Antibodies and related molecules and uses thereof

Abstract

The present invention relates to an isolated antibody, which selectively binds to CLEC14A, wherein said antibody (a) comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises: (i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO. 105, preferably of SEQ ID NO: 2 or 42; (ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO. 106, preferably of SEQ ID NO: 3 or 43; and/or (iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO. 107, preferably of SEQ ID NO: 4 or 44; and/or wherein said light chain variable region comprises: (iv) a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO. 108, preferably of SEQ ID NO: 6 or 46; (v) a VL CDR2 that has the amino acid sequence of SEQ ID NO. 109, preferably of SEQ ID NO: 7 or 47; and/or (vi) a VL CDR3 that has the amino acid sequence of SEQ ID NO. 1 10, preferably of SEQ ID NO: 8 or 48; or (b) comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises: (i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO: 22; (ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 23; and/or (iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 24; and/or wherein said light chain variable region comprises: (iv) a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO: 26; (v) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 27; and/or (vi) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 28; or (c) is an antibody which can compete with antibody (a) or (b) for binding to CLEC14A. The invention further provides chimeric antigen receptors, nucleic acid molecules encoding the antibodies of the invention or the chimeric antigen receptors, vectors, cells and methods/uses of the antibodies and chimeric antigen receptors.

Claims

1. An isolated antibody, which selectively binds to CLEC14A, wherein said antibody: (a) comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises: (i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: 2, or SEQ ID NO: 42; (ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 106, SEQ ID NO: 3, or SEQ ID NO: 43; and (iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 107, SEQ ID NO: 4, or SEQ ID NO: 44; and wherein said light chain variable region comprises: (iv) a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO: 108, SEQ ID NO: 6, or SEQ ID NO: 46; (v) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 109, SEQ ID NO: 7, or SEQ ID NO: 47; and (vi) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 110, SEQ ID NO: 8, or SEQ ID NO: 48; or (b) comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises: (i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO: 22; (ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 23; and (iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 24; and wherein said light chain variable region comprises: (iv) a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO: 26; (v) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 27; and (vi) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 28.

2. The antibody of claim 1, wherein said antibody: (a) has a VH domain having the amino acid sequence of either SEQ ID NO: 1 or SEQ ID NO: 41 and/or a VL domain having the amino acid sequence of either SEQ ID NO: 5 or SEQ ID NO: 45; or (b) has a VH domain having the amino acid sequence of SEQ ID NO: 21 and/or a VL domain having the amino acid sequence of SEQ ID NO: 25.

3. The antibody of claim 1, wherein: (a) said antibody is a mouse or humanised antibody; (b) said antibody comprises all or a portion of an antibody heavy chain constant region and/or all or a portion of an antibody light chain constant region; (c) said antibody is an IgG antibody; (d) said antibody comprises: a heavy chain that comprises the amino acid sequence of either SEQ ID NO: 1 or SEQ ID NO: 41 and a light chain that comprises the amino acid sequence of either SEQ ID NO: 5 or SEQ ID NO: 45; or (ii) a heavy chain that comprises the amino acid sequence of SEQ ID NO: 21 and a light chain that comprises the amino acid sequence of SEQ ID NO: 25; (g) said antibody is an antigen binding fragment of an antibody; and/or (h) said antibody is an antigen binding fragment of an antibody, which is a Fab′, Fab, F(ab′)2, TandAbs dimer, Fv, scFv, dsFv, ds-scFv, minibody, diabody, bispecific antibody fragment, bibody, tribody, sc-diabody, BiTE, DVD-Ig or DART.

4. An immunoconjugate comprising the antibody of claim 1 conjugated to a therapeutic, diagnostic or imaging agent.

5. A composition comprising an antibody of claim 1 or an immunoconjugate comprising an antibody of claim 1, and at least one physiologically acceptable carrier or excipient, wherein said composition is a therapeutic or pharmaceutical composition.

6. The composition of claim 5, wherein said composition comprises at least one further therapeutic agent.

7. The composition of claim 5, wherein said composition is provided as a combined preparation with one or more additional therapeutic agents for separate, simultaneous or sequential use or administration.

8. The composition of claim 6, wherein said therapeutic agent is an anti-cancer and/or anti-angiogenesis agent.

9. The composition of claim 8, wherein said anti-cancer agent is an alkylating agent, topoisomerase I inhibitor, topoisomerase II inhibitor, RNA/DNA antimetabolite, DNA antimetabolite, an antimitotic agent or a cytotoxic moiety.

10. The composition of claim 9, wherein said cytotoxic moiety is a directly cytotoxic chemotherapeutic agent, a directly cytotoxic polypeptide, a moiety which is able to convert a prodrug into a cytotoxic drug, a radiosensitizer, a directly cytotoxic nucleic acid, a nucleic acid molecule that encodes a directly or indirectly cytotoxic polypeptide, or a radioactive atom.

Description

(1) The invention will now be described in more detail in the following non-limited examples with reference to the Figures in which:

(2) FIG. 1 shows a graph showing the relative expression of CLEC14A in HUVECs and other primary cells. CLEC14A was expressed specifically in endothelial cells (HUVEC), and not in human aortic smooth muscle cells (HASMC), human lung fibroblasts (MRC5), human bronchial epithelial cells (HBE), hepatocytes, or peripheral blood mononuclearcells (PBMC).

(3) FIG. 2 shows [A] siRNA duplex targeting CLEC14A can efficiently knockdown CLEC14A mRNA expression in HUVEC, as determined by qPCR. Relative expression was determined by normalising 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 after 16 hours for control or clec14a targeted siRNA treated HUVEC. [D] Quantitation of sprouts for 27 spheroids (9 spheroids from 3 cords) for control and CLEC14A knockdown HUVEC; Mann-Whitney statistical test p<0.001. [E] Representative images of sprout outgrowth after 24 hours for mixed control (green) and clec14a targeted siRNA treated HUVEC (red). [F] Quantitation of the percentage of tip and stalk cells derived from control (CON) and CLEC14A knockdown (KD) HUVEC; two-way ANOVA statistical test with Bonferroni post-tests ***=p<0.001, ns=not significant.

(4) FIG. 3 shows [A] a schematic diagram of clec14a gene in C57BL/6 (clec14a +/+) or C57BL/6(Clec14atm1(KOMP)Vlcg) (clec14a −/−) mice. [B] Quantitative PCR analysis of cDNA generated from three clec14a +/+ mice (white bars) and three clec14a −/− mice (black bars) for the 5′ untranslated region (UTR), coding sequence (CDS) and 3′ UTR of clec14a. Relative expression was determined by normalising expression to flotilin2. [C] Western blot analysis of CLEC14A protein expression in lung lysates from clec14a +/+ and clec14a −/− mice using polyclonal antisera against murine CLEC14A. Tubulin was used as a loading control. [D] Representative images of the aortic ring sprouting assay from clec14a +/+ and clec14a −/− mice. Quantitation of tubes formed per ring [E], and quantitation of the maximal distance migrated by an endothelial tube from the aortic ring [F], data from 48 rings per genotype, 6 mice for each genotype; Mann-Whitney statistical test p<0.001. [G] Representative images of haematoxylin and eosin stained sections of sponge implant from clec14a +/+ and clec14a −/− mice, sections at the centre of the sponge were analysed. [H] Quantitation of cellular invasion into the sponge implants shown in G; Mann-Whitney statistical test p<0.05. [I] Quantitation of vessel density; Mann-Whitney statistical test p<0.001. [J] Sections of liver and sponge tissue stained with x-gal from clec14a −/− mice, counterstained with haematoxylin and eosin.

(5) FIG. 4 shows [A] Lewis lung carcinoma (LLC) tumour growth in clec14a +/+ (black line with dots) and clec14a −/− (black line with squares) mice; two-way ANOVA statistical analysis, *=p<0.05, **=p<0.01, ***=p<0.001. [B] Representative images of LLC tumours. [C] Endpoint tumour weight for 7 clec14a +/+ (dots) and 7 clec14a −/− (squares) mice; Mann-Whitney statistical test p<0.001. [D] Representative images of immunofluorescent staining of LLC tumour sections stained for murine CD31. Quantitation of vessel density [E] and percentage endothelial coverage [F] from clec14a +/+ and clec14a −/− mice; Mann-Whitney statistical test p<0.0001. [G] Sections of liver and LLC tumour tissue from clec14a −/− mice stained with x-gal, counterstained with haematoxylin and eosin.

(6) FIG. 5 shows (A) a light microscopy image of the results of a HUVEC scratch wound healing assay with anti-CLEC14A monoclonal antibody CRT-3 showing a retardation of wound closure. (B) Graphical representation of the results from (A).

(7) FIG. 6 shows analysis of tubule formation assays with CLEC14A antibody treated HUVECS. HUVECS were treated with 20 μg/ml CRT2, 3 or 4 or mouse IgG isotype control. Images of tubules were taken at 16 hours and analysed for total tubule length, number of junctions, number of branches, branch length, number of meshes and total mesh area. Data shown represents three experiments with five data points analysed for each. Error bars show SEM. *p, 0.05. **p<0.01.

(8) FIG. 7 shows graphs of flow cytometry analysis of CRT-2 and CRT-3 binding to (A) HEK293T transfected with CLEC14A and (B) HEK293T transfected with thrombomodulin.

(9) FIG. 8 shows graphs of flow cytometry analysis of CRT-2 and CRT-3 binding to (A) HEK293T transfected with a chimera containing CTLD of thrombomodulin and the remainder of CLEC14A, (B) HEK293T transfected with the a chimera containing the sushi-like domain of thrombomodulin and the remainder of CLEC14A and (C) HEK293T transfected with a chimera containing loop residues 97-108 of thrombomodulin and the remainder of CLEC14A.

(10) FIG. 9 shows the alignment of CLEC14A regions 1-42 of CD141; CLEC14A regions 97-108 of CD141; and CLEC14A regions 122-142 of CD141.

(11) FIG. 10 shows CAR expression vector design and expression of the CAR in transduced human T cells wherein (A) shows a retroviral CAR vector (based on pMP71) that co-expresses a truncated CD34 marker gene and an scFv fragment/CD3 zeta chain chimeric receptor. Expression is driven from the LTR promoter and the 2A peptide linker ensures equimolar expression of both the CD34 and the CAR. Second generation CAR constructs included the CD28 co-stimulatory domain. (B) shows CD34 staining analysed by flow cytometry demonstrating successful transduction of T cells using retroviral constructs that co-express a CLEC14A-specific CAR. First generation CARs based on the antibody CRT-3 is referred to as CRT3.z. Second generation CARs based on the antibody CRT-3 is referred to as CRT3.28z. (C). shows cells analysed by flow cytometry stained directly for expression of CAR using CLEC14A-Fc (% values show specific binding of CLEC14A-Fc having subtracted background staining with Fc alone).

(12) FIG. 11 shows CAR-transduced T cells respond to CLEC14A in vitro. T cells transduced to express 1st or 2nd generation CARs based on antibody CRT-3 or mock-transduced (control) T cells were tested for their ability to respond to CLEC14A expressed either as (A) plate-bound recombinant Fc fusion protein, (B) expressed on engineered CHO cells, or (C) expressed on human umbilical vein endothelial cells (HUVECs) which naturally express CLEC14A when grown in static culture. T cell response was measured using an ELISA for interferon gamma production. Data shown are representative of that obtained from 3-7 repeat experiments. T cells were adjusted to equalise the frequency of transgene expressing cells. All histograms show mean response+SD.

(13) FIG. 12 shows further in vitro functional testing of CLEC14A-specific CAR-transduced T cells. T cells transduced to express 1st or 2nd generation CARs based on antibody CRT-3, 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 (having subtracted background levels of lysis of CHO alone (control cells)). Data shown are representative of 5 repeat experiments. (B) Proliferation, using CFSE-labelled CAR-transduced T cells we measured the proliferation of CAR+ (CD34+) and CAR− (CD34−) cell subsets when co-cultured for 4 days with HUVECs. Data shown are representative of 2 repeat experiments. (C) The response of (CLEC14A-specific CAR-transduced T cells to both human and mouse CLEC14A was assessed using interferon gamma release. T cells were adjusted to equalise the frequency of transgene expressing cells. Data shown are representative of 6 repeat experiments. All histograms show mean response+SD.

(14) FIG. 13 shows histological pictures of tissue samples following toxicity testing in vivo using healthy C57/BL6 mice injected with CLEC14A-specific CAR-transduced mouse T cells.

(15) FIG. 14 shows graphs of Lewis Lung carcinoma tumour volume from mice treated with T cells transduced to express 2nd generation CARs based on 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 on 2.2 million of these cells. Tumour growth was then monitored using (A) Bioluminescence or (B) Calipers.

(16) FIG. 15 shows bar charts depicting tumour weight (A), percentage of tumour tissue area covered by vessels (B) and percentage of tumour tissue that stained for fibrinogen (C) in Lewis Lung carcinoma tumours from mice injected with T cells transduced to express 2nd generation CARs based on antibody CRT-3 or mock-transduced (control) T cells.

(17) FIG. 16 shows (A) images of HUVEC treated with CRT-3 at (i) 0 minutes and (ii) 90 minutes and demonstrates that the antibody is internalised; and (B) a graph showing cell viability of HUVEC treated with a CRT-3-antibody drug conjugate (immunoconjugate).

(18) FIG. 17 shows pictures of mouse lung tissue (A) 24 hours after treatment with a control ADC (B12-ADC) and (B) 24 hours after treatment with a CRT-3-ADC.

(19) FIG. 18 shows CRT1, 3 and 5 CAR (with CD28 costimulatory domain) T cell response to titrated concentrations of human and mouse recombinant CLEC14A.

(20) FIG. 19 shows the design of CARs with different costimulatory domains 1) tCD34-F2A-scFv-CD28 TM-CD28 signal-CD3zeta, 2) tCD34-F2A-scFv-CD8 TM-4-1BB signal-CD3 zeta, 3) tCD34-F2A-scFv-CD8 TM-OX40 signal-CD3 zeta, 4) tCD34-F2A-scFv-CD28 TM-CD28 signal-4-1BB signal-CD3zeta, 5) tCD34-F2A-scFv-CD28 TM-CD28 signal-OX40 signal-CD3 zeta, 6) tCD34-F2A-scFv-CD8 TM-4-1BB signal-OX40signal-CD3zeta. The tCD34 is included to identify successfully transduced cells and thus constructs may exclude this and F2A. A hinge or spacer region may additionally be included e.g. one from CD8α.

(21) FIG. 20 shows the results of a cytotoxicity assay with CRT1, 3 and 5 CARs vs mouse endothelial cells expressing CLEC14A (FIG. 20A). The results of a proliferative assay for CRT 1, 3 and 5 CARs are shown in FIG. 20B.

(22) FIG. 21 shows the functional testing of CRT3 CAR T cells comprising different costimulatory domains and shows the IFNgamma production in response to titrated numbers of CHO cells expressing human CLEC14A.

(23) FIG. 22 shows the IFN gamma release by CRT1, 3 and 5 CAR (CD28 costimulatory domain) T cells after incubated with 293 or SEND cells engineered to express CLEC14A chimera (A1-human CLEC14A with mouse intracellular domain, B1-human CLEC14A with mouse transmembrane and intracellular domains, huCLEC-human CLEC14A). Cytotoxicity data are shown in FIG. 22B for the CAR T cells after incubation with SEND cells.

(24) FIG. 23 shows a schematic of a suitable vector to generate RNA for electroporation by in vitro transcription.

(25) FIG. 24 shows constructs which encode CARs which can be used to transduce murine T cells. The constructs comprise transmembrane, costimulatory and intracellular signalling sequences from murine proteins (see SEQ ID NOs 116-121). The constructs may further comprise a hinge or spacer domain from murine CD8α.

(26) TABLE-US-00001 Sequence name SEQ ID (sequence type) NO: Sequence VH (aa)   1 M A E V Q L Q Q S G T V L A R P G A S V K M S C K A S G Y T F T S Y W M H W V K Q R P G Q G L E W I G A I Y P G N S D T S Y N Q K F K G K A K L T A V T S T S T A Y M E L S S L T N E D S A V F Y C T H Y Y G S D Y A M D Y W G Q G T S V T V VH CDR1 (aa)   2 GYTFTSYW VH CDR2 (aa)   3 IYPGNSDT VH CDR3 (aa)   4 THYYGSDYAMDY VL (aa)   5 Q I V L T Q S P A I M S A S L G E R V T M T C T A S S S V S S S Y L H W Y Q Q K P G S S P K L W I Y S T S N L A S G V P A R F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C H Q Y H R S P R T F G G G T K L E I K R A A A VL CDR1 (aa)   6 SSVSSSY VL CDR2 (aa)   7 STS VL CDR3 (aa)   8 HQYHRSPRT ScFv (aa)   9 M A E V Q L Q Q S G T V L A R P G A S V K M S C K A S G Y T F T S Y W M H W V K Q R P G Q G L E W I G A I Y P G N S D T S Y N Q K F K G K A K L T A V T S T S T A Y M E L S S L T N E D S A V F Y C T H Y Y G S D Y A M D Y W G Q G T S V T V S S G G G G S G G G G S G G G G S Q I V L T Q S P A I M S A S L G E R V T M T C T A S S S V S S S Y L H W Y Q Q K P G S S P K L W I Y S T S N L A S G V P A R F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C H Q Y H R S P R T F G G G T K L E I K R A A A CAR3 full-aa  10 M G V L L T Q R T L L S L V L A L L F P S M A S M A E V Q L Q Q S G T V L A R P G A S V K M S C K A S G Y T F T S Y W M H W V K Q R P G Q G L E W I G A I Y P G N S D T S Y N Q K F K G K A K L T A V T S T S T A Y M E L S S L T N E D S A V F Y C T H Y Y G S D Y A M D Y W G Q G T S V T V S S G G G G S G G G G S G G G G S Q I V L T Q S P A I M S A S L G E R V T M T C T A S S S V S S S Y L H W Y Q Q K P G S S P K L W I Y S T S N L A S G V P A R F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C H Q Y H R S P R T F G G G T K L E I K R A A A I E V M Y P P P Y L D N E K S N G T I I H V K G K H L C P S P L F P G P S K P F W V L V V V G G V L A C Y S L L V T V A F I I F W V R S K R S R L L H S D Y M N M T P R R P G P T R K H Y Q P Y A P P R D F A A Y R S R V K F S R S A D A P A Y Q Q G Q N Q L Y N E L N L G R R E E Y D V L D K R R G R D P E M G G K P Q R R K N P Q E G L Y N E L Q K D K M A E A Y S E I G M K G E R R R G K G H D G L Y Q G L S T A T K D T Y D A L H M Q A L P P R VH (nt)  11 atggccgaggtccagctgcagcagtctgggactgtgctggcaaggcctgg ggcttcagtgaagatgtcctgcaaggcttctggctacacctttaccagct actggatgcactgggtaaaacagaggcctggacagggtctggaatggatt ggcgctatttatcctggaaatagtgatactagctacaaccagaagttcaa gggcaaggccaaactgactgcagtcacatccaccagcactgcctacatgg agctcagcagcctgacaaatgaggactctgcggtcttttactgtacacat tactacggtagtgactatgctatggactactggggtcaaggaacctcagt cactgtc VH CDR1 (nt)  12 ggctacacctttaccagctactgg VH CDR2 (nt)  13 atttatcctggaaatagtgatact VH CDR3 (nt)  14 acacattactacggtagtgactatgctatggactac VL (nt)  15 caaattgttctcacccagtctccagcaatcatgtctgcatctctagggga acgggtcaccatgacctgcactgccagctcaagtgtaagttccagttact tgcactggtaccagcagaagccaggatcctcccccaaactctggatttat agcacatccaacctggcttctggagtcccagctcgcttcagtggcagtgg gtctgggacctcttactctctcacaatcagcagcatggaggctgaagatg ctgccacttattactgccaccagtatcatcgttccccacggacgttcggt ggaggcaccaagctggaaatcaaacgt VL CDR1 (nt)  16 tcaagtgtaagttccagttac VL CDR2 (nt)  17 agcacatcc VL CDR3 (nt)  18 caccagtatcatcgttccccacggacg ScFv (nt)  19 atggccgaggtccagctgcagcagtctgggactgtgctggcaaggcctgg ggcttcagtgaagatgtcctgcaaggcttctggctacacctttaccagct actggatgcactgggtaaaacagaggcctggacagggtctggaatggatt ggcgctatttatcctggaaatagtgatactagctacaaccagaagttcaa gggcaaggccaaactgactgcagtcacatccaccagcactgcctacatgg agctcagcagcctgacaaatgaggactctgcggtcttttactgtacacat tactacggtagtgactatgctatggactactggggtcaaggaacctcagt cactgtctcctcaggtggaggcggttcaggcggaggtggctctggcggtg gcggatcgcaaattgttctcacccagtctccagcaatcatgtctgcatct ctaggggaacgggtcaccatgacctgcactgccagctcaagtgtaagttc cagttacttgcactggtaccagcagaagccaggatcctcccccaaactct ggatttatagcacatccaacctggcttctggagtcccagctcgcttcagt ggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggc tgaagatgctgccacttattactgccaccagtatcatcgttccccacgga cgttcggtggaggcaccaagctggaaatcaaacgtgcggccgca CAR3 full-nt  20 atgggcgtgctgctgacccagaggaccctgctgagcctggtgctggccct gctgtttccatctatggcatcgatggccgaggtccagctgcagcagtctg ggactgtgctggcaaggcctggggcttcagtgaagatgtcctgcaaggct tctggctacacctttaccagctactggatgcactgggtaaaacagaggcc tggacagggtctggaatggattggcgctatttatcctggaaatagtgata ctagctacaaccagaagttcaagggcaaggccaaactgactgcagtcaca tccaccagcactgcctacatggagctcagcagcctgacaaatgaggactc tgcggtcttttactgtacacattactacggtagtgactatgctatggact actggggtcaaggaacctcagtcactgtctcctcaggtggaggcggttca ggcggaggtggctctggcggtggcggatcgcaaattgttctcacccagtc tccagcaatcatgtctgcatctctaggggaacgggtcaccatgacctgca ctgccagctcaagtgtaagttccagttacttgcactggtaccagcagaag ccaggatcctcccccaaactctggatttatagcacatccaacctggcttc tggagtcccagctcgcttcagtggcagtgggtctgggacctcttactctc tcacaatcagcagcatggaggctgaagatgctgccacttattactgccac cagtatcatcgttccccacggacgttcggtggaggcaccaagctggaaat caaacgtgcggccgcaattgaagttatgtatcctcctccttacctagaca atgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgt ccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggt ggttggtggagtcctggcttgctatagcttgctagtaacagtggccttta ttattttctgggtgaggagtaagaggagcaggctcctgcacagtgactac atgaacatgactccccgccgccccgggcccacccgcaagcattaccagcc ctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttca gcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctat aacgagctcaatctaggacgaagagaggagtacgatgttttggacaagag acgtggccgggaccctgagatggggggaaagccgcagagaaggaagaacc ctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcc tacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacga tggcctttaccagggtctcagtacagccaccaaggacacctacgacgccc ttcacatgcaggccctgccccctcgctaataaaagcttaacacgagcca VH (aa)  21 VH CDR1 (aa)  22 GFTFNTYA VH CDR2 (aa)  23 IRSKSNNYAT VH CDR3 (aa)  24 VREGVYYYGSSGYYAMDY VL (aa)  25 EIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGT SPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDA ATYYCQQRSSYPLTFGAPGKLELKRAA VL CDR1 (aa)  26 SYMHWF VL CDR2 (aa)  27 LWIYSTSNLA VL CDR3 (aa)  28 QQRSSYPL ScFv (aa)  29 CAR2 full-aa  30 M P R G W T A L C L L S L L P S G F M S L D N N G T A T P E L P T Q G T F S N V S T N V S Y Q E T T T P S T L G S T S L H P V S Q H G N E A T T N I T E T T V K F T S T S V I T S V Y G N T N S S V Q S Q T S V I S T V F T T P A N V S T P E T T L K P S L S P G N V S D L S T T S T S L A T S P T K P Y T S S S P I L S D I K A E I K C S G I R E V K L T Q G I C L E Q N K T S S C A E F K K D R G E G L A R V L C G E E Q A D A D A G A Q V C S L L L A Q S E V R P Q C L L L V L A N R T E I S S K L Q L M K K H Q S D L K K L G I L D F T E Q D V A S H Q S Y S Q K T L I A L V T S G A L L A V L G I T G Y F L M N R R S W S P T G E R L E L E P V D R V K Q T L N F D L L K L A G D V E S N P G P G N M G V L L T Q R T L L S L V L A L L F P S M A SM A E V Q G V E S G G G L V Q P K G S L K L S C A A S G F T F N T Y A M H W V C Q A P G K G L E W V A R I R S K S N N Y A T Y Y A D S V K D R F T I S R D D S Q S M L Y L Q M N N L K T E D T A M Y Y C V R E G V Y Y Y G S S G Y Y A M D Y W G Q G T S V T V S S GGGGGSGGGGSGGGGSEIVLTQSPAIMSASPGEKVT ITCSASSSVSYMHWFQQKPGTSPKLWIYSTSNLASG VPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSS YPLTFGAPGKLELKRAA I E V M Y P P P Y L D N E K S N G T I I H V K G K H L C P S P L F P G P S K P F W V L V V V G G V L A C Y S L L V T V A F I I F W V R S K R S R L L H S D Y M N M T P R R P G P T R K H Y Q P Y A P P R D F A A Y R S R V K F S R S A D A P A Y Q Q G Q N Q L Y N E L N L G R R E E Y D V L D K R R G R D P E M G G K P Q R R K N P Q E G L Y N E L Q K D K M A E A Y S E I G M K G E R R R G K G H D G L Y Q G L S T A T K D T Y D A L H M Q A L P P R VH (nt)  31 VH CDR1 (nt)  32 VH CDR2 (nt)  33 VH CDR3 (nt)  34 VL (nt)  35 VL CDR1 (nt)  36 VL CDR2 (nt)  37 VL CDR3 (nt)  38 0 ScFv (nt)  39 CAR2 full-nt  40 atgcctcgcggctggacagccctgtgcctgctgtctctgctgccatcc ggcttcatgagcctggataataacggcacagccaccccagagctgc ctacacagggcaccttcagcaatgtgtccacaaacgtgagctatcag gagaccacaaccccttctaccctgggatccacaagcctgcaccccgt gtctcagcacggcaacgaagccaccaccaacatcaccgagaccac agtgaagtttacctccacctctgtgattacctctgtgtacggaaatacaa actccagcgtgcagtctcagacatctgtgatctccacagtgtttacaac acctgccaatgtgtccaccccagagacaaccctgaagcccagcctg tctcctggaaatgtgtccgatctgtctaccacctccaccagcctggcca cctctcccaccaagccctatacctcctcttctcccatcctgagcgatat caaagccgagatcaaatgcagcgggattcgggaagtgaaactgaca cagggcatctgcctggaacagaataagacatccagctgcgccgagtt taagaaagatagaggagagggactggccagggtgctgtgtggcga agagcaggccgacgccgatgccggcgcccaggtgtgttccctgctg ctggcccagtctgaggtgcgcccccagtgcctgctgctggtgctggc caatcggacagaaattagcagcaagctgcagctgatgaaaaaacac cagagcgatctgaaaaagctgggcatcctggactttaccgagcagg acgtggcctctcaccagagctacagccagaaaacactgatcgccct ggtgaccagcggagccctgctggccgtgctgggcatcaccggatatt tcctgatgaataggcgcagctggagccccaccggcgagcggctgg agctggagcctgtcgaccgagtgaagcagaccctgaactttgatctg ctgaagctggccggcgacgtggagtccaaccccgggccagggaat atgggcgtgctgctgacccagaggaccctgctgagcctggtgctgg ccctgctgtttccatctatggcatcgGACGCTTATCGATGGCC GAGGTGCAGGGGGTGGAGTCTGGTGGAGGATTGG TGCAGCCTAAAGGATCATTGAAACTCTCATGTGCC GCCTCTGGTTTCACCTTCAATACCTATGCCATGCAC TGGGTCTGCCAGGCTCCAGGAAAGGGTTTGGAAT GGGTTGCTCGCATAAGAAGTAAAAGTAATAATTAT GCAACATATTATGCCGATTCAGTGAAAGACAGATT CACCATCTCCAGAGATGATTCACAAAGCATGCTCT ATCTGCAAATGAACAACCTGAAAACTGAGGACACA GCCATGTATTACTGTGTGAGAGAAGGGGTTTATTA CTACGGTAGTAGTGGGTACTATGCTATGGACTACT GGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGGT tcctcaggtggaggcggttcaggcggaggtggctctggcggtggcg gatcgGAAATTGTTCTCACCCAGTCTCCAGCAATCAT GTCTGCATCTCCAGGGGAGAAGGTCACC ATAACCTGCAGTGCCAGCTCAAGTGTAAGTTACAT GCACTGGTTCCAGCAGAAG CCAGGCACTTCTCCCAAACTCTGGATTTATAGCAC ATCCAACCTGGCTTCTGGAGTCCCT GCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTA CTCTCTCACAATCAGCCGAATGGAG GCTGAAGATGCTGCCACTTATTACTGCCAGCAAAG GAGTAGTTACCCCCTCACGTTCGGT GCTGGGACCAAGCTGGAGCTGAAACGTGCGGCCG Caattgaagttatgtatcctcctccttacctagacaatgagaagagcaa tggaaccattatccatgtgaaagggaaacacctttgtccaagtccccta tttcccggaccttctaagcccttttgggtgctggtggtggttggtggagt cctggcttgctatagcttgctagtaacagtggcctttattattttctggg tgaggagtaagaggagcaggctcctgcacagtgactacatgaacatga ctccccgccgccccgggcccacccgcaagcattaccagccctatgc cccaccacgcgacttcgcagcctatcgctccagagtgaagttcagca ggagcgcagacgcccccgcgtaccagcagggccagaaccagctc tataacgagctcaatctaggacgaagagaggagtacgatgttttggac aagagacgtggccgggaccctgagatggggggaaagccgcagag aaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagat aagatggcggaggcctacagtgagattgggatgaaaggcgagcgc cggaggggcaaggggcacgatggcctttaccagggtctcagtacag ccaccaaggacacctacgacgcccttcacatgcaggccctgccccc tcgctaataa VH* (aa)  41 MAEVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHW VKQRPGQGLEWIGAIYPGNSDTSYNQKFKGKAKLTAVTST STAYMELSSLTNEDSAVFYCTHYGSDYAMDYWGQGTSVTI SSG VH CDR1* (aa)  42 TSYWMH VH CDR2* (aa)  43 WIGAIYPGNSDTS VH CDR3* (aa)  44 THYYGSDYAMD VL* (aa)  45 QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKP GSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAE DAATYYCHQYHRSPRTFGGGTKLEIKRAA VL CDR1* (aa)  46 SSSYLHWY VL CDR2* (aa)  47 LWIYSTSNLA VL CDR3* (aa)  48 HQYHRSPR ScFv* (aa)  49 MAEVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHW (VH*) VKQRPGQGLEWIGAIYPGNSDTSYNQKFKGKAKLTAVTST STAYMELSSLTNEDSAVFYCTHYGSDYAMDYWGQGTSVTI SSG S S G G G G S G G G G S G G G G S Q I V L T Q S P A I M S A S L G E R V T M T C T A S S S V S S S Y L H W Y Q Q K P G S S P K L W I Y S T S N L A S G V P A R F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C H Q Y H R S P R T F G G G T K L E I K R A A A ScFv* (aa)  50 M A E V Q L Q Q S G T V L A R P G A S V K M (VL*) S C K A S G Y T F T S Y W M H W V K Q R P G Q G L E W I G A I Y P G N S D T S Y N Q K F K G K A K L T A V T S T S T A Y M E L S S L T N E D S A V F Y C T H Y Y G S D Y A M D Y W G Q G T S V T V S S G G G G S G G G G S G G G G SQIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQK PGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEA EDAATYYCHQYHRSPRTFGGGTKLEIKRAA ScFv* (aa)  51 MAEVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHW (VH*VL*) VKQRPGQGLEWIGAIYPGNSDTSYNQKFKGKAKLTAVTST STAYMELSSLTNEDSAVFYCTHYGSDYAMDYWGQGTSVTI SSG S S G G G G S G G G G S G G G G S QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKP GSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAE DAATYYCHQYHRSPRTFGGGTKLEIKRAA CLEC14A (aa)  52 MRPAFALCLL WQALWPGPGG GEHPTADRAG CSASGACYSL HHATMKRQAA EEACILRGGA LSTVRAGAEL RAVLALLRAG PGPGGGSKDL LFWVALERRR SHCTLENEPL RGFSWLSSDP GGLESDTLQW VEEPQRSCTA RRCAVLQATG GVEPAGWKEM RCHLRANGYL CKYQFEVLCP APRPGAASNL SYRAPFQLHS AALDFSPPGT EVSALCRGQL PISVTCIADE IGARWDKLSG DVLCPCPGRY LRAGKCAELP NCLDDLGGFA CECATGFELG KDGRSCVTSG EGQPTLGGTG VPTRRPPATA TSPVPQRTWP IRVDEKLGET PLVPEQDNSV TSIPEIPRWG SQSTMSTLQM SLQAESKATI TPSGSVISKF NSTTSSATPQ AFDSSSAVVF IFVSTAVVVL VILTMTVLGL VKLCFHESPS SQPRKESMGP PGLESDPEPA ALGSSSAHCT NNGVKVGDCD LRDRAEGALL AESPLGSSDA CLEC14A (nt)  53 CTCCTCTTGC TCTAAGCAGG GTGTTTGACC TTCTAGTCGA CTGCGTCCCC TGTACCCGGC GCCAGCTGTG TTCCTGACCC CAGAATAACT CAGGGCTGCA CCGGGCCTGG CAGCGCTCCG CACACATTTC CTGTCGCGGC CTAAGGGAAA CTGTTGGCCG CTGGGCCCGC GGGGGGATTC TTGGCAGTTG GGGGGTCCGT CGGGAGCGAG GGCGGAGGGG AAGGGAGGGG GAACCGGGTT GGGGAAGCCA GCTGTAGAGG GCGGTGACCG CGCTCCAGAC ACAGCTCTGC GTCCTCGAGC GGGACAGATC CAAGTTGGGA GCAGCTCTGC GTGCGGGGCC TCAGAGAATG AGGCCGGCGT TCGCCCTGTG CCTCCTCTGG CAGGCGCTCT GGCCCGGGCC GGGCGGCGGC GAACACCCCA CTGCCGACCG TGCTGGCTGC TCGGCCTCGG GGGCCTGCTA CAGCCTGCAC CACGCTACCA TGAAGCGGCA GGCGGCCGAG GAGGCCTGCA TCCTGCGAGG TGGGGCGCTC AGCACCGTGC GTGCGGGCGC CGAGCTGCGC GCTGTGCTCG CGCTCCTGCG GGCAGGCCCA GGGCCCGGAG GGGGCTCCAA AGACCTGCTG TTCTGGGTCG CACTGGAGCG CAGGCGTTCC CACTGCACCC TGGAGAACGA GCCTTTGCGG GGTTTCTCCT GGCTGTCCTC CGACCCCGGC GGTCTCGAAA GCGACACGCT GCAGTGGGTG GAGGAGCCCC AACGCTCCTG CACCGCGCGG AGATGCGCGG TACTCCAGGC CACCGGTGGG GTCGAGCCCG CAGGCTGGAA GGAGATGCGA TGCCACCTGC GCGCCAACGG CTACCTGTGC AAGTACCAGT TTGAGGTCTT GTGTCCTGCG CCGCGCCCCG GGGCCGCCTC TAACTTGAGC TATCGCGCGC CCTTCCAGCT GCACAGCGCC GCTCTGGACT TCAGTCCACC TGGGACCGAG GTGAGTGCGC TCTGCCGGGG ACAGCTCCCG ATCTCAGTTA 1021 CTTGCATCGC GGACGAAATC GGCGCTCGCT GGGACAAACT CTCGGGCGAT GTGTTGTGTC CCTGCCCCGG GAGGTACCTC CGTGCTGGCA AATGCGCAGA GCTCCCTAAC TGCCTAGACG ACTTGGGAGG CTTTGCCTGC GAATGTGCTA CGGGCTTCGA GCTGGGGAAG GACGGCCGCT 1201 CTTGTGTGAC CAGTGGGGAA GGACAGCCGA CCCTTGGGGG GACCGGGGTG CCCACCAGGC GCCCGCCGGC CACTGCAACC AGCCCCGTGC CGCAGAGAAC ATGGCCAATC AGGGTCGACG AGAAGCTGGG AGAGACACCA CTTGTCCCTG AACAAGACAA TTCAGTAACA TCTATTCCTG VH* (nt)  54 VH CDR1* (nt)   55 VH CDR2* (nt)  56 VH CDR3* (nt)  57 VL* (nt)  58 VL CDR1* (nt)  59 VL CDR2* (nt)  60 VL CDR3* (nt)  61 ScFv* (nt)  62 (VH*) 0 ScFv* (nt)  63 (VL*) ScFv* (nt)  64 (VH*VL*) (G.sub.4S).sub.3 linker  65 GGGGSGGGGSGGGGS Hinge domain   66 FVPVFLPAKP TTTPAPRPPT PAPTIASQPL SLRPEACRPA of CD8α AGGAVHTRGL DFACD shortened IgG   67 AEPKSPDKTH TCP hinge Linker   68 KDPK sequence shortened IgG  69 AEPKSPDKTH TCPKDPK hinge + linker CD8α  70 IYIWAPLAGT CGVLLLSLVI TLYCNHRN transmembrane domain CD28   71 FWVLVVVGGV LACYSLLVTV AFIIFWV transmembrane domain CH2CH3 hinge  72 DPAEPKSPDK THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV  VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR  EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK  VSNKALPAPI EKTISKAKGQ PREPQVYTLP  PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE  SNGQPENNYKTTPPVLDSDG SFFLYSKLTV  DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS  LSPGKKDPK  CD3ζ  73 RVKFSRSADA PAYQQGQNQL YNELNLGRRE domain EYDVLDKRRG RDPEMGGKPR RKNPQEGLYNELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT YDALHMQALP PR intracellular  74 RFSVVKRGRK KLLYIFKQPF MRPVQTTQEE domain of 4-1BB DGCSCRFPEE EEGGCEL intracellular  75 RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY domain of APPRDFAAYR S human CD28 OX40 (CD134)  76 RDQRLPPDAH KPPGGGSFRT PIQEEQADAH STLAKI co-stimulatory domain Truncated CD34  77 atgcctcgcggctggacagccctgtgcctgctgtctctgctgccatccgg molecule (nt) cttcatgagcctggataataacggcacagccaccccagagctgcctacac agggcaccttcagcaatgtgtccacaaacgtgagctatcaggagaccaca accccttctaccctgggatccacaagcctgcaccccgtgtctcagcacgg caacgaagccaccaccaacatcaccgagaccacagtgaagtttacctcca cctctgtgattacctctgtgtacggaaatacaaactccagcgtgcagtct cagacatctgtgatctccacagtgtttacaacacctgccaatgtgtccac cccagagacaaccctgaagcccagcctgtctcctggaaatgtgtccgatc tgtctaccacctccaccagcctggccacctctcccaccaagccctatacc tcctcttctcccatcctgagcgatatcaaagccgagatcaaatgcagcgg gattcgggaagtgaaactgacacagggcatctgcctggaacagaataaga catccagctgcgccgagtttaagaaagatagaggagagggactggccagg gtgctgtgtggcgaagagcaggccgacgccgatgccggcgcccaggtgtg ttccctgctgctggcccagtctgaggtgcgcccccagtgcctgctgctgg tgctggccaatcggacagaaattagcagcaagctgcagctgatgaaaaaa caccagagcgatctgaaaaagctgggcatcctggactttaccgagcagga cgtggcctctcaccagagctacagccagaaaacactgatcgccctggtga ccagcggagccctgctggccgtgctgggcatcaccggatatttcctgatg aataggcgcagctggagccccaccggcgagcggctggagctggagcctgt cgaccgagtgaagcagaccctgaactttgatctgctgaagctggccggcg acgtggagtccaaccccgggccagggaatatgggcgtgctgctgacccag aggaccctgctgagcctggtgctggccctgctgtttccatctatggcatc g Truncated CD34  78 M P R G W T A L C L L S L L P S G F M S L D molecule (aa) N N G T A T P E L P T Q G T F S N V S T N V S Y Q E T T T P S T L G S T S L H P V S Q H G N E A T T N I T E T T V K F T S T S V I T S V Y G N T N S S V Q S Q T S V I S T V F T T P A N V S T P E T T L K P S L S P G N V S D L S T T S T S L A T S P T K P Y T S S S P I L S D I K A E I K C S G I R E V K L T Q G I C L E Q N K T S S C A E F K K D R G E G L A R V L C G E E Q A D A D A G A Q V C S L L L A Q S E V R P Q C L L L V L A N R T E I S S K L Q L M K K H Q S D L K K L G I L D F T E Q D V A S H Q S Y S Q K T L I A L V T S G A L L A V L G I T G Y F L M N R R S W S P T G E R L E L E P V D R V K Q T L N F D L L K L A G D V E S N P G P G N M G V L L T Q R T L L S L V L A L L F P S M A S CLEC14A primer  79 CTGGGACCGAGGTGAGTG CLEC14A probe  80 CGCGATGCAAGTAACTGAGA Flotillin 2 primer  81 TGTTGTGGTTCCGACTATAAACAG Flotillin 2 probe  82 GGGCTGCAACGTCATAATCT CLEC14A fwd primer  83 TAGTAGGAATTCGAGAGAATGAGGCCGGCGTTCGCCCT G CLEC14A rev primer  84 AGAACCGCGGCCGCTGGAGGAGTCGAAAGCCTGAGGA GT Murine CLEC14A fwd  85 TAGTAGGAATTCGAGAGAATGAGGCCAGCGCTTGCCCT primer G Murine CLEC14A rev  86 CTACTAGCGGCCGCTCGTGGAAGAGGTGTCGAAAGT primer Human CLEC14A fwd  87 TAGTAGTTAATTAAGAGAGAATGAGGCCGGCGTTC primer Murine CLEC14A fwd  88 TAGTAGTTAATTAAGAGAGAATGAGGCCAGCGCTT primer Human fc rev primer  89 CTACTAGTTTAAACTCATTTACCCGGAGACAGGGA 5′ UTR Fwd  90 TTCCTTTTCCAGGGTTTGTG 5′ UTR rev  91 GCCTACAAGGTGGCTTGAAT CDS fwd  92 AAGCTGTGCTCCTGCTCTTG CDS rev  93 TCCTGAGTGCACTGTGAGATG 3′ UTR fwd  94 CTGTAGAGGGCGGTGACTTT 3′ UTR rev  95 AGCTGCTCCCAAGTCCTCT mACTB fwd  96 CTAAGGCCAACCGTGAAAAG mACTB rev  97 ACCAGAGGCATACAGGGACA CD141 1-42 aa  98 MLGVLVLGALALAGLGFPAPAEPQPGGSQCVEHDCFALYP GP CD141 97-108  99 QLPPGCGDPKRL CD141 122-142 100 TSYSRWARLDLNGAPLCGPL Codon optimised sequences Human codon 101 ATGGCCGAGGTGCAGCTGCAGCAGTCTGGCACCGTGCT optimised scFv GGCCAGGCCCGGAGCAAGCGTGAAGATGTCCTGCAAGG nucleotide CCTCTGGCTACACCTTCACAAGCTATTGGATGCACTGGG sequence of TGAAGCAGCGCCCAGGACAGGGCCTGGAGTGGATCGG CRT3 AGCAATCTACCCCGGCAACTCCGACACCTCTTATAATCA GAAGTTCAAGGGCAAGGCCAAGCTGACAGCCGTGACCT CTACAAGCACCGCCTACATGGAGCTGAGCAGCCTGACC AACGAGGATAGCGCCGTGTTTTATTGCACACACTACTAT GGCTCCGACTACGCTATGGACTATTGGGGCCAGGGCAC CTCCGTGACAGTGTCTAGCGGAGGAGGAGGCAGCGGA GGAGGAGGCTCCGGCGGCGGCGGCTCTCAGATCGTGC TGACCCAGAGCCCTGCCATCATGTCCGCCTCTCTGGGC GAGCGGGTGACAATGACCTGTACAGCCTCCTCTAGCGT GTCCTCTAGCTACCTGCACTGGTATCAGCAGAAGCCCG GCTCCTCTCCTAAGCTGTGGATCTACAGCACCTCCAATC TGGCATCCGGCGTGCCTGCAAGGTTCTCTGGCAGCGGC TCCGGCACCTCTTACAGCCTGACAATCAGCAGCATGGAG GCAGAGGACGCAGCAACATACTATTGTCACCAGTATCAC CGGAGCCCAAGAACCTTTGGCGGCGGCACAAAGCTGGA GATCAAGCGGGCGGCCGCA Murine codon 102 ATGGCCGAGGTGCAGCTGCAGCAGTCTGGCACCGTGCT optimised scFv GGCTCGGCCCGGAGCTAGCGTGAAGATGTCCTGCAAGG nucleotide CTTCTGGCTACACCTTCACAAGCTACTGGATGCACTGGG sequence of  TGAAGCAGCGCCCAGGACAGGGCCTGGAGTGGATCGG CRT3 CGCCATCTACCCCGGAAACTCCGACACCTCTTACAACCA GAAGTTCAAGGGCAAGGCTAAGCTGACAGCCGTGACCT CTACAAGCACCGCTTACATGGAGCTGAGCAGCCTGACC AACGAGGATAGCGCCGTGTTTTACTGCACACACTACTAC GGCTCCGACTACGCTATGGATTACTGGGGACAGGGCAC CTCCGTGACAGTGTCTAGCGGAGGAGGAGGAAGCGGC GGAGGcGGCAGCGGAGGAGGAGGATCTCAGATCGTGCT GACCCAGTCTCCTGCTATCATGTCCGCCTCTCTGGGCGA GAGGGTGACAATGACCTGTACAGCCTCCTCTAGCGTGTC CTCTAGCTACCTGCACTGGTATCAGCAGAAGCCCGGCTC CTCTCCTAAGCTGTGGATCTACAGCACCTCCAACCTGGC TTCCGGAGTGCCTGCTCGGTTCTCTGGAAGCGGCTCCG GAACCTCTTACAGCCTGACAATCAGCAGCATGGAGGCTG AGGACGCCGCTACATACTACTGTCACCAGTACCACAGGA GCCCAAGAACCTTTGGCGGAGGCACAAAGCTGGAGATC AAGAGGGCGGCCGCA Human codon 103 ATGGCAGAGGTGCAGGGAGTGGAGAGCGGAGGCGGCC optimised scFv TGGTGCAGCCTAAGGGCTCCCTGAAGCTGTCTTGCGCC nucleotide GCCAGCGGCTTCACCTTTAACACATATGCAATGCACTGG sequence of  GTGTGCCAGGCACCAGGCAAGGGCCTGGAGTGGGTGG CRT2 CACGGATCAGAAGCAAGTCCAACAATTATGCCACCTACT ATGCCGACAGCGTGAAGGATAGGTTCACAATCTCCCGC GACGATTCTCAGAGCATGCTGTACCTGCAGATGAACAAT CTGAAGACCGAGGACACAGCCATGTACTATTGCGTGCG GGAGGGCGTGTACTATTACGGCAGCTCCGGCTATTACG CTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG TCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTG GCGGCGGCGGCAGCGAGATCGTGCTGACCCAGTCCCC AGCAATCATGTCCGCCTCTCCAGGAGAGAAGGTGACCAT CACATGCTCCGCCTCCTCTAGCGTGTCTTATATGCACTG GTTCCAGCAGAAGCCCGGCACCTCTCCTAAGCTGTGGA TCTACAGCACATCCAATCTGGCATCCGGCGTGCCCGCAA GGTTTTCTGGCAGCGGCTCCGGCACCTCTTATAGCCTGA CAATCAGCCGGATGGAGGCAGAGGACGCAGCAACCTAT TACTGTCAGCAGAGATCCTCTTACCCTCTGACCTTTGGC GCCGGCACAAAGCTGGAGCTGAAGCGCGCGGCCGCA Murine codon  104 ATGGCTGAGGTGCAGGGAGTGGAGAGCGGAGGAGGCC optimised scFv TGGTGCAGCCTAAGGGCTCCCTGAAGCTGTCTTGCGCC nucleotide GCTAGCGGATTCACCTTTAACACATACGCTATGCACTGG sequence of  GTGTGCCAGGCTCCAGGAAAGGGCCTGGAGTGGGTGG CRT2 CCAGGATCAGAAGCAAGTCCAACAACTACGCTACCTACT ACGCCGACAGCGTGAAGGATCGGTTCACAATCTCCCGC GACGATTCTCAGAGCATGCTGTACCTGCAGATGAACAAC CTGAAGACCGAGGACACAGCTATGTACTACTGCGTGCG GGAGGGCGTGTACTACTACGGCAGCTCCGGATACTACG CTATGGACTACTGGGGACAGGGCACCTCCGTGACAGTG TCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTG GAGGCGGAGGCAGCGAGATCGTGCTGACCCAGTCTCCA GCTATCATGTCCGCCTCTCCCGGCGAGAAGGTGACCAT CACATGCTCCGCCTCCTCTAGCGTGTCTTACATGCACTG GTTCCAGCAGAAGCCCGGCACCTCTCCTAAGCTGTGGA TCTACAGCACATCCAACCTGGCTAGCGGAGTGCCCGCT CGGTTTTCTGGAAGCGGCTCCGGAACCTCTTACAGCCTG ACAATCTCCAGGATGGAGGCTGAGGACGCCGCTACATA CTACTGTCAGCAGAGATCCTCTTACCCTCTGACCTTTGG CGCCGGAACAAAGCTGGAGCTGAAGCGCGCGGCCGCA Consensus of  105 ((GYTF)/X) TSYW ((MH)/X) CRT3 Variants  (1 and 2) heavy chain CDR1 (SEQ ID NOs 2  and 42) Consensus of  106 ((WIGA)/X)IYPGNSDT(S/X) CRT3 Variants (1 and 2) heavy chain CDR2 (SEQ ID NOs 3  and 43) Consensus of  107 THYYGSDYAMD(Y/X) CRT3 Variants (1 and 2) heavy chain CDR3 (SEQ ID NOs 4  and 44) Consensus of  108 ((SSV)/X) SSSY ((LHWY)/X) CRT3 Variants (1 and 2) light chain CDR1 (SEQ ID NO. 6  and 46) Consensus of  109 ((LWIY)/X) STS ((NLA)/X) CRT3 Variants (1 and 2) light chain CDR2 (SEQ ID NO. 7  and 47) Consensus of  110 HQYHRSPR(T/X) CRT3 Variants (1 and 2) light chain CDR3 (SEQ ID NO. 8  and 48) Nucleotide 111 actactaccaagccagtgctgcgaactccctcacctgtgcaccctaccgg sequence gacatctcagccccagagaccagaagattgtcggccccgtggctcagtga encoding Hinge  aggggaccggattggacttcgcctgtgatatttacatctgggcacccttg and gccggaatctgcgtggcccttctgctgtccttgatcatcactctcatctg transmembrane ctaccacaggagccga regions of mouse CD8α Nucleotide 112 aatagtagaaggaacagactccttcaaagtgactacatgaacatgactcc sequence ccggaggcctgggctcactcgaaagccttaccagccctacgcccctgcca encoding mouse  gagactttgcagcgtaccgcccc intracellular signalling sequences from mouse CD28 Nucleotide 113 aaatggatcaggaaaaaattcccccacatattcaagcaaccatttaagaa sequence gaccactggagcagctcaagaggaagatgcttgtagctgccgatgtccac encoding mouse aggaagaagaaggaggaggaggaggctatgag ctg 4-1BB domain Nucleotide sequence encoding mouse CD3 zeta chain 114 Nucleotide 115 cggaaggcttggagattgcctaacactcccaaaccttgttggggaaacag sequence cttcaggaccccgatccaggaggaacacacagacgcacactttactctgg encoding mouse ccaagatc OX40 domain Nucleotide sequence encoding murine CD8α hinge and transmembrane regions, CD28  intracellular signalling domain and CD3ζ intracellular signalling domain 116 Nucleotide sequence encoding murine CD8α hinge and transmembrane domains, 4-1BB   intracellular signalling domain and CD3ζ intracellular signalling domain 117 Nucleotide sequence encoding murine CD8α hinge and transmembrane domains, OX40   intracellular signalling domain and CD3ζ intracellular signalling domain 118 Nucleotide sequence encoding murine CD8α hinge and transmembrane domains, CD28  and 4-1BB  intracellular signalling domains and CD3ζ intracellular signalling domain 119 Nucleotide sequence encoding murine CD8α hinge and transmembrane domains, CD28   and OX40  intracellular signalling domains and CD3ζ intracellular signalling domain 120 Nucleotide sequence encoding murine CD8α hinge and transmembrane domains, 4-1BB  and OX40  intracellular signalling domains and CD3ζ intracellular signalling domain 121 *refers to variant sequence X refers to no amino acid being present

EXAMPLES

Example 1

Analysis of CLEC14A Expression

(27) HUVEC Preparation and Culture

(28) Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords donated by the UK National Health Service after informed consent of the donors. Cords were dissected from placentas and the vein was washed in sterile PBS to remove blood. 1 mg/ml of collagenase diluted in M199 medium (Sigma) was injected into the vein and then incubated at 37° C. for 20 minutes to detach the endothelial cells. 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 1 gelatin from porcine skin (Sigma) coated dishes.

(29) Primary Cells Source

(30) Human aortic smooth muscle cells (HASMC) and human bronchial epithelial cells (HBE) 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 Studies at the University of Birmingham. Hepatocytes were a gift from Professor David Adams, School of Immunity and Infection, University of Birmingham.

(31) RNA Extraction and Real Time PCR

(32) Total RNA was isolated from primary cells in culture using TRI reagent (Sigma) followed by cDNA synthesis using a High-Capacity cDNA Archive kit (Applied Biosystems) with supplied random primers. ProbeLibrary Real-time PCR Assay System (Exiqon) was employed in the primary cell screening of CLEC14A expression. Flotillin 2 was chosen as the housekeeping gene to which the expression of CLEC14A was normalized. Primer and probe sets for CLEC14A and Flotillin 2 were designed by ProbeFinder software (Roche). For CLEC14A, primer and probe set was:

(33) TABLE-US-00002 (SEQ ID NO: 79) 5′-CTGGGACCGAGGTGAGTG-3′, and (SEQ ID NO: 80) 5′-CGCGATGCAAGTAACTGAGA-3′,
with probe number 24.

(34) For Flotillin 2, primer and probe set was:

(35) TABLE-US-00003 (SEQ ID NO: 81) 5′-TGTTGTGGTTCCGACTATAAACAG-3′, and (SEQ ID NO: 82) 5′-GGGCTGCAACGTCATAATCT-3′,
with probe number 28. Quantitative PCR reactions were performed on the Rotor-Gene RG3000 thermal cycler (Corbett Research). A reaction mix was prepared in triplicate for each primary cell type and 5 ng of cDNA was applied in each reaction. The fold change was calculated using the ΔΔCt method.

(36) HUVEC Immunofluorescence

(37) HUVECs were grown in glass micro-well chambers (Nunc) fixed in ice-cold methanol, washed with PBST blocked in 10% FCS 3% BSA in PBST. Cells were then stained with CLEC14A antibody following the same protocol used for paraffin embedded sections or co-stained with 5 g/m mouse monoclonal IgG antibody against human VE-cadherin, kindly donated by Professor Maria Grazia Lampugnani, Fire Institute for Molecular Oncology, Milan. Sections staining were analyzed with a 510 laser scanning confocal microscope (Carl Zeiss).

(38) Results

(39) FIG. 1 is a graph showing the relative expression of CLEC14A in HUVECs and other primary cells. CLEC14A was expressed specifically in endothelial cells. This confirms previous findings that CLEC14A is endothelial-specific.

(40) The expression of CLEC14A in sections of solid tumours and normal tissue was examined using CLEC14A-specific probes. CLEC14A expression was measured by immunofluorescence in human ovarian, bladder, liver, breast, colon, rectal, oesophagus, kidney, lung, prostate, stomach, pancreatic and thyroid tumour tissues. Endothelial specificity of CLEC14A expression was confirmed by co-localisation with Ulex europeaus agglutinin I (UEAI) which binds specific fucose residues on endothelial cells. CLEC14A expression was seen in the blood vessels in all tumour tissues analysed. Ovarian, bladder, liver, breast, kidney and prostate tumours were strongly positive for CLEC14A expression, whereas stomach, oesophagus, lung, colon, rectal, pancreatic and thyroid tumour tissues showed a lower level of specific CLEC14A expression. CLEC14A expression was not detected in any of the corresponding normal control (non-tumour) tissues.

(41) Taken together, these results demonstrate that the transmembrane protein CLEC14A is specifically expressed in tumour vasculature and may therefore be used as tumour endothelial marker.

Example 2

Analysis of CLEC14A Function In Vitro and In Vivo

(42) Materials and Methods

(43) 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 antibodies: rabbit polyclonal anti-murine PECAM (Santa Cruz); secondary antibodies: 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 described below); secondary antibodies: goat polyclonal anti-mouse IgG conjugated to Alexa Fluor488 (Invitrogen).

(44) 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 was generated by initial PCR subcloning from clec14a IMAGE clone (Origene) into pcDNA3-Fc plasmid. The primers used were as follows:

(45) TABLE-US-00004 human CLEC14A fwd (SEQ ID NO: 83) 5′TAGTAGGAATTCGAGAGAATGAGGCCGGCGTTCGCCCTG3′; human CLEC14A rev (SEQ ID NO: 84) 5′AGAACCGCGGCCGCTGGAGGAGTCGAAAGCCTGAGGAGT3′; murine CLEC14A fwd (SEQ ID NO: 85) 5′TAGTAGGAATTCGAGAGAATGAGGCCAGCGCTTGCCCTG3′; murine CLEC14A rev (SEQ ID NO: 86) 5′CTACTAGCGGCCGCTCGTGGAAGAGGTGTCGAAAGT3′.

(46) EcoR1 and Not1 restriction sites were used to insert CLEC14A. A further round of PCR subcloning was performed to transfer the CLEC14A-Fc fusion into pWPI. The primers used were as follows:

(47) TABLE-US-00005 human CLEC14A fwd (SEQ ID NO: 87) 5′TAGTAGTTAATTAAGAGAGAATGAGGCCGGCGTTC3′; murine CLEC14A fwd (SEQ ID NO: 88) 5′TAGTAGTTAATTAAGAGAGAATGAGGCCAGCGCTT3′; human Fc rev (SEQ ID NO: 89) 5′CTACTAGTTTAAACTCATTTACCCGGAGACAGGGA3′.

(48) For this step, Pac1 and Pme1 restriction sites were used.

(49) Human Umbilical Vein Endothelial Cells were isolated as described previously. Umbilical cords were obtained from Birmingham Women's Health Care NHS Trust with informed consent. HUVECs were used between passages 1-6 and were cultured in M199 complete medium (cM199) containing 10% fetal calf 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 were seeded on plates coated in 0.1% type 1 gelatin from porcine skin. HEK293T cells were cultured in DMEM (Sigma) complete medium (cDMEM) containing 10% fetal calf serum (PAA), 4 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen).

(50) SiRNA transfections in HUVEC were performed as previously described. Lentivirus was produced in HEK293T cells by transient transfection with the lentiviral packaging, envelope and expression plasmids above. Plasmids were incubated in OptiMEM (Invitrogen) with polyethylenimine (36 μg/ml) at a 1:4 ratio for 10 minutes at room temperature prior to adding to HEK293T cells in cDMEM. Media 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 polyethylenimine transient transfection as above using pHL-Avitag3 hMMRN2.

(51) Quantitative PCR

(52) cDNA was prepared using the High-Capacity cDNA Archive kit (Applied Biosystems), from 1 μg of extracted total RNA. qPCR reactions were performed with Express qPCR supermix (Invitrogen) on a RG-3000 (Corbett/Qiagen, Manchester, UK) thermocycler. Primers for human clec14a and flotillin-2 were as previously described. Primers for murine clec14a 5′ UTR, CDS and 3′ UTR and murine beta-actin, are as follows:

(53) TABLE-US-00006 5′UTR fwd- (SEQ ID NO: 90) TTCCTTTTCCAGGGTTTGTG; 5′ UTR rev- (SEQ ID NO: 91) GCCTACAAGGTGGCTTGAAT; CDS fwd- (SEQ ID NO: 92) AAGCTGTGCTCCTGCTCTTG; CDS rev- (SEQ ID NO: 93) TCCTGAGTGCACTGTGAGATG; 3′ UTR fwd- (SEQ ID NO: 94) CTGTAGAGGGCGGTGACTTT; 3′ UTR rev- (SEQ ID NO: 95) AGCTGCTCCCAAGTCCTCT; mACTB fwd- (SEQ ID NO: 96) CTAAGGCCAACCGTGAAAAG; mACTB rev- (SEQ ID NO: 97) ACCAGAGGCATACAGGGACA.

(54) Relative expression ratios were calculated according to the efficiency adjusted mathematical model.

(55) Western Blotting and Immunoprecipitation

(56) Whole cell protein lysates were made and co-immunoprecipitation experiments were performed in which protein was extracted from 2×10.sup.7 HUVECs. For initial isolation of CLEC14A interacting proteins 5 μg CLEC14A-Fc or an equimolar amount of hFc was used. For endogenous immunoprecipitation experiments 0.4 μg anti-CLEC14A antibody or sheep IgG was used. For blocking experiments 5 μg CLEC14A-Fc or hFc were bound to protein G beads overnight in PBS. Beads were blocked for 5-6 hours in PBS containing 20% FCS (PAA). Bound CLEC14A-Fc or hFc protein was blocked with increasing concentrations of mIgG or anti-CLEC14A antibody (CRT-2, described below) in binding buffer overnight. Standard protocols were used for western blotting and SDS-PAGE. Primary antibodies were used as indicated in the text with corresponding HRP conjugated secondary antibodies.

(57) Flow Cytometry

(58) Cells were detached with cell dissociation buffer (Invitrogen), rinsed in PBS before incubation in blocking buffer (PBS, 3% BSA, 1% NaN3) for 15 minutes. Subsequent staining using 10 μg/ml anti-HA tag (CRUK), 10 μg/ml anti-CLEC14A (CRT-2, described below), as primary antibodies, in blocking buffer for 30 minutes. Cells were rinsed in PBS and stained with goat polyclonal anti-mouse IgG conjugated to Alexa Fluor488 (Invitrogen) in blocking buffer. Data (15,000 events/sample) were collected using a FACSCalibur apparatus (Becton Dickinson, Oxford, UK), and results were analysed with Becton Dickinson Cell Quest software.

(59) Huvec Spheroid Sprouting Assay and In Vitro Matrigel Tube Forming Assay

(60) Generation of HUVEC spheroids and induction of endothelial sprouting was performed in a collagen gel using 1000 HUVECs per spheroid. Quantification was performed 16 hours after embedding. To quantify sprout growth, the number of sprouts was counted, the cumulative sprout length and the maximal sprout length was assessed. For two colour sprouting experiments, HUVECs were pre-labelled with orange and green CellTracker dyes (Invitrogen). After 24 hours spheroids were fixed in 4% formaldehyde and mounted with Vectorshield (Vector labs). Slides were imaged with an Axioskop2 microscope and AxioVision SE64 Rel4.8 software (Zeiss, Cambridge, UK).

(61) For the Matrigel tube forming assays 1.4×10.sup.5 HUVECs were seeded onto 70 μl basement membrane extract (Matrigel, BD Bioscience, Oxford, UK) in a 12 well plate. After 16 hours, images were taken of 5 fields of view per well using a Leica DM IL microscope (Leica, Milton Keynes, UK) with a USB 2.0 2M Xli digital camera (XL Imaging LLC, Carrollton, Tex., USA) at 10× magnification. Images were analysed with the Angiogenesis analyser plugin for Image J (Carpentier G. et al., Angiogenesis Analyzer for ImageJ. 4th ImageJ User and Developer Conference proceedings) and available at the NIH website (http://imagej.nih.gov/ij/macros/toolsets/Angiogenesis%20Analyzer.txt).

(62) Protein Production

(63) Culture media (CM) from CLEC14A-Fc expressing HEK293T cells was collected. CM was flowed over a HiTrap protein A HP column (GE healthcare, Amersham, UK) and protein eluted using a 0-100% gradient of 100 mM sodium citrate (pH 3) before neutralising with 1 M Tris base. Fractions were run on a SDS-PAG and assessed for protein purity and specificity by Coomassie staining and Western blotting. Fractions containing similar concentrations of protein were combined and dialysed in PBS prior to functional assays.

(64) Monoclonal Antibody Generation

(65) Mouse monoclonal antibodies were commercially prepared by Serotec Ltd (Oxford, UK) using the following protocol to break tolerance supplied by us. Purified mouse CLEC14A-Fc fusion protein was given at 50 μg in Freund's complete adjuvant subcutaneously. Two weeks later mice were given another 50 μg subcutaneously but this time in Freund's adjuvant. Mice were culled and spleens harvested for fusion two weeks later.

(66) Generation of clec14a −/− mice

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

(68) Aortic Ring and Murine Subcutaneous Sponge Angiogenesis Assay

(69) Aortas were isolated and processed for aortic ring assays in collagen. Tube/sprout outgrowth, maximal endothelial migration and total endothelial outgrowth was quantitated. Male C57 black mice were implanted with a subcutaneous sterile polyether sponge disc (10×5×5 mm) under the dorsal skin of each flank at day 0. 100 μl bFGF (40 ng/ml; R&D systems) was injected through the skin directly into the sponges every other day for 14 days. Sponges were excised on day 14, fixed in 10% formalin, and paraffin embedded. Sections were stained with haematoxylin and eosin, sponge cross-sections were taken using a Leica MZ 16 microscope (Leica, Milton Keynes, UK) with a USB 2.0 2M Xli digital camera (XL Imaging LLC, Carrollton, Tex., USA) at ×1 magnification for cellular invasion analysis. Images captured by Leica DM E microscope (Leica, Milton Keynes, UK) at 40× magnification were analysed for vessel density. Vessel counts were assessed in five fields per section per sponge. All animal experimentation was carried out in accordance with Home Office License number PPL 40/3339 held by RB.

(70) Tumour Implantation Assays

(71) 10.sup.6 Lewis lung carcinoma cells were injected subcutaneously into the flank of male mice at 8-10 weeks of age. Tumour growth was monitored by daily calliper measurements and after two-four weeks growth, tumour mass was determined by weight, fixed in 4% PFA, paraffin embedded and serial sections cut at 6 μm.

(72) CLEC14A Regulates Sprouting Angiogenesis In Vitro

(73) To investigate the role of CLEC14A in sprouting angiogenesis in vitro, HUVEC spheroids were generated from HUVECs treated with siRNA targeting clec14a or a non-complementary siRNA duplex. Knockdown of clec14a expression was confirmed at the mRNA level by qPCR with an average reduction of 74% across three experiments (FIG. 2A) and at the protein level by Western blot analysis of protein extracts probed with an anti-CLEC14A polyclonal antisera (FIG. 2B). VEGF induced sprouting from CLEC14A knockdown spheroids was impaired, knockdown spheroids produced on average 6.9 sprouts per spheroid, compared to 13.2 for control cells (FIGS. 2C and 2D). To determine the role of CLEC14A in tip/stalk cell formation, control HUVECs and knockdown HUVECs were stained either red or green and mixed, prior to spheroid formation and induced sprouting (FIG. 2E). Knockdown of CLEC14A reduced 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 that were derived from CLEC14A knockdown HUVECs (FIG. 2F). These data suggest CLEC14A has a role in sprout initiation and migration.

(74) CLEC14A Regulates Sprouting Angiogenesis In Vivo

(75) To investigate the role of CLEC14A in vivo and ex vivo, mice were generated to replace the clec14a coding sequence with a lacZ reporter (FIG. 3A). Breeding of heterozygotes (clec14a −/+) produced equal proportions of male and female mice (49.5%/50.5% respectively) and a Mendelian ratio of wildtype: heterozygote: homozygote mice (26.4%: 47.2%: 26.4% respectively). As clec14a is an endothelial-restricted gene, aortas were isolated from clec14a +/+ and clec14a −/− mice. Extracted cDNA was analysed by qPCR and confirmed loss of the clec14a coding region but expression of the 5′ and 3′ untranslated regions were retained (FIG. 2B). Loss of CLEC14A at the protein level was also confirmed by Western blot analysis of lung tissue lysates (FIG. 3C).

(76) To confirm the role of CLEC14A in sprouting angiogenesis in multicellular three dimensional co-culture, aortas were isolated, cut into rings and embedded in collagen. Cellular outgrowth was stimulated by VEGF and monitored over 7 days before end-point quantitation of endothelial sprouting. Again, loss of CLEC14A impaired endothelial sprout outgrowth and migration (FIG. 3D). Aortic rings from wildtype mice produced over double the number of tubes compared to that observed for CLEC14A knockout mice (30.6 tubes compared to 13.4 tubes respectively) (FIG. 3E). In addition, the maximum migration, which is defined by the furthest distance migrated away from each aortic ring, was also reduced in knockout cultures (FIG. 3F). To assess whether CLEC14A has a similar function in vivo, sponge barrels were implanted subcutaneously into CLEC14A knockout mice. Cellular infiltration and neo-angiogenesis were stimulated using bFGF injections into the sponge every two days for two weeks. Macroscopic analysis of sponge sections stained with haematoxylin and eosin revealed impaired infiltration of cells into the sponge in clec14a −/− animals (FIGS. 3G and 3H). In addition, vascularity was significantly reduced (p<0.01) for clec14a −/− animals (FIG. 3I). To confirm the endothelial cells lining the neoangiogenic vessels express clec14a in this model, sponges and livers from CLEC14A KO mice were stained with x-gal. Strong x-gal staining was observed on blood vessels within the sponge compared to matched liver sections (FIG. 3J). From these data we can conclude that mouse CLEC14A expression regulates endothelial migration and angiogenic sprouting in vivo, as well as in vitro, and CLEC14A is upregulated on sprouting endothelium.

(77) CLEC14A Promotes Tumour Growth

(78) CLEC14A expression is found highly up-regulated on human tumour vessels compared to vessels from healthy tissue, suggesting that cancer therapies could be targeted against CLEC14A. Therefore, to investigate whether loss of CLEC14A effects tumour growth we used the syngeneic Lewis lung carcinoma (LLC) model. For this 1×10.sup.6 LLC cells were injected subcutaneously into the right flank of either clec14a +/+ or clec14a −/− mice. Tumour growth was impaired in the clec14a −/− mice compared to clec14a +/+ littermates (FIG. 4A). This was confirmed by three independent experiments. Excised tumours taken from clec14a −/− mice were smaller in size (FIG. 4B) and smaller in weight (FIG. 4C) than clec14a +/+ littermates. To determine whether the vascular density within these tumours was also effected, tissue sections were stained with an anti-CD31 antibody. Analysis shows a reduced density of discrete vessels (FIGS. 4D and 4E) and reduced percentage endothelial coverage (FIG. 4F). Furthermore, x-gal staining of tumour and liver sections taken from clec14a −/− mice reveals high expression of clec14a on both mature vessels, with erythrocyte filled lumens (FIG. 4G, black arrows), and immature microvessels within the tumour (FIG. 4G), confirming clec14a is upregulated on tumour vessels.

Example 3

Preparation of Anti-CLEC14A Monoclonal Antibodies and Their Effect on Angiogenesis

(79) Preparation of Monoclonal Antibodies

(80) The antigens used for the preparation of monoclonal antibodies were murine CLEC14AFc (CM) and human CLEC14A-Fc (CH), optionally conjugated with adjuvant protein (AP). These four antigens (CM, CH, CM-AP, CH-AP) were used for mice immunisation using the following protocol:

(81) Day Operation

(82) 0 Pre-immune sample taken

(83) Immunisation of 100 ug of antigen in complete Freund's adjuvant (foot pads)

(84) 14 Immunisation of 100 ug of antigen in incomplete Freund's adjuvant (foot pads)

(85) 17 Test bleed

(86) 18 Popliteal lymph node harvest for fusion

(87) Sera were tested by ELISA against three antigens: CM, CH and Fc. A non-immune serum was taken as a negative control.

(88) The fusion protocol was as follows:

(89) (1) Popliteal lymph nodes were harvested from the immune mice and homogenised.

(90) (2) Cells were washed with warm DMEM.

(91) (3) Cells were mixed with sp2/0 myeloma cells.

(92) (4) The mixture was centrifuged (1000 g)

(93) (5) The pellet was suspended in 50% PEG 500 and incubated for 1 min.

(94) (6) The suspension was slowly diluted with warm DMEM.

(95) (7) Suspension was centrifuged (1000 g).

(96) (8) Cells were seeded into plates with peritoneal macrophages.

(97) (9) Cells were cultivated at 37° C. and 5% C0 2

(98) More than 500 HAT-resistant hybridoma clones from each mouse were obtained. All of the clone supernatants were tested twice with 4 days interval by ELISA against three absorbed antigens (CM, CH and Fc). Testing resulted in 5 clones, 2 of which, CRT-2 and CRT-3 (both subclass IgG1), were further studied and shown to react with both CM and CH and did not react with Fc. The positives were cloned 2-4 times by the limiting dilution method, propagated in culture flasks and injected into mice for ascites. One clone (CRT-3) was the result of immunisation with CLEC14a human-AP (CHAP), and the other clone (CRT-2) was the result of immunisation with CLEC14a mouse-AP (CM-AP).

(99) Scratch Wound Healing Assay with CLEC14A Monoclonal Antibodies

(100) A scratch with a 10 ml pipette tip was made in confluent HUVECs. New medium containing 1 g/m or 10 g/l of a monoclonal CLEC14A antibody raised in mice against the extracellular domain of CLEC14A was applied. Chemokinetic migration of HUVECs was assessed by acquiring images of wound closure at time zero, 4, 6, 12 hours with a Leica DM 1000 light microscope and USB 2.0 2M Xli camera. The open area of the wound was quantitated using Image J software.

(101) The ability of CLEC14A inhibitors to inhibit angiogenesis was examined. Scratch wound healing assays using monoclonal antibodies described above show that the anti-CLEC14A monoclonal antibodies inhibit endothelial cell migration in a HUVEC scratch wound healing assay. As shown in FIGS. 5A and B, when HUVECs were treated with 10 g/ml of monoclonal antibody CRT-3, 25% of the wound area remained open at 12 h compared to 13% in the control.

(102) These results show that anti-CLEC14A monoclonal antibody CRT-3 has an inhibitory effect on endothelial cell migration. Endothelial cell migration is an essential feature of angiogenesis. Accordingly, these assays provide evidence that the monoclonal antibodies of the invention inhibit angiogenesis directly.

(103) To further characterise the functional effects of CLEC14A antibody treatment on endothelial cells, tube formation assays were performed with 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 mesh area per field. The results show that CRT2, 3 and 4 all have a differing negative effect on tube formation.

Example 4

Characterisation of Anti-CLEC14A Monoclonal Antibodies

(104) Various polypeptide constructs were generated and expressed in cells to map the binding sites of the monoclonal antibodies of the invention. All constructs have a C-terminus GFP tag so green cells were gated and stained red. Binding of CRT antibodies was analysed using flow cytometry.

(105) FIG. 7A shows that CRT-2 and CRT-3 both bind to cells expressing CLEC14A and FIG. 7B demonstrates that the antibodies do not bind to cells expressing thrombomodulin.

(106) A chimera comprising the C-type lectin domain (CTLD) of thrombomodulin (CD141) and remainder of the CLEC14A molecule was produced. Cells expressing this antigen are not recognised by either of the CRT antibodies (FIG. 8A), although a slight shift in fluorescence was observed with CRT-2. A chimera comprising CLEC14A in which the Sushi-like domain has been replaced with the Sushi-like domain of thrombomodulin (CD141) (to ensure correct folding of CTLD of CLEC14A) was also generated and results in binding of CRT-3, but not CRT-2 (FIG. 8B). Both CRT antibodies bind WT CLEC14A and, as expected, neither binds to WT CD141 (FIG. 7).

(107) These data suggest that the binding site of the antibody CRT-3 is within the C-type lectin domain and that CRT-2 binds on a region between the CTLD and sushi-like domain.

(108) To further determine the binding regions of the antibodies, chimeric loop constructs were made. This was based on the structural predictions of CLEC14A CTLD.

(109) CLEC14A with regions 1-42 of CD141

(110) TABLE-US-00007 CD141 sequence- (SEQ ID NO: 98) MLGVLVLGALALAGLGFPAPAEPQPGGSQCVEHDCFALYPGP

(111) CLEC14A with regions 97-108 of CD141

(112) TABLE-US-00008 CD141 sequence- (SEQ ID NO: 99) QLPPGCGDPKRL

(113) CLEC14A with regions 122-142 of CD141

(114) TABLE-US-00009 CD141 sequence- (SEQ ID NO: 100) TSYSRWARLDLNGAPLCGPL

(115) The alignment is shown in FIG. 9. Unfortunately 1-42 and 122-142 chimeras did not fold correctly. This is thought due to the fact they are present on the cell surface (stain positive for CLEC14A polyclonal antibodies, but they do not stain for either of the antibodies.

(116) The 97-108 chimera does bind CRT-2 and CRT-3 showing that this mutant is correctly folded. Residues 97-108 were swapped with corresponding regions from thrombomodulin. This resulted in correct folding as CRT-2 and CRT-3 can still bind (FIG. 8C). These data suggest that CRT-2 and CRT-3 do not bind to residues 97-108 of CLEC14A.

(117) This experiment has been repeated three times with the same result.

(118) The CRT-2 and CRT-3 encoding sequences were clones from their respective hybridomas using standard techniques and sequenced. The CDRs have been predicted using standard software. The polypeptide and nucleotide sequences are set out in the table below. In view of different in prediction software, sequence variants, including CDR variants, are also shown (marked with a “*”).

(119) TABLE-US-00010 Sequence name SEQ ID (sequence type) NO: Sequence CRT-3 VH CDR1 (aa)  2 GYTFTSYW VH CDR2 (aa)  3 IYPGNSDT VH CDR3 (aa)  4 THYYGSDYAMDY VH CDR1* (aa) 42 TSYWMH VH CDR2* (aa) 43 WIGAIYPGNSDTS VH CDR3* (aa) 44 THYYGSDYAMD VL CDR1 (aa)  6 SSVSSSY VL CDR2 (aa)  7 STS VL CDR3 (aa)  8 HQYHRSPRT VL CDR1* (aa) 46 SSSYLHWY VL CDR2* (aa) 47 LWIYSTSNLA VL CDR3* (aa) 48 HQYHRSPR VH (aa)  1 M A E V Q L Q Q S G T V L A R P G A S V K M S C K A S G Y T F T S Y W M H W V K Q R P G Q G L E W I G A I Y P G N S D T S Y N Q K F K G K A K L T A V T S T S T A Y M E L S S L T N E D S A V F Y C T H Y Y G S D Y A M D Y W G Q G T S V T V VH* (aa)  41 MAEVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHW VKQRPGQGLEWIGAIYPGNSDTSYNQKFKGKAKLTAVTST STAYMELSSLTNEDSAVFYCTHYGSDYAMDYWGQGTSVTI SSG VL (aa)  5 Q I V L T Q S P A I M S A S L G E R V T M T C T A S S S V S S S Y L H W Y Q Q K P G S S P K L W I Y S T S N L A S G V P A R F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C H Q Y H R S P R T F G G G T K L E I K R A A A VL* (aa) 45 QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHVVYQQKP GSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAE DAATYYCHQYHRSPRTFGGGTKLEIKRAA ScFv (aa)  9 M A E V Q L Q Q S G T V L A R P G A S V K M S C K A S G Y T F T S Y W M H W V K Q R P G Q G L E W I G A I Y P G N S D T S Y N Q K F K G K A K L T A V T S T S T A Y M E L S S L T N E D S A V F Y C T H Y Y G S D Y A M D Y W G Q G T S V T V S S G G G G S G G G G S G G G G S Q I V L T Q S P A I M S A S L G E R V T M T C T A S S S V S S S Y L H W Y Q Q K P G S S P K L W I Y S T S N L A S G V P A R F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C H Q Y H R S P R T F G G G T K L E I K R A A A ScFv* (aa) 49 MAEVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHW (VH*) VKQRPGQGLEWIGAIYPGNSDTSYNQKFKGKAKLTAVTST STAYMELSSLTNEDSAVFYCTHYGSDYAMDYWGQGTSVTI SSG S S G G G G S G G G G S G G G G S Q I V L T Q S P A I M S A S L G E R V T M T C T A S S S V S S S Y L H W Y Q Q K P G S S P K L W I Y S T S N L A S G V P A R F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C H Q Y H R S P R T F G G G T K L E I K R A A A ScFv* (aa) 50 M A E V Q L Q Q S G T V L A R P G A S V K M (VL*) S C K A S G Y T F T S Y W M H W V K Q R P G Q G L E W I G A I Y P G N S D T S Y N Q K F K G K A K L T A V T S T S T A Y M E L S S L T N E D S A V F Y C T H Y Y G S D Y A M D Y W G Q G T S V T V S S G G G G S G G G G S G G G G SQIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHVVYQQK PGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEA EDAATYYCHQYHRSPRTFGGGTKLEIKRAA ScFv* (aa) 51 MAEVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHW (VH*VL*) VKQRPGQGLEWIGAIYPGNSDTSYNQKFKGKAKLTAVTST STAYMELSSLTNEDSAVFYCTHYGSDYAMDYWGQGTSVTI SSG S S G G G G S G G G G S G G G G S QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHVVYQQKP GSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAE DAATYYCHQYHRSPRTFGGGTKLEIKRAA VH CDR1 (nt) 12 ggctacacctttaccagctactgg VH CDR2 (nt) 13 atttatcctggaaatagtgatact VH CDR3 (nt) 14 acacattactacggtagtgactatgctatggactac VL CDR1 (nt) 16 tcaagtgtaagttccagttac VL CDR2 (nt) 17 agcacatcc VL CDR3 (nt) 18 caccagtatcatcgttccccacggacg VHC(nt) 11 atggccgaggtccagctgcagcagtctgggactgtgctggcaaggcctggggcttc agtgaagatgtcctgcaaggcttctggctacacctttaccagctactggatgcactgg gtaaaacagaggcctggacagggtctggaatggattggcgctatttatcctggaaat agtgatactagctacaaccagaagttcaagggcaaggccaaactgactgcagtc acatccaccagcactgcctacatggagctcagcagcctgacaaatgaggactctg cggtcttttactgtacacattactacggtagtgactatgctatggactactggggtcaa ggaacctcagtcactgtc VL (nt) 15 caaattgttctcacccagtctccagcaatcatgtctgcatctctaggggaacgggtca ccatgacctgcactgccagctcaagtgtaagttccagttacttgcactggtaccagc agaagccaggatcctcccccaaactctggatttatagcacatccaacctggcttctg gagtcccagctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcag cagcatggaggctgaagatgctgccacttattactgccaccagtatcatcgttcccc acggacgttcggtggaggcaccaagctggaaatcaaacgt ScFv (nt) 19 atggccgaggtccagctgcagcagtctgggactgtgctggcaaggcctggggcttc agtgaagatgtcctgcaaggcttctggctacacctttaccagctactggatgcactgg gtaaaacagaggcctggacagggtctggaatggattggcgctatttatcctggaaat agtgatactagctacaaccagaagttcaagggcaaggccaaactgactgcagtc acatccaccagcactgcctacatggagctcagcagcctgacaaatgaggactctg cggtcttttactgtacacattactacggtagtgactatgctatggactactggggtcaa ggaacctcagtcactgtctcctcaggtggaggcggttcaggcggaggtggctctgg cggtggcggatcgcaaattgttctcacccagtctccagcaatcatgtctgcatctcta ggggaacgggtcaccatgacctgcactgccagctcaagtgtaagttccagttacttg cactggtaccagcagaagccaggatcctcccccaaactctggatttatagcacatc caacctggcttctggagtcccagctcgcttcagtggcagtgggtctgggacctcttac tctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaccag tatcatcgttccccacggacgttcggtggaggcaccaagctggaaatcaaacgtgc ggccgca CRT-2 VH CDR1 (aa) 22 GFTFNTYA VH CDR2 (aa) 23 IRSKSNNYAT VH CDR3 (aa) 24 VREGVYYYGSSGYYAMDY VL CDR1 (aa) 26 SYMHWF VL CDR2 (aa) 27 LWIYSTSNLA VL CDR3 (aa) 28 QQRSSYPL VH (aa) 21 M A E V Q G V E S G G G L V Q P K G S L K L 0 S V K D R F T I S R D D S Q S M L Y L Q M N VL (aa) 25 EIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGT SPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDA ATYYCQQRSSYPLTFGAPGKLELKRAA ScFv (aa) 29 M A E V Q G V E S G G G L V Q P K G S L K L S V K D R F T I S R D D S Q S M L Y L Q M N GSGGGGSGGGGSEIVLTQSPAIMSASPGEKVTITCSASSSV SYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGT SYSLTISRMEAEDAATYYCQQRSSYPLTFGAPGKLELKRAA VH CDR1 (nt) 32 VH CDR2 (nt) 33 VH CDR3 (nt) 34 0 VL CDR1 (nt) 36 VL CDR2 (nt) 37 VL CDR3 (nt) 38 VH (nt) 31 GACGCTTATCGATGGCCGAGGTGCAGGGGGTGGAGTCT GGTGGAGGATTGGTGCAGCCTAAAGGATCATTGAAACTC TCATGTGCCGCCTCTGGTTTCACCTTCAATACCTATGCC A TGCACTGGGTCTGCCAGGCTCCAGGAAAGGGTTTGGAA CAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAA CTCCTCAGGT VL (nt) 35 GAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCAT CTCCAGGGGAGAAGGTCACC 0 GCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCT CTCACAATCAGCCGAATGGAG GCTGGGACCAAGCTGGAGCTGAAACGTGCGGCCGC ScFv (nt) 39 GACGCTTATCGATGGCCGAGGTGCAGGGGGTGGAGTCT GGTGGAGGATTGGTGCAGCCTAAAGGATCATTGAAACTC ATGCACTGGGTCTGCCAGGCTCCAGGAAAGGGTTTGGA CCAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGA 0 TCTCCTCAGGTtcctcaggtggaggcggttcaggcggaggtggctctggcg gtggcggatcgGAAATTGTTCTCACCCAGTCTCCAGCAATCAT GTCTGCATCTCCAGGGGAGAAGGTCACC ATAACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCAC TGGTTCCAGCAGAAG CCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCC AACCTGGCTTCTGGAGTCCCT GCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCT CTCACAATCAGCCGAATGGAG GCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGT AGTTACCCCCTCACGTTCGGT GCTGGGACCAAGCTGGAGCTGAAACGTGCGGCCGC

Example 5

Design and Analysis of Chimeric Antigen Receptors Based on the Antigen-Binding Domains of the anti-CLEC14A Monoclonal Antibodies

(120) Generation of CAR Constructs

(121) Hybridomas expressing CLEC14A-specific monoclonal antibodies that cross react with human and mouse forms of the protein were obtained as described above. Gene constructs encoding an scFv were then isolated from each of the mouse hybridomas by RT-PCR using degenerate primer sets designed to amplify all mouse V-gene families. The scFv genes were then subcloned 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) as a ClaI, NotI fragment, replacing the CD19-specific scFv region. These vectors were originally constructed using the MP71 retroviral expression plasmid (a kind gift from C. Baum, Hannover) and co-expressed a truncated CD34 marker gene.

(122) Transduction of Human and Mouse T-Cells

(123) To generate recombinant retrovirus for transducing human T cells, Phoenix amphotropic packaging cells were transfected with an MP71 retroviral vector and pCL ampho (Imgenex) using FuGENE HD (Roche) according to the manufacturer's instructions. Recombinant retrovirus for transducing mouse T cells was generated in the same way but using Phoenix ecotropic packaging cells and pCL eco. 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 hours 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, Calif.) using standard medium (RPMI1640 (Sigma) containing 10% foetal bovine serum (FBS; PAA, Pasching Austria), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 pg/ml streptomycin) plus 1% human AB serum (TCS Biosciences, Buckingham, UK). Transduction of mouse T cells was conducted using mouse splenocytes pre-activated for 48 hours with concanavalin A (2 ug/ml; Sigma) and mouse interleukin 7 (1 ng/ml; eBioscience) in standard medium. Preactivated human and mouse T cells were subsequently transduced (or mock-transduced with conditioned supernatant from non-transfected phoenix cells) by spinfection in retronectin (Takara)-coated plates according to the manufacturer's instructions. Human T cells were then cultured in standard medium plus 1% human AB serum with IL2 (100 U/ml). After spinfection, mouse T cells were cultured for 24 hrs in standard medium with IL2 (100 U/ml), 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. Studies with human donors were approved by the National Research Ethics Service Committee West Midlands (Solihull) and all donors gave written informed consent.

(124) Cell Lines and Recombinant Proteins

(125) Phoenix A or E, CHO and Lewis lung carcinoma cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS; PAA, Pasching Austria), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 pg/ml streptomycin. CHO cells had been transduced with the pWPI vector (Addgene) expressing full length human CLEC14A (or vector alone). Human umbilical vein endothelial cells (HUVECs) were isolated as described above using umbilical cords obtained from Birmingham Women's Health Care NHS Trust with informed consent and with ethical approval of the south Birmingham research ethics committee. HUVECs were maintained in M199 complete medium containing 10% FBS, 4 mM L-glutamine, 10% large vessel endothelial cell growth supplement (TCS Cellworks) and cultured in plates coated with 0.1% type 1 gelatin from porcine skin (Sigma). Human and murine CLEC14A proteins with a human Fc tag were expressed in HEK293T cells and purified on a protein A column.

(126) Flow Cytometry

(127) HUVECs were trypsinised and stained for 1 hr on ice with CLEC14A-specific mouse monoclonal antibodies described above (10 ug/ml) or IgG1 isotype control (Dako) in 5% normal goat serum/PBS. Cells were washed and bound antibody detected by incubating with R-PE-conjugated goat-anti mouse antibody (Serotec). Dead cells were identified by staining with propidium iodide. Human T-cells were washed with PBS and stained with Live/Dead Fixable Violet Dead Cell Stain Kit (Life Technologies) for 20 mins in the dark. Cells were then washed with flow buffer (0.5% w/v BSA+2 mM EDTA in PBS; pH7.2) and stained with anti-human CD4 (PE-conjugated), anti-human CD8 (FITC-conjugated) (all from BD Pharmingen) and anti-human CD34 (Pe-Cy5) (BioLegend) for 30 mins on ice in the dark. Alternatively rather than staining for CD34, CAR expression was detected directly by firstly blocking cells with human Fc fragment (10 ug/ml), then incubating them with 10 ug/ml recombinant human CLEC14A-Fc fusion protein (or Fc control) followed by sheep anti CLEC14A polyclonal antibody (R&D systems, 10 ug/ml). Finally cells were stained with FITC-conjugated rabbit anti-sheep antibody (Invitrogen, diluted 1:10). All incubations were conducted for 1 hour on ice.

(128) When staining mouse T cells from heparinized tail bleeds they were first subject to red blood cell lysis using BD Pharm lyse (Becton Dickinson) before staining as described above but using anti mouse CD4-FITC, CD8-PE and CD45.1 (PE-Cy7 conjugated) (all BD Biosciences). Cells were analyzed using a BD LSR II flow cytometer and FlowJo software (TreeStar Inc, Ashland, Oreg.).

(129) CFSE Labelling

(130) T-cells were washed twice with PBS and incubated with 2.5 μM Carboxyfluorescein succinimidyl ester (CFSE) for 10 minutes at 37° C. The labelling reaction was quenched by addition of RPMI-1640 containing 10% FBS. Cells were washed, resuspended in standard medium plus 1% human AB serum and IL2 (10 IU/ml) at 1.5×10.sup.6 cells/ml and added to wells containing HUVECs to give a T-cell:HUVEC ratio of 10:1. After 5 days incubation at 37° C./5% CO.sub.2, cells were analysed by flow cytometry as described above using anti-human CD34 (Pe-Cy5).

(131) IFNγ Release Assay

(132) Stimulator cells (2.5×10.sup.4/well) were co-cultured in triplicate with CD34+ CAR-T-cells at responder:stimulator ratios indicated. Alternatively 2×10.sup.4 CD34+ CAR-T cells were incubated in wells precoated with recombinant protein (1 ug/ml). Cells were incubated at 37° C./5% CO.sub.2 in 100 μl/well of standard medium supplemented with IL2 (25 U/ml). After 18 hours, culture supernatant was tested for secreted IFNγ using an ELISA (Pierce Endogen, Rockford, Ill.) according to the manufacturer's instructions.

(133) Cytotoxicity Assays

(134) Chromium release assays were set up at known effector:target ratios (1250 targets/well) and harvested after 7.5 hours.

(135) Toxicity Testing

(136) Six to eight week old C57BL6 mice (Charles River Laboratories) received 4 Gy total body irradiation (TBI). Eighteen hours later, each mouse was injected into the tail vein with 2×10.sup.7 CAR- or Mock-transduced T cell preparations from CD45.1+ congenic BoyJ mice. Mice were monitored for signs of toxicity and immune monitoring was conducted by weekly tail bleeds. Mice were eventually culled 45 days later and major organs removed for histological analysis.

(137) RipTag2 Transgenic Mouse Tumour Model

(138) RIP-Tag2 mice are a model of pancreatic islet cell carcinogenesis. RIP-Tag2 mice were maintained on a C57BL/6J background (The Jackson Laboratory). Cryopreserved CAR-transduced and mock transduced T cells were thawed, washed and 15 million T cells/mouse injected intravenously into the tail vein on a single occasion into 12-week old mice that had been conditioned with 4 Gy TBI the day before. From 12 weeks of age, all RIP-Tag2 mice received 50% sugar food (Harlan Teklad) to relieve hypoglycaemia induced by the insulin-secreting tumours. Total tumour burden in culled CAR-T cell-treated mice was quantified at 16 weeks of age using calipers to measure individually excised macroscopic tumours (>1 mm3) using the formula: volume=a×b2×0.52, where a and b represent the longer and shorter diameter of the tumour, respectively. The volumes of all tumours from each mouse were added to give the total tumour burden per animal. There are no age-matched control comparisons for the 16-week CAR-treated mice, since untreated RIP-Tag2 mice do not survive to 16 weeks, and thus the comparison was made to 14-week old Mock-treated mice.

(139) Lewis Lung Carcinoma (LLC) Mouse Model

(140) 6-8 week old female C57BL6 mice were inoculated subcutaneously on the flank with 10.sup.6 LLC cells. Three days later mice received 4 Gy TBI and 18 hrs after this each mouse was injected into the tail vein with 2×10.sup.7 CAR or Mock T cell preparations from CD45.1+ congenic BoyJ mice. Tumour growth was measured with calipers (using the formula: volume=length×width2×0.5) and bioluminescence imaging (IVIS Spectrum, Caliper Life Sciences). Immune monitoring was conducted by weekly tail bleeds.

(141) Tissue Preparation and Immunofluorescence Analysis

(142) Tissues from mouse experiments were embedded in OCT (Bio Optica), frozen in dry ice and stored at −80° C. Tissue preparation and histology analysis were carried out with the following primary antibodies: purified rat monoclonal anti-panendothelial cell antigen (550563, clone Meca32, BD Pharmingen, USA), diluted 1:100; rabbit monoclonal anti-cleaved caspase 3 (asp175, clone 5A1, Cell Signaling, USA), diluted 1:100; rabbit polyclonal anti-Fibrinogen (A0080, Dako), diluted 1:100; and rabbit monoclonal anti-CD34 (ab174720, Abcam) diluted 1:50; sheep polyclonal anti-CLEC14A (AF4968, R&D) diluted 1:50. After incubation and washing, samples were incubated with secondary antibodies anti Rabbit Alexa Fluor-488 and Alexa Fluor-555; anti Rat Alexa Fluor-488 and Alexa Fluor-555; and anti Sheep Alexa Fluor-488 (Molecular Probes) and counterstained with DAPI Nucleic Acid Stain (Invitrogen). To detect CAR-transduced T cells tissues were stained with rabbit monoclonal anti-CD34 (ab174720, Abcam) diluted 1:50 in PBS. After incubation and washing, samples were stained with anti Rabbit Alexa Fluor-555 (Molecular Probes) and counterstained with DAPI.

(143) Human tumour tissue arrays (SuperBiochips Inc., Seoul, Korea) were stained using sheep polyclonal anti-CLEC14A (AF4968, R&D systems) diluted 1:20 and Ulex europaeus agglutinin I conjugated to rhodamine (Vectorlabs, UK) for 1 hour, followed by anti-sheep FITC antibody (10 μg/ml, Invitrogen, UK).

(144) For analysis of RipTag2 tumour tissue, the surface area occupied by vessels was quantified through the ImageJ software as the area occupied by Meca32-positive structures, compared with the total tissue area visualised by DAPI. For each animal, the total vessel area of at least four field/images was quantified. To determine the amount of fibrinogen extravasation (red channel) in each image, we drew a region of interest (ROI) close to each blood vessel (Meca32, green channel), and then quantified the mean fluorescence intensity (MFI) of red and green channels using the Leica Confocal Software Histogram Quantification Tool. In order to normalize the vessel number values obtained, we calculated the ratio between red and green channel MFI; values are expressed as percentage of red-green co-staining. To determine the expression levels of caspase 3 (green channel) in each analysed image, we considered 5 random ROIs of the same size. Then we measured the MFI of the green channel, and we normalized the values by comparing caspase 3-stained area with the total cells present in the tissue area. At least 10 images of five mice per treatment group were analyzed for each sample. Tissue from RipTag2 mice were analyzed using a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems). All other tissues were analysed using an Axiovert 100M laser scanning confocal microscope (Carl Zeiss, Welwyn Garden City, UK).

(145) Statistical Analysis

(146) Statistical analyses of data were conducted using the tests indicated and GraphPad Prism software. A p value <0.05 was considered significant.

(147) Results

(148) A retroviral CAR vector was generated (based on pMP71) that co-expresses a truncated CD34 marker gene and an scFv fragment/CD3 zeta chain chimeric receptor. Expression is driven from the LTR promoter and the 2A peptide linker ensures equimolar expression of both the CD34 and the CAR. Second generation CAR constructs included the CD28 co-stimulatory domain (see FIG. 10).

(149) FIG. 10B shows CD34 staining analysed by flow cytometry demonstrated successful transduction of T cells using retroviral constructs that co-express a CLEC14A-specific CAR. A first generation CAR based on the antibody CRT-3 is referred to as CRT3.z. A second generation CAR based on the antibody CRT-3 is referred to as CRT3.28z. Note equivalent expression was seen in CD4 and CD8 T cell subsets (data not shown).

(150) FIG. 10C shows cells stained directly for expression of CAR using CLEC14A-Fc (% values show specific binding of CLEC14A-Fc having subtracted background staining with Fc alone).

(151) FIG. 11 shows CAR-transduced T cells respond to CLEC14A in vitro. T cells transduced to express 1st or 2nd generation CARs based on antibody CRT-3 or mock-transduced (control) T cells were tested for their ability to respond to CLEC14A expressed either as (A) plate-bound recombinant Fc fusion protein, (B) expressed on engineered CHO cells, or (C) expressed on human umbilical vein endothelial cells (HUVECs) which naturally express CLEC14A when grown in static culture. T cell response was measured using an ELISA for interferon gamma production. Data shown in FIG. 11 are representative of that obtained from 3-7 repeat experiments. T cells were adjusted to equalise the frequency of transgene expressing cells. All histograms show mean response+SD.

(152) FIG. 12 shows further in vitro functional testing of CLEC14A-specific CAR-transduced T cells. T cells transduced to express 1st or 2nd generation CARs based on antibody CRT-3, 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 (having subtracted background levels of lysis of CHO alone (control cells)). Data shown are representative of 5 repeat experiments. (B) Proliferation, using CFSE-labelled CAR-transduced T cells we measured the proliferation of CAR+ (CD34+) and CAR− (CD34−) cell subsets when co-cultured for 4 days with HUVECs. Data shown are representative of 2 repeat experiments. (C) The response of (CLEC14A-specific CAR-transduced T cells to both human and mouse CLEC14A was assessed using interferon gamma release. T cells were adjusted to equalise the frequency of transgene expressing cells. Data shown are representative of 6 repeat experiments. All histograms show mean response+SD.

(153) FIG. 13 shows toxicity testing in vivo using healthy C57/BL6 mice injected with CLEC14A-specific CAR-transduced mouse T cells. At the end of the experiment mice were culled and major organs harvested. Histological examination revealed no evidence of pathology.

(154) FIG. 14 shows the anti-tumour response of CLEC14A-specific CAR-transduced mouse T cells when injected into mice carrying Lewis Lung carcinoma tumours. C57BL6 mice were injected subcutaneously with Lewis Lung Carcinoma cells (1 million cells/mouse) and 4 days later mice received 4 Gy total body irradiation to aid T cell engraftment. T cells transduced to express 2nd generation CARs based on antibody CRT-3, or mock-transduced (control) T cells, were then injected into the tail vein. Mice received a total of 20 million T cells (CD8:CD4=5:2) with CRT3.28z expressed on 2.2 of these cells. Tumour growth was then monitored using (A) Bioluminescence or (B) Calipers.

(155) FIG. 15 shows the anti-tumour response of CLEC14A-specific CAR-transduced mouse T cells when injected into mice carrying Lewis Lung carcinoma tumours. At the end of the experiment tumours were excised and weighed (A). Histological analysis demonstrated that tumours from mice treated with the CARs showed significantly reduced vascular density (B, staining for MECA-32) and greater levels of vascular leakage (C, staining for fibrinogen).

(156) The polypeptide and nucleotide sequences for the CAR derived from CRT-3 are set out in the table below:

(157) TABLE-US-00011 CAR3 full-aa 10 M G V L L T Q R T L L S L V L A L L F P S M A S M A E V Q L Q Q S G T V L A R P G A S V K M S C K A S G Y T F T S Y W M H W V K Q R P G Q G L E W I G A I Y P G N S D T S Y N Q K F K G K A K L T A V T S T S T A Y M E L S S L T N E D S A V F Y C T H Y Y G S D Y A M D Y W G Q G T S V T V S S G G G G S G G G G S G G G G S Q I V L T Q S P A I M S A S L G E R V T M T C T A S S S V S S S Y L H W Y Q Q K P G S S P K L W I Y S T S N L A S G V P A R F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C H Q Y H R S P R T F G G G T K L E I K R A A A I E V M Y P P P Y L D N E K S N G T I I H V K G K H L C P S P L F P G P S K P F W V L V V V G G V L A C Y S L L V T V A F I I F W V R S K R S R L L H S D Y M N M T P R R P G P T R K H Y Q P Y A P P R D F A A Y R S R V K F S R S A D A P A Y Q Q G Q N Q L Y N E L N L G R R E E Y D V L D K R R G R D P E M G G K P Q R R K N P Q E G L Y N E L Q K D K M A E A Y S E I G M K G E R R R G K G H D G L Y Q G L S T A T K D T Y D A L H M Q A L P P R CAR3 full-nt 20 atgggcgtgctgctgacccagaggaccctgctgagcctggtgctggccctgctgttt ccatctatggcatcgatggccgaggtccagctgcagcagtctgggactgtgctggc aaggcctggggcttcagtgaagatgtcctgcaaggcttctggctacacctttaccag ctactggatgcactgggtaaaacagaggcctggacagggtctggaatggattggc gctatttatcctggaaatagtgatactagctacaaccagaagttcaagggcaaggc caaactgactgcagtcacatccaccagcactgcctacatggagctcagcagcctg acaaatgaggactctgcggtcttttactgtacacattactacggtagtgactatgctat ggactactggggtcaaggaacctcagtcactgtctcctcaggtggaggcggttcag gcggaggtggctctggcggtggcggatcgcaaattgttctcacccagtctccagca atcatgtctgcatctctaggggaacgggtcaccatgacctgcactgccagctcaagt gtaagttccagttacttgcactggtaccagcagaagccaggatcctcccccaaactc tggatttatagcacatccaacctggcttctggagtcccagctcgcttcagtggcagtg ggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgcca cttattactgccaccagtatcatcgttccccacggacgttcggtggaggcaccaagct ggaaatcaaacgtgcggccgcaattgaagttatgtatcctcctccttacctagacaat gagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtc ccctatttcccggaccttctaagcccttttgggtgctggtggtggttggtggagtcctgg cttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagagga gcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccac ccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcca gagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaa ccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggaca agagacgtggccgggaccctgagatggggggaaagccgcagagaaggaaga accctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcct acagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatgg cctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgc aggccctgccccctcgctaataaaagcttaacacgagcca

Example 6

Design and Analysis of Anti-CLEC14A Monoclonal Antibody-Drug Conjugates (Immunoconjugates)

(158) Internalisation of CRT-3 Antibody-Drug Conjugate (Immunoconjugate)

(159) Monoclonal anti-CLEC14A antibody (CRT-3) drug conjugates (CRT-3-ADC) were generated, wherein the antibody was attached to tirapazamine.

(160) FIG. 16A shows HUVEC cells treated with CRT-3 and shows the localisation of the antibody after 0 minutes (FIG. 16A(i)) and 90 minutes (FIG. 16A(ii)) and demonstrates that the antibody is internalised in HUVEC cells. FIG. 16B shows a graph of cytotoxicity measures by Cell Titre Glo luminescent cell viability assay in HUVEC treated CRT-3-ADC and demonstrates that the CRT-3 antibody shows cytotoxicity as an immunoconjugate.

(161) LLC ADC 24 Hour Pilot Experiment

(162) 1 million Lewis lung carcinoma cells were injected subcutaneously into the right flank of 2 mice and allowed to grow to a visible size. Next, 1 mg/kg of CRT-3-ADC or B12-ADC (control) was administered through tail vein injections. The mice were observed for an hour and culled 24 hours later.

(163) Results

(164) Treatment with CRT-3-ADC for 24 h had no effect on the overall health of the mouse. Extensive haemorrhage at the site of the tumour was observed only in the

(165) CRT-3-ADC treated mouse and not the control, demonstrating tumour-specific disruption of angiogenesis (FIG. 17).

Example 7

Titration of CRT1, 3 and 5 Against CLEC14A

(166) CLEC14A was expressed as an Fc fusion protein for incubation with CRT1, 3 and 5 CAR (CD28 costimulatory domain) T cells. All CAR-T cell lines were diluted with Mock T cells to equalise for transduction efficiencies. The results can be seen in FIG. 18 where it is shown that all of the tested CAR T cells respond well to CLEC14A (data shown are means of triplicate cultures +SD).

Example 8

CRT1, 3 and 5 CAR T Cell Cytotoxicity and Proliferation Assay

(167) A cytotoxicity study was carried out using CRT1, 3 and 5 CAR (with CD28 costimulatory domain) T cells. The T cells were diluted with Mock T cells to equalise for transduction efficiencies and were incubated with mouse endothelial cells expressing human CLEC14A. The results are shown in FIG. 20 which demonstrate that all three tested CARs can mediate cytotoxicity. The data shown are means of triplicate cultures +SD.

(168) Further, a proliferation assay was carried out (CFSE labelling) with CRT1, 3 and 5 CAR (CD28 costimulatory domain) T cells stimulated with plate-bound recombinant CLEC14A-Fc fusion proteins. All the CAR T cell lines were diluted with Mock T cells to equalise for transduction efficiencies, where all three tested CARs were capable of proliferating after stimulation.

Example 9

CARs with Different Costimulatory and Transmembrane Regions

(169) The following CARs have been cloned and engineered into T cells from a single donor using a retroviral vector:

(170) 1) CRT3-CD28 TM-CD28 costim signal-CD3 (CRT3.28z)

(171) 2) CRT3-CD8 TM-4-1BB costim signal-CD3 (CRT3.BBz)

(172) 3) CRT3-CD28 TM-CD28 and 4-1BB costim signals-CD3 (CRT3.28BBz)

(173) 4) CRT3-CD28 TM-CD28 and OX40 costim signals-CD3 (CRT3.28Oxz)

(174) 5) CRT3-CD8 TM-4-1BB and OX40 costim signals-CD3 (CRT3.BBOxz) All constructs generated transduced well into T cells. The function of the different constructs was assessed in vitro, analysing cytokine production, cytotoxicity and proliferative response (see FIG. 21). Cytokine release indicated strong antigen specific responses especially by T cells expressing CRT3.28z and CRT3.28Oxz. Cytokine production was analysed by measuring IFNgamma production in response to titrated numbers of CHO cells expressing human CLEC14A (or vector only control). All CAR T cell lines were diluted with Mock T cells to equalise for transduction efficiencies. Data shown are means of triplicate cultures +SD. Cytotoxic activity was measured against mouse endothelial cells (SEND) engineered to express CLEC14A and the proliferative response was measured following stimulation with plate-bound recombinant CLEC14A-Fc fusion proteins (data not shown).

Example 10

Determination of Cytokine Release from CAR T Cells Following Stimulation with Chimeric CLEC14A

(175) Chimeric forms of CLEC14A that contain the human sequence but with the transmembrane and/or intracellular domains of mouse origin were expressed in 293 and SEND cells. These cells were sorted using GFP co-expressed from a lentiviral vector to equalise for CLEC expression and then tested using CAR T cells (CRT1, 3 and 5 with CD28 costimulatory domain). The release of IFN gamma was measured after incubation of the CAR T cells with both the 293 and SEND cells. The results can be seen in FIG. 22. Additionally, the cytotoxicity of the T cells when incubated with the CLEC14A chimera expressing SEND cells was determined. All CAR T cell lines were diluted with Mock T cells to equalise for transduction efficiencies. Data shown are means of triplicate cultures.

(176) As can be seen from FIG. 22, all of the tested CRT1, 3 and 5 CAR T cells result in the release of IFN gamma from 293 and SEND cells expressing either human CLEC14A (huCLEC), human CLEC14A with mouse intracellular domain (A1) and human CLEC14A with mouse transmembrane and intracellular domain (B1).