Amino acid sequences directed against the angiopoietin/tie system and polypeptides comprising the same for the treatment of diseases and disorders related to angiogenesis

Abstract

The present invention relates to amino acid sequences that are directed against proteins from the group of the Angiopoietin/Tie family such as Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Angptl5, Angptl6, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more of such amino acid sequences.

Claims

1. An isolated polypeptide comprising at least one single variable domain that binds to Ang1, wherein said at least one single variable domain consists of the structure FR1 CDR1 FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR 1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, in which CDR1 is the amino acid sequence of SEQ ID NO: 190; and CDR2 is the amino acid sequence of SEQ ID NO: 284; and CDR3 is the amino acid sequence of SEQ ID NO: 378; or CDR1 is the amino acid sequence of SEQ ID NO: 191; and CDR2 is the amino acid sequence of SEQ ID NO: 285; and CDR3 is the amino acid sequence of SEQ ID NO: 379; or CDR1 is the amino acid sequence of SEQ ID NO: 192; and CDR2 is the amino acid sequence of SEQ ID NO: 286; and CDR3 is the amino acid sequence of SEQ ID NO: 380; and wherein the single variable domain is capable of forming a single antigen binding unit.

2. The isolated polypeptide according to claim 1, which is purified at least 2 fold relative to the source or medium from which it has been obtained.

3. The isolated polypeptide according to claim 1, wherein the single variable domain binds to human Ang1.

4. The isolated polypeptide according to claim 1, comprising at least two single variable domains.

5. The isolated polypeptide according to claim 1, further comprising a single variable domain that binds human Tie2.

6. A pharmaceutical composition comprising an isolated polypeptide according to claim 1 and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant.

7. A single variable domain that binds to Ang1, wherein said single variable domain consists of the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR 1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, in which CDR1 is the amino acid sequence of SEQ ID NO: 190; and CDR2 is the amino acid sequence of SEQ ID NO: 284; and CDR3 is the amino acid sequence of SEQ ID NO: 378; or CDR1 is the amino acid sequence of SEQ ID NO: 191; and CDR2 is the amino acid sequence of SEQ ID NO: 285; and CDR3 is the amino acid sequence of SEQ ID NO: 379; or CDR1 is the amino acid sequence of SEQ ID NO: 192; and CDR2 is the amino acid sequence of SEQ ID NO: 286; and CDR3 is the amino acid sequence of SEQ ID NO: 380.

8. The single variable domain according to claim 7, wherein the single variable domain has at least 80% sequence identity with any of SEQ ID NOs: 472-474.

9. The single variable domain according to claim 7, wherein the single variable domain has 100% sequence identity with any of SEQ ID NOs: 472-474.

10. The isolated polypeptide according to claim 1, wherein the single variable domain has at least 80% sequence identity with any of SEQ ID NOs: 472-474.

11. The isolated polypeptide according to claim 1, wherein the single variable domain has 100% sequence identity with any of SEQ ID NOs: 472-474.

Description

(1) The invention will now be further described by means of the following non-limiting figures:

(2) FIG. 1. Tie2 binding assay for a selection of clones. Negative controls are addition of irrelevant phage selected against a viral antigen and no phage addition.

(3) FIG. 2. Tie2-Ang1 blocking assay of selected P.E. Negative controls are addition of irrelevant P.E. selected against a viral antigen and no P.E. addition. 5 clones (family I, II, III and IV) show significant blocking of Ang-1 binding.

(4) FIG. 3. Tie2-Ang1 blocking assay of purified nanobodies in a dilution series. Negative controls are addition of irrelevant nanobody selected against a viral antigen and no nanobody addition.

(5) FIG. 4. Tie2-Ang2 blocking assay of purified nanobodies in a dilution series. Negative controls are addition of irrelevant nanobody selected against a viral antigen and no nanobody addition. None of the Tie2-Ang1 blocking nanobodies is able to block binding of Ang2 to Tie2.

(6) FIG. 5. Ang2 binding assay for a selection of clones. Negative controls are addition of irrelevant phage selected against a viral antigen and no phage addition.

(7) FIG. 6. Ang2-Tie2 blocking assay for a selection of clones. Negative controls are addition of irrelevant P.E. selected against a viral antigen and no P.E. addition. 6 clones (family I) show significant blocking of Ang-2 binding to Tie2.

(8) FIG. 7. Ang2-Tie2 blocking assay of purified nanobodies in a dilution series. Negative controls are addition of irrelevant nanobody selected against a viral antigen and no nanobody addition.

(9) FIG. 8. Ang1 binding assay for a selection of clones. Negative controls are addition of irrelevant phage selected against a viral antigen and no phage addition.

(10) FIG. 9. Ang4 binding assay for a selection of clones. Negative controls are addition of irrelevant phage selected against a viral antigen and no phage addition.

(11) FIG. 10. Angptl4 binding assay for a selection of clones. Negative controls are addition of irrelevant phage selected against a viral antigen and no phage addition.

(12) FIGS. 11&12. Ratio of phospho-Akt to Akt (FIG. 11) and phospho-ERK to ERK (FIG. 12) is reported. Ø indicate non Ang-1 stimulated samples. Among anti-Tie2 NBs tested, only Nanobody 163E9 was able to block the Ang1-induced Akt and Erk phosphorylation both at 7.5 ug/ml (˜500 nM) and 1 ug/ml (˜67 nM). None of the others Tie-2 Nanobodies inhibited phosphorylation of AKt and Erk.

(13) FIGS. 13&14. Ratio of phospho-Akt to Akt (FIG. 13) and phospho-ERK to ERK (FIG. 14) is reported. Nanobody 163E9 dose-dependedtly inhibited Ang-1 induced phosphorylation of Akt and Erk.

(14) FIG. 15. The Tie-2 Nanobody 163E9 reverses the anti-apoptotic effect of Ang-1

(15) FIG. 16. Nanobody 163E9 dose-dependently inhibits Ang-1 induced phosphorylation of Tie-2

(16) FIG. 17. Nanobody 163E9 dose-dependently inhibits Ang-1 induced sprouting of endothelial cells.

(17) The invention will now be further described by means of the following non-limiting experimental part.

EXPERIMENTAL PART

Example 1: Animal Immunizations

(18) Two llamas (161 and 166) are immunized, according to standard protocols, with 6 boosts of a cocktail 121 containing recombinant human Tie2/Fc Chimera (R&D Systems Cat No 313-TI, Lot No BKC06). Blood is collected from these animals 5 and 8 days after boost 6. In addition, approximately 1 g of lymph node is collected from each animal 5 days after boost 6.

Example 2: Library Construction

(19) Peripheral blood mononuclear cells are prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA is extracted from these cells and lymph node tissue and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments are cloned into phagemid vector pAX50 (see below). Phage is prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).

(20) pAX50—An expression vector is used derived from pUC119 which contains the LacZ promoter, a coliphage pIII protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multicloning site and the gen3 leader sequence. In frame with the Nanobody® coding sequence, the vector codes for a C-terminal c-myc tag and a (His)6 tag.

Example 3: Selections of Phage Displaying Tie2 Binding Nanobodies

(21) Phage libraries 161 and 166 are used for selections on recombinant human Tie2/Fc Chimera (R&D Systems Cat No 313-TI, Lot No BKC06). Tie2/Fc is immobilized directly on Maxisorp 96 well microtiter plates (Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control). To minimize the number of phage binding to the Fc-portion of Tie2/Fc the phage is pre-incubated with 250 ug/ml human IgG. Following incubation with the phage libraries and extensive washing, bound phage was eluted with trypsin. The eluted phage are amplified and applied in a second round of selection on 2 ug/ml, 0.2 ug/ml, 0.02 ug/ml and 0 ug/ml (control) immobilized Tie2/Fc. To minimize the number of phage binding to the Fc-portion of Tie2/Fc the phage is pre-incubated with 100 ug/ml human IgG plus 100 ug/ml rh B7.2/Fc (R&D Systems Cat No 141-B2, Lot No BOT 075031). Individual colonies obtained from the eluted phage pools are grown and i) induced for new phage production and ii) induced with IPTG for Nanobody expression and extraction (periplasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 4: Screening for Tie2 Binding Nanobodies

(22) In order to determine binding specificity to Tie2, the clones are tested in an ELISA binding assay setup, using the monoclonal phage pools. Phage binding to Tie2/Fc Chimera (R&D Systems Cat No 313-TI, Lot No BKC06) is tested. Shortly, 0.2 ug/ml receptor is immobilized on Maxisorp ELISA plates (Nunc) and free binding sites are blocked using 4% Marvel skimmed milk in PBS. Next, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ul 2% Marvel PBS are allowed to bind to the immobilized antigen. After incubation and a wash step, phage binding is revealed using a HRP-conjugated monoclonal-anti-M13 antibody (Gentaur Cat#27942101). Binding specificity is determined based on OD values compared to controls having received no phage and to controls where in a similar ELISA binding assay the same monoclonal phage are tested for binding to 0.2 ug/ml of immbolized human IgG and 0.2 ug/ml of rh B7.2/Fc.

(23) FIG. 1 and Table B-1 show a selection of clones binding to Tie2 (see Table B-1 for definition of clones).

(24) TABLE-US-00025 TABLE B-1 Nanobodies against Tie2. SEQ ID Name NO: Sequence 162-E1 455 EVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGW YQQAPGKQRELVAFITSVGTTNYADSVKGRFIISRD NAKNTVYLQMNSLKPEDTAVYYCAADLHYSGPNYWG QGTQVTVSS 162-E9 456 EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGW FRQAPGKEREAVSCISSVDGSTHYADSVKGRFTISR DNAKDTVYLQMNSLKPEDTAAYYCAVQGYSGGYYYT CEDSADFGFWGQGTQVTVSS 162-F11 457 EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGW FRQAPGKEREGVACISSSDGSTYYADSVKGRFTISS DNAKNTVYLQMNSLKPEDTAVYSCSAGSVAGCIPYY WGQGTQVTVSS 162-F3 458 EVQLVESGGGLVQAGDSLRLSCTTSGRTFSDDTMGW FRQAPRKEREFVAAILWDSIKTYYADSVKGRFTISR DNAKNTVYLQMDSLKPEDTAVYYCAATPTAYGTDWY RNNYHYWGQGTQVTVSS 162- 459 EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAVGW H10 FRQAPGKEREGVSCIGSSYGSTYYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCAVQGYSGGYYYT CEDSADFGFWGQGTQVTVSS 163-E7 460 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYSMSW VRQAPGKGLEWVSAISGGGEVTTYADSVKGRFTISR DNAKNTLYLQMSSLKPEDTALYYCAEHLNFYSVSVR SSPTSQGTQVTVSS 163-E9 461 EVQLVESGGGLVQPGDSLRLSCAASGFTFGSNGMRW VRQAPGKGPEWVSSINSDGTSTYYADSVKGRFTISR DNAKNTLCLQMNSLKPEDTAVYYCTTTEDPYPRGQG TQVTVSS 163-G8 462 EVQLVESGGGLVQPGGSLRLSCAASGFTFGSNGMRW VRQAPGKGPEWVSSINSDGTSAFYAESVKGRFTISR DNAKNTLYLQMNSLKPEDTAVYYCTTTMNPNPRGQG TQVTVSS 163-H8 463 EVQLVESGGGLVQPGGSLRLSCAASGFTFGSNGMRW VRQAPGKGPEWVSSINSDGTSTYYAESVKGRFTISR DNAKNTLYLQMHSLKPEDTAVYYCTTTENPNPRGPG TQVTVSS

Example 5: Screening for Nanobodies Blocking Tie2-Ang1 Interaction

(25) Clones tested positive in the Tie2 binding assay are screened for their ability to block Ang1 binding to Tie2/Fc. For this, Nanobody-containing periplasmic extracts (P.E.) are used in an ELISA-based ligand competition setup. In short, 0.75 ug/ml human Ang1 (R&D Systems Cat No 923-AN/CF Lot No FHW073091) is coated in 96 well Maxisorp microtiter plates (Nunc) and blocked with 4% Marvel skimmed milk in PBS. In parallel, 0.2 ug/ml Tie2/Fc is incubated with 10 ul of periplasmic extract P.E. containing nanobody of the different clones in 100 ul 2% Marvel/PBS. After 1 hour, the receptor-Nanobody pre-mixes are incubated 1 hour with the coated ligand. Bound Tie2/Fc is detected using HRP-conjugated goat anti-human IgG (Jackson Immunoresearch, Cat #109-035-098). Blocking activity is determined as loss of OD signal, as compared to wells where no P.E., or irrelevant P.E., has been added.

(26) FIG. 2 shows results of this blocking assay using a selection of clones binding to Tie2.

(27) 162-E1, 162-E9, 162-F11, 162-H10, 163-E7 (see Table B-2 below) show significant blocking of Ang1 binding to Tie2.

(28) TABLE-US-00026 TABLE B-2 Nanobodies against Tie2 and able to block Ang1 binding to Tie2. SEQ ID Name NO: Sequence 162-E1 455 EVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGW YQQAPGKQRELVAFITSVGTTNYADSVKGRFIISRD NAKNTVYLQMNSLKPEDTAVYYCAADLHYSGPNYWG QGTQVTVSS 162-E9 456 EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGW FRQAPGKEREAVSCISSVDGSTHYADSVKGRFTISR DNAKDTVYLQMNSLKPEDTAAYYCAVQGYSGGYYYT CEDSADFGFWGQGTQVTVSS 162-F11 457 EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGW FRQAPGKEREGVACISSSDGSTYYADSVKGRFTISS DNAKNTVYLQMNSLKPEDTAVYSCSAGSVAGCIPYY WGQGTQVTVSS 162- 459 EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAVGW H10 FRQAPGKEREGVSCIGSSYGSTYYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCAVQGYSGGYYYT CEDSADFGFWGQGTQVTVSS 163-E7 460 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYSMSW VRQAPGKGLEWVSAISGGGEVTTYADSVKGRFTISR DNAKNTLYLQMSSLKPEDTALYYCAEHLNFYSVSVR SSPTSQGTQVTVSS

(29) Because above Nanobodies are able to block Ang1 binding to Tie2 they are thus considered antagonists of Tie2. Functional assay to confirm this function can be found later in this experimental part.

Example 6: Determining Tie2-Ang1 Interaction Blocking Efficiency by Titration of Purified Nanobody

(30) In order to determine the receptor blocking efficiency of clones tested positive for Ang1 competition, a dilution series of purified Nanobodies are tested in the ELISA-based ligand competition setup. In short, 0.75 ug/ml human Ang1 (R&D Systems Cat No 923-AN/CF Lot No FHW073091) is coated in 96 well Maxisorp microtiter plates (Nunc) and blocked with 4% Marvel skimmed milk in PBS. In parallel, 0.2 ug/ml Tie2/Fc is incubated with a dilution series of purified Nanobodies. After 1 hour, the receptor-Nanobody pre-mixes are incubated 1 hour with the coated ligand. Bound Tie2/Fc is detected using HRP-conjugated goat anti-human IgG (Jackson Immunoresearch, Cat #109-035-098). Blocking activity is determined as loss of OD signal, as compared to wells where no P.E., or irrelevant P.E., has been added. FIG. 3 shows the results of this assay.

Example 7: Screening for Tie2-Ang2 Blocking Among the Purified Tie2-Ang1 Blocking Nanobodies

(31) In order to investigate whether the clones tested positive for Ang1 competition can also block binding of Ang2 to the receptor the pervious purified Nanobodies are tested in a new ELISA-based ligand competition setup. In short, 0.75 ug/ml human Ang2 (R&D Systems Cat No 923-AN/CF) is coated in 96 well Maxisorp microtiter plates (Nunc) and blocked with 4% Marvel skimmed milk in PBS. In parallel, 0.2 ug/ml Tie2/Fc is incubated with 150 nM of purified Nanobodies. After 1 hour, the receptor-Nanobody pre-mixes are incubated 1 hour with the coated ligand. Bound Tie2/Fc was detected using HRP-conjugated goat anti-human IgG (Jackson Immunoresearch, Cat #109-035-098). Blocking activity is determined as loss of OD signal, as compared to wells where no P.E., or irrelevant P.E., has been added. FIG. 4 shows the result of this example. None of the Tie2-Ang1 blocking nanobodies is able to block binding of Ang2 to Tie2.

(32) Sequences Alignments of Tie2 Binding Nanobodies (FRs in Small Letters, CDRs in Capital Letters):

(33) TABLE-US-00027 163-G8 evqlvesggglvqpggslrlscaasgftfgSNGMRwvrqapgkgpewvsSINSDGTSAFY 163-H8 evqlvesggglvqpggslrlscaasgftfgSNGMRwvrqapgkgpewvsSINSDGTSTYY 163-E9 evqlvesggglvqpgdslrlscaasgftfgSNGMRwvrqapgkgpewvsSINSDGTSTYY 163-E7 evqlvesggglvqpggslrlscaasgftfsDYSMSwvrqapgkglewvsAISGGGEVTTY 162-E1* evqlvesggglvqaggslrlscaasgsifsINAMGwyqqapgkgrelvaFITSVG-TTNY 162-F3 evqlvesggglvqagdslrlscttsgrtfsDDTMGwfrqaprkerefvaAILWDSIKTYY 162-E9 evqlvesggglvqpggslrlscaasgftldDYAIGwfrqapgkereaysCISSVDGSTHY 162-H10 evqlvesggglvqpggslrlscaasgftldDYAVGwfrqapgkeregvsCIGSSYGSTYY 162-F11 evqlvesggglvqaggslrlscaasgftfdDYAIGwfrqapgkeregvaCISSSDGSTYY 163-G8 AESVKGrftisrdnakntlylqmnslkpedtavyycttTM-----NPN----------Pr 163-H8 AESVKGrftisrdnakntlylqmhslkpedtavyycttTE-----NPN----------Pr 163-E9 ADSVKGrftisrdnaknticlqmnslkpedtavyycttTE-----DPY----------Pr 163-E7 ADSVKGrftisrdnakntlylqmsslkpedtalyycaeHL-----NFYSV---SVRSSPt 162-E1 ADSVKGrfiisrdnakntvylqmnslkpedtavyycaa-------DLHYS-----GPNYw 162-F3 ADSVKGrftisrdnakntvylqmdslkpedtavyycaaTPTAYGTDWYRN-----NYHYw 162-E9 ADSVKGrftisrdnakdtvylqmnslkpedtaayycavQG--YSGGYYYTCEDSADFGFw 162-H10 ADSVKGrftisrdnakntvylqmnslkpedtavyycavQG--YSGGYYYTCEDSADFGFw 162-F11 ADSVKGrftissdnakntvylqmnslkpedtavyscsaGS--VAGCIPY---------Yw 163-G8 gqgtqvtvss 163-H8 gpgtqvtvss 163-E9 gqgtqvtvss 163-E7 sqgtqvtvss 162-E1 gqgtqvtvss 162-F3 gqgtqvtvss 162-E9 gqgtqvtvss 162-H10 gqgtqvtvss 162-F11 gqgtqvtvss *q in FR2 of 162-E1 from an Amber stop codon
Members:
Families of Binders (One Family of Nanobodies has Same CDR3):

(34) TABLE-US-00028 members: I 162-E1 II 162-E9, 162-H10 III 163-E7 IV 162-F11 V 162-F3 VI 163-E9, 163-G8, 163-H8

Example 8: Animal Immunizations

(35) Two llamas (171 and 172) are immunized, according to standard protocols, with 6 boosts of a cocktail 121 containing:

(36) Recombinant human Angiopoietin-1 (R&D Systems Cat No 923-AN/CF),

(37) Recombinant human Angiopoietin-2 (R&D Systems Cat No 623-AN/CF),

(38) Recombinant human Angiopoietin-4 (R&D Systems Cat No 964-AN/CF),

(39) Recombinant human Angiopoietin-like-4 (R&D Systems Cat No 3485-AN)

(40) Blood is collected from these animals 8 days after boost 6.

Example 9: Library Construction

(41) Peripheral blood mononuclear cells are prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA is extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments are cloned into phagemid vector pAX50. Phage is prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein). Example results in phage libraries 171 (from Llama 171) and 172 (from Llama 172).

Example 10: Selections of Phage Displaying Ang2 Binding Nanobodies

(42) Phage libraries 171 and 172 are used for selections on recombinant human Ang2 (R&D Systems Cat No 623-AN/CF). Ang2 is immobilized directly on Maxisorp 96 well microtiter plates (Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control). Following incubation with the phage libraries and extensive washing, bound phage is eluted with trypsin. The eluted phage are amplified and applied in a second round of selection on 2 ug/ml, 0.2 ug/ml, 0.02 ug/ml and 0 ug/ml (control) immobilized Ang2. Individual colonies obtained from the eluted phage pools are grown and i) induced for new phage production and ii) induced with IPTG for Nanobody expression and extraction (periplasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 11: Screening for Ang2 Binding Nanobodies

(43) In order to determine binding specificity to Ang2, the clones are tested in an ELISA binding assay setup, using the monoclonal phage pools. Phage binding to Ang2 (R&D Systems Cat No 623-AN/CF) is tested. Shortly, 0.2 ug/ml Ang2 is immobilized on Maxisorp ELISA plates (Nunc) and free binding sites are blocked using 4% Marvel skimmed milk in PBS. Next, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ul 2% Marvel PBS are allowed to bind to the immobilized antigen. After incubation and a wash step, phage binding is revealed using a HRP-conjugated monoclonal-anti-M13 antibody (Gentaur Cat#27942101). Binding specificity is determined based on OD values compared to controls having received an irrelevant phage or no phage.

(44) FIG. 5 and Table B-3 shows a selection of clones binding to Ang2.

(45) TABLE-US-00029 TABLE B-3 Nanobodies against Ang2 SEQ ID Name NO: Sequence 166-C1 464 EVQLVESGGGLVQAGGSLRLSCAASGFTFGSTTIGW FRQAPGKEREGVSCISTGDGSTYYAESVKGRFTISS DNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWS APYEYDYWGQGTQVTVSS 166- 465 EVQLVESGGGLVQAGGSLRLSCAASGFTFGTTTIGW C10 FRQAPGKEREGVSCISTGDGSTNYAESVKGRFTISS DNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWS APYEYDYWGQGTQVTVSS 166-D7 466 EVQLVESGGGLVQAGGSLRLSCAASGFTFSDTTIGW FRQAPGKEREGISCISTGDGSTYYAESVKGRFTISS DNAKNTVYLQMNSLNPEDTAVYYCALDQAPLWSTWS APYEYDYWGQGTQVTVSS 166-F8 467 EVQLVESGGGLVQAGGSLRLSCAASGFTFGTTTIGW FRQAPGKEREVVSCISTGGGSTYYTESVKGRFTISS DNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSNWS APYEYDYWGQGTQVTVSS 166-G4 468 EVQLVESGGGLVQAGGSLRLSCAASGFTFSDTTIGW FRQAPGKEREGISCISTGDGSTYYAESVKGRFTISS DNAKNTVYLQMNSLNPEDTAVYYCALDQAPLWSTWS APYEYDYWGQGTQVTVSS 166-H4 469 EVQLVESGGDLVQAGGSLRLSCAASGFTFGDFTIGW FRQAPGKEREGVSCINTGDGSTNYAESVKGRFTISS DNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWS APYEYDYWGQGTQVTVSS 166-E12 470 KVQLVESGGGLVQAGGSLRLSCAASGFTFGSTTIGW FRQAPGKEREGVSCISTGDGSTYYAESVKGRFTISS DNAKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWS APYEYDYWGQGTQVTVSS 166-D4 471 EVQLVESGGGLVQAGGSLRLSCVASGRIFTNTAMGW YRQAPGKWRELVATIYSGGSTKYIDSVKGRFIISRD NTRNTVHLQMNSLKPEDTAVYYCNTVGAGSYWGQGA QVTVSS

Example 12: Screening for Nanobodies Blocking Ang2-Tie2 Interaction

(46) Clones tested positive in the Ang2 binding assay are screened for their ability to block Ang2 binding to Tie2/Fc. For this, Nanobody-containing periplasmic extracts (P.E.) are used in an ELIS A-based ligand competition setup. In short, 4 ug/ml human Tie2/Fc Chimera (R&D Systems Cat No 313-TI, Lot No BKC06)) is coated in 96 well Maxisorp microtiter plates (Nunc) and blocked with 4% Marvel skimmed milk in PBS. In parallel, 0.05 ug/ml biotinylated rh Ang2 (R&D Systems Cat No BT623, Lot No BNR174091) is incubated with 10 ul of periplasmic extract containing nanobody of the different clones in 100 ul 2% Marvel/PBS. After 1 hour, the biotinylated Ang2-Nanobody pre-mixes are incubated 1 hour with the coated receptor. Bound biotinylated Ang2 is detected using HRP-conjugated extravidin (SIGMA E2886-1ML, 126K4801). Blocking activity is determined as loss of OD signal, as compared to wells where no P.E., or irrelevant P.E., has been added. FIG. 6 shows results of this blocking assay using a selection of clones binding to Ang2.

(47) 166-D7, 166-G4, 166-H4, 166-C10, 166-C1, 166-F8 (see Table B-4 below) show significant blocking of Ang2 binding to Tie2.

(48) TABLE-US-00030 TABLE B-4 Nanobodies against Ang2 and able to block Ang2 binding to Tie2. SEQ ID Name NO: Sequence 166- 464 EVQLVESGGGLVQAGGSLRLSCAASGFTFGSTTIGWF C1 RQAPGKEREGVSCISTGDGSTYYAESVKGRFTISSDN AKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWSAPY EYDYWGQGTQVTVSS 166- 465 EVQLVESGGGLVQAGGSLRLSCAASGFTFGTTTIGWF C10 RQAPGKEREGVSCISTGDGSTNYAESVKGRFTISSDN AKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWSAPY EYDYWGQGTQVTVSS 166- 466 EVQLVESGGGLVQAGGSLRLSCAASGFTFSDTTIGWF D7 RQAPGKEREGISCISTGDGSTYYAESVKGRFTISSDN AKNTVYLQMNSLNPEDTAVYYCALDQAPLWSTWSAPY EYDYWGQGTQVTVSS 166- 467 EVQLVESGGGLVQAGGSLRLSCAASGFTFGTTTIGWF F8 RQAPGKEREVVSCISTGGGSTYYTESVKGRFTISSDN AKNTVYLQMNSLKPEDTAVYYCALDQAPMWSNWSAPY EYDYWGQGTQVTVSS 166- 468 EVQLVESGGGLVQAGGSLRLSCAASGFTFSDTTIGWF G4 RQAPGKEREGISCISTGDGSTYYAESVKGRFTISSDN AKNTVYLQMNSLNPEDTAVYYCALDQAPLWSTWSAPY EYDYWGQGTQVTVSS 166- 469 EVQLVESGGDLVQAGGSLRLSCAASGFTFGDFTIGWF H4 RQAPGKEREGVSCINTGDGSTNYAESVKGRFTISSDN AKNTVYLQMNSLKPEDTAVYYCALDQAPMWSSWSAPY EYDYWGQGTQVTVSS

Example 13: Determining Ang2-Tie2 Interaction Blocking Efficiency by Titration of Purified Nanobody

(49) In order to determine the receptor blocking efficiency of clones tested positive for Ang2 blocking, a dilution series of purified Nanobodies are tested in the ELISA-based ligand competition setup. In short, 4 ug/ml human Tie2/Fc Chimera (R&D Systems Cat No 313-TI, Lot No BKC06)) is coated in 96 well Maxisorp microtiter plates (Nunc) and blocked with 4% Marvel skimmed milk in PBS. In parallel, 0.05 ug/ml biotinylated rh Ang2 (R&D Systems Cat No BT623, Lot No BNR174091) is incubated with a dilution series of purified Nanobodies. After 1 hour, the biotinylated Ang2-Nanobody pre-mixes are incubated 1 hour with the coated receptor. Bound biotinylated Ang2 is detected using HRP-conjugated extravidin (SIGMA E2886-1ML, 126K4801). Blocking activity is determined as loss of OD signal, as compared to wells where no P.E., or irrelevant P.E., has been added. FIG. 7 shows the results of this assay.

(50) Sequences Alignments of Ang2 Binding Nanobodies (FRs in Small Letters, CDRs in Capital Letters):

(51) TABLE-US-00031 166-D7 evqlvesggglvgaggslrlscaasgftfsDTTIGwfrgapgkeregisCISTGDGETYY 166-G4 evqlvesggglvgaggslrlscaasgftfsDTTIGwfrgapgkeregisCISTGDGETYY 166-H4 evqlvesggdlvgaggslrlscaasgftfgDFTIGwfrgapgkeregysCINTGDGETNY 166-E12  kvqlvesggglvgaggslrlscaasgftfgETTIGwfrgapgkeregysCISTGDGETYY 166-C10 evqlvesggglvgaggslrlscaasgftfgTTTIGwfrgapgkeregysCISTGDGETNY 166-C1 evqlvesggglvgaggslrlscaasgftfgETTIGwfrgapgkeregysCISTGDGETYY 166-F8 evqlvesggglvgaggslrlscaasgftfgTTTIGwfrgapgkerevvsCISTGGGSTYY 166-D4 evqlvesggglvgaggslrlscvasgriftNTAMGwyrqapgkwrelva.TIYEGGSTKY 166-H5 evqlvesggglvgaggslslacvvsgrfsrINSMAwsrqvpgnqrelva.SVTSGGYTNY 166-D7 AESVEGrftissdnakntvylqmnslnpedtavyycalDQAPLWSTWSAPYEYDYwgqgt 166-G4 AESVEGrftissdnakntvylqmnslnpedtavyycalDQAPLWSTWSAPYEYDYwgqgt 166-H4 AESVEGrftissdnakntvylqmnslkpedtavyycalDQAPMWSSWSAPYEYDYwgqgt 166-E12 AESVEGrftissdnakntvylqmnslkpedtavyycalDQAPMWSSWSAPYEYDYwgqgt 166-C10 AESVEGrftissdnakntvylqmnslkpedtavyycalDQAPMWSSWSAPYEYDYwgqgt 166-C1 AESVEGrftissdnakntvylqmnslkpedtavyycalDQAPMWSSWSAPYEYDYwgqgt 166-F8 TESVEGrftissdnakntvylqmnslkpedtavyycalDQAPMWSNWSAPYEYDYwgqgt 166-D4 IDSVEGrfiisrdntrntvhlqmnslkpedtavyycnt.......VGAGSY....wgqga 166-H5 VDSVEGrftisrdnaknaiylqmnslksedtavyycna...RVVVRTAHGFEDNYwgqgt 166-D7 qvtvss 166-G4 qvtvss 166-H4 qvtvss 166-E12 qvtvss 166-C10 qvtvss 166-C1 qvtvss 166-F8 qvtvss 166-D4 qvtvss 166-H5 qvtvss
Members:
Families of Binders (One Family of Nanobodies has Same CDR3):

(52) TABLE-US-00032 members: I 166-D7, 166-G4, 166-H4, 166-E12, 166-C10, 166-C1, 166-F8 II 166-D4

Example 14: Selections of Phage Displaying Ang1 Binding Nanobodies

(53) Phage libraries 171 and 172 (Example 9) are used for selections on recombinant human Ang1 (R&D Systems Cat No 923-AN/CF, Lot No FHW073091). Ang1 is immobilized directly on Maxisorp 96 well microtiter plates (Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control). Following incubation with the phage libraries and extensive washing, bound phage is eluted with trypsin. The eluted phage are amplified and applied in a second round of selection on 2 ug/ml, 0.2 ug/ml, 0.02 ug/ml and 0 ug/ml (control) immobilized Ang1. In this second round, and following incubation with the phage libraries and extensive washing, bound phage is eluted with trypsin and 100 fold excess (nM compared to coated Ang1) recombinant human Tie2/Fc Chimera (R&D Systems Cat No 313-TI, Lot No BKC06). Individual colonies obtained from the eluted phage pools are grown and i) induced for new phage production and ii) induced with IPTG for Nanobody expression and extraction (periplasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 15: Screening for Ang1 Binding Nanobodies

(54) In order to determine binding specificity to Ang1, the clones are tested in an ELISA binding assay setup, using the monoclonal phage pools. Phage binding to Ang1 (R&D Systems Cat No 923-AN/CF, Lot No FHW073091) is tested. Shortly, 0.2 ug/ml Ang1 is immobilized on Maxisorp ELISA plates (Nunc) and free binding sites are blocked using 4% Marvel skimmed milk in PBS. Next, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ul 2% Marvel PBS are allowed to bind to the immobilized antigen. After incubation and a wash step, phage binding is revealed using a HRP-conjugated monoclonal-anti-M13 antibody (Gentaur Cat#27942101). Binding specificity is determined based on OD values compared to controls having received an irrelevant phage or no phage.

(55) FIG. 8 and Table B-5 show a selection of clones binding to Ang1.

(56) TABLE-US-00033 TABLE B-5 NANOBODIES AGAINST ANG1 SEQ ID Name NO: Sequence 173- 472 EVQLVESGGGLVQPGGSLRLSCAASGFTLSGNWMYWLR H9 QAPGKGLEWISTITPRGLTAYADSVKGRFTISRDIAEN TLYLQMNSLKSGDTAVYYCARDKTGERRGQGTQVTVSS 184- 473 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMTWVR B6 QAPGKGLEWVSDISWDGDITTYAASVKGRFTISRDNAK KTLYLQMNSLKPEDSAVYYCNTYGYDSGRYYSYWGQGT QVTVSS 185- 474 EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFR H5 QAPGKEREGVSYISSSDGRTYYADSVKGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCATDLSGRGDVSEYEYDYW GQGTQVTVSS
Sequences Alignments of Ang1 Binding Nanobodies (FRs in Small Letters, CDRs in Capital Letters):

(57) TABLE-US-00034 185-H5 evqlvesggglvqpggslrlscaasgftld.YYAIGwfrqapgkeregvsYISSSDGRTY 173-H9 evqlvesggglvqpggslrlscaasgftlsGNWMY.wlrqapgkglewis.TITPRGLTA 184-B6 evqlvesggglvqpggslrlscaasgftfs.NYAMTwvrqapgkglewvsDISWDGDITT 185-H5 YADSVKGrftisrdnakntvylqmnslkpedtavyycatDLSGRGDVSEYEYDYwgqgtq 173-H9 YADSVKGrftisrdiaentlylqmnslksgdtavyycarDKTGER.........rgqgtq 184-B6 YAASVKGrftisrdnakktlylqmnslkpedsavyycnt..YGYDSGRYYSY..wgqgtq 185-H5 vtvss 173-H9 vtvss 184-B6 vtvss
Members:
Families of Binders (One Family of Nanobodies has Same CDR3):

(58) TABLE-US-00035 Members: I 173-H9 II 184-B6 III 185-H5

Example 16: Selections of Phage Displaying Ang4 Binding Nanobodies

(59) Phage libraries 171 and 172 (see example 9) are used for selections on recombinant human Angiopoietin-4 (R&D Systems Cat No 964-AN/CF). Ang4 is immobilized directly on Maxisorp 96 well microtiter plates (Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control). Following incubation with the phage libraries and extensive washing, bound phage is eluted with trypsin. The eluted phage are amplified and applied in a second round of selection on 2 ug/ml, 0.2 ug/ml, 0.02 ug/ml and 0 ug/ml (control) immobilized Ang4. In this second round, and following incubation with the phage libraries and extensive washing, bound phage is eluted with trypsin. Individual colonies obtained from the eluted phage pools are grown and i) induced for new phage production and ii) induced with IPTG for Nanobody expression and extraction (periplasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 17: Screening for Ang4 Binding Nanobodies

(60) In order to determine binding specificity to Ang4, the clones are tested in an ELISA binding assay setup, using the monoclonal phage pools. Phage binding to Angiopoietin-4 (R&D Systems Cat No 964-AN/CF) was tested. Shortly, 0.2 ug/ml Ang1 is immobilized on Maxisorp ELISA plates (Nunc) and free binding sites are blocked using 4% Marvel skimmed milk in PBS. Next, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ul 2% Marvel PBS are allowed to bind to the immobilized antigen. After incubation and a wash step, phage binding is revealed using a HRP-conjugated monoclonal-anti-M13 antibody (Gentaur Cat#27942101). Binding specificity is determined based on OD values compared to controls having received an irrelevant phage or no phage.

(61) FIG. 9 and Table B-6 show a selection of clones binding to Ang4.

(62) TABLE-US-00036 TABLE B-6 Nanobodies against Ang4 SEQ ID Name NO: Sequence 168- 475 EVQLVESGGGLVQPGGSLRLSCAASGFTLSGNWMYWLR A3 GQAPGKLEWISTITPRGLTAYADSVKGRFTISRDIAEN TLYLQMNSLKSGDTAVYYCARDKTGERRGQGTQVTVSS 168- 476 EVQLVESGGGLVQPGGSLRLSCAASGFTLSSNWMYWLR E5 QAPGKGLEWISTITPRDLTAYADSVKGRFTISRDNAEN TLYLQMNSLKSEDTAVYYCAKDKAGERRGQGTQVTVSS 168- 477 EVQLVESGGGLVQPGGSLRLSCAASGSTLDYYAIGWYR G3 QAPGKEREWVSCISSSNYGITTYADSVKGRFTISRDNA KNTVYLQMNSLKPEDTAIYYCATNTRRKYGRLCDLNAD YWGQGTQVTVSS 169- 478 EVQLVESGGGLVQPGGSLRLSCATSGFTFSPSWMYWLR A10 QAPGKGLEWVSTITPRGLTEYANSVKGRFTISKDNAKN TLYLQMNSLKSEDTAVYYCTRDKNGPPMGQGTQVTVSS 169- 479 EVQLVESGGGLVQPGGSLRLSCVASGSIRSIIHMGWYR A12 QAPGNERDLVAVIIDSRTTKYSESVKGRFTISRDNAKN TVYLQMNSLKPEDTAVYYCNALALGTDQSSTFDSWGQG TQVTVSS 169- 480 EVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYR B12 QAPGNQRDLVAAITSGDSTKYADFVKGRFTISRDNAKN TVYLQMNSLKPEDTAVYYCAAELLGKWYWGQGTQVTVS S 169- 481 EVQLVESGGGLVQPGGSLRLSCAASGSIRSIIHMGWYR C12 QTPGNERDMVAVIIDSRTTKYAESVKGRFTISRDNAKN TVYLQMNSLKPEDTAVYYCNALALGTDQSSTFDSWGQG TQVTVSS 169- 482 EVQLVESGGGLVQPGGSLRLSCATSGFTFSTSWMYWLR C8 QAPGKGLEWVSTITPRGLTDYTDSVKGRFTISRDSAKN TLYLQMNSLKSEDTADYYCTRDKNGPPMGQGTQVTVSS 169- 483 EVQLVESGGGLVQAGGSLRLSCAASGSIFSINTMGWYR E12 QAPGNQRDLVAAITNGGSTKYVDSVKGRFTISRDNAKN TVYLQMNSLKPEDTAVYYCAAESLGRWGWGQGTQVTVS S 169- 484 EVQLVESGGGLVQPGGSLRLSCATSGFTFSTSWMYWLR F11 QAPGKGLEWVSTITPRGLTDYTNSVKGRFTVSRDNAKN TLYLQMNSLKSEDTAVYYCTKDKNGPPMGQGTQVTVSS
Sequences Alignments of Ang4 Binding Nanobodies (FRs in Small Letters, CDRs in Capital Letters):

(63) TABLE-US-00037 169-F11 evqlvesggglvqpggslrlscatsgftfsTSWMYwlrqapgkglewvs..TITPRGLTD 169-C8 evqlvesggglvqpggslrlscatsgftfsTSWMYwlrqapgkglewvs..TITPRGLTD 169-A10 evqlvesggglvqpggslrlscatsgftfsPSWMYwlrqapgkglewvs..TITPRGLTE 168-E5 evqlvesggglvqpggslrlscaasgftlsSNWMYwlrqapgkglewis..TITPRDLTA 168-A3 evqlvesggglvqpggslrlscaasgftlsGNWMYwlrqapgkglewis..TITPRGLTA 168-G3 evqlvesggglvqpggslrlscaasgstldYYAIGwyrqapgkerewvsCISSSNYGITT 169-B12 evqlvesggglvqaggslrlscaasgsifsINAMGwyrqapgnqrdlva..AITSGDSTK 169-E12 evqlvesggglvqaggslrlscaasgsifsINTMGwyrqapgnqrdlva..AITNGGSTK 169-A12 evqlvesggglvqpggslrlscvasgsirsIIHMGwyrqapgnerdlva..VIIDSRTTK 169-C12 evqlvesggglvqpggslrlscaasgsirsIIHMGwyrqtpgnerdmva..VIIDSRTTK 169-F11 YTNSVKGrftvsrdnakntlylqmnslksedtavyyctk..........DKNGPP..... 169-C8 YTDSVKGrftisrdsakntlylqmnslksedtadyyctr..........DKNGPP..... 169-A10 YANSVKGrftiskdnakntlylqmnslksedtavyyctr..........DKNGPP..... 168-E5 YADSVKGrftisrdnaentlylqmnslksedtavyycak..........DKAGER..... 168-A3 YADSVKGrftisrdiaentlylqmnslksgdtavyycar..........DKTGER..... 168-G3 YADSVKGrftisrdnakntvylqmnslkpedtaiyycatNTRRKYGRLCDLNADY..... 169-B12 YADFVKGrftisrdnakntvylqmnslkpedtavyycaa..........ELLGKWY.... 169-E12 YVDSVKGrftisrdnakntvylqmnslkpedtavyycaa..........ESLGRWG.... 169-A12 YSESVKGrftisrdnakntvylqmnslkpedtavyycna..........LALGTDQSSTF 169-C12 YAESVKGrftisrdnakntvylqmnslkpedtavyycna..........LALGTDQSSTF 169-F11 ..mgqgtqvtvss 169-C8 ..mgqgtqvtvss 169-A10 ..mgqgtqvtvss 168-E5 ..rgqgtqvtvss 168-A3 ..rgqgtqvtvss 168-G3 ..wgqgtqvtvss 169-B12 ..wgqgtqvtvss 169-E12 ..wgqgtqvtvss 169-A12 DSwgqgtqvtvss 169-C12 DSwgqgtqvtvss
Members:
Families of Binders (One Family of Nanobodies has Same CDR3):

(64) TABLE-US-00038 Members: I 169-F11, 169-C8, 169-A10 II 168-A3, 168-E5 III 168-G3 IV 169-B12, 169-E12 V 169-A12, 169-C12

Example 18: Selections of Phage Displaying Angptl4 Binding Nanobodies

(65) Phage libraries 171 and 172 (see Example 9) are used for selections on recombinant human Angiopoietin-like-4 (R&D Systems Cat No 3485-AN).

(66) Angptl4 is immobilized directly on Maxisorp 96 well microtiter plates (Nunc) at 5 ug/ml, 0.5 ug/ml and 0 ug/ml (control). Following incubation with the phage libraries and extensive washing, bound phage is eluted with trypsin. The eluted phage are amplified and applied in a second round of selection on 2 ug/ml, 0.2 ug/ml, 0.02 ug/ml and 0 ug/ml (control) immobilized Angptl4. In this second round, and following incubation with the phage libraries and extensive washing, bound phage is eluted with trypsin. Individual colonies obtained from the eluted phage pools are grown and i) induced for new phage production and ii) induced with IPTG for Nanobody expression and extraction (periplasmic extracts) according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 19: Screening for Angptl4 Binding Nanobodies

(67) In order to determine binding specificity to Angptl4, the clones are tested in an ELISA binding assay setup, using the monoclonal phage pools. Phage binding to recombinant human Angiopoietin-like-4 (R&D Systems Cat No 3485-AN) is tested. Shortly, 0.2 ug/ml Ang1 was immobilized on Maxisorp ELISA plates (Nunc) and free binding sites are blocked using 4% Marvel skimmed milk in PBS. Next, 10 ul of supernatant from the monoclonal phage inductions of the different clones in 100 ul 2% Marvel PBS are allowed to bind to the immobilized antigen. After incubation and a wash step, phage binding is revealed using a HRP-conjugated monoclonal-anti-M13 antibody (Gentaur Cat#27942101). Binding specificity is determined based on OD values compared to controls having received an irrelevant phage or no phage.

(68) FIG. 10 and Table B-7 show a selection of clones binding to Angptl4.

(69) TABLE-US-00039 TABLE B-7 Nanobodies against Angpt14 SEQ ID Name NO: Sequence 170- 485 EVQLVESGGGLVQAGGSLRLSCAASESIFSLYVTGWYRQ B1 APGKQRELVASITSGGSLTYADSVKGRFTISRDNAKNTV HLQMHSLKPEDTAVYFCNGRSIGVDDMPYVYWGQGTQVT VSS 170- 486 EVQLVESGGGLVQPGGSLRLSCAASGFTFSLNAMTWVRQ C2 APGKGLEWVSTISSGGWTTSYADSVKGRFTISRDNAKNT LYLQMNSLKPEDMAVYYCAKGSEFNGYEVRGQGTQVTVS S 170- 487 EVQLVESGGGLVQAGGSLRLSCAASGSISSINVMGWYRQ E2 APGKQRDLVATITRALNTAYATSVKGRFTISRDNFTNTV YLQMNSLEPEDTAVYYCNAGGYYTNLRTGGNYWGQGTQV TVSS 170- 488 EVQLVESGGGLVQAGGSLRLSCAASGIFIIDTMGWYRQA F2 PGKQRELVASITPTGNTNYVDSVKGRFAISRDNNKNTMH LQMNSLKPEDTAVYYCNAVYPRYYGDDDRPPVDSWGQGT RVTVSS 170- 489 EVQLVESGGGLAQAGGSLRLSCAASGSISSINVMGWYRQ H1 APGKQRDLVAVITRALNTNYATSVKGRFTISRDDFKDTV YLQMNSLEPEDTAVYYCNAGGYYTNLRTGGNYWGQGTQV TVSS 171- 490 EVQLVESGGGQVQAGDSLRLSCKASRRTISTYGMGWFRQ A2 APGDKRDLVSSISASGASTYYVDSVKGRFTISRDNIKNT VYLQMNSLKPEDAAVYYCAAAPNGRFITMSAHVDSWGQG TQVTVSS 171- 491 EVQLVESGGGQVQAGDSLRLSCKASRRTISTYGMGWFRQ A3 APGDKRDLVSSISASGASTYYVDSVKGRFTISRDNIKNT VYLQMNSLKPEDAAVYYCAAAPNGRFITMSTHVDYWGQG TQVTVSS 171- 492 EVQLVESGGGLVQPGGSLRLSCAASGRTFSTFNTYSMGW C4 FRQAPGKEREFVAAISRGGNVTPYADSVKGRFAISRDNA KNTVALQMNSLKPEDTAVYYCAASKIGIASTIRYYDYWG QGTQVTVSS 171- 493 EVQLVESGGGLVQAGGSLRLSCAASVLTFGTYTVGWFRQ D2 APGKEREFVSIITGSGTYNDYADSVKGRFTVSRDNAKNT VYLQMNSLKSEDTAVYYCAARHWGMFSRSENDYNYWGQG TQVTVSS 171- 494 EVQLVESGGGLVQAGASLRLSCVDSGDTFSWYAMGWFRQ E2 QAPGKEREFVSSISGGGSNTVYADSVKGRFTVSRDRAKN TVYLQMNSLKPEDSGVYYCAADKRWGSPATSRSTHDYDF WGQGTQVTVSS 171- 495 EVQLVESGGGLVQPGGSLRLSCAASGRTFSTFNTYSMGW E4 FRQAPGKEREFVAAISRSGNVTPYADSVKGRFAISRDNA KNTLTLQMNSLKPEDTAVYYCAASKIGIASTIRYYDYWG QGTQVTVSS 171- 496 EVQLVESGGGLVQTGGSLRLSCAASGRSFNLYYMGWFRQ F3 APGREREFVAGISGSGGSTFYGDSVKGRFTISRDNLKNT MYLQMNSLKPEDTAVYYCQSSRRIITNPREYGYWGQGTQ VTVSS 171- 497 EVQLVESGGGLVQAGGSLRLSCTASGLTFSMYAMAWIRL G2 APGKEREVIAAIDWSGGSTFYGDSVKGRFTISRDNAKNT VYLEMNSLKPEDTAVYYCAANRRIYSSGSSLSDNSLYNF WGQGTQVTVSS 171- 498 EVQLVESGGGLVQAGGSLRLSCVASGDTFNWYAMGWFRQ G4 QAPGKEREFVSAISGGGSNIVYVDSVKGRFTVSRDRIKN TVYLQMNSLKPEDSGVYYCAVDKRWGSPATSRSTHDYDF WGQGTQVTVSS 170- 499 EVQLVESGGGLVQAGGSLRLSCAASETIFASAMGWYRQP G3 PGKQRELVARITRGGSTNYAESVKGRFAISRDNADSTLY LRMNNLKPEDTAVYYCNADTIGHSSSYITYWGQGTQVTV SS 171- 500 EVQLVESGGGLVQAGGSLRLSCAASGRPFSMYAMGWFRQ H2 APGKEREFVTVITWSGGSTYYADSVKGRFTISKDIAKNT VYLQMNSLKPDDMAVYYCAAARRYGNLYNTNNYDYWGQG TQVTVSS 171- 501 EVQLVESGGGQVQAGDSLRLSCKASRRTISTYGMGWFRQ H4 APGDKRDLVSSISASGASTYYVDSVKGRFTISRDNIKNT VYLQMNSLKPEDAAVYYCAAAPNGRFITMSTHVDSWGQG TQVTVSS
Sequences Alignments of Angptl4 Nanobodies (FRs in Small Letters, CDRs in Capital Letters):

(70) TABLE-US-00040 171-G4 evqlvesggglvgaggslrlscvasgdtfn...WYAMGwfrqqapgkerefv.SAISGGG 171-E2 evqlvesggglvgagaslrlscvdsgdtfs...WYAMGwfrqqapgkerefv.SSISGGG 170-H1 evqlvesggglaqaggslrlscaasgsiss...INVMGwyr.qapgkqrdlva..VITRA 170-E2 evqlvesggglvgaggslrlscaasgsiss...INVMGwyr.qapgkqrdlva..TITRA 171-H2 evqlvesggglvgaggslrlscaasgrpfs...MYAMGwfr.qapgkerefvt.VITWEG 171-E4 evqlvesggglvqpggslrlscaasgrtfsTENTYSMGwfr.qapgkerefva.ATERSG 171-C4 evqlvesggglvqpggslrlscaasgrtfsTENTYSMGwfr.qapgkerefva.ATERGG 170-F2 evqlvesggglvgaggslrlscaasgifii....DTMGwyr.qapgkgrelva..SITPT 170-B1 evqlvesggglvgaggslrlscaasesifs...LYVTGwyr.qapgkgrelva..SITSG 171-F3 evqlvesggglvqtggslrlscaasgrsfn...LYYMGwfr.qapgrerefva.GISGSG 171-H4 evqlvesgggqvgagdslrlsckasrrtis...TYGMGwfr.qapgdkrdlvs.SISASG 171-A2 evqlvesgggqvgagdslrlsckasrrtis...TYGMGwfr.qapgdkrdlvs.SISASG 171-A3 evqlvesgggqvgagdslrlsckasrrtis...TYGMGwfr.qapgdkrdlvs.SISASG 171-D2 evqlvesggglvgaggslrlscaasvltfg...TYTVGwfr.qapgkerefvs.ITTGSG 170-G3 evqlvesggglvgaggslrlscaasetifa....SAMGwyr.qppgkgrelva..RITRG 170-C2 evqlvesggglvqpggslrlscaasgftfs...LNAMTwvr.qapgkglewvs.TISEGG 171-G2 evqlvesggglvgaggslrlsctasgltfs...MYAMAwir.lapgkerevia.AIDWEG 171-G4 SNIVYVDSVKGrftvsrdrikntvylqmnslkpedsgvyycay...DKRWGSPATERSTH 171-E2 ENTVYADSVKGrftvsrdrakntvylqmnslkpedsgvyycaa...DKRWGSPATERSTH 170-H1 LNTNYATSVKGrftisrddfkdtvylqmnslepedtavyycna......GGYYTNLRTGG 170-E2 LNTAYATSVKGrftisrdnftntvylqmnslepedtavyycna......GGYYTNLRTGG 171-H2 GSTYYADSVKGrftiskdiakntvylqmnslkpddmavyycaa......ARRYGNLYNTN 171-E4 NVTPYADSVKGrfaisrdnakntltlqmnslkpedtavyycaa....SKIGIASTIRYYD 171-C4 NVTPYADSVKGrfaisrdnakntvalqmnslkpedtavyycaa....SKIGIASTIRYYD 170-F2 GNTNYVDSVKGrfaisrdnnkntmhlqmnslkpedtavyycna...VYPRYYGDDDRPPV 170-B1 GELTYADSVKGrftisrdnakntvhlgmhslkpedtavyfcng......RSIGVDDMPYV 171-F3 GSTFYGDSVKGrftisrdnlkntmylqmnslkpedtavyycqs.....SRRITTNPREYG 171-H4 ASTYYVDSVKGrftisrdnikntvylqmnslkpedaavyycaa....APNGRFITMETHV 171-A2 ASTYYVDSVKGrftisrdnikntvylqmnslkpedaavyycaa....APNGRFITMSAHV 171-A3 ASTYYVDSVKGrftisrdnikntvylqmnslkpedaavyycaa....APNGRFITMETHV 171-D2 TYNDYADSVKGrftvsrdnakntvylqmnslksedtavyycaa....RHWGMFERSENDY 170-G3 GSTNYAESVKGrfaisrdnadstlylrmnnlkpedtavyycna.......DTIGHESSYT 170-C2 WTTSYADSVKGrftisrdnakntlylqmnslkpedmavyycak.......GSEFNGYEV. 171-G2 GSTFYGDSVKGrftisrdnakntvylemnslkpedtavyycaaNRRTYSSGSSLEDNELY 171-G4 DYDFwgqgtqvtvss 171-E2 DYDFwgqgtqvtvss 170-H1 NY..wgqgtqvtvss 170-E2 NY..wgqgtqvtvss 171-H2 NYDYwgqgtqvtvss 171-E4 Y...wgqgtqvtvss 171-C4 Y...wgqgtqvtvss 170-F2 DS..wgqgtrvtvss 170-B1 Y...wgqgtqvtvss 171-F3 Y...wgqgtqvtvss 171-H4 DS..wgqgtqvtvss 171-A2 DS..wgqgtqvtvss 171-A3 DY..wgqgtqvtvss 171-D2 NY..wgqgtqvtvss 170-G3 TY..wgqgtqvtvss 170-C2 ....rgqgtqvtvss 171-G2 NF..wgqgtqvtvss
Members:
Families of Binders (One Family of Nanobodies has Same CDR3):

(71) TABLE-US-00041 members: I 171-A3, 171-A2 II 170-E2, 170-H1 III 171-H2 IV 171-E2, 171-G4 V 171-E4, 171-C4 VI 170-G3 VII 170-B1 VIII 170-F2 IX 171-F3 X 171-D2 XI 170-C2 XII 171-G2

Example 20: List of General In Vitro, Cell-Based or In Vivo Assays

(72) In vitro binding assays: ELISA, Biacore

(73) In vivo binding assay: Flow cytometry

(74) Solid-phase receptor binding and blocking assays (Onliner et al., 2004, supra): ELISA-based assays with either immobilized ligand or receptor, where inhibition of binding of receptor/ligand is determined. E.g. suitable cell-based assay for Tie2, Ang1 or/and Ang2 Nanobodies.

(75) Receptor activation/inactivation assays (Fiedler et al., 2003, Harfouche and Hussain, 2006; both supra): Western blot detection of phophorylated receptor (activated) or phosphorylation of components of the downstream signalling pathways. E.g. suitable cell-based assay for Tie2, Ang1 or/and Ang2 Nanobodies.

(76) Cell proliferation assays (Onliner et al., 2004, supra): Inhibition of tumour endothelial cell (e.g. specific tumor cell lines or “general” endothelial cells such as human umbilical cord endothelial cells (HUVECs) proliferation is assayed on tumour cells stimulated with or without addition of the neutralizing nanobody. Cell proliferation is determined by counting the number of live cells by FACS analysis.

(77) In vivo angiogenesis assay (Onliner et al., 2004, supra): Assay determining the effect on the tumour growth by addition of neutralizing nanobodies in xenografts studies. E.g. suitable in vivo assay for Tie2, Ang1 or/and Ang2 Nanobodies.

(78) In vivo direct anti angiogenic effect (Onliner et al., 2004, supra): Assay determining a direct antineovascular effect in vivo by rat corneal angiogenesis model. E.g. suitable in vivo assay for Tie2, Ang1 or/and Ang2 Nanobodies.

(79) Lipoprotein lipase (LPL) assay: Measurement of LPL activity using .sup.3H-oleic acid as substrate (Yoshida et al., 2002, supra). E.g. suitable in vitro assay for Angptl4.

(80) In vivo .CAM (chick chorioallantoric membrane) assay: Assay determining inhibition or not of vascularisation by addition of Angptl4 binding nanobodies using a CHO-Angptl4 expressing cell line (Le Jan et al., 2003, supra). E.g. suitable in vivo assay for Angptl4.

(81) In vivo animal model studies: Assay determine the effect of injecting Angptl4 nanobodies on the lipid metabolism of transgenic mice overexpressing h Angptl4 (Koster et al., 2005, supra). E.g. suitable in vivo assay for Angptl4.

Example 21: List of Particularly Preferred Embodiments of Amino Acid Sequences of the Invention

(82) Amino acid sequence comprising e.g. 2 Nanobodies with antagonistic effect for the same target, e.g. Tie2, either being directed against two different epitopes, or being against the same epitope. Amino acid sequence comprising a Nanobody against the Tie2 receptor and a Nanobody against angiopoietin 1. Amino acid sequence comprising a Nanobody against the Tie2 receptor and a Nanobody against angiopoietin 2. Amino acid sequence comprising a cytotoxic compound (e.g. peptidic toxin, e.g. immunotoxin) and a Nanobody wherein the said Nanobody is able to disrupt at least one of the Tie/Ang or Angptl interactions, e.g. Ang1/Tie2 or Ang2/Tie2 interactions. The amino acid sequences of the invention such as those presented e.g. in SEQ ID NOs: 455 to 501 may be used for targeting specific types of cancers.

Example 22: List of Target Proteins of the Invention (Links to Nucleic and Amino Acid Sequence)

(83) TABLE-US-00042 Tie1, Tie2, Ang1, Ang2, Ang3, Ang4, Angptl1, Angptl2, Angptl3, Angptl4, Sequences from various species found e.g. on Angptl5, and Angptl6 http://www.ncbi.nlm.nih.gov/sites/entrez Human Tie1 NM_005424 Human Tie2 NM_000459 Human Ang1 NM_001146 Human Ang2 NM_001147 Human Ang3 AF074332 Human Ang4 NM_015985 Human Angptl1 NM_004673 Human Angptl2 BC012368 Human Angptl3 NM_014495 Human Angptl4 NM_001039667 Human Angptl5 NM_178127 Human Angptl6 NM_031917

Example 23: Further Analysis of Tie-2 Nanobody 163E9

(84) Reagents Used

(85) Recombinant Human Angiopoietin-1 (R&D SYSTEM catalog number: 923-AN); Recombinant Human Angiopoietin-2 (R&D SYSTEM catalog number: 623-AN); Anti-total Erk p44/42 MAPK (Cell Signaling Technology catalog number #9102); Anti-Phospho Erk p44/42 MAPK (Thr 202/Tyr 204) (E10) (Cell Signaling Technology catalog number #9106S); Anti-total Akt (C67E7) (Cell Signaling Technology catalog number #4691); Anti-Phospho-Akt (Ser473) (D9E) (Cell Signaling Technology catalog number #4060); Anti Tie-2/TEK, clone Ab33 (UPSTATE catalog number #05-584); Anti-Phosphotyrosine, 4G10 (Platinum Millipore)

Example 23a: The Tie-2 Nanobody 163E9 Inhibits Ang-1 Induced Phosphorylation of Akt and Erk as Determined by Bioplex Analysis

(86) To identify Tie-2 Nanobodies that inhibits Ang-1-induced activation of Tie-2, signalling pathways, the Bio-Plex phosphoprotein and total target assays was used. With this assay the phosphorylation and expression of proteins in lysates derived from cell culture or tissue samples, respectively are determined. The Bio-Plex total target assay reports the abundance of the target protein in one well, while the Bio-Plex phosphoprotein assay reports the phosphorylation level of the same protein in a separate well.

(87) Method:

(88) The Bio-Plex assay used a selection of beads with different spectral addresses, each coupled to antibodies against a different target, (in total target assay Akt and ERK ½; in phosphoprotein assay Akt (Ser.sup.473) and ERK ½ (Thr.sup.202/Tyr.sup.204, Thr.sup.185/Tyr.sup.187)). The coupled beads were added to wells of a 96-well plate. Cell lysates, in a protein range concentration of 200-900 μg/ml derived from HUVECs appropriately treated, were added to the wells containing coupled beads. The incubation was left for 15-18 hr. Biotin-labeled detection antibodies specific for secondary epitopes on each target are added to wells. The incubation was left for 30 min. Fluorescently labeled streptavidin reporter, able to bind to biotin-labeled detection antibodies, was added to the wells. The incubation was left for 10 min. After rinse, the complex was resuspended in assay buffer. In The Bio-Plex array reader, a red classification laser and a green reporter laser illuminated individual beads to identify each bead's spectral address and associated reporter signal. Dyed beads were identified by their internal fluorescent signature, the level of target bound to beads was indicated by intensity of reporter signal. Multiplex data were reported simultaneously.

(89) HUVECs (Human umbilical vein endothelial cells) were obtained by treating human umbilical cord vein with collagenase and cultured in M199 containing 20% FCS (2% Penicillin˜Streptomycin, brain extract and 25 μg Heparin sodium sulfate). After starvation for 3-4 hr in M199 containing 0.5% BSA, the cells were treated with indicated concentration of Tie-2 Nanobodies for 10 min and then stimulated with 100 ng/ml h-Ang-1 for 10 min. Cells were rinsed in ice-cold cell wash buffer and lysed in buffer with protease and phosphatase inhibitors. Proteins concentration were measured through BCA (Bicinchoninic acid) assay and an equal amount of protein for each sample, ranging between 200-900 μg/ml, was used for Bio-plex analysis. Ratio of phospho-Akt to Akt and phospho-ERK to ERK is reported in FIGS. 11 and 12 respectively. Ø indicate non Ang-1 stimulated samples. Among anti-Tie2 NB s tested, only Nanobody 163E9 was able to block the Ang1-induced Akt and Erk phosphorylation both at 7.5 ug/ml (˜500 nM) and 1 ug/ml (˜67 nM). None of the others Tie-2 Nanobodies inhibited phosphorylation of AKt and Erk.

Example 23b: Nanobody 163E9 Dose-Dependently Inhibits Ang-1 Induced Phosphorylation of Akt and Erk as Determined by Western Blotting

(90) HUVEC (Human umbilical vein endothelial cells) were obtained by treating human umbilical cord vein with collagenase and cultured in M199 containing 20% FCS (2% Penicillin˜Streptomycin, brain extract and 25 μg Heparin sodium sulfate).

(91) HUVECs were plated in 6-well plates and used in subconfluent condition (1, 5±2 10.sup.5/9.6 mm dishes). After starvation in M199 containing 0.5% BSA for 3-4 hr, the cells were treated with indicated concentration (of Nanobodies for 10 min and then stimulated with 100 ng/ml h-Ang-1 for 10 min. Cells were rinsed in ice-cold PBS and lysed in boiling buffer (500 mM Tris HCl, ph 6.8; 10% SDS, Glycerol). Lysates were clarified by centrifugation and proteins concentration was measured through BCA (Bicinchoninic acid) assay. 10 μg proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane and subject to Western Blot analysis with anti-total Erk ½, anti-phospho-Erk ½, anti total Akt and anti-phospho-Akt. The corresponding chemiluminescent signal is acquired and quantified by a CCD camera. Ratio of phospho-Akt to Akt (FIG. 13) and phospho-ERK to ERK (FIG. 14) is reported. Nanobody 163E9 dose-dependedtly inhibited Ang-1 induced phosphorylation of Akt and Erk.

Example 23c: The Tie-2 Nanobody 163E9 Reverses the Anti-Apoptotic Effect of Ang-1

(92) Serum starvation of HUVECS is known to result in apoptotic cell death, a process that can be inhibited by Ang-1. To further demonstrate that the Tie-2 Nanobody 163E9 interferes with Ang-1 induced activities through Tie-2, it was investigated if Nanobody 163E9 would be able to reverse the anti-apoptotic activity of Ang-1.

(93) Apoptosis experiments were performed using the Cell Death Detection ELISA.sup.PLUS kit (Roche) evaluating the level of nucleosome associated DNA fragments. HUVECs cells were seeded in 24 wells (2 10.sup.4 cells/well) or 6 wells (9.8 10.sup.4 cells/well) and treated over-night with different growth factors indicated. Buffers and reagents used in the procedure are supplied with the kit. Cells were lysed with 200 μl or 980 μl of Lysis Buffer for 30 min at room temperature and lysates were centrifuged at 200 g for 10 min. ELISA assay was performed with 20 μl of the sample supernatant and 80 μl of the immunoreagent. The immunoreagent was prepared by mixing 1/20 volume of Anti-DNA-HRP and 1/20 Anti-histon-biotin with 18/20 volumes of incubation Buffer. The immunoassay binding reaction was allowed to proceed for 2 hours after which the excess of reagent was removed with two washes of Incubation Buffer (200 μl each). The quantitative determination of the amount of nucleosome was assessed by the evaluation of HRP (Horse Readish Peroxidase) retained by the immunocomplex which is photometrically measured with ABTS as substrate. Finally the colorimetric reaction is blocked after 10-15 min with ABST Stop Solution.

(94) Nanobodies against Tie-2 were tested in HUVECs cells, stimulated over-night with Ang-1 (300 ng/ml) or growth factors-starved (SF) as a control. As shown in FIG. 15, Ang-1 strongly inhibited apoptosis following serum starvation. Importantly, Nanobody 163E9 dose-dependently inhibited the anti-apoptotic activity of Ang-1. Indeed lowering the Nanobody concentrations resulted in reduced cell apoptotsis.

Example 23d: Nanobody 163E9 Dose-Dependently Inhibits Ang-1 Induced Phosphorylation of Tie-2

(95) Following binding of Ang-1 to Tie-2, the cytoplasmic tail of Tie-2 becomes phosphorylated. To further demonstrate that the Tie-2 Nanobody 163E9 interferes with Ang-1-induced activities through Tie-2, it was investigated if Nanobody 163E9 would be able to inhibit phosphorylation of Tie-2.

(96) HUVEC (Human umbilical vein endothelial cells) were obtained by treating human umbilical cord vein with collagenase and cultured in M199 containing 20% FCS (2% Penicillin˜Streptomycin, brain extract and 25 μg Heparin sodium sulfate).

(97) After starvation in M199 containing 0, 5% BSA for 3-4 hr, the cells were treated with indicated concentration (ng/ml) of Nanobodies for 10 min and then stimulated with h-Ang-1 for 10 min. Cells were rinsed in ice-cold PBS 1× and lysed at 4° C. in EB buffer (10 mM TrisHCl, ph 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X100, 10% Glycerol) with protease and phosphatase inhibitors (50 μg/ml pepstatin, 50 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 100 μM ZnCl.sub.2, 1 mM Na.sub.3VO.sub.4). Lysates (450-800 μg) were incubated with protein G-Sepharose and anti-Tie-2 antibody (1 μg) for 2 hr at 4° C. After washes, immunoprecipitates were resolved in 6% SDS-PAGE and immunoblotted for P-Tyr and Tie-2. As shown in FIG. 16, at the highest concentration of Nanobody 163E9 used, phosphorylation of Tie-2 was indeed reduced.

Example 23e: Nanobody 163E9 Dose-Dependently Inhibits Ang-1 Induced Sprouting of Endothelial Cells

(98) HUVECS were trypsinized, counted, and suspended at a density of 4 cells/μl in culture medium containing 20% Methocel (Sigma) (20 ml of Methocel stock with 80 ml of M-199 20% FCS, 0.1 mg/ml heparin, and 0.1 mg/ml brain extract). 800 cells were seeded into non-adherent round-bottom 96-well plates, and cultured overnight at 37° C. The following day the formed spheroids were harvested, centrifuged for 15′ at 300 g at room temperature, and embedded into Collagen gels. A diluted collagen-I (Sigma, from rat tail) solution was prepared before use by mixing 7 vol collagen (equilibrated to 3 mg/ml in sterile 0.2% acetic acid pH 3, 4° C.), 1 vol 10×M-199, 1 vol 0.1 N NaOH, and 1 vol 0.2 M HEPES pH 7.3. The EC spheroids were suspended in 200 μl of M-199 medium containing 40% FCS with or without 100 ng/ml Ang1 and Nanobody 163E9 at concentrations indicated, and mixed with an equal volume of diluted collagen solution. The spheroids were rapidly transferred into 96-well plates (400 μl/well) to allow polymerizing.

(99) Capillary-like sprouts were examined with inverted-phase contrast microscope (Leica Microsystem, Heerbrugg, Switzerland) and photographed. The lengths and projected areas of the capillary-like structures were quantified with the imaging software winRHIZO Pro (Regent Instruments Inc.).

(100) As shown in FIG. 17, Nanobody 163E9 dose-dependently inhibited sprouting of HUVEC cells induced by Ang-1.

(101) Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

(102) All references disclosed herein are incorporated by reference in their entirety for the purpose and information indicated in the specification.