Polypeptides and polypeptide constructs comprising single domain antibodies directed against von willebrand factor

Abstract

The present invention relates to polypeptides comprising at least one single domain antibody directed against vWF, vWF A1 domain, A1 domain of activated vWF, vWF A3 domain, gpIb and/or collagen, homologues of said polypeptides, and/or functional portions of said polypeptides, for the treatment for conditions which require a modulation of platelet-mediated aggregation and which overcomes the problems of the prior art. A further aspect of the invention is methods of production of said polypeptides, methods to coat devices with such polypeptides used in medical procedures (e.g. PCTA, stenting), methods and kits for screening for agents that modulate platelet-mediated aggregation and kits for the diagnosis of diseases related to platelet-mediated aggregation.

Claims

1. A nucleic acid encoding a polypeptide construct comprising at least one single domain antibody that specifically binds von Willebrand Factor (vWF), wherein the at least one single domain antibody has complementary determining regions (CDRs) and framework regions (FRs), and wherein the at least one single domain antibody comprises: a sequence represented by any one of SEQ ID NOs: 1 to 7, 23 to 31, and 62 to 65; or a homologous sequence of any one of SEQ ID NOs: 1 to 7, 23 to 31, and 62 to 65, wherein the FRs have a sequence identity of more than 85% with the FRs of the parent sequence; or a homologous sequence of any one of SEQ ID NOs: 1 to 7, 23 to 31, and 62 to 65, wherein the FRs have up to 10 amino acid substitutions compared to the FRs of the parent sequence; and wherein the polypeptide construct is able to inhibit at least 50% of platelet aggregation at high shear (1600 s.sup.−1) at a concentration of between 0.08 and 0.3 μg/ml.

2. A method of producing a polypeptide construct comprising: (a) culturing host cells comprising a nucleic acid according to claim 1 under conditions allowing the expression of the polypeptide construct; and (b) recovering the produced polypeptide construct from the culture.

3. The nucleic acid according to claim 1, wherein the at least one single domain antibody comprises a sequence represented by any one of SEQ ID NOs: 1 to 7, 23 to 31, and 62 to 65.

4. A method of producing a polypeptide construct, comprising: (a) culturing host cells comprising a nucleic acid according to claim 3 under conditions allowing the expression of the polypeptide construct; and (b) recovering the produced polypeptide construct from the culture.

Description

FIGURES

(1) FIG. 1. Interactions involved in the first steps of platelet aggregation.

(2) FIG. 2. Interactions involved in the first steps of platelet aggregation. A VHH is indicated inhibiting the interaction between vWF and collagen.

(3) FIG. 3. Binding to vWF as determined by ELISA, by purified VHH as described in Example 7.

(4) FIG. 4. ELISA to test inhibition by VHH of binding of vWF to collagen as described in Example 9.

(5) FIG. 5. Western blot showing expression of A3 domain of vWF as a fusion with Oprl on the surface of E. coli as described in Example 11.

(6) FIG. 6. Restriction map of multiple cloning site of PAX011 for construction of bivalent or bispecific nanobodies.

(7) FIG. 7. Binding in ELISA to purified vWF, for monovalent versus bivalent and bispecific VHH as described in Example 13.

(8) FIG. 8. Stability of bispecifc VHH in human plasma upon incubation at 37° C. for up to 24 hours as described in Example 15.

(9) FIG. 9. Interactions involved in the first steps of platelet aggregation. A VHH is indicated inhibiting the interaction between vWF and platelets.

(10) FIG. 10. Western blot showing expression of A1 domain of vWF as a fusion with Oprl on the surface of E. coli as described in Example 18.

(11) FIG. 11. Binding to vWF as determined by ELISA, by purified VHH as described in Example 20.

(12) FIG. 12. Inhibition of binding of gpIb to VWF by A50 and A38 (negative control) as described in Example 21.

(13) FIG. 13. Interactions involved in the first steps of platelet aggregation. A bispecific constructs is indicated with one VHH specific for vWF and inhibiting the interaction between vWF and collagen and the second VHH specific for vWF but inhibiting the interaction between vWF and platelets.

(14) FIG. 14. Binding in ELISA to vWF as described in Example 28.

(15) FIG. 15. Interactions involved in the first steps of platelet aggregation. A VHH is indicated specific for collagen and inhibiting the interaction between vWF and collagen.

(16) FIG. 16. Binding of purified VHH to collagen type I and type III in ELISA as described in Example 34.

(17) FIG. 17. Phage ELISA to show that HSA-specific nanobodies are present in the library as described in Example 44.

(18) FIG. 18. Binding of phages expressing the albumin binders, to plasma blotted on nitrocellulose as described in Example 48.

(19) FIG. 19. Coomassie staining of plasma samples on SDS-PAGE as described in Example 48.

(20) FIG. 20. Binding of purified nanobodies to mouse albumin as determined by ELISA as described in Example 50.

(21) FIG. 21. Bispecific constructs with one VHH binding to albumin and a second VHH binding to vWF for improvement of half-life as described in Example 51.

(22) FIG. 22. Sandwich ELISA showing the functionality of both VHHs in a bispecific construct as described in Example 53.

(23) FIG. 23. Interactions involved in the first steps of platelet aggregation. A VHH is indicated specific for gpIb and inhibiting the interaction between vWF and platelets.

(24) FIG. 24. Residual activity for C37 stored at −20° C. as compared to C37 incubated at 37° C. for up to 194 hours. C37 stability is compared to stability of a scFv specific for B3 antigen and a stabilized form, dsFv (stabilized by 2 disulphide bonds) as described in Example 61.

(25) FIG. 25. Inhibitory activity for C37 stored at −20° C. as compared to C37 incubated at 37° C. for 1 year as described in Example 61.

(26) FIG. 26. Binding of vWF from human plasma to C37 immobilized in acrylamide as described in Example 62.

(27) FIG. 27. Amino acid alignment of C37 with human germline sequence DP-47 as described in Example 63.

(28) FIG. 28. Inhibition of binding of vWF to collagen as determined by ELISA for C37 and C37 hum as described in Example 64.

(29) FIG. 29. Binding of A11, A12, A13, A14, A15 and A16 clones to rA1 as measured in ELISA

(30) FIG. 30. Binding of A11, A12, A13, A14, A15 and A16 clones to vWF as measured in ELISA

TABLES

(31) Table 1. Immunization scheme used for llama 002 according to Example 1.

(32) Table 2. Plaque forming units (pfu) after one or two round(s) of panning on vWF as compared to PBS-casein as described in Example 4. Pfu vWF (antigen) divided by pfu casein (a specific binding)=enrichment.

(33) Table 3. Number of inhibitors versus the number of clones tested after the first and the second round of panning as described in Example 5.

(34) Table 4. Yield (mg/liter culture) after expression and purification of VHH grown in WK6 E. coli cells as described in Example 6.

(35) Table 5. OD 405 nm for binding of VHH in ELISA to vWF and 3 antigens that were also immunized in llama002 according to Example 8.

(36) Table 6. Concentration of VHH (nM) needed to inhibit binding of vWF to collagen by 50% (IC50) as described in Example 9.

(37) Table 7. Epitope mapping of VHH binding to vWF and inhibiting the interaction with collagen as described in example 11.

(38) Table 8. Yields of purified protein (mg) per liter of culture for bivalent and bispecific VHHs as described in Example 12.

(39) Table 9. IC50 values for monovalent as compared to bivalent and bispecific VHHs. Inhibition was tested with human, pig and baboon plasma as described in Example 14.

(40) Table 10. Inhibition of platelet aggregation at high shear (1600 s.sup.−1) as described in Example 16.

(41) Table 11. Inhibition of platelet aggregation at low shear (300 s.sup.−1) as described in Example 16.

(42) Table 12. Plaque forming units (pfu) after one round of panning on vWF as described in Example 17. Pfu vWF (antigen) divided by pfu casein (a-specific binding)=enrichment.

(43) Table 13. Results of screening in ELISA of individual colonies for binding to vWF and to the A1 domain of vWF as described in Example 18.

(44) Table 14. Results after one round of MATCHM on pBAD-Oprl-A1 cells as described in Example 19.

(45) Table 15. Inhibition of platelet aggregation at high shear (1600 s.sup.−1) as described in Example 23.

(46) Table 16. Inhibition of platelet aggregation at low shear (300 s.sup.−1) as described in Example 23.

(47) Table 17. Inhibition of platelet aggregation at high shear (1600 s.sup.−1) as described in Example 25.

(48) Table 18. Inhibition of platelet aggregation at low shear (300 s.sup.−1) as described in Example 25.

(49) Table 19. Yields after expression and purification of bispecific constructs as described in Example 27.

(50) Table 20. IC50 values for bispecific nanobodies for the A1 and A3 domain of vWF as described in Example 29.

(51) Table 21. Inhibition of platelet aggregation at high shear (1600 s.sup.−1) as described in Example 30.

(52) Table 22. Inhibition of platelet aggregation at low shear (300 s.sup.−1) as described in Example 30.

(53) Table 23. Plaque forming units (pfu) after one round of panning on collagen type I as described in Example 31. Pfu vWF (antigen) divided by pfu casein (a-specific binding)=enrichment.

(54) Table 24. Number of clones binding to collagen type I and type III after one round of selection as described in Example 32.

(55) Table 25. Immunization scheme for human serum albumin according to example 41.

(56) Table 26. Results after one and two rounds of panning on mouse serum albumin as described in Example 45.

(57) Table 27. Clones were selected after one and two rounds of selection and periplasmic extracts were prepared. These clones were analyzed in ELISA for binding to human and mouse albumin as described in Example 46.

(58) Table 28. IC50 values for bispecific nanobodies against albumin and against vWF as described in Example 54.

(59) Table 29. Sequences of the primers used for humanization of C37 as described in Example 64.

(60) Table 30. Amino acid sequence listing of the peptides of the present invention and of human von Willebrand factor (vWF). The sequence of human vWF indicates A1 and A3 domains respectively in bold lettering.

(61) Table 31. Results after two panning rounds on rA1 domain of vWF as described in Example 66.

(62) Table 32. ELISA analyses of selected clones for binding to rA1 and vWF as described in Example 67.

(63) TABLE-US-00004 TABLE 1 Immunization scheme used for llama 002 according to Example 1. Llama002 Collagen Collagen Day of immunization vWF Type I Type III 0 100 μg  100 μg  100 μg  7 100 μg  100 μg  100 μg  14 50 μg 50 μg 50 μg 21 50 μg 50 μg 50 μg 28 50 μg 50 μg 50 μg 35 50 μg 50 μg 50 μg

(64) TABLE-US-00005 TABLE 2 Plaque forming units (pfu) after one or two round(s) of panning on vWF as compared to PBS-casein as described in example 4. Pfu vWF (antigen) divided by pfu casein (a specific binding) = enrichment. round Pfu vWF Pfu casein Enrichment First 1 × 10.sup.7 2.5 × 10.sup.5 40 Second 5 × 10.sup.8 2.5 × 10.sup.6 200

(65) TABLE-US-00006 TABLE 3 Number of inhibitors versus the number of clones tested after the first and the second round of panning as described in Example 5. Number of inhibitors versus round number of clones tested First 4/800 Second 4/96 

(66) TABLE-US-00007 TABLE 4 Yield (mg/liter culture) after expression and purification of VHH grown in WK6 E.coli cells as described in Example 6. Name VHH Yield (mg/liter culture) 22-2L-34 1.4 T76 2.9 AM-4-15-3 2.2 22-4L-16 2.8 C37 3.8 AM-2-75 3.6

(67) TABLE-US-00008 TABLE 5 OD 405 nm for binding of VHH in ELISA to vWF and 3 antigens that were also immunized in llama002 according to Example 8. OD405 nm vWF Antigen 1 Antigen 2 Antigen 3 nM 670 67 6.7 670 67 6.7 670 67 6.7 670 67 6.7 T76 0.77 0.36 0.13 0.05 0.05 0.05 0.06 0.06 0.04 0.04 0.04 0.03 22-2L-34 1.30 0.63 0.20 0.06 0.0 0.10 0.10 0.07 0.05 0.06 0.05 0.03 22-4L-16 1.41 0.86 0.81 0.08 0.10 0.11 0.15 0.11 0.05 0.08 0.07 0.03 C37 1.51 1.09 1.06 0.10 0.10 0.12 0.12 0.11 0.08 0.10 0.08 0.06 AM-2-75 1.57 1.10 1.04 0.09 0.11 0.12 0.14 0.11 0.09 0.10 0.13 0.05 AM-4-15-3 1.32 1.06 0.56 0.09 0.12 0.12 0.12 0.11 0.10 0.10 0.10 0.08

(68) TABLE-US-00009 TABLE 6 Concentration of VHH (nM) needed to inhibit binding of vWF to collagen by 50% (IC50) as described in Example 9. IC50 (nM) human IC50 (nM) undiluted Name VHH plasma 1/60 human plasma 22-2L-34 10 — T76 30 — AM-4-15-3 7 200 22-4L-16 4 1000 C37 3 — AM-2-75 2 100

(69) TABLE-US-00010 TABLE 7 Epitope mapping of VHH binding to vWF and inhibiting the interaction with collagen as described in Example 11. Name VHH Binding to A3 domain of vWF 22-2L-34 Yes T76 No 22-4L-16 No C37 Yes AM-2-75 Yes

(70) TABLE-US-00011 TABLE 8 Yields of purified protein (mg) per liter of culture for bivalent and bispecific VHHs as described in Example 12. NH2-terminal VHH COOH-terminal VHH Yield mg/liter culture AM-2-75 AM-4-15-3 3.2 AM-4-15-3 AM-4-15-3 2.3 AM-4-15-3 AM-2-75 4.0 AM-2-75 AM-2-75 1.0 AM-2-75 22-4L-16 3.0

(71) TABLE-US-00012 TABLE 9 IC50 values for monovalent as compared to bivalent and bispecific VHHs. Inhibition was tested with human, pig and baboon plasma as described in Example 14. IC50 (ng/ml) IC50 (ng/ml) Second IC50 (ng/ml) baboon pig First VHH VHH human plasma plasma plasma AM-2-75 150 400 50 AM-4-15-3 50 200 40 22-4L-16 15 70 7 AM-2-75 AM-4-15-3 3 5 6 AM-4-15-3 AM-2-75 2 8 3 AM-4-15-3 AM-4-15-3 5 10 7 AM-2-75 22-4L-16 8 20 10 AM-2-75 AM-2-75 5

(72) TABLE-US-00013 TABLE 10 Inhibition of platelet aggregation at high shear (1600 s.sup.−1) as described in Example 16. Concentration μg/ml] % inhibition AM-2-75 0.2 0 AM-2-75 0.3 12 AM-2-75 0.4 56 AM-2-75 0.6 97 AM-2-75 0.8 96 AM-4-15-3 0.05 0 AM-4-15-3 0.1 75 AM-4-15-3 0.25 74 AM-4-15-3 0.5 86 AM-4-15-3 1 91 22-4L-16 0.1 32 22-4L-16 0.5 54 22-4L-16 0.75 86 22-4L-16 2 97 22-4L-16 10 99 AM-4-15-3/AM-4-15-3 0.05 0 AM-4-15-3/AM-4-15-3 0.075 23 AM-4-15-3/AM-4-15-3 0.1 37 AM-4-15-3/AM-4-15-3 0.15 56 AM-4-15-3/AM-4-15-3 0.2 98 AM-4-15-3/AM-4-15-3 1.9 100 AM-4-15-3/AM-2-75 1.9 100 AM-2-75/AM-4-15-3 0.05 2 AM-2-75/AM-4-15-3 0.1 36 AM-2-75/AM-4-15-3 0.2 96 AM-2-75/AM-4-15-3 0.35 91 AM-2-75/AM-4-15-3 0.4 98 AM-2-75/AM-2-75 0.04 5 AM-2-75/AM-2-75 0.1 26 AM-2-75/AM-2-75 0.2 52 AM-2-75/AM-2-75 0.3 80 AM-2-75/AM-2-75 0.4 99 AM-2-75/AM-2-75 0.83 100 AM-2-75/22-4L-16 1.17 99

(73) TABLE-US-00014 TABLE 11 Inhibition of platelet aggregation at low shear (300 s.sup.−1) as described in Example 16. Concentration [μg/ml] % inhibition AM-2-75 10 20 AM-4-15-3 10 17 22-4L-16 10 22 AM-4-15-3/AM-4-15-3 10 23 AM-4-15-3/AM-2-75 10 21 AM-2-75/AM-4-15-3 10 18 AM-2-75/AM-2-75 2 32 AM-2-75/22-4L-16 10 13

(74) TABLE-US-00015 TABLE 12 Plaque forming units (pfu) after one round of panning on vWF as described in Example 17. Pfu vWF (antigen) divided by pfu casein (a-specific binding) = enrichment Pfu vWF Pfu casein Enrichment 1.5 × 10.sup.7 1 × 10.sup.4 1.500

(75) TABLE-US-00016 TABLE 13 Results of screening in ELISA of individual colonies for binding to vWF and to the A1 domain of vWF as described in Example 18. No. clones +ve for vWF/No. tested No. clones +ve for A1/No. tested 344/380 5/570

(76) TABLE-US-00017 TABLE 14 Results after one round of MATCHM on pBAD-Oprl-A1 cells as described in Example 19. No. clones +ve for No. clones +ve for Round vWF/No. tested A1/No. tested First — 1/96 second 45/348 12/348

(77) TABLE-US-00018 TABLE 15 Inhibition of platelet aggregation at high shear (1600 s.sup.−1) as described in Example 23. Concentration % inhibition [μg/ml] 2A1-4L-129 13.5 26 2A1-4L-129 20 50 2L-A1-15 9.7 30 2L-A1-15 25 45 A50 10.2 20 2A1-4L-79 11.1 20 2A1-4L-34 11.1 3 Z29 10.6 0 I53 9.7 0 M53 10.6 0

(78) TABLE-US-00019 TABLE 16 Inhibition of platelet aggregation at low shear (300 s.sup.−1) as described in Example 23. Concentration [μg/ml] % inhibition 2A1-4L-129 10 0 2L-A1-15 10 3 A50 25 0 2A1-4L-79 25 15

(79) TABLE-US-00020 TABLE 17 Inhibition of platelet aggregation at high shear (1600 s.sup.−1) as described in Example 25. Concentration [μg/ml] % inhibition 2A1-4L-79/2A1-4L-79 25 54 2LA1-15/2LA1-15 25 45

(80) TABLE-US-00021 TABLE 18 Inhibition of platelet aggregation at low shear (300 s.sup.−1) as described in Example 25. Concentration [μg/ml] % inhibition 2A1-4L-79/2A1-4L-79 25 0 2LA1-15/2LA1-15 25 23

(81) TABLE-US-00022 TABLE 19 Yields after expression and purification of bispecific constructs as described in Example 27. NH2 terminal VHH COOH-terminal VHH Yield mg/liter culture 2A1-4L-79 AM-4-15-3 7.5 2A1-4L-79 AM-2-75 2 2A1-4L-79 22-4L-16 2.5

(82) TABLE-US-00023 TABLE 20 IC50 values for bispecifici nanobodies for the A1 and A3 domain of vWF as described in example 29. NH2-terminal VHH COOH-terminal VHH IC50 (ng/ml) 2A1-4L-79 AM-4-15-3 10 AM-4-15-3 — 45 2A1-4L-79 AM-2-75 12 AM-2-75 — 40 2A1-4L-79 22-4L-16 10 22-4L-16 — 10 2A1-4L-79 — >10000

(83) TABLE-US-00024 TABLE 21 Inhibition of platelet aggregation at high shear (1600 s.sup.−1) as described in Example 30. Concentration [μg/ml] % inhibition 2A1-4L-79/AM-4-15-3 12 100 2A1-4L-79/AM-2-75 0.02 0 2A1-4L-79/AM-2-75 0.1 28 2A1-4L-79/AM-2-75 0.5 79 2A1-4L-79/AM-2-75 1 95 2A1-4L-79/22-4L-16 12 96

(84) TABLE-US-00025 TABLE 22 Inhibition of platelet aggregation at low shear (300 s.sup.−1) as described in Example 30. Concentration [μg/ml] % inhibition 2A1-4L-79/AM-4-15-3 10 15 2A1-4L-79/AM-2-75 10 25 2A1-4L-79/22-4L-16 10 27

(85) TABLE-US-00026 TABLE 23 Plaque forming units (pfu) after one round of panning on collagen type I as described in Example 31. Pfu vWF (antigen) divided by pfu casein (a-specific binding) = enrichment. Phages eluted from collagen type I 5 × 10.sup.6 Phages eluted from casein 4 × 10.sup.4 Enrichment 100

(86) TABLE-US-00027 TABLE 24 Number of clones binding to collagen type I and type III after one round of selection as described in Example 32. Collagen Type I 15/32 Collagen Type III  7/32 Casein  0/32

(87) TABLE-US-00028 TABLE 25 Immunization scheme for human serum albumin according to Example 41. HSA Day of immunization Llama006 0 100 μg 7 100 μg 14  50 μg 21  50 μg 28  50 μg 35  50 μg

(88) TABLE-US-00029 TABLE 26 Results after one and two rounds of panning on mouse serum albumin as described in Example 45. First round Second round Pfu mouse serum albumin 2.5 × 10.sup.7 2.5 × 10.sup.7 Pfu casein   5 × 10.sup.3 2.5 × 10.sup.3 Enrichment 5.000 10.000

(89) TABLE-US-00030 TABLE 27 Clones were selected after one and two rounds of selection and periplasmic extracts were prepared. These clones were analyzed in ELISA for binding to human and mouse albumin as described in Example 46. First round Second round ELISA mouse serum albumin 1/16 15/16 ELISA human serum albumin 1/16 15/16 ELISA casein 0/16  0/16

(90) TABLE-US-00031 TABLE 28 IC50 values for bispecific nanobides against albumin and against vWF as described in Example 54. IC50 (ng/ml) AM-2-75 100 MSA21/AM-2-75 60 AM-4-15-3 155 MSA21/AM-4-15-3 245 22-4L-16 100 MSA21/22-4L-16 140

(91) TABLE-US-00032 TABLE 29 Sequences of the primers used for humanization  of C37 as described in Example 64. SEQ Muta- ID tion Template Primer sequence NO A74S+ Wild 5′-AGA GAC AAC TCC AAG AAC ACG   76 N75K+ type CTG TAT CTG CAA ATG AAC AGC CTG  AGA GCT GAG GAC ACG-3′ P84A Arg Asp Asn Ser Lys Asn Thr Leu   77 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr A74S+ A74S+ 5′-AT TAC TGT GCT AAA GGG GCC   78 GGT ACT AGT T-3′ N75K+ N75K+ Tyr Cys Ala Lys Gly Ala Gly Thr  79 P84A+ P84A Ser R94K N28T+ A74S+ 5′-TCC TGT GCA GCC TCC GGA TTC   80 N30S N75K+ ACT TTC AGT TGG TA-3′ A74S+ P84A+ Ser Cys Ala Ala Ser Gly Phe Thr   81 N75K+ R94K Phe Ser Trp P84A+ R94K

(92) TABLE-US-00033 TABLE 30 Amino acid sequence listing of the peptides of the present invention and of human von Willebrand factor (vWF). The sequence of human vWF indicates A1 and A3 domains re- spectively in bold lettering. SEQ ID NAME NO SEQUENCE Anti-vWF A3 VHH C37  1 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTIS TYGEPRYADSVKGRFTISRDNANNTLYLQMNSLRPEDTAVYYCARGAGTSSY LPQRGNWDQGTQVTISS C37-  2 QVQLQESGGGLVQPGGSLRLSCAASGFTFSWYPMSWVRQAPGKGLEWVSTIS hum TYGEPRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAGTSSY LPQRGNWDQGTQVTISS AM-2-  3 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTIS 75 TYGEPRYADSVKGRFTISRDNANNTLYLQMNSLRPEDTAVYYCARGAGTSSY LPQRGNWDQGTQVTVSS 22-2L-  4 QVQLQDSGGGLVQAGGSLRLSCAASVRIFTSYAMGWFRQAPGKEREFVAAIN 34 RSGKSTYYSDSVEGRFTISRDNAKNTVSLQMDSLKLEDTAVYYCAADYSGSY TSLWSRPERLDWGQGTQVTVFS 22-4L-  5 QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAIS 16 WSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVADTGGIS WIRTQGYNYWGQGTQVTVSS T76  6 QVQLQESGGGLVQPGESLRLSCAASGSIFSINTMGWYGQAPGKQRELVASIT FGGVTNYADSVKGRFTISRDNTNDTVYLQMNSLKPEDTAVYICNAVTWGGLT NYWGQGTQVTVSS AM-4-  7 QVQLQDSGGGLVQPGGSLRLACAASGSIFSINSMGWYRQAPGKQRELVAHAL 15-3 ADGSASYRDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTVPSSVTK GYWGQGTQVTVSS Anti-vWF A3 domain VHH: bivalent or bispecific AM-4-  8 QVQLQDSGGGLVQPGGSLRLACAASGSIFSINSMGWYRQAPGKQRELVAHAL 15- ADGSASYRDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTVPSSVTK 3/AM- GYWGQGTQVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQPGGSLRLACAASGS 4-15-3 IFSINSMGWYRQAPGKQRELVAHALADGSASYRDSVKGRFTISRDNAKNTVY LQMNSLKPEDTAVYYCNTVPSSVTKGYWGQGTQVTVSS AM-4-  9 QVQLQDSGGGLVQPGGSLRLACAASGSIFSINSMGWYRQAPGKQRELVAHAL 15- ADGSASYRDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTVPSSVTK 3/AM- GYWGQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGF 2-75 NFNWYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKADSPSSETTPTTRCI CNEQPETEDTAVYYCARGAGTSSYLPQRGNWDQGTQVTVSS AM-2- 10 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTIS 75/AM- TYGEPRYADSVKGRFTISRDNANNTLYLQMNSLRPEDTAVYYCARGAGTSSY 4-15-3 LPQRGNWDQGTQVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQPGGSLRLACA ASGSIFSINSMGWYRQAPGKQRELVAHALADGSASYRDSVKGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCNTVPSSVTKGYWGQGTQVTVSS AM-2- 11 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTIS 75/AM- TYGEPRYADSVKGRFTISRDNANNTLYLQMNSLRPEDTAVYYCARGAGTSSY 2-75 LPQRGNWDQGTQVTVSSQVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMS WVRQAPGKGLEWVSTISTYGEPRYADSVKGRFTISRDNANNTLYLQMNSLRP EDTAVYYCARGAGTSSYLPQRGNWDQGTQVTVSS AM-2- 12 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTIS 75/22- TYGEPRYADSVKGRFTISRDNANNTLYLQMNSLRPEDTAVYYCARGAGTSSY 4L-16 LPQRGNWDQGTQVTVSSEPKTPKPQPAAAQVQLVESGGGLVQAGGSLRLSCA ASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNA KNTVYLQMNSLKPEDTAVYYCVADTGGISWIRTQGYNYWGQGTQVTVSS Anti-vWF VHH + anti-mouse serum albumin VHH MSA21/ 13 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGIS AM-2- SLGDSTLYADSVKGRFTSRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPG 75 GQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGFNFN WYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKADSPSSETTPTTRCICNE QPETEDTAVYYCARGAGTSSYLPQRGNWDQGTQVTVSS MSA21/ 14 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGIS AM-4- SLGDSTLYADSVKGRFTSRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPG 15-3 GQGTQVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQPGGSLRLACAASGSIFS INSMGWYRQAPGKQRELVAHALADGSASYRDSVKGRFTISRDNAKNTVYLQM NSLKPEDTAVYYCNTVPSSVTKGYWGQGTQVTVSS MSA21/ 15 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGIS 22-4L- SLGDSTLYADSVKGRFTSRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPG 16 GQGTQVTVSSEPKTPKPQPAAAQVQLVESGGGLVQAGGSLRLSCAASGRTFS SYAMGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNAKNTVYLQ MNSLKPEDTAVYYCVADTGGISWIRTQGYNYWGQGTQVTVSS Anti mouse serum albumin VHH MSA21 16 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGIS SLGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNP GGQGTQVTVSS MSA24 17 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSWVRQAPGKEPEWVSSIS GSGSNTIYADSVKDRFTISRDNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSR SSQGTQVTVSS MSA210 18 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLEWVSAIS SDSGTKNYADSVKGRFTISRDNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPS SQGTQVTVSS MSA212 19 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSWVRQAPGKGLEWVSAIS ADGSDKRYADSVKGRFTISRDNGKKMLTLDMNSLKPEDTAVYYCVIGRGSPA SQGTQVTVSS MSAc16 49 AVQLVESGGGLVQAGDSLRLSCVVSGTTFSSAAMGWFRQAPGKEREFVGAIK WSGTSTYYTDSVKGRFTISRDNVKNTVYLQMNNLKPEDTGVYTCAADRDRYR DRMGPMTTTDFRFWGQGTQVTVSS MSAcl1 50 QVKLEESGGGLVQTGGSLRLSCAASGRTFSSFAMGWFRQAPGREREFVASIG 2 SSGITTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGLCYCAVNRYGIP YRSGTQYQNWGQGTQVTVSS MSAcl1 51 EVQLEESGGGLVQPGGSLRLSCAASGLTFNDYAMGWYRQAPGKERDMVATIS 0 IGGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCVAHRQTVVR GPYLLWGQGTQVTVSS MSAcl1 52 QVQLVESGGKLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVAGSG 4 RSNSYNYYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASTNLWP RDRNLYAYWGQGTQVTVSS MSAcl1 53 EVQLVESGGGLVQAGDSLRLSCAASGRSLGIYRMGWFRQVPGKEREFVAAIS 6 WSGGTTRYLDSVKGRFTISRDSTKNAVYLQMNSLKPEDTAVYYCAVDSSGRL YWTLSTSYDYWGQGTQVTVSS MSAcl1 54 QVQLVEFGGGLVQAGDSLRLSCAASGRSLGIYKMAWFRQVPGKEREFVAAIS 9 WSGGTTRYIDSVKGRFTLSRDNTKNMVYLQMNSLKPDDTAVYYCAVDSSGRL YWTLSTSYDYWGQGTQVTVSS MSAcl5 55 EVQLVESGGGLVQAGGSLSLSCAASGRTFSPYTMGWFRQAPGKEREFLAGVT WSGSSTFYGDSVKGRFTASRDSAKNTVTLEMNSLNPEDTAVYYCAAAYGGGL YRDPRSYDYWGRGTQVTVSS MScl11 56 AVQLVESGGGLVQAGGSLRLSCAASGFTLDAWPIAWFRQAPGKEREGVSCIR DGTTYYADSVKGRFTISSDNANNTVYLQTNSLKPEDTAVYYCAAPSGPATGS SHTFGIYWNLRDDYDNWGQGTQVTVSS MSAcl1 57 EVQLVESGGGLVQAGGSLRLSCAASGFTFDHYTIGWFRQVPGKEREGVSCIS 5 SSDGSTYYADSVKGRFTISSDNAKNTVYLQMNTLEPDDTAVYYCAAGGLLLR VEELQASDYDYWGQGIQVTVSS MSAcl8 58 AVQLVDSGGGLVQPGGSLRLSCTASGFTLDYYAIGWFRQAPGKEREGVACIS NSDGSTYYGDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCATADRHYS ASHHPFADFAFNSWGQGTQVTVSS MSAcl7 59 EVQLVESGGGLVQAGGSLRLSCAAYGLTFWRAAMAWFRRAPGKERELVVARN WGDGSTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVRTYGS ATYDIWGQGTQVTVSS MSAcl2 60 EVQLVESGGGLVQDGGSLRLSCIFSGRTFANYAMGWFRQAPGKEREFVAAIN 0 RNGGTTNYADALKGRFTISRDNTKNTAFLQMNSLKPDDTAVYYCAAREWPFS TIPSGWRYWGQGTQVTVSS MSAcl4 61 DVQLVESGGGWVQPGGSLRLSCAASGPTASSHAIGWFRQAPGKEREFVVGIN RGGVTRDYADSVKGRFAVSRDNVKNTVYLQMNRLKPEDSAIYICAARPEYSF TAMSKGDMDYWGKGTLVTVSS Anti vWF A1 domain VHH + anti vWF A3 domain VHH 2A1- 20 QVQLQDSGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIG 4L- SDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSS 79/AM- GIYYREGSYAYWGQGTQVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQPGGSL 4-15-3 RLACAASGSIFSINSMGWYRQAPGKQRELVAHALADGSASYRDSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCNTVPSSVTKGYWGQGTQVTVSS 2A1- 21 QVQLQDSGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIG 4L- SDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSS 79/AM- GIYYREGSYAYWGQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL 2-75 RLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKADSPSS ETTPTTRCICNEQPETEDTAVYYCARGAGTSSYLPQRGNWDQGTQVTVSS 2A1- 22 QVQLQDSGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIG 4L- SDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSS 79/22- GIYYREGSYAYWGQGTQVTVSSEPKTPKPQPAAAQVQLVESGGGLVQAGGSL 4 RLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTI L-16 SRDNAKNTVYLQMNSLKPEDTAVYYCVADTGGISWIRTQGYNYWGQGTQVTV SS Anti vWF A1 domain VHH A50 23 QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAAIS RRGDNVYYADSVKGRFAISRDNAESTLYLQMNSLKPEDTAVYYCAAHVTVSA ITLSTSTYDYWGQGTQVTVSS I53 24 QVQLQDSGGGLVQAGGSLRLSCAASGRTKDMAWFRQPPGKEREFVAVIYSSD GSTLVAASVKGRFTISRDNAKNTVYLQMTSLKPADTAVYYCATSRGYSGTYY STSRYDYWTGGTQVTVSS Z29 25 QVQLQESGGGSVQAGDSLTLSCAASGRTFSMHAMGWFRQAPGKEREFVAAIS PSAFTEYADSLKGRFTVSRDNAKKLVWLQMNGLKPEDTAAYYCAARRGAFTA TTAPLYDYWGQGTQVTVSS M53 26 QVQLQDSGGGLVQAGESLRLSCGTSGRTFGRRAMAWFRQAPGKERQFVAWIA RYDGSTLYADSVKGRFTISRDDNKNTMYLHMNNLTPEDTAVYYCAAGPRGLY YESRYEYWGQGTLVTVSS 2A1- 27 QVQLQDSGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIG 4L-79 SDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSS GIYYREGSYAYWGQGTQVTVSS 2A1- 28 QVQLQESGGGLVQAGASLRLSCAASGRSFSSYPMAWFRQAPGKEREFVVFIG 4L-129 SDHSTLYSTSVRGRFTISRDNAKNTVYLQMMNLKPEDTAVYYCAARNSAWSS GIYYRETSYDYWGQGTQVTVSS 2A1- 29 QVQLQDSGGGSVQAGASLRLSCAASGGTFSSYAMAWFRQAPGKEREFVGFIG 4L-34 SDGSTLYSSSVRGRFTISRDNAKNTVALQMMNLKPEDTAVYYCAARARYSGI YYRETDYPYWGQGTQVTVSS 2A1- 30 QVQLQESGGGLVQAGASLRLSCTASGRSFGGFPMGWFRQAPGKEREFVSGLT 4L-78 RSLFTVYADSVKGRFTVSTDNTKNTVYLQMNSLKPEDTAVYYCAARPDLYAY SRDPNEYDYWGQGTQVTVSS 2LA1- 31 QVQLQDSGGGLVQSGGSLRLACAASGRIVSTYAMGWFRQSPGKEREFVATVK 15 GRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAKTKRTGIFTTARMVDYWGQG TQVTVSS Anti vWF A1 domain VHH: bispecific and bivalent VHH 2A1- 32 QVQLQDSGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIG 4L- SDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSS 79/2A1- GIYYREGSYAYWGQGTQVTVSSEPKTPKPQPAAAQVQLQDSGGRLVKAGASL 4L-79 RLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIGSDSSTLYTSSVRGRFTIS RDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSSGIYYREGSYAYWGQGTQV TVSS 2LA1- 33 QVQLQDSGGGLVQSGGSLRLACAASGRIVSTYAMGWFRQSPGKEREFVATVK 15/2LA GRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAKTKRTGIFTTARMVDYWGQG 1-15 TQVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQSGGSLRLACAASGRIVSTYA MGWFRQSPGKEREFVATVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAK TKRTGIFTTARMVDYWGQGTQVTVSS A50/A5 34 QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAAIS 0 RRGDNVYYADSVKGRFAISRDNAESTLYLQMNSLKPEDTAVYYCAAHVTVSA ITLSTSTYDYWGQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGGSLR LSCAASGRTFSSYRMGWFRQAPGKEREFVAAISRRGDNVYYADSVKGRFAIS RDNAESTLYLQMNSLKPEDTAVYYCAAHVTVSAITLSTSTYDYWGQGTQVTV SS Anti collagen VHH 3P1-31 35 QVQLQESGGGLVQAGGSLRLSCAASGRTFRRYAMGWYRQAPGKQRELVAAIT SGGRTSVADTVKGRFTISSDNAKNTVYLQMNSLKPEDAAVYYCTLYNSTTNY YNQSPSSWGQGTQVTVSS 3L-41 36 QVQLQDSGGGLVQAGGSLRLSCAASGRTFRRYAMGWYRQAPGKQRVLVAAIT SNGRPSVADSVKGRFTISSDTAKNTVYLQMNSLKPEDTALYYCTLYNTSADY YNQSPSSWGQGTQVTVLS 3P2-31 37 QVQLQESGGGLVQAGDSLRLSCAASGRTFTMGWFRQAPGKERQFVAALTWTG GSPVYADSVKGRFTTWRVLDNNTVYLHMNSLKPEDTAVYHCAAARTYYGNIS EYYDYWGQGTQVTVSS Anti-vWF VHH: humanized C37-3 38 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTIS TYGEPRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGAGTSSY LPQRGNWDQGTQVTISS C37-4 39 QVQLQESGGGLVQPGGSLRLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTIS TYGEPRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAGTSSY LPQRGNWDQGTQVTISS C37-8 40 EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYPMSWVRQAPGKGLEWVSTIS TYGEPRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAGTSSY LPQRGNWDQGTQVTISS C37-10 41 EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYPMSWVRQAPGKGLEWVSTIS TYGEPRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGAGTSSY LPQRGNWDQGTLVTVSS Humanised anti- vWF VHH + anti-mouse serum albumin VHH MSA21/ 42 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGIS C37- SLGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNP hum GGQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGFTF SWYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGAGTSSYLPQRGNWDQGTQVTISS MSA24/ 43 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSWVRQAPGKEPEWVSSIS C37- GSGSNTIYADSVKDRFTISRDNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSR hum SSQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGFTF SWYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGAGTSSYLPQRGNWDQGTQVTISS MSA210/ 44 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLEWVSAIS C37- SDSGTKNYADSVKGRFTISRDNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPS hum SQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGFTFS WYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKGAGTSSYLPQRGNWDQGTQVTISS MSA212/ 45 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSWVRQAPGKGLEWVSAIS C37- ADGSDKRYADSVKGRFTISRDNGKKMLTLDMNSLKPEDTAVYYCVIGRGSPA hum SQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGFTFS WYPMSWVRQAPGKGLEWVSTISTYGEPRYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKGAGTSSYLPQRGNWDQGTQVTISS Anti collagen VHH: bispecific 3P1- 46 QVQLQESGGGLVQAGGSLRLSCAASGRTFRRYAMGWYRQAPGKQRELVAAIT 31/3P2- SGGRTSVADTVKGRFTISSDNAKNTVYLQMNSLKPEDAAVYYCTLYNSTTNY 31 YNQSPSSWGQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGDSLRLSC AASGRTFTMGWFRQAPGKERQFVAALTWTGGSPVYADSVKGRFTTWRVLDNN TVYLHMNSLKPEDTAVYHCAAARTYYGNISEYYDYWGQGTQVTVSS 3L- 47 QVQLQDSGGGLVQAGGSLRLSCAASGRTFRRYAMGWYRQAPGKQRVLVAAIT 41/3P2- SNGRPSVADSVKGRFTISSDTAKNTVYLQMNSLKPEDTALYYCTLYNTSADY 31 YNQSPSSWGQGTQVTVLSEPKTPKPQPAAAQVQLQESGGGLVQAGDSLRLSC AASGRTFTMGWFRQAPGKERQFVAALTWTGGSPVYADSVKGRFTTWRVLDNN TVYLHMNSLKPEDTAVYHCAAARTYYGNISEYYDYWGQGTQVTVSS Conformation-specific Anti-vWF VHH A11 62 EVQLVESGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKEREFVAFIG SDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCAARSSAFSS GIYYREGSYAYWGQGTQVTVSS A12 63 QVQLVESGGGLVQAGGSLRLSCTASGRTFSTYALGWFRQVPGKGREFIAVIY WRDGSSLYSDSVKGRFTISKDNAKNTVYLQMNSLKPEDTAVYYCANRHDSRG TYYSSRGYDYWGQGTQVTVSS A13 64 QVQLVESGGGLVQAGGSLRLSCAASGRTKDMAWFRQPPGKEREFVAVIYSSD GSTLVAASVKGRFTISRDNAKNTVYLQMTSLKPADTAVYYCATSRGYSGTYY STSRYDYWGQGTQVTVSS A15 65 QVQLVESGGGLVQAGGSLRLSCAASGRTKDMAWFRQPPGKEREFVAVIYSSD GSTLVAASVTGRFTISRDNAKNMVYLQMTSLKPADTAVYYCASSRGYSGTYY STSRYDYWGQGTQVTVSS Human vWF Human 48 MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYS vWF FAGYCSYLLAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQ GDQRVSMPYASKGLYLETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFN KTCGLCGNFNIFAEDDFMTQEGTLTSDPYDFANSWALSSGEQWCERASPPSS SCNISSGEMQKGLWEQCQLLKSTSVFARCHPLVDPEPFVALCEKTLCECAGG LECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGMEYRQCVSPCARTC QSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLS RDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLAR DCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDG QDIQLPLLKGDLRIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKT CGLCGNYNGNQGDDFLTPSGLAEPRVEDFGNAWKLHGDCQDLQKQHSDPCAL NPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCG ALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPD EECNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTM CYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADN LRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQ GKEYAPGETVKIGCNTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYL FPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIEL FDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTY QEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLD SSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCE SIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEW RYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDC PVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPT DAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFV VDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGS QVASTSEVLKYTLFQIFSKIDRPEASRIALLLMASQEPQRMSRNFVRYVQGL KKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSY LCDLAPEAPPPTLPPHMAQVTVGPGLRNSMVLDVAFVLEGSDKIGEADFNRS KEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIR YQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPG DIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGE GLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGP RLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFA VRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGD RYDAAQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMD EDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSP VKVEETCGCRWTCPCVCTGSSTRHIVTEDGQNFKLTGSCSYVLFQNKEQDLE VILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGN MEVNVYGAIMHEVRENHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICD ENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQV LLLPLFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDW RTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVMLE GSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCP TAKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLT NPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCCDEYECACNCVNS TVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTC TDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGS PRGDSQSSWKSVGSQWASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVC PSGFQLSCKTSACCPSCRCERMEACMLNGTVIGPGKTVMIDVCTTCRCMVQV GVISGFKLECRKTTCNPCPLGYKEENNTGECCGRCLPTACTIQLRGGQIMTL KRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHKCLAEGGKIMKIPG TCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDIN DVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK

(93) TABLE-US-00034 TABLE 31 Results after two panning rounds on rA1 domain of vWF as described in Example 66 First library Second library Third library Pfu rA1 1 × 10.sup.8 2 × 10.sup.7 4 × 10.sup.9 Pfu casein 2 × 10.sup.4 2 × 10.sup.4 2 × 10.sup.4 Enrichment 5.000 1.000 200.000

(94) TABLE-US-00035 TABLE 32 ELISA analyses of selected clones for binding to rA1 and vWF as described in example 67 First library Second library Third library ELISA rA1 54/64 51/64 49/64 ELISA vWF 36/64 35/64 33/64