14A is specifically expressed in endothelial cells. This confirms the previous finding that CLEC14A is endothelial-specific.

[0607] CLEC14A was expressed in sections of solid tumors and normal tissues using a CLEC14A-specific probe. CLEC14A expression was measured by immunofluorescence in human ovarian, bladder, liver, breast, colon, rectal, esophageal, kidney, lung, prostate, stomach, pancreatic, and thyroid tumor tissues. Endothelial specificity of CLEC14A expression was confirmed by co-localization with Ulex europeaus agglutinin I (UEAI), which binds to specific fucose residues on endothelial cells. CLEC14A expression was seen in the blood vessels of all tumor tissues analyzed. Ovarian, bladder, liver, breast, kidney, and prostate tumors were strongly positive for CLEC14A expression, while stomach, esophageal, lung, colon, rectal, pancreatic, and thyroid tumor tissues showed lower levels of specific CLEC14A expression. CLEC14A expression was not detected in any corresponding normal control (non-tumor) tissues.

[0608] In summary, these results demonstrate that the transmembrane protein CLEC14A is specifically expressed in the tumor vasculature and can therefore be used as a tumor endothelial marker.

[0609] Example 2: Analysis of CLEC14A Function In Vitro and In Vivo

[0610] Materials and Methods

[0611] For Western blotting and immunoprecipitation; primary antibodies: sheep polyclonal anti-human CLEC14A (R&D systems), mouse monoclonal anti-human Tubulin (Sigma); secondary antibodies: goat polyclonal anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Dako), donkey polyclonal anti-sheep IgG conjugated to HRP (R&D systems). For immunofluorescence; primary antibody: rabbit polyclonal anti-mouse PECAM (Santa Cruz); secondary antibody: donkey polyclonal anti-rabbit conjugated to Alexa Fluor488 (Invitrogen). For flow cytometry; primary antibodies: mouse monoclonal anti-HA tag (CRUK), mouse monoclonal anti-CLEC14A (C2, C4 as described below); secondary antibody: goat polyclonal anti-mouse IgG conjugated to Alexa Fluor488 (Invitrogen).

[0612] For protein production; lentiviral plasmids psPAX2 (lentiviral packaging; Addgene), pMD2G (envelope plasmid; Addgene), and pWPI hCLEC14A-ECD-Fc (lentiviral mammalian expression plasmid containing IRES-EGFP; Addgene) were used. pWPI hCLEC14A-Fc and mCLEC14A-Fc were generated by initial PCR subcloning of the clec14a IMAGE clone (Origene) into the pcDNA3-Fc plasmid. The primers used were as follows:

[0613] Human CLEC14A fwd

[0614] 5’TAGTAGGAATTCGAGAGAATGAGGCCGGCGTTCGCCCTG3’ (SEQ ID NO:83);

[0615] Human CLEC14A rev

[0616] 5’AGAACCGCGGCCGCTGGAGGAGTCGAAAGCCTGAGGAGT3’ (SEQ ID NO:84);

[0617] Mouse CLEC14A fwd

[0618] 5’TAGTAGGAATTCGAGAGAATGAGGCCAGCGCTTGCCCTG3’ (SEQ ID NO:85);

[0619] Mouse CLEC14A rev

[0620] 5’CTACTAGCGGCCGCTCGTGGAAGAGGTGTCGAAAGT3’ (SEQ ID NO:86).

[0621] CLEC14A was inserted using the EcoR1 and Not1 restriction sites, and further rounds of PCR subcloning were performed to transfer the CLEC14A-Fc fusion into pWPI. The primers used were as follows:

[0622] Human CLEC14A fwd

[0623] 5’TAGTAGTTAATTAAGAGAGAATGAGGCCGGCGTTC3’ (SEQ ID NO: 87);

[0624] Mouse CLEC14A fwd

[0625] 5’TAGTAGTTAATTAAGAGAGAATGAGGCCAGCGCTT3’(SEQ ID NO: 88);

[0626] Human Fc rev

[0627] 5’CTACTAGTTTAAACTCATTTACCCGGAGACAGGGA3’(SEQ ID NO: 89).

[0628] For this step, Pac1 and Pme1 restriction sites are used.

[0629] Human umbilical vein endothelial cells (HUVECs) were isolated as described previously. Umbilical cords were obtained from the Birmingham Women's Health Care NHS Trust with informed consent. HUVECs between passages 1 - 6 were used and cultured in complete M199 medium (cM199) containing 10% fetal bovine serum (PAA), 1% bovine brain extract, 90 μg / ml heparin, and 4 mM L - glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin (Invitrogen), and seeded on plates coated with 0.1% type I gelatin from porcine skin. HEK293T cells were cultured in complete DMEM (cDMEM) containing 10% fetal bovine serum (PAA), 4 mM L - glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin (Invitrogen).

[0630] SiRNA transfection in HUVECs was performed as described previously. Lentiviruses were produced in HEK293T cells by transient transfection with the above lentiviral packaging, envelope, and expression plasmids. The plasmids were incubated with polyethyleneimine (36 μg / ml) at a 1:4 ratio in OptiMEM (Invitrogen) for 10 minutes at room temperature and then added to HEK293T cells in cDMEM. The culture medium supernatant was used to transduce fresh HEK293T cells. GFP - positive HEK293T cells were sorted and used for protein production. Expression of MMRN2 in HEK293T cells was achieved by transient transfection with pHL - Avitag3hMMRN2 using polyethyleneimine as described above.

[0631] Quantitative PCR

[0632] cDNA was prepared from 1 μg of total RNA extracted using the High-Capacity cDNA Archive kit (Applied Biosystems). qPCR reactions were performed using the Express QPCR supermix (Invitrogen) on an RG-3000 (Corbett / Qiagen, Manchester, UK) thermal cycler. Primers for human clec14a and flotillin-2 were as described previously. Primers for mouse clec14a 5'UTR, CDS, and 3'UTR, and murine β-actin were as follows:

[0633] 5’UTR fwd–TTCCTTTTCCAGGGTTTGTG (SEQ ID NO:90);

[0634] 5’UTR rev–GCCTACAAGGTGGCTTGAAT (SEQ ID NO:91);

[0635] CDS fwd–AAGCTGTGCTCCTGCTCTTG (SEQ ID NO:92);

[0636] CDS rev–TCCTGAGTGCACTGTGAGATG (SEQ ID NO:93);

[0637] 3’UTR fwd–CTGTAGAGGGCGGTGACTTT (SEQ ID NO:94);

[0638] 3’UTR rev–AGCTGCTCCCAAGTCCTCT (SEQ ID NO:95);

[0639] mACTB fwd–CTAAGGCCAACCGTGAAAAG (SEQ ID NO:96);

[0640] mACTB rev–ACCAGAGGCATACAGGGACA (SEQ ID NO:97).

[0641] Relative expression ratios were calculated using a mathematical model adjusted for efficiency.

[0642] Western blotting and immunoprecipitation

[0643] Whole cell protein lysates were prepared and co-immunoprecipitation experiments were performed, in which from 2x10 7Extract proteins from individual HUVECs. For the initial isolation of CLEC14A interacting proteins, 5 μg of CLEC14A-Fc or an equimolar amount of hFc was used. For endogenous immunoprecipitation experiments, 0.4 μg of anti-CLEC14A antibody or sheep IgG was used. For blocking experiments, 5 μg of CLEC14A-Fc or hFc was bound to protein G beads in PBS overnight. The beads were blocked in PBS containing 20% FCS (PAA) for 5 - 6 hours. The bound CLEC14A-Fc or hFc proteins were blocked overnight in binding buffer with increasing concentrations of mIgG or anti-CLEC14A antibody (CRT-2, described below). Standard protocols were used for Western blotting and SDS-PAGE. Primary antibodies were used with corresponding HRP-conjugated secondary antibodies as shown in the text.

[0644] Flow cytometry

[0645] Cells were detached with cell dissociation buffer (Invitrogen), rinsed in PBS, and then incubated in blocking buffer (PBS, 3% BSA, 1% NaN3) for 15 minutes. Subsequently, 10 μg / ml of anti-HA tag (CRUK) and 10 μg / ml of anti-CLEC14A (CRT-2, described below) were used as primary antibodies and the cells were stained in blocking buffer for 30 minutes. The cells were rinsed in PBS and stained with goat polyclonal anti-mouse IgG conjugated to Alexa Fluor 488 (Invitrogen) in blocking buffer. Data were collected (15,000 events / sample) using a FACSCalibur device (Becton Dickinson, Oxford, UK) and the results were analyzed using Becton Dickinson Cell Quest software.

[0646] HUVEC spheroid sprouting assay and in vitro Matrigel tube formation assay

[0647] Generation of HUVEC spheroids and induction of endothelial sprouting in collagen gels were performed using 1000 HUVECs per spheroid. Quantification was performed 16 hours after embedding. To quantify sprout growth, the number of sprouts was counted and the cumulative sprout length and maximum sprout length were evaluated. For two-color sprout experiments, HUVECs were pre-labeled with orange and green CellTracker dyes (Invitrogen). After 24 hours, the spheroids were fixed in 4% formaldehyde and mounted with Vectorshield (Vector labs). The slides were imaged using an Axioskop2 microscope and AxioVision SE64Rel4.8 software (Zeiss, Cambridge, UK).

[0648] For the Matrigel tube formation assay, 1.4x10 5 HUVECs were seeded onto 70 μl of basement membrane extract (Matrigel, BD Bioscience, Oxford, UK) in a 12-well plate. After 16 h, images of five fields per well were taken at 10x magnification using a Leica DM IL microscope (Leica, Milton Keynes, UK) and a USB 2.0 2M Xli digital camera (XL Imaging LLC, Carrollton, TX, USA). The images were analyzed using Image J (Carpentier G. et al., Angiogenesis Analyzer for ImageJ. 4 th ImageJ User and Developer Conference proceedings) and the Angiogenesis Analyzer plugin available at the NIH website (http: / / imagej.nih.gov / ij / macros / toolsets / Angiogenesis%20Analyzer.txt).

[0649] Protein production

[0650] Conditioned medium (CM) was collected from HEK293T cells expressing CLEC14A-Fc. The CM was passed over a HiTrap Protein A HP column (GE healthcare, Amersham, UK), and the protein was eluted using a 0-100% gradient of 100 mM sodium citrate (pH 3), and then neutralized with 1 M Tris base. Fractions were run on SDS-PAG, and protein purity and specificity were evaluated by Coomassie staining and Western blotting. Fractions containing similar concentrations of protein were pooled and dialyzed against PBS prior to functional assays.

[0651] Monoclonal antibody production

[0652] Mouse monoclonal antibodies were commercially prepared by Serotec Ltd (Oxford, UK) using the following protocol for breaking tolerance provided by us. Purified mouse CLEC14A-Fc fusion protein was administered subcutaneously at 50 μg in complete Freund's adjuvant. Two weeks later, another 50 μg was administered subcutaneously to the mouse, but this time in incomplete Freund's adjuvant. The mouse was culled and the spleen was harvested two weeks later for fusion.

[0653] Generation of clec14a- / - mice

[0654] Mice were housed at the Birmingham Biomedical Services Unit (Birmingham, UK). C57BL / 6N VGB6 feeder cell-dependent embryonic stem cells containing the CLEC14A deletion cassette (Clec14atm1(KOMP)Vlcg; project ID VG10554) were obtained from the Knockout Mouse Project (University of California, Davis, USA). The Transgenic Mouse Facility at the University of Birmingham generated chimeric mice by injecting embryonic stem cells into albino C57BL / 6 mice and bred them with C57BL / 6 females to produce mice heterozygous for the cassette.

[0655] Aortic ring and murine subcutaneous sponge angiogenesis assays

[0656] Aortas were isolated and processed for aortic ring assays in collagen. Quantification of tube / bud outgrowth, maximal endothelial migration, and total endothelial outgrowth was performed. On day 0, subcutaneous sterile polyether sponge discs (10 x 5 x 5 mm) were implanted subcutaneously under the dorsal skin on each side of the body of male C57 black mice. Every other day, 100 μl bFGF (40 ng / ml; R&D systems) was injected directly through the skin into the sponge for 14 days. On day 14, the sponge was excised, fixed in 10% formalin, and paraffin-embedded. Sections were stained with hematoxylin and eosin, and cross-sections of the sponge were photographed at 1x magnification using a Leica MZ 16 microscope (Leica, Milton Keynes, UK) and a USB 2.0 2M Xli digital camera (XL Imaging LLC, Carrollton, TX, USA) for cell invasion analysis. The vascular density of images captured at 40x magnification using a Leica DM E microscope (Leica, Milton Keynes, UK) was analyzed. Vessel counts were evaluated in five fields per section of each sponge. All animal experiments were conducted in accordance with Home Office license number PPL 40 / 3339 held by RB.

[0657] Tumor implantation assay

[0658] At 8 - 10 weeks of age, 10 6 Lewis lung carcinoma cells were injected subcutaneously into the flanks of male mice. Tumor growth was monitored by daily caliper measurements, and after 2 - 4 weeks of growth, tumor mass was determined by weight, fixed in 4% PFA, paraffin-embedded, and serially sectioned at 6 μm.

[0659] CLEC14A regulates sprouting angiogenesis in vitro

[0660] To study the role of CLEC14A in in vitro sprouting angiogenesis, HUVEC spheroids were generated from HUVECs treated with siRNA targeting clec14a or non-complementary siRNA duplexes. Knockdown of clec14a expression was confirmed by qPCR at an average reduction of 74% among three experiments ( Figure 2 A) at the mRNA level and by Western blot analysis of protein extracts probed with anti-CLEC14A polyclonal antiserum at the protein level ( Figure 2 B). VEGF-induced sprouting from CLEC14A knockdown spheroids was impaired, with knockdown spheroids generating an average of 6.9 sprouts per spheroid compared to 13.2 for control cells ( Figure 2 C and 2D). To determine the role of CLEC14A in tip / stalk cell formation, control HUVECs and knockdown HUVECs were stained red or green and mixed before spheroid formation and induction of sprouting ( Figure 2 E). Knockdown of CLEC14A decreased the percentage of cells at the tip position (33%) compared to control cells (67%); however, there was no effect on the percentage of stalk cells derived from CLEC14A knockdown HUVECs ( Figure 2 F). These data suggest that CLEC14A plays a role in sprout initiation and migration.

[0661] CLEC14A regulates sprouting angiogenesis in vivo

[0662] To study the in vivo and ex vivo roles of CLEC14A, mice were generated to replace the clec14a coding sequence with a lacZ reporter ( Figure 3 A). Breeding of heterozygotes (clec14a- / + ) produced equal proportions of male and female mice (49.5% / 50.5% respectively) and a Mendelian ratio of wild-type:heterozygous:homozygous mice (26.4%:47.2%:26.4% respectively). Since clec14a is an endothelial cell-restricted gene, aortas were isolated from clec14a + / + and clec14a- / - mice. Extracted cDNA was analyzed by qPCR and the loss of the clec14a coding region was confirmed, but expression of the 5' and 3' untranslated regions was retained ( Figure 2 B). Loss of CLEC14A at the protein level was also confirmed by Western blot analysis of lung tissue lysates ( Figure 3 C).

[0663] To confirm the role of CLEC14A in sprouting angiogenesis in multicellular three-dimensional co-cultures, aortas were isolated, cut into rings and embedded in collagen. VEGF-stimulated cells were allowed to grow out and were monitored for 7 days prior to quantification at the endpoint of endothelial sprouting. Similarly, loss of CLEC14A impaired endothelial sprout outgrowth and migration( Figure 3 D). Aortic rings from wild-type mice produced more than a doubling in the number of tubes compared to observations on CLEC14A knockout mice (30.6 tubes compared to 13.4 tubes)( Figure 3 E). In addition, maximum migration (defined as the furthest distance away from each aortic ring) was also reduced in knockout cultures( Figure 3 F). To evaluate whether CLEC14A has a similar function in vivo, sponge barrels were implanted subcutaneously into CLEC14A knockout mice. bFGF was injected into the sponges every two days to stimulate cell infiltration and neovascularization for two weeks. Macroscopic analysis of sponge sections stained with hematoxylin and eosin revealed impaired infiltration of the sponge by cells in clec14a− / − animals( Figure 3 G and 3H). In addition, the vascular supply was significantly reduced in clec14a− / − animals (p < 0.01)( Figure 3 I). To confirm that endothelial cells, which line the newly formed blood vessels in this model, express clec14a, sponges and livers from CLEC14A KO mice were stained with x-gal. Strong x-gal staining was observed on blood vessels within the sponges compared to matched liver sections( Figure 3 J). From these data, we can conclude that mouse CLEC14A expression regulates endothelial migration and angiogenic sprouting in vivo and in vitro, and that CLEC14A is upregulated on sprouting endothelium.

[0664] CLEC14A promotes tumor growth

[0665] CLEC14A expression was found to be highly upregulated on human tumor blood vessels compared to blood vessels from healthy tissues, suggesting that cancer therapies could target CLEC14A. Therefore, to investigate whether loss of CLEC14A affects tumor growth, we used the syngeneic Lewis lung carcinoma (LLC) model. For this, 1 x 10 6 LLC cells were injected subcutaneously into the right flanks of clec14a+ / + or clec14a− / − mice. Tumor growth was impaired in clec14a− / − mice compared to clec14a + / + littermate mice( Figure 4 A). This was confirmed by three independent experiments. Excised tumors harvested from clec14a− / − mice were smaller in size( Figure 4 B) and lighter in weight( Figure 4C). To determine whether the vascular density within these tumors was also affected, tissue sections were stained with anti-CD31 antibody. Analysis revealed a decrease in the density of discrete blood vessels (Figures 4D and 4E) and a decrease in the percentage of endothelial coverage ( Figure 4 F). In addition, x-gal staining of tumor and liver sections harvested from clec14a− / − mice revealed high expression of clec14a on both mature blood vessels with lumens filled with red blood cells ( Figure 4 G, black arrows) and immature microvessels within the tumors ( Figure 4 G), confirming upregulation of clec14a on tumor blood vessels.

[0666] Example 3: Preparation of Anti-CLEC14A Monoclonal Antibody and Its Effect on Angiogenesis

[0667] Preparation of monoclonal antibodies

[0668] The antigens used for the preparation of monoclonal antibodies were murine CLEC14A-FC (CM) and human CLEC14A-Fc (CH), optionally conjugated with an adjuvant protein (AP). These four antigens (CM, CH, CM-AP, CH-AP) were used for mouse immunization using the following protocol:

[0669] Day Procedure

[0670] 0 Collection of pre-immunization samples

[0671] Immunization with 100 μg of antigen in complete Freund's adjuvant (footpad)

[0672] 14 Immunization with 100 μg of antigen in incomplete Freund's adjuvant (footpad)

[0673] 17 Bleeding test

[0674] 18 Popliteal lymph node harvest for fusion

[0675] Serum was tested by ELISA against three antigens: CM, CH, and Fc. Non-immunized serum was collected as a negative control.

[0676] The fusion protocol was as follows:

[0677] (1) Harvest popliteal lymph nodes from immunized mice and homogenize.

[0678] (2) Wash the cells with warm DMEM.

[0679] (3) Mix the cells with sp2 / 0 myeloma cells.

[0680] (4) Centrifuge the mixture (1000 g)

[0681] (5) Suspend the pellet in 50% PEG 1500 and incubate for 1 minute.

[0682] (6) Slowly dilute the suspension with warm DMEM.

[0683] (7) Centrifuge the suspension (1000 g).

[0684] (8) Seed the cells into plates with peritoneal macrophages.

[0685] (9) Culture the cells at 37 °C and 5% CO2

[0686] More than 500 HAT-resistant hybridoma clones were obtained from each mouse. All clone supernatants were tested twice at 4-day intervals against three absorbed antigens (CM, CH, and Fc) by ELISA. Testing yielded 5 clones, and 2 of them, namely CRT-2 and CRT-3 (both subclass IgG1), were further studied and shown to react with both CM and CH and not with Fc. All positive clones were diluted 2- to 4-fold by the limiting dilution method, propagated in culture flasks, and injected into mice to obtain ascites. One clone (CRT-3) was the result of immunization with CLEC14a human-AP (CHAP), while the other clone (CRT-2) was the result of immunization with CLEC14a mouse-AP (CM-AP).

[0687] Scratch Wound Healing Assay with Anti-CLEC14A Monoclonal Antibody

[0688] Scratches were made in confluent HUVECs with a 10-μl pipette tip. Fresh medium was applied, which contained monoclonal CLEC14A antibodies produced against the extracellular domain of CLEC14A in mice at 1 g / m or 10 g / l. The chemokinetic migration of HUVECs was evaluated by acquiring images of wound closure at 0, 4, 6, and 12 hours using a Leica DM1000 optical microscope and a USB 2.0 2M Xli camera. The open area of the wound was quantified using Image J software.

[0689] The ability of the CLEC14A monoclonal antibody to inhibit angiogenesis was detected. The scratch wound healing assay using the monoclonal antibody described below showed that the anti-CLEC14A monoclonal antibody inhibited endothelial cell migration in the HUVEC scratch wound healing assay. As Figure 5 shown in FIGS. 5A and 5B, compared with 13% in the control, when HUVECs were treated with 10 g / ml monoclonal antibody CRT-3, 25% of the wound area remained open at 12 hours.

[0690] These results show that the anti-CLEC14A monoclonal antibody CRT-3 inhibits endothelial cell migration, which is an essential feature of angiogenesis. Thus, this assay provides evidence that the monoclonal antibody of the present invention directly inhibits angiogenesis.

[0691] To further characterize the functional effects of CLEC14A antibody treatment on endothelial cells, a tube formation assay was performed using HUVECs treated with 20 μg / ml of CRT2, CRT3, or CRT4. Treatment with CRT2 and CRT3 gave a significant reduction in tubule length and the number of junctions. CRT2 treatment also significantly reduced the grid area per field. The results showed that CRT2, 3, and 4 all had different negative effects on tube formation.

[0692] Example 4: Characterization of Anti-CLEC14A Monoclonal Antibody

[0693] Various polypeptide constructs were generated and expressed in cells to localize the binding site of the monoclonal antibody of the present invention. All constructs had a C-terminal GFP tag, so green cells were gated and stained red. Flow cytometry was used to analyze the binding of the CRT antibodies.

[0694] Figure 7 A shows that both CRT-2 and CRT-3 bind to cells expressing CLEC14A, while Figure 7 B demonstrates that the antibody does not bind to cells expressing thrombomodulin.

[0695] Chimeras were generated that contain the C-type lectin domain (CTLD) of thrombomodulin (CD141) and the remainder of the CLEC14A molecule. Although a slight change in fluorescence was observed with CRT-2, cells expressing this antigen were not recognized by either CRT antibody ( Figure 8 A). Chimeras were also generated that resulted in the binding of CRT-3, but not CRT-2 ( Figure 8 B), which contain CLEC14A in which the sushi-like domain has been replaced with the sushi-like domain of thrombomodulin (CD141) to ensure proper folding of the CTLD of CLEC14A. Both of these CRT antibodies bind to WT CLEC14A and, as expected, neither binds to WT CD141 ( Figure 7 ).

[0696] These data suggest that the binding site of antibody CRT-3 is within the C-type lectin domain and that CRT-2 binds to the region between the CTLD and the sushi-like domain.

[0697] To further define the binding region of the antibody, chimeric loop constructs were prepared. This was based on the structural prediction of the CLEC14A CTLD.

[0698] CLEC14A with regions 1 - 42 of CD141

[0699] CD141 sequence -

[0700] MLGVLVLGALALAGLGFPAPAEPQPGGSQCVEHDCFALYPGP (SEQ ID NO:98)

[0701] CLEC14A with regions 97 - 108 of CD141

[0702] CD141 sequence – QLPPGCGDPKRL (SEQ ID NO:99)

[0703] CLEC14A with regions 122 - 142 of CD141

[0704] CD141 sequence – TSYSRWARLDLNGAPLCGPL (SEQ ID NO:100)

[0705] Figure 9 The alignment is shown. Unfortunately, the 1 - 42 and 122 - 142 chimeras are incorrectly folded. This is thought to be due to the fact that they are present on the cell surface (positive staining with polyclonal CLEC14A antibody, but they do not stain with either antibody).

[0706] The 97 - 108 chimera does bind to CRT - 2 and CRT - 3, indicating that this mutant is correctly folded. Residues 97 - 108 were exchanged with the corresponding region from thrombomodulin. This results in correct folding because CRT - 2 and CRT - 3 can still bind ( Figure 8 C). These data suggest that CRT - 2 and CRT - 3 do not bind to residues 97 - 108 of CLEC14A.

[0707] This experiment was repeated three times with the same results.

[0708] The coding sequences of CRT - 2 and CRT - 3 were cloned from their respective hybridomas and sequenced using standard techniques. The CDRs have been predicted using standard software. The polypeptide and nucleotide sequences are listed in the following table. Given different prediction software, sequence variants, including CDR variants (marked with “*”), are also shown.

[0709]

[0710]

[0711]

[0712]

[0713]

[0714]

[0715] Example 5: Design and Analysis of Chimeric Antigen Receptor Based on the Antigen-Binding Domain of Anti-CLEC14A Monoclonal Antibody

[0716] Generation of CAR constructs

[0717] As described above, hybridomas that produce CLEC14A - specific monoclonal antibodies that cross - react with human and murine forms of the protein were obtained. Then, gene constructs encoding scFv were isolated from each murine hybridoma by RT - PCR using a degenerate primer set designed to amplify all murine V gene families.

[0718] Then, the scFv gene was subcloned as a ClaI, NotI fragment into two previously described CAR vectors, pMP71.tCD34.2A.CD19ζ and pMP71.tCD34.2A.CD19.IEVζ (Cheadle et al., J. Immunol., 2014, 192(8), 3654 - 65), replacing the CD19 - specific scFv region. These vectors were initially constructed using the MP71 retroviral expression plasmid (a kind gift from C. Baum, Hannover) and co - express a truncated CD34 marker gene.

[0719] Transduction of human and murine T cells

[0720] To generate recombinant retroviruses for transducing human T cells, Phoenix amphotropic packaging cells were transfected with the MP71 retroviral vector and pCL ampho (Imgenex) using FuGENE HD (Roche) according to the manufacturer's instructions. Recombinant retroviruses for transducing murine T cells were generated in the same manner, but using Phoenix ecotropic packaging cells and pCLeco. Human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density gradient centrifugation on Lymphoprep (Axis Shield, Oslo, Norway). PBMCs were pre-activated for 48 h using anti-CD3 antibody (OKT3, eBioscience; 30 ng / ml), anti-CD28 antibody (R&D Systems; 30 ng / ml), and interleukin-2 (IL2; 300 U / ml; Chiron, Emeryville, CA) in standard medium (RPMI 1640 (Sigma) containing 10% fetal bovine serum (FBS; PAA, Pasching, Austria), 2 mM L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin) plus 1% human AB serum (TCS Biosciences, Buckingham, UK). Murine T cells were transduced using murine splenocytes pre-activated for 48 h with concanavalin A (2 μg / ml; Sigma) and murine interleukin 7 (1 ng / ml; eBioscience) in standard medium. Subsequently, pre-activated human and murine T cells were transduced (or mock-transduced with conditioned supernatant from untransfected phoenix cells) by spinfection in retronectin (Takara)-coated plates according to the manufacturer's instructions. Human T cells were then cultured in standard medium containing IL2 (100 U / ml) plus 1% human AB serum. After spinfection, murine T cells were cultured in standard medium containing IL2 (100 U / ml) for 24 h and then purified using Lymphoprep (Axis Shield). Where indicated, transduced cells were enriched by immunomagnetic selection using anti-CD34 microbeads (Miltenyi Biotec, Germany) according to the manufacturer's instructions. The study of human donors was approved by the National Research Ethics Service Committee West Midlands (Solihull), and all donors gave written informed consent.

[0721] Cell lines and recombinant proteins

[0722] Maintain Phoenix A or E, CHO, and Lewis lung carcinoma cells in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS; PAA, Pasching, Austria), 2 mM L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin. Transduce CHO cells with the pWPI vector (Addgene) expressing full-length human CLEC14A (or the vector alone). Isolate human umbilical vein endothelial cells (HUVEC) using umbilical cords obtained from the Birmingham Women’s HealthCare NHS Trust under informed consent and ethical approval from the south Birmingham research ethics committee as described above. Maintain HUVEC in complete M199 medium containing 10% FBS, 4 mM L-glutamine, and 10% large vessel endothelial cell growth supplement (TCS Cellworks), and culture them in plates coated with 0.1% type I gelatin (Sigma) from porcine skin. Express human and murine CLEC14A proteins with human Fc tags in HEK293T cells and purify them on a protein A column.

[0723] Flow cytometry

[0724] HUVECs were treated with trypsin and stained for 1 hour on ice with the above-mentioned CLEC14A-specific mouse monoclonal antibody (10 μg / ml) or IgG1 isotype control (Dako) in 5% normal goat serum / PBS. Cells were washed and bound antibodies were detected by incubation with goat anti-mouse antibody conjugated to R-PE (Serotec). Dead cells were identified by staining with propidium iodide. Human T cells were washed with PBS and stained for 20 minutes in the dark with the Live / Dead Fixable Violet Dead Cell Staining Kit (Life Technologies). Cells were then washed with flow buffer (0.5% w / v BSA + 2 mM EDTA in PBS; pH 7.2) and stained for 30 minutes on ice in the dark with anti-human CD4 (conjugated to PE), anti-human CD8 (conjugated to FITC) (both from BD Pharmingen) and anti-human CD34 (Pe-Cy5) (BioLegend). Alternatively, different from staining for CD34, CAR expression was directly detected by first blocking cells with human Fc fragment (10 μg / ml), then incubating them with 10 μg / ml recombinant human CLEC14A-Fc fusion protein (or Fc control), and then incubating with sheep anti-CLEC14A polyclonal antibody (R&D systems, 10 μg / ml). Finally, cells were stained with rabbit anti-sheep antibody conjugated to FITC (Invitrogen, 1:10 dilution). All incubations were carried out on ice for 1 hour.

[0725] When staining mouse T cells from heparinized tail bleedings, they were first subjected to red blood cell lysis using BD Pharm lyse (Becton Dickinson), and then stained as described above, but using anti-mouse CD4-FITC, CD8-PE and CD45.1 (conjugated to PE-Cy7) (all from BD Biosciences). Cells were analyzed using a BD LSR II flow cytometer and FlowJo software (TreeStar Inc, Ashlan...

Claims

1. An isolated antibody that selectively binds to CLEC14A, wherein the antibody: (a) comprises at least one heavy chain variable region comprising 3 CDRs and at least one light chain variable region comprising 3 CDRs, wherein the heavy chain variable region comprises: (i) variable heavy chain (VH) CDR1, which consists of the amino acid sequence SEQ ID NO:2; (ii) VH CDR2, which consists of the amino acid sequence SEQ ID NO:3; and (iii) VH CDR3, which consists of the amino acid sequence SEQ ID NO:4; and wherein the light chain variable region comprises: (iv) variable light chain (VL) CDR1, which consists of the amino acid sequence SEQ ID NO:6; (v) VL CDR2, which consists of the amino acid sequence SEQ ID NO:7; and (vi) VL CDR3, which consists of the amino acid sequence SEQ ID NO:8; or (b) comprises at least one heavy chain variable region comprising 3 CDRs and at least one light chain variable region comprising 3 CDRs, wherein the heavy chain variable region comprises: (i) variable heavy chain (VH) CDR1, which consists of the amino acid sequence SEQ ID NO:42; (ii) VH CDR2, which consists of the amino acid sequence SEQ ID NO:43; and (iii) VH CDR3, which consists of the amino acid sequence SEQ ID NO:44; and wherein the light chain variable region comprises: (iv) variable light chain (VL) CDR1, which consists of the amino acid sequence SEQ ID NO:46; (v) VL CDR2, which consists of the amino acid sequence SEQ ID NO:47; and (vi) VL CDR3, which consists of the amino acid sequence SEQ ID NO:48; or (c) comprises at least one heavy chain variable region comprising 3 CDRs and at least one light chain variable region comprising 3 CDRs, wherein the heavy chain variable region comprises: (i) variable heavy chain (VH) CDR1, which consists of the amino acid sequence SEQ ID NO:22; (ii) VH CDR2, which consists of the amino acid sequence SEQ ID NO:23; and (iii) VH CDR3, which consists of the amino acid sequence SEQ ID NO:24; and wherein the light chain variable region comprises: (iv) variable light chain (VL) CDR1, which consists of the amino acid sequence SEQ ID NO:26; (v) VL CDR2, which consists of the amino acid sequence SEQ ID NO:27; and (vi) VL CDR3, which consists of the amino acid sequence SEQ ID NO:

28.

2. The antibody of claim 1, wherein the antibody has the VH domain of SEQ ID NO:1 and / or the VL domain of SEQ ID NO:5 or the antibody has the VH domain of SEQ ID NO:41 and / or the VL domain of SEQ ID NO:

45.

3. The antibody of claim 1, wherein the antibody: A VH domain having SEQ ID NO:21 and / or a VL domain having SEQ ID NO:

25.

4. The antibody of any one of claims 1 to 3, wherein the antibody is a murine, human or humanized antibody.

5. The antibody of claim 1, wherein the antibody comprises the whole or part of the antibody heavy chain constant region and / or the whole or part of the antibody light chain constant region.

6. The antibody of claim 1, wherein the antibody is an IgG antibody.

7. The antibody of claim 1, wherein the antibody comprises: (a) a heavy chain and a light chain, the heavy chain comprising the amino acid sequence SEQ ID NO:1 or a sequence homologous thereto, and the light chain comprising the amino acid sequence SEQ ID NO:5 or a sequence homologous thereto; or (b) a heavy chain and a light chain, the heavy chain comprising the amino acid sequence SEQ ID NO:41 or a sequence homologous thereto, and the light chain comprising the amino acid sequence SEQ ID NO:45 or a sequence homologous thereto; or (c) a heavy chain and a light chain, the heavy chain comprising the amino acid sequence SEQ ID NO:21 or a sequence homologous thereto, and the light chain comprising the amino acid sequence SEQ ID NO:25 or a sequence homologous thereto.

8. The antibody of claim 1, wherein the antibody is an antigen-binding fragment of an antibody.

9. The antibody of claim 8, wherein the antigen-binding fragment of the antibody is a Fab', Fab, F(ab')2, Fv, scFv, dsFv, ds-scFv, minibody, or diabody, which comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), the antibody light chain variable region (VL) comprising three CDR domains and the antibody heavy chain variable region (VH) comprising three CDR domains.

10. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody of any one of claims 1-9 or a chimeric antigen receptor (CAR) against the antigen CLEC14A, wherein the CAR is capable of binding the antigen CLEC14A expressed on the surface of a target cell when expressed on the surface of an immune effector cell, and comprises an antigen-binding domain, which comprises: (a) at least one variable heavy chain (VH) sequence comprising 3 CDRs and at least one variable light chain (VL) sequence comprising 3 CDRs, wherein the VH sequence comprises: (i) VH CDR1, which is composed of the amino acid sequence SEQ ID NO:2; (ii) VH CDR2, which is composed of the amino acid sequence SEQ ID NO:3; and (iii) VH CDR3, which is composed of the amino acid sequence SEQ ID NO:4; and wherein the VL sequence comprises: (iv) VL CDR1, which is composed of the amino acid sequence SEQ ID NO:6; (v) VL CDR2, which is composed of the amino acid sequence SEQ ID NO:7; and (vi) VL CDR3, which is composed of the amino acid sequence SEQ ID NO:8; or (b) At least one variable heavy chain (VH) sequence comprising 3 CDRs and at least one variable light chain (VL) sequence comprising 3 CDRs, wherein the VH sequence comprises: (i) VH CDR1, which is composed of the amino acid sequence SEQ ID NO:42; (ii) VH CDR2, which is composed of the amino acid sequence SEQ ID NO:43; and (iii) VH CDR3, which is composed of the amino acid sequence SEQ ID NO:44; and wherein the VL sequence comprises: (iv) VL CDR1, which is composed of the amino acid sequence SEQ ID NO:46; (v) VL CDR2, which is composed of the amino acid sequence SEQ ID NO:47; and (vi) VL CDR3, which is composed of the amino acid sequence SEQ ID NO:48; or (c) At least one VH sequence comprising 3 CDRs and at least one VL sequence comprising 3 CDRs, wherein the VH sequence comprises: (i) VH CDR1, which is composed of the amino acid sequence SEQ ID NO:22; (ii) VH CDR2, which is composed of the amino acid sequence SEQ ID NO:23; and (iii) VH CDR3, which is composed of the amino acid sequence SEQ ID NO:24; and wherein the VL sequence comprises: (iv) VL CDR1, which is composed of the amino acid sequence SEQ ID NO:26; (v) VL CDR2, which is composed of the amino acid sequence SEQ ID NO:27; and (vi) VL CDR3, which is composed of the amino acid sequence SEQ ID NO:

28.

11. The nucleic acid molecule of claim 10, wherein the nucleotide sequence is as shown in any one of SEQ ID NO:11-19, 31-39, 54-64, 101-104 or a nucleotide sequence having at least 60% sequence identity therewith.

12. An expression vector comprising the nucleic acid molecule of claim 10 or 11.

13. A host cell comprising the nucleic acid molecule of claim 10 or 11 or the expression vector of claim 12.

14. A virus comprising the nucleic acid molecule of claim 10 or 11 or the expression vector of claim 12.

15. A composition comprising the antibody of any one of claims 1-9, the nucleic acid of claim 10 or 11, the expression vector of claim 12, the host cell of claim 13 or the virus of claim 14 and at least one physiologically acceptable carrier or excipient.

16. The composition of claim 15, wherein the composition is a therapeutic agent or a pharmaceutical composition.

17. The composition of claim 15 or 16, wherein the composition comprises at least one additional therapeutic agent.

18. A kit comprising: (a) The antibody of any one of claims 1-9; (b) The nucleic acid molecule of claim 10 or 11; (c) The expression vector of claim 12; (d) The host cell of claim 13; (e) The virus of claim 14; and / or (f) The composition of claim 15, 16 or 17.

19. A product, comprising: (a) An antibody of any one of claims 1-9; (b) A nucleic acid molecule of claim 10 or 11; (c) An expression vector of claim 12; (d) A host cell of claim 13; (e) A virus of claim 14; and / or (f) The composition of claim 15, 16 or 17.

20. The composition of claim 17, the kit of claim 18 or the product of claim 19, wherein the additional therapeutic agent is an anti-cancer and / or anti-angiogenic agent.

21. The composition, kit or product of claim 20, wherein the anti-cancer agent is an alkylating agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an RNA / DNA antimetabolite, a DNA antimetabolite or an anti-mitotic agent.

22. The composition, kit or product of claim 20, wherein the anti-cancer agent is a cytotoxic drug.

23. The composition, kit or product of claim 22, wherein the cytotoxic drug is a direct cytotoxic chemotherapeutic agent, a direct cytotoxic polypeptide, a moiety capable of converting a prodrug into a cytotoxic drug, a radiosensitizer, a direct cytotoxic nucleic acid, a nucleic acid molecule encoding a direct or indirect cytotoxic polypeptide, or a radioactive atom.

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