ANG2-BINDING MOLECULES
20230203146 · 2023-06-29
Inventors
- Rene Georg Ott (Perchtoldsdorf, AT)
- Eric Borges (Moedling, AT)
- Andreas Gschwind (Ingelheim am Rhein, DE)
- Joachim Boucneau (De Pinte, BE)
- Marie-Ange Buyse (Merelbeke, BE)
- Erik Depla (Destelbergen, BE)
- Pascal Merchiers (Kasterlee, BE)
- Frederik STEVENAERT (Maarkedal, BE)
Cpc classification
A61K45/06
HUMAN NECESSITIES
C07K2317/569
CHEMISTRY; METALLURGY
C07K16/3069
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
A61K39/3955
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/22
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/22
CHEMISTRY; METALLURGY
A61K39/395
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
Abstract
Ang2-binding molecules, preferably Ang2-binding immunoglobulin single variable domains like VHHs and domain antibodies, pharmaceutical compositions containing same and their use in the treatment of diseases that are associated with Ang2-mediated effects on angiogenesis. Nucleic acids encoding Ang2-binding molecules, host cells and methods for preparing same.
Claims
1. An Ang2-binding molecule comprising an immunoglobulin single variable domain, wherein said immunoglobulin single variable domain comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein CDR1 has an amino acid sequence selected from amino acid sequences shown in SEQ ID Nos: 168 to 170, CDR2 has an amino acid sequence selected from amino acid sequences shown in SEQ ID Nos: 171 to 173 and CDR3 has an amino acid selected from amino acid sequences shown in SEQ ID NOs: 174 to 177.
2. The Ang2-binding molecule according to claim 1, wherein (a) CDR1 has an amino acid sequence shown in SEQ ID NO: 168, CDR2 has an amino acid sequence shown in SEQ ID NO: 171 and CDR3 has an amino acid sequence shown in SEQ ID NO: 174, or wherein (b) CDR1 has an amino acid sequence shown in SEQ ID NO: 168, CDR2 has an amino acid sequence shown in SEQ ID NO: 171 and CDR3 has an amino acid sequence shown in SEQ ID NO: 175, or wherein (c) CDR1 has an amino acid sequence shown in SEQ ID NO: 169, CDR2 has an amino acid sequence shown in SEQ ID NO: 172 and CDR3 has an amino acid sequence shown in SEQ ID NO: 176, or wherein (d) CDR1 has an amino acid sequence shown in SEQ ID NO: 170, CDR2 has an amino acid sequence shown in SEQ ID NO: 173 and CDR3 has an amino acid sequence shown in SEQ ID NO: 177.
3. The Ang2-binding molecule according to claim 1, wherein said immunoglobulin single variable domain is a VHH or a domain antibody.
4. The Ang2-binding molecule according to claim 1, wherein said immunoglobulin single variable domain is a VHH.
5. The Ang2-binding molecule according to claim 4, wherein said VHH consists of an immunoglobulin single variable domain having a sequence selected from a group consisting of SEQ ID NOs: 167, 166, 129 and 138.
6. An Ang2-binding molecule consisting of the immunoglobulin single variable domain according to claim 5.
7. The Ang2-binding molecule according to claim 5 wherein said VHH consisting of an immunoglobulin single variable domain has a modification or exchange on N terminus, wherein said modification is a deletion of a first amino acid and said exchange is a replacement of the first aminom acid by another amino acid.
8. A nucleic acid molecule encoding the Ang2 binding molecule according to claim 1.
9. An expression vector comprising said nucleic acid molecule according to claim 8.
10. A host cell comprising one or more expression vectors according to claim 9.
11. A method for producing the Ang2-binding molecule according to claim 1, comprising the steps of: (a) transfecting a host cell with one or more said vectors comprising said nucleic acid molecule encoding the Ang2 binding molecule according to claim 1, (b) culturing said host cell, and (c) recovering and purifying said Ang2 binding molecule.
12. A pharmaceutical composition comprising, as the active ingredient, one or more said Ang2-binding molecules according to claim 1, and at least a physiologically acceptable carrier.
13. The pharmaceutical composition according to claim 12, further comprising one or more additional therapeutic agents, selected from chemotherapeutic agents like DNA damaging agents and/or anti-mitotic drugs in cancer cells (e.g. taxol), or therapeutically active compounds that inhibit angiogenesis (an anti angiogenic drug such as anti VEGF/VEGF receptor inhibitor, e.g. avastin, nintedanib or sunitinib), or signal transduction pathway inhibitors such as mTOR inhibitors (e.g. temsirolimus), or a hormonal therapy agent (e.g. tamoxifen).
14. A method of treating a disease that is associated with Ang 2 mediated effects on angiogenesis comprising administering an effective amount of a pharmaceutical composition according to claim 12, to a patient in need thereof.
15. The method according to claim 14 wherein the disease is cancer and cancerous diseases selected from breast cancer, renal cell carcinoma, ovarian cancer and pancreatic cancer.
16. The method according to claim 14 wherein the disease is eye diseases selected from age-related macular degeneration and diabetic retinopathy.
17. The method according to claim 14 wherein the disease is chronic kidney disease, selected from diabetic nephropathy, postrenal failure, prerenal azotemia and intrinsic renal failure.
18. A method of treating a disease comprising administering to a patient in need, an effective amount of one or more Ang2-binding molecules according to claim 1 or pharmaceutical compositions thereof.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
[0265] Materials and Methods
[0266] a) Generation of HEK293H Stable Cell Lines Overexpressing Human or Mouse Tie2 Receptor
[0267] The cDNAs encoding human Tie2 (NM_000459.3; SEQ ID NO:182), mouse Tie2 (NM_013690.2; SEQ ID NO:183) and cyno Tie2 (SEQ ID NO:184); are cloned in pcDNA3.1-neo expression vector (Invitrogen, Carlsbad, Calif., USA). To establish Human Embryonic Kidney (HEK) cells overexpressing human Tie2 or mouse Tie2, parental HEK293H cells undergo lipid mediated transfection with Fugene (Roche) with pcDNA3.1-neo-hTie2 or pcDNA3.1-neo-mTie2, respectively. For all conditions, transfectants are selected 2 days post-transfection by adding 1 mg/mL geneticin (Invitrogen, Carlsbad, Calif., USA). For human, mouse and cyno Tie2, final high expressing clones are selected by single cell sorting clones binding to PE labeled anti-human Tie2 (R&D Systems, Minneapolis, Minn., US), PE labeled anti-mouse Tie2 (eBioscience, San Diego, Calif., USA) and a 2-step goat-anti-human Tie2 (R&D Systems, Minneapolis, Minn., US) followed by PE labeled donkey-anti-goat (Jackson ImmunoResearch, West Grove, Pa., USA), respectively, using the FACSAria Cell Sorter (BD Biosciences, San Jose, Calif., USA).
[0268] b) Generation of HEK293T Cell Lines Overexpressing Mouse or Cynomolgus Ang2 and Production of Recombinant Mouse and Cynomolgus Ang2 Conditioned Medium
[0269] The cDNAs encoding N-terminally FLAG-tagged mouse Ang2 (NM_007426.3; SEQ ID SEQ ID NO: 185) and cynomolgus Ang2 (AB172643.1; SEQ ID NO: 186) are cloned in a pSecTag2B expression vector (Invitrogen, Carlsbad, Calif., USA). Human Embryonic Kidney (HEK) cells transiently overexpressing mouse Ang2 or cynomolgus Ang2 are generated by lipid-mediated transfection (Fugene; Roche) of pSecTag2B-mAng2 or pSecTag2B-cAng2, respectively, in the HEK293T parental cell line. Productions are performed in 1.5 liter CF10 Bag, and 1.5 L conditioned medium (CM) is collected 5 days post-transfection.
[0270] c) Production of Recombinant Cynomolgus Tie2/Fc Chimera in HEK293-F Cells
[0271] The cDNA encoding for the extracellular domain of Tie-2 is subcloned into the expression plasmid pSecTag2b using appropriate restriction sites to generate an Fc-fusion protein. Transfection into HEK293-F cells (Invitrogen) is performed as described by the manufacturer using Megaprep (Qiagen) preparations of plasmids, Optimem-Medium (Invitrogen), 293-fectin (Invitrogen) at an initial cell density of 1×10.sup.6 viable cells/mL with 1 μg plasmid DNA/10.sup.6 cells. Transfected cells are cultivated in shaker flasks for 7 days at 37° C. Conditioned Medium (CM) is harvested by centrifugation at 4000 g for 10 min and filtered through a sterile filter (0.45 μm membrane).
[0272] Fc-fusion proteins are purified using affinity chromatography by loading the CM at 5 ml/min onto a 5 ml MabSelect SuRe Protein A column equilibrated with DPBS. After a washing step with DPBS, bound Fc-protein is eluted with 10 mM sodium citrate buffer pH 3.0 and subsequently neutralized to pH 7.0 by adding 1 M Tris/HCl pH 8.0. The purified protein is concentrated and buffer exchanged to DPBS with a Millipore Amicon Ultra (10 kDa molecular weight cuttoff) centrifugal concentrator. Presence of the protein is confirmed with standard analytical methods (electrophoresis with Experion Pro 260 kit-BioRad; mass spectrometry). The protein is further analysed by size-exclusion chromatography and the endotoxin-content is determined (Endosafe PTS kit—Charles River).
[0273] d) Production of Recombinant Human, Cynomolgus, Mouse and Rat Ang2-FLD in HEK293-F Cells
[0274] Molecular cloning and cell culture is performed as described for Tie2-Fc-fusion protein. For purification of His-tagged proteins the CM is loaded at 5 ml/min on a 2 ml Ni.sup.2+ chelating sepharose fast flow column (His-Trap—GE Healthcare Life Sciences) equilibrated with DPBS. After loading in the presence of 4% elution buffer (DPBS+0.5% imidazol) to prevent unspecific binding, the column is washed with DPBS. Ang2-FLD-proteins are eluted from the column with DPBS containing 0.5% imidazol. Subsequently an ultrafiltration step was done for concentration and buffer exchange (10 kDa molecular withgt cut off). An aliquot of the protein is retained for analytical characterization as described for Tie2-Fc.
Example 1
[0275] Immunization with Recombinant Human Ang2 Induces a Humoral Immune Response in Llama
[0276] 1.1. Immunizations
[0277] After approval of the Ethical Committee of the faculty of Veterinary Medicine (University Ghent, Belgium), 4 llamas (designated No. 406, 408, 454, 455) are immunized with 4 intramuscular injections (day 0: 50 μg, day 14: 20 μg, day 28: 17.5 μg and day 42: 17.5 μg dose) of recombinant human Ang2 (R&D Systems, Minneapolis, Minn., US). The antigen is formulated in Complete Freund's Adjuvant for the prime injection at day 0 (Difco, Detroit, Mich., USA) and in Incomplete Freund's Adjuvant for the booster injections (Difco, Detroit, Mich., USA).
[0278] 1.2. Evaluation of Induced Immune Responses in Llama
[0279] To evaluate the induction of an immune response in the animals against human Ang2 by ELISA, sera are collected at day 0 (pre-immune), day 35 and day 46 (time of peripheral blood lymphocyte [PBL] collection). In short, 1 μg/mL of recombinant human Ang2 (R&D Systems, Minneapolis, Minn., USA) is immobilized overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (PBS+1% casein). After addition of serial serum dilutions, specifically bound immunoglobulins are detected using a horseradish peroxidase (HRP)-conjugated goat anti-llama immunoglobulin (Bethyl Laboratories Inc., Montgomery, Tex., USA) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA), showing that a significant antibody dependent immune response against human Ang2 is induced. The antibody response is mounted both by conventional and heavy-chain only antibody expressing B-cell repertoires, since bound immunoglobulins can be detected with antibodies specifically recognizing the conventional llama IgG1 antibodies or the heavy chain only llama IgG2 or IgG3 antibodies. In all llamas injected with human Ang2, an antibody response is mounted by conventional and heavy chain only antibody expressing B-cells specifically against human Ang2. The Ang2 serum titer responses for each llama are depicted in Table 1.
TABLE-US-00005 TABLE 1 Antibody mediated specific serum response against recombinant human Ang2. IgG reponse Llama Immunogen IgG1 IgG2 IgG3 406 rec. human Ang2 +++ ++ ++ 408 rec. human Ang2 +++ ++ ++ 454 rec. human Ang2 +++ ++ ++ 455 rec. human Ang2 +++ ++ +++ Legend: (*) Low (or +/−): 1,000 ≥ HSD.sub.S/N≥2 < 1,500 Moderate (or +): 1,500 ≥ HSD.sub.S/N≥2 < 13,500 Good (or ++): 13,500 ≥ HSD.sub.S/N≥2 < 365,000 Excellent (or +++): HSD.sub.S/N≥2 ≥ 365,000 (*) HSD, Highest Serum Dilution; S/N ≥ 2, signal-to-noise ratio ≥ 2
Example 2
[0280] Cloning of the Heavy-Chain Only Antibody Fragment Repertoires and Preparation of Phage
[0281] Following the final immunogen injection, immune tissues as the source of B-cells that produce the heavy-chain only antibodies are collected from the immunized llamas. Typically, two 150 mL blood samples collected 4 and 10 days after the last antigen injection, and one lymph node biopsy, collected 4 days after the last antigen injection are collected per animal. From the blood samples, peripheral blood mononuclear cells (PBMCs) are prepared using Ficoll-Hypaque according to the manufacturer's instructions (Amersham Biosciences, Piscataway, N.J., USA). From the PBMCs and the lymph node biopsy (not prelevated from animal No. 406), total RNA is extracted, which is used as starting material for RT-PCR to amplify the VHH encoding DNA segments, as described in Example 3 (page 46) of WO 05/044858. For each immunized llama, a library is constructed by pooling the total RNA isolated from all collected immune tissues of that animal. In short, the PCR-amplified VHH repertoire is cloned via specific restriction sites into a vector designed to facilitate phage display of the VHH library. The vector is derived from pUC119 and contains the LacZ promoter, a M13 phage gIII protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multiple cloning site and a hybrid gIII-pelB leader sequence. In frame with the VHH coding sequence, the vector encodes a C-terminal c-myc tag and a His6 tag. Phage are prepared according to standard protocols and stored after filter sterilization at 4° C. for further use.
Example 3
[0282] Selection of Ang2 Specific VHHs Via Phage Display
[0283] VHH repertoires obtained from all llamas and cloned as phage library are used in different selection strategies, applying a multiplicity of selection conditions. Variables include i) the Ang2 protein format: biotinylated C-terminally His-tagged full length recombinant human Ang2 (R&D Systems, Minneapolis, Minn., USA) and C-terminally His-tagged full length mouse Ang2 (produced at GeneArt, now Invitrogen, Carlsbad, Calif., USA), ii) the Ang2 presentation method: plates directly coated with mouse Ang2 or incubation in solution with biotinylated human Ang2 followed by capturing on neutravidin-coated plates, and iii) the antigen concentration. All selections are done in 96 well MaxiSorp plates (Nunc, Wiesbaden, Germany).
[0284] Multi-round selections are performed as follows: Ang2 preparations for solid and solution phase selection formats are presented as described above at multiple concentrations (biotinylated human Ang2: 50, 5, 0.5, 0.05 and 0.005 nM; mouse Ang2: 10, 1, 0.1 and 0.01 μg/mL). After 2 h incubation with the phage libraries, followed by extensive washing, bound phages are eluted with trypsin (1 mg/mL) for 15-30 minutes at room temperature. Trypsin activity is then immediately neutralized by applying 0.8 mM protease inhibitor ABSF. As background control, selections w/o antigen are performed in parallel. Phage outputs that show enrichment over background are used to infect E. coli. Infected E. coli cells are either used to prepare phage for the next selection round (phage rescue) or plated on LB agar plates (ampicillin+glucose.sup.2%) for analysis of individual VHH clones. In order to screen a selection output for specific binders or blockers, single colonies are picked from the agar plates and grown in 1 mL 96-deep-well plates. LacZ-controlled VHH expression is induced by adding IPTG (0.1-1 mM final) in the absence of glucose. Periplasmic extracts (in a volume of ˜80 uL) are prepared according to standard protocols (as disclosed in for example WO 2006/040153 cited herein). Briefly, cultures were centrifuged for 15 minutes at 4,500 rpm. The pellet was frozen overnight or for 1 hour at −20° C. Next, the pellet was thawed at room temperature for 40 minutes, re-suspended in 15 ml peri buffer (50 mM NaHPO4, 300 mM NaCl) and shaken for 1 hour. Periplasmic fraction was isolated by centrifugation for 20 minutes at 14000 rpm.
Example 4
[0285] Screening of Periplasmic Extracts in Ang2-Tie2 and Ang1-Tie2 ELISA and AlphaScreen Competition Assays
[0286] Periplasmic extracts containing expressed VHHs are screened in a human Ang2-human Tie2 AlphaScreen competition assay to assess their blocking capacity. In brief, human Tie2/Fc chimera (R&D Systems, Minneapolis, Minn., USA) is biotinylated using N-hydroxysulfosuccinimide ester of biotin (Thermo Fisher Scientific, Rockford, Ill., USA). FLAG tagged human Ang2 (Alexis Biochemicals, San Diego, Calif., USA) is captured using Acceptor beads (Perkin Elmer, Waltham, Mass., US) coated with anti-FLAG M2 antibody (Sigma, St Louis, Mo., USA). To evaluate the capacity of the VHHs to inhibit binding of human Ang2 to its receptor human Tie2, 1:25 dilutions of the periplasmic extracts containing expressed VHHs are incubated with 0.1 nM FLAG tagged human Ang2. To this mixture, the Acceptor beads and 0.3 nM biotinylated human Tie2/Fc chimeras are added and further incubated for 2 hours at room temperature. Finally, streptavidin conjugated Donor beads (Perkin Elmer, Waltham, Mass., US) are added and the mixture is incubated for an additional 2 hours at room temperature. Assay buffer is PBS+0.03% Tween-20+0.1% BSA. Fluorescence is measured using the Envision Multilabel Plate reader (Perkin Elmer, Waltham, Mass., USA) using an excitation wavelength of 680 nm and an emission wavelength of 520 nm. Decrease in fluorescence signal indicates that the binding of human Ang2 to human Tie2 is blocked by the VHH expressed in the periplasmic extract. VHHs able to block the human Ang2-human Tie2 interaction for at least 50% are screened in a confirmatory ELISA based competition assay. Additionally, cross-reactivity for binding to mouse Ang2 and selectivity over human Ang1 is also assessed in a competition ELISA. In brief, human or mouse Tie2/Fc chimera (R&D Systems, Minneapolis, Minn., USA) are immobilized at 2 μg/mL overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a 1% casein solution. A 1:5 dilution of periplasmic extract containing expressed VHHs is incubated with the following Ang species according to the type of assay: 0.02 nM FLAG tagged human Ang2, a 1:3,000 dilution of HEK293 conditioned medium containing FLAG tagged mouse Ang2 or 0.02 nM FLAG-tagged human Ang1 (Alexis Biochemicals, San Diego, Calif., USA). This mixture is added to the Tie2/Fc coated well and incubated for 2 hours at room temperature. Residual binding of Ang is detected using HRP-conjugated anti-FLAG M2 antibody (Sigma, St Louis, Mo., USA).
[0287] In a second screening cycle, periplasmic extracts containing expressed VHHs of selection outputs that yielded a high diversity of mouse Ang2 cross-reactive blocking VHHs are screened at a 1:300 dilution. VHHs inhibiting the binding of human Ang2 to human Tie2, mouse Ang2 to mouse Tie2 and showing no inhibition of human Ang1 binding to human Tie2 are selected. Sequence analysis revealed 86 unique VHHs belonging to 38 different B-cell lineages. The total number of unique sequence variants found for each B-cell lineage, a representative VHH and the selection condition used, is depicted in Table 2. An overview of AlphaScreen and ELISA based screening data is given in Table 3. The amino acid sequences of all unique VHHs are shown in the Sequence Listing (SEQ ID NOs: 1 to 86) and in Table 4.
TABLE-US-00006 TABLE 2 Selection parameters used for the identification of Ang2 specific VHH B-cell lineages. B-cell Representative # Unique Selection Lineage no. VHH ID variants Library Selection format rounds 1 2F04 3 408 biot-hAng2 1 or 2 biot-hAng2 > biot-hAng2 rmAng2 > rmAng2 2 1D01 2 406 biot-hAng2 1 or 2 biot-hAng2 > rmAng2 3 10H02 7 408 biot-hAng2 > biot-hAng2 1 or 2 rmAng2 > rmAng2 4 3A07 3 454 biot-hAng2 1 or 2 biot-hAng2 > rmAng2 5 7G08 3 454 biot-hAng2 > biot-hAng2 1 or 2 6 2G01 1 408 biot-hAng2 1 7 8A11 1 455 biot-hAng2 > biot-hAng2 2 8 16A03 4 455 biot-hAng2 1 or 2 biot-hAng2 > rmAng2 9 14A09 2 408 biot-hAng2 1 or 2 biot-hAng2 > rmAng2 10 11B07 18 454 biot-hAng2 1 or 2 biot-hAng2 > biot-hAng2 biot-hAng2 > rmAng2 rmAng2 > rmAng2 11 1E01 1 406 biot-hAng2 1 12 13A03 2 406 biot-hAng2 > rmAng2 2 13 15A06 1 454 biot-hAng2 > rmAng2 2 14 11A03 2 454 rmAng2 > rmAng2 2 15 14H02 3 408 biot-hAng2 > rmAng2 2 rmAng2 > rmAng2 16 14A08 1 408 biot-hAng2 > rmAng2 2 17 15H04 1 454 biot-hAng2 > rmAng2 2 18 16G09 3 455 biot-hAng2 > rmAng2 2 19 13A02 1 406 biot-hAng2 > rmAng2 2 20 10C06 4 408 rmAng2 > rmAng2 2 21 12A08 1 455 rmAng2 > rmAng2 2 22 12B03 2 455 biot-hAng2 > rmAng2 2 rmAng2 > rmAng2 23 10A03 1 408 rmAng2 > rmAng2 2 24 16A02 2 455 biot-hAng2 > rmAng2 2 rmAng2 > rmAng2 25 10A09 3 408 biot-hAng2 > rmAng2 2 rmAng2 > rmAng2 26 22C07 1 408 rmAng2 > rmAng2 2 27 21G10 2 408 biot-hAng2 > rmAng2 2 rmAng2 > rmAng2 28 19A03 1 406 biot-hAng2 > rmAng2 2 29 23C10 1 454 biot-hAng2 > rmAng2 2 30 25B01 1 455 biot-hAng2 > rmAng2 2 31 25F01 1 455 biot-hAng2 > rmAng2 2 32 25D08 1 455 biot-hAng2 > rmAng2 2 33 24B05 1 454 rmAng2 > rmAng2 2 34 22G11 1 408 rmAng2 > rmAng2 2 35 25G04 1 455 biot-hAng2 > rmAng2 2 36 28D10 1 408 biot-hAng2 > rmAng2 2 37 32H10 1 408 rmAng2 > rmAng2 2 38 29B08 1 408 biot-hAng2 > rmAng2 2
TABLE-US-00007 TABLE 3 Screening of periplasmic extracts containing expressed anti-Ang2 VHH.sup.(a) B-cell Represen- # AlphaScreen ELISA Lineage tative Unique hAng2 hAng2 mAng2 hAng1 No. VHH ID variants (% inh) (% inh) (% inh) (% inh) 1 2F04 3 79-84 50-86 13-27 0-4 2 1D01 2 77-83 97-101 85-102 0 3 10H02 7 54-94 65-101 42-98 0-3 4 3A07 3 53-80 0-75 1-27 0-4 5 7G08 3 96-96 101 101 0 6 2G01 1 55 39 2 0 7 8A11 1 57 67 13 0 8 16A03 4 54-96 0 0-4 0-2 9 14A09 2 0-69 6-13 0-2 0 10 11B07 18 46-92 26-107 9-93 0-2 11 1E01 1 0 2 3 4 12 13A03 2 50-53 28-40 0 0-2 13 15A06 1 72 0 0 0 14 11A03 2 57-74 0-3 0 0 15 14H02 3 54-63 54-56 36-48 0-7 16 14A08 1 52 5-5 4 0 17 15H04 1 57 82 38 0 18 16G09 3 55-95 57-99 21-96 1-7 19 13A02 1 91 96 97 1 20 10C06 4 64-85 84-90 43-50 0-2 21 12A08 1 98 0 2 3 22 12B03 2 83-92 92-94 11-99 0-7 23 10A03 1 67 0 0 0 24 16A02 2 67-90 0 0-1 0-1 25 10A09 3 52-65 0-11 0-1 0-4 26 22C07 1 73 78 82 0 27 21G10 2 55-85 45-72 13-30 0 28 19A03 1 74 74 22 0 29 23C10 1 57 37 24 0 30 25B01 1 61 5 24 4 31 25F01 1 67 85 42 14 32 25D08 1 78 94 6 10 33 24B05 1 97 102 98 3 34 22G11 1 96 100 98 0 35 25G04 1 90 87 9 0 36 28D10 1 89 118 93 4 37 32H10 1 55 112 58 17 38 29B08 1 60 112 63 0 .sup.(a)if multiple unique VHH variants within a B-cell lineage are identified, the range (min-max) of % inhibition is given.
TABLE-US-00008 TABLE 4 Amino acid sequence of unique anti-Ang2 VHHs identified during screening VHH ID/ SEQ ID NOs: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 001D01/1 EVQLVES DYAL WFRQA CIRCSD RFTISSDNAK SIVPRSKLE WGQ GGGLVQA G AGKERE GSTYYA NTVYLQMNSL PYEYDA GTQV GGSLRLS GVS DSVKG KPEDTAVYYC TVSS CAASGFT AA FD 001E01/2 EVQLVES THAM WYRQA TFTNRG RFTISRDNAK GPY WGQ GGGLVQP G PGKQR STYYAG NTMYLQMNSL GTQV GGSLRLS EHVA SVKG KPEDTAVYYC TVSS CVHSGTIS NT S 002A01/3 EVQLVES SSVM WFRQA AISGSG RFTISRDNAK GRAFLARDT WGQ GGGLVQA G PGKERE SSTDSA NTVYLQMNSL FYYDI GTQV GGSLRLS FVA QG KPEDTAVYYC TVSS CAASGRT AA FS 002F04/4 EVQLVES DHLI WFRQA CISRSA RFTISSDNAK GPAWGRPA WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL SPLPYEYDY GTQV GGSLRLS AVS DSVKG KADDTAVYYC TVSS CAASGFA AA FD 002G01/5 EVQLVES SYAM WFRQA AISLSG RFTISRDNAK TDWDFEDIP WGQ GGGLVQA A PGKERE DSTYYA NTVYLQMNSL EYYCSGYG GTQV GGSLRLS FVA DSVKG KPEDTAEYYC CDESLFDS TVSS CAASGRTI AA S 003A07/6 EVQLVES YDAI WFRQA CITSSD RFTISRDNAK GNRRIYYSD WGQ GGGLVQP G PGKERE GITYYA NTVYLQMNSL YALACFPYE GTQV GGSLRLS GVS DSVKG KPEDTAVYYC YDY TVSS CAASGFT AT LD 003D01/7 EVQLVES TYLM WFRQA VIWSSG RFTISRDSAK SYDGNYYIP WGQ GGGLVQV VG PGKERE DTAYAD NTVYLQMNSL GFYKD GTQV GDSLRLS FAA SVKG KAEDTAVYYC TVSS CAASGRT AG FS 003E10/8 EVQLVES YYAV WFRQA CISSSD RFTISRDNAK GNLRIYYSD WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL YALACFPYE GTQV GGSLRLS GVS DSVKG KPEDTAVYYC YDY TVSS CTTSGFTL AT D 003F02/9 EVQLVES TYLM WFRQA GIWSSG RFTISRDNAK SYDGNYYIP WGQ GGGLVQV VG PGKERE DTAYAD NTVYLQMNSL GFYKD GTQV GDSLRLA FAA SVKG KTEDTAVYYC TVSS CAASGRT AG FS 003F07/10 EVQLVES TYLM WFRQA AMWSS RFTISRDNAK SYGGNYYIP WGQ GGGLVQV VG PGKERE GVPAYA NTVYLQMNSL GFYED GTQV GDSLRLS FAA DSVKG KAEDTAVYYC TVSS CAASGRT AG FS 004B06/11 EVQLVES RYAM WFRRV HITWNR RFTISRDKAS QIKYGAVTH WGQ GGGLVQA G PGKERE GSTYYA NTLYLQMNSL PEEYSY GTQV GGSLRLS FVT DSVKG KPEDTAVYYC TVSS CAASGHT AA FS 006F05/12 KVQLVES DHLI WFRQA CISRSA RFTISSDNAK GPAWGRPA WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL SPLPYEYDY GTQV GGSLRLS AVS DSVKG KADDTAVYYC TVSS CAASGFA AA FD 006H05/13 EVQLVES DYAI WFRQA CISSSD RFTISSDNAK VPATRRTPQ WGQ GGGLVQA G PGKERE GSTAYA NTVYLQMNSL MVAANVCW GTQV GGSLRLS GVS DSVKG KPEDTAVYYC LAEYEYDY TVSS CAASGFT TA FD 007B09/14 EVQLVES TYLM WFRQA GIWSSG RFTVSRDNAK SYDGNYYIP WGQ GGGLVQV VG PGKERE GTAYAD NTVYLQMNSL GFYKD GTQV GDSLRLS FAA SVKG KAEDTAVYYC TVSS CAASGRT AG FS 007C01/15 EVQLVEF TYLM WFRQA GIWSSG RFTVSRDNDK SYDGNYYIP WGQ GGGLVQV VG PGKERE GTAYAD NTVYLQMNSL GFYKD GTQV GDSLRLS FAA SVKE QAEDTAVYYC TVSS CAASGRT AG FS 007C07/16 EVQLVES TYLM WFRQT VIWSSG RFTISRDNAK SYGGNYYIP WGQ GGGLVQV VG PGKERE DTAYAD NTVYLQMNSL GFYED GTQV GDSLRLS FAA SVKG KAEDTAVYYC TVSS CAASGHT AG FS 007G08/17 EVQLVES YYAI WFRQV CISSSD RFTISRDNAK DSGGYIDYD WGQ GGGLVQP G PGKERE GITYYV NTVYLQMNSL CMGLGYDY GTQV GGSLRLS GVS DSVKG KPEDTAVYYC TVSS CAASGFA AT LD 008A11/18 EVQLVES SYAM WFRQA AVSWS RFTISRDSAK QSTIVEVTTL WGQ GGGLVQA G PGKELE GGSTYY NTVYLQMNSL EAYDY GTQV GGSLRLS FVT ADSVKG KPEDTAVYYC TVSS CAASGRT AA FS 010A03/19 EVQLVES PYAM WFRQA HITWSA RFTISRDNAK KRRYGIVDR WGQ GGGSVQA G PGKERE GSTYYA NTVYLQMNSL DYND GTQV GGSLRLS FVA DSVKG KPEDTAVYYC TVSS CAASERT AA FS 010A09/20 EVQLVES SVSA WYRQA GISNIGA RFTISRDNAK LLWSGNL WGQ GGGLVQA TG PGKQR TKFADS NTVYLQMNSL GTQV GGSLRLA EFVA VKG KPEDTAVYYC TVSS CIASGRDI NV F 010A10/21 EVQLVES SITAI WYRQA GISNIGA RFTISGDNAE LLWSANY WGQ GGGLVQA G PGKQR TKYTDS NTVYLQMNSL GTQV GGSLRLS EFVA VKG KPEDTAVYYC TVSS CIASGRDI NV F 010B02/22 EVQLVES DHLI WFRQA CISRSA RFTISGDNAK GPAWGRPA WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL SPLPYEYDY GTQV GGSLRLS AVS DSVKG KADDTAVYYC TVSS CAASGFA AA FD 010B08/23 EVQLVES DYAI WFRQA CISSSD RFTISSDNAK VPATRRTPQ WGQ GGGLVQA G PGKERE GSTAYA NTVYLQMNSL MVVANVCW GTQV GGSLRLS GVS DSVKG KPEDTAVYYC LAEYEYDY TVSS CAASGFT TA FD 010C06/24 EVQLVES DYAI WFRQA CISSSD RFTISGDNAK GITPCSDYT WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL QTYEYDV GTQV GGSLRLS GVS DSVKG KPEDTAVYYC TVSS CVASGFT AA FD 010C07/25 EVQLVES DYAI WFRQA CISRSD RFTISSDNAK VPATRRTPQ WGQ GGGLVQA G PGKERE GSTSYA NTVYLVMNSL MVVANMCW GTQV GGSLRLS GVS DSVKG KPEDTAVYYC LAEYEYDY TVSS CAASGFT TA FD 010D04/26 EVQLVES DYAI WFRQA CISSSD RFTISSDNAK GITPCSDYT WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL QTYEYDV GTQV GGSLRLS GVS DSVKG KPEDTAVYYC TVSS CAASGFT AA FD 010E02/27 EVQLVES DYAI WFRQA CISSSD RFTISSDNAK VPATRRTPQ WGQ GGGLVQA G PGKERE GSTAYA NTVYLQMNSL MVVANVCW GTQV GGSLRLS GVS DSVKG KPEDTAVYYC LAEYEYDY TVSS CAVSGFT TA FD 010F10/28 EVQLVES DYAI WFRQA YISSSD RFTSSSDNAK RPTLRVRLD WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL NDRHHLLYE GTQV GGSLRLS GVS DSVKG KPEDTAVYYC YEYDY TVSS CAASGFT AA FD 010G02/29 EVQLVES DYTI WFRQA CISSSD RFTISSDNAK VPATRRTPQ WGQ GGGLVQA G PGKERE GSTSYA NTVYLQMNSL MVVLNMCW GTQV GGSLRLS GVS DSVKG KPEDTAVYYC LAEYEYDY TVSS CAASGFT TA FD 010G11/30 EVQLVES DYAI WFRQA CISSSD RFTISSDNAK GITPCSDYT WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL QTYEYDV GTQV GGSLRLS GVS DSVKG KPEDTAVYYC TVSS CVASGFT AA FD 010H02/31 EVQLVES DYAI WFRQA CISSSD RFTISSDTAKN VPATRRTPQ WGQ GGGSVQA G PGKERE GSTNYA TVYLQMNSLK MVDANMCW GTQV GGSLRLS GVS DSVKG PEDTAVYYCT LAEYEYDY TVSS CAASGFT A FD 011A02/32 EVQLVES GHA WFRQA TIYWTS RFTISRDNAE IKDFQLRVD WGQ GGGLAQA MG PGEERE GMTRYA NTVFLQMNSL VTSASAYDY GTQV GGSLRLS FVA GSVKG KPEDTAVYYC TVSS CAASGRR AV FG 011A03/33 GVQLVES GHA WFRQA TIYWTT RFTISRDNAE IRDFNIRLDV WGQ GGGLAQA MG PGKDRE GMTRYA NTVFLQMNSL TSASAYGY GTQV GGSLRLS FVA DSVKG KPEDTAVYYC TVSS CAASGRR AL FG 011B07/34 EVQLVES TYLM WFRQA GIWSSG RFTISRDNAK SYDGNYYIP WGQ GGGLVQV VG PGKERE DTAYAD NTVYLQMNSL GFYKD GTQV GDSLRLS FAA SVRG KTEDTAVYYC TVSS CAASGRT AG FS 011C01/35 EVQLVES TYLV WFRQA AIWSSG RFTISRDNAK SYGGNYYIP WGQ GGGLVQV VG PGKERE DTAYAD NTVYLQMNSL GFYED GTQV GDSLRLS FAA SVKG KAEDTAVYYC TVSS CAASGRT AG FS 012A02/36 EVQLVES RNA WFRQV GIRWNV RFTISRDNAE YAGLVFSGI WGQ GGGLVQA MA PGKVRE GRLDYA NTVYLQMNDL PDY GTQV GGSLRLS FVA DSVKG KTEDTAVYYC TVSS CAASGRT AA FS 012A08/37 EVQLVES INAM WYRQA AITSGG RFTISRDNAK DSDYSSDYY WGQ GRGLVQA L PGKQR STNYAD NTVYLQMNSL Y GTQV GGSLRLS ELVA SVKG KPEDTAVYYC TVSS CAASGSIF AA S 012B03/38 EVQLVES YYTI WFRQA CISGGD RFTISRDNAK DSAGVPAG WGQ GGGLVQS G PGKERE TSTYYA NTVYLQMNSL PAAVYGSTC GAQV GGSLRLS GVS DSVKG KPEDTAVYYC SRLEYDY TVSS CAASGFA AT LD 013A02/39 EVQLVES SYSM WFRQA AINWNG RFTVSRDNAK TGWGRAYE WGQ GGGLVQA G PGKERE DSTYYE NTVYLQMNSL QAYEYDV GTQV GGSLRLS FIA DSVKG KPEDTAVYYC TVSS CAASGGT AA FS 013A03/40 EVQLVES DYAM WVRQA TISWND RFTISRDNAK GGSRLYDY WGQ GGGLVQP S PGKGLE EYTYYA NTLYLQMNSL HY GTQV GGSLRLS WVS ESMKG KSEDTAVYYC TVSS CAASGFT AK VD 014A08/41 EVQLVES DYAI WFRQA CISSSD RFTISSDNAK RPPFHSCSE WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL YENDY GTQV GGSLRLS GVS DSVKG KPEDTAVYYC TVSS CASSGFT AA FD 014A09/42 EVQLVES SSVL WFRQA AISGSG RFTISRDNAK GRAFLTRDP WGQ GGGLVQA G PGKERE SSTDSA NTVYLQMNSL FYYDI GTQV GGSLRLS FVA KD KPEDTAVYYC TVSS CAASGRTI AA S 014A11/43 EVQLVES SVTA WYRQA GLSNIG RFTVSGDAAK LLWSGNY WGQ GGGLVQA MG PGKQR ATKYAD NTVYLQMNSL GTQV GGSLRLT EFVA SVKG KPEDTAVYYC TVSS CIASGRDI NV F 014D03/44 EVQLVES DYAI WFRQA YISKSD RFTSSSDNAK RPTLRVRLD WGQ GGGLVQA G PGKERE GTTYYA NTVYLQINSLK NDRHHLLYE GTQV GGSLRLS GVS DSVKG PEDTAVYYCA YEYDY TVSS CAASGFT A FD 014H02/45 EVQLVES EYAI WFRQA YISSSD RVTSSSDNAK RPTLRVRLD WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL NDRHHLLYE GTQV GGSLRLS GVS DSVKG KPEDTAVYYC YEYDY TVSS CAASGFT AA FD 015A06/46 EVQLVES SYAM WFRQA RISWNG RFTISRDNPK SIALVGGVT WGQ GGGLVQA G PGKERE GSTYHA NTVYLQMDSL PHSYDY GTQV GGSLRLS FVA DSVKG KPEDAAIYYC TVSS CAASGRT AA FS 015C05/47 EVQLVES TYLM WFRQA VIWSSG RFTISRDNAK SYGGNYYIP WGQ GGGLVQV VG PGKERE DTDYAD NTVYLQMNSL GFYKD GTQV GDSLRLS FAA SVKG KAEDTAVYYC TVSS CAASGRT AG FS 015D05/48 EVQLVES TYLM WFRQA GIWSSG RFTISRDNAK SYDGNYYIP WGQ GGGLVQV VG PGKERE GTAYAD NTVYLQMNSL GFYKD GTQV GDSLRLS FAA SVKG KAEDTAVYYC TVSS CAASGRT AG FS 015H04/49 EVQLVES SYVM WFRQA GISWSS RFTISRDAAE NSVSEPTLH WGQ GGGLVQA G PGKELE GRTYYT NTWYLQMNS TWQYEASY GTQV GGSLGLS FVA DSVKG LKPEDTAVYY DY TVSS CAASERT CAS LP 016A01/50 EVQLVES RYAM WFRRV HITWNR RFTISRDKAS QIKYGEITHP WGQ GGGLVQA G PGEERE GSTYYA NTLYLQMNSL EEYSY GTQV GGSLRLS FVT DSVKG KPEDTAVYYC TVSS CAASGHT AA FS 016A02/51 EVQLVES RNA WFRQV AIRWNV RFAISRDNAE YAGLVYSGI WGQ GGGLVQA MG PGKARE GRLDYA NTVYLQMNDL PDY GTQV GGSLRLS FVA DSVKG KTEDTAVYYC TVSS CAASGRT AA FS 016A03/52 EVQLVES RYAM WFRRV HITWNR RFTISRDKAS QIKYGEITHP WGQ GGGLVQA G PGKERE GSTYYA NTLYLQMNSL EEYSY GTQV GGSLRLS FVT DSVKG KPEDTAVYYC TVSS CAASGHT AA FS 016A05/53 EVQLVES RYAM WFRRV HITWNR RFTISRDKAS QTKYGEITR WGQ GGGLVQA G PGKERE GSTYYA NTLYLQMNSL PEEYSY GTQV GDSLRLS FVT DSVKG KSEDTAVYYC TVSS CAASGHT AA FS 016G09/54 EVQLVES INAM WYRQA FMINDS RFTISRDSTKN AYEQHTY WGQ GGGLVQA A PGKQR STDYTD ILYLQMNNLN GTQV GGSLRLV EWVA SVKG VEDTAVYYCN TVSS CSASGIDF T S 019A03/55 EVQLVES DYAI WFRQA CITPSD RFIISSDNAKN VPRLRGLGY WGQ GGGLVQA A PGKERE DRTYYA TVYLQMNSLK WPYPEYEY GTQV GGSLRLS GIS DSVKG PEDTADYYCA DY TVSS CAASGFT A FD 019G07/56 KVQLVES DYAL WFRQA CIRCSD RFTISSDNAK SIVPRSKLE WGQ GGGLVQA G AGKERE GSTYYA NTVYLQMNSL PYEYDA GTQV GGSLRLS GVS DSVKG KPEDTAVYYC TVSS CAASGFT AA FD 019G08/57 EVQLVES DYAM WVRQA TISWND RFTISRDNAK GGSRLYDY WGQ GGGLVQP S PGKGLE EYTYYA NTLYLQMNSL HY GTQV GGSLRLS WVS ESMKG KSEDTAVYYC TVSS CAVSGFT AK VD 021G10/58 EVQLVES DYAI WFRQA CISSSD RFTVSSDNAK GLRGRYYR WGQ GGGLVQA G PGKERE GSTTYA NTVYLQMNSL GTYSLVCAP GTQV GGSLRLS GVS DSVKG KPEDTAVYYC YEYDF TVSS CAASGFT AA LD 022B09/59 EVQLVES DYAI WFRQA CISSSD RFTISADNAK GLRGRYYS WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMHSL GSNYLVCAP GTQV GGSLRLS GVS DSVKG KPEDTAVYYC YEYDY TVSS CAASGFT AA LD 022C07/60 EVQLVES DYAI WFRQA CISSSD RFTISSDNAK DFIISSKRLC RGQG GGGLVQA G PGKERE GSTYYA NTVYLQMNSL LDLFGS TQVT GGSLRLS GVS DSVKG KPEDTAVYYC VSS CAASGFT AA FD 022G03/61 EVQLVES DYAI WFRQA CISSSD RFTISSDNAK GITPCSDYT WGQ GGGSVQA G PGKERE GSTYYA NTVYLQMNSL QTYEYDV GTQV GGSLRLS GVS DSVKG KPGDTAVYYC TVSS CAASGFT AA FD 022G05/62 EVQLVES DYAI WFRRA CITSSD RFTISSDSAKN VPATRRNP WGQ GGGLVQA G PGKERE GSTSYA TVYLQMNSLK QMVVAKKC GTQV GGSLRLS GVS DSVKG PEDTAVYYCS WLAEYEYD TVSS CAASGFT V Y FD 022G11/63 EVQLVES DYAI WFRQA CISRSD RFTISSDNAK SWSGAYYS WGQ GGGLVQA G PGKERE GSPYYA GTVYLQMSSL GTYYCDRLY GTQV GGSLRLS GVS DSVKG KPEDTAVYYC EYDA TVSS CAVSGFT AA FD 023A04/64 EVQLVES TYLM WFRQA TMWSS RFTISRDNAK SYGGNYYIP WGQ GGGLVQV VG PGKERE GDTAYA NTVYLQMNSL GFYED GTQV GDSLRLS FAA DSVKG KAEDTAVYYC TVSS CAASERT AG FS 023C10/65 EVQLVES YYAI WFRQA CISSSD RFTISRDNAK DSLGYGSS WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL CRMAPYEY GTQV GGSLRLS GVS DSVKG KPEDTAVYYC DY TVSS CAASGFA AT LD 023D01/66 EVQLVES TYLM WFRQA VIWSSG RFTISRDNAK SYDGNYYIP WGQ GGGLVQV VG PGKERE GTAYAD NTVYLQMNSL GFYKD GTQV GDSLRLS FAA SVKG KAEDTAVYYC TVSS CAASGRT AG FS 023E02/67 EVQLVES DYAI WFRQA CISSSD RFTISRDNAK GNRRIYYSD WGQ GGGLVQP G PGKERE GITYYA NTVYLQMNSL YALACFPYE GTQV GGSLRLS GVS DSVKG KPEDTAVYYC YDY TVSS CAASGFT AT SD 023E08/68 EVQLVES TYLM WFRQA GIWSSG RFTISRDNAK SYDGNYYIP WGQ GRRLVQV VG PGKERE DTAYAD NTVYLQMNSL GFYKD GTQV GDSLRLA FAA SVKG KTEDTAVYYC TVSS CAASGRT AG FS 023F10/69 EVQLVES TYLM WFRQA TMWVS RFTISRDNPK SYGGNYYIP WGQ GGGLVQV VG PGKERE GDTAYA NTVYLQMNSL GFYKD GTQV GDSLRLS FAA DSVKG KAEDTAVYYC TVSS CAASERT AG FS 023F11/70 EVQLVES TYLM WFRQA AIWSSG RFTISRDNAK SYGGNYYIP WGQ GGGLVQI VG PGKERE DTAVAD NTVYLQMNSL GFYED GTQV GDSLRLS FAA SVKG KAEDTAVYYC TVSS CAASGRT AG FS 024B05/71 EVQLVES YYAI WFRQA CISSSD RFTISRDNAK DSIVCGSYY WGK GGGLVQP G PGKEW GSTYYA NTVYLQMNSL GMDY GTQV GGSLRLS EGVS DSVKG KPEDTAVYYC TVSS CAASGFT AT LD 024G05/72 EVQLVES TYLM WFRQA GIWSSG RFTVSRDNDK SYDGNYYIP WGQ GGASVQP VG PGKERE GTAYAD NTVYLQMNSL GFYKD GTQV GGSLRLS FAA SVKE QAEDTAVYYC TVSS CAASGRT AG FS 025B01/73 EVQLVES DYAI WFRQA CISSSD RFTISSDNAK AWGASRLPI WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL GTMPPYEY GTQV GGSLRLS GVS DSVKG KPEDTAVYYC DY TVSS CAASGFT AA FD 025C06/74 EVQLVES INVM WYRQA FIGSGG RFTISRDSTKN AYEQHTY WGQ GGGLVQA G PGKQR STDYID ILYLRMNNLNV GTQV GGSLRLV EWVA YTDSVK EDTAVYYCNT TVSS CSASGIDF G S 025D08/75 EVQLVES DYAI WFRQA CISSSD RFTISSDNAK VNGLGPFSV WGQ GGGLVQA G PGKDLE GSTYYA NTVYLQMNSL PVPVYDF GTQV GGSLRLS GVS DSVKG KPEDTAVYYC TVSS CAASGFT AA FD 025F01/76 EVQLVES DYVI WFRQA CISSSD RFTISSDNAK GGPRINIAT WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL MTCSHDEY GTQV GGSLRLS GVS DSVKG KPEDTAAYYC EYDY TVSS CAVSGLP AA FD 025F07/77 EVQLVES YYAI WFRQA CIESSD RFTISRDNAK DSAGVPAG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL PAAVYGSSC GTQV GGSLRLS GVS DSVKG KPEDTAVYYC SRLEYDY TVSS CAASGFA AT LD 025G04/78 EVQLVES SYDM WVRQA AINSRG RFTISRDNAK DPYSLSYYG WGQ GGGLVQP S PGKGPE GSTYYA NTLYLQMNSL YPLYDY GTQV GGSLRLS WVS DSVKG KPEDTAVYYC TVSS CAASGFT AT FG 025G10/79 EVQLVES INVM WYRQA FIGSGS RFSISRDSTK AYEQHTY WGQ GGGLVQA G PGKQR STGYTD NILYLQMNNL GTQV GGSLRLV EWVA SVKG NVEDTAVYYC TVSS CSASGIDF NT S 028D10/80 EVQLVES DYAI WFRQA CIRDSD RTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTQV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD 029B08/81 EVQLVES SYTM WFRQT AISSSLG RFQILRDNAK SRSLNLAYT WGQ GGGVVQA A PGKERD RTYYAD ETVWLQMNSL TKPYDY GTQV GDSVRLS IVA SVKG KPEDTAVYIC TVSS CAASGPT AA FR 032H10/82 EVQLVES DYD WFRQA YISSSD RFTISSDNAK RPWTRRVY WGQ GGGLVQA MG PGKERE GSTYYT NTVYLQMNSL GSSWLARS GTQV GGSLRLS GVS DSVKG IPEDTAVYYCA LDEYEYDY TVSS CAASGFD A FE 036H10/83 EVQLVES TYLM WFRQT VIWSSG RFTISRDNAK SYGGNYYIP WGQ GGGLVQV VG PGKERE DTAYAD NTVYLQMNSL GFYED GTQV GDSLRLS FAA SAKG KAEDTAVYYC TVSS CAASGHT AG FS 037A09/84 EVQLMES YYAI WFRQV CISSSD RFTISRDNAK DSGGYIDYD WGQ GGGLVQP G PGKERE GITYYV NTVYLQMNSL CMGLGYDY GTQV GGSLRLS GVS DSVKG KPGDTAVYYC TVSS CAASGFA AT LD 037F02/85 EVQLVES YYAI WFRQA CISSSD RFTISRDNAK DSGGYIDYD WGQ GGGLVQP G PGKERE GITYYV NTVYLQMNSL CMGLGYDY GTQV GGSLRLS GVS DSVKG KPEDTAVYYC TVSS CAASGFA AT LD 043E10/86 EVQLVES TYLM WFRQA VIWSSG RFTISRDNAK SYDGNYYIP WGQ GGGLVQV VG PGKERE DTAYAD NTVYLQMNSL GFYKD GTQV GDSLRLS FAA SVKG KAEDTAVYYC TVSS CAASGRT AG FS
Example 5
[0288] Characterization of Purified Anti-Ang2 VHHs
[0289] A subset of inhibitory anti-Ang2 VHHs selected from the screening described in Example 4 are further purified and characterized. Selected VHHs are expressed in E. coli TG1 as c-myc, His6-tagged proteins. Expression is induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37° C. After spinning the cell cultures, periplasmic extracts are prepared by freeze-thawing the pellets. These extracts are used as starting material and VHHs are purified via IMAC and size exclusion chromatography (SEC) resulting in ≥95% purity as assessed via SOS-PAGE.
[0290] 5.1. Evaluation of hAng2 Blocking VHHs in ELISA
[0291] The blocking capacity of the VHHs is evaluated in a human Ang2-human Tie2 blocking ELISA. In brief, 2 μg/mL of Tie2/Fc chimera (R&D Systems, Minneapolis, Minn., USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). A fixed concentration of 0.02 nM FLAG-tagged human Ang2 (Alexis Biochemicals, San Diego, Calif., USA) is added to a dilution series of the purified VHH (diluted in PBS+0.1% casein+0.05% Tween-20), and incubated on the coated human Tie2 receptor for 2 hours. Residual binding of human Ang2 is detected using horseradish peroxidase (HRP) conjugated anti-FLAG M2 (Sigma, St. Louis, Mo., USA) (
TABLE-US-00009 TABLE 5-1 IC.sub.50 (nM) values of purified VHHs blocking the hAng2/hTie2 interaction (competition ELISA; VHH: n = 2-3; Fab Ab536: n = 6) VHH ID IC.sub.50 (nM) 1D01 3.4 2F04 2.8 3A07 21.0 3F02 9.1 6H05 5.3 7G08 0.07 8A11 30.2 10C06 7.7 10H02 3.0 11B07 5.4 12B03 4.6 13A02 4.1 14H02 64.4 15H04 18.6 16G09 11.3 21G10 6.2 22C07 11.0 24B05 1.0 25F01 6.5 Fab Ab536 39.3
TABLE-US-00010 TABLE 5-2 IC.sub.50 (nM) values of purified VHHs blocking the hAng2-hTie2 interaction (competition ELISA; VHH: n = 1-3; AMG386 peptide: n = 3) VHH ID IC.sub.50 (nM) 1D01 6.2 7G08 0.04 10H02 8.7 11B07 14.0 13A02 23.0 24B05 1.1 28D10 1.3 32H10 4.0 37A09 0.1 37F02 0.08 AMG386 peptide 3.4
[0292] 5.2. Evaluation of Cross-Reactivity Towards Mouse and Cynomolgus Ang2 in Blocking ELISA
[0293] In order to determine if the VHH inhibits binding of mouse Ang2 to mouse Tie2 and cyno Ang2 to cyno Tie2, a competition ELISA is performed. In brief, 2 μg/mL of recombinant mouse Tie2-Fc or cyno Tie2-Fc is coated overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Coated wells are blocked with a 1% casein solution. FLAG-tagged mouse Ang2 (1:3,000 dilution of conditioned medium from transient HEK transfection) or FLAG-tagged cyno Ang2 (1:800 dilution of conditioned medium from transient HEK transfection) and a dilution series of purified VHH (diluted in PBS+0.1% casein+0.05% Tween-20) are incubated on the coated Tie2-Fc receptor for 2 hours at room temperature to reach binding equilibrium. Residual binding of FLAG-mAng2 or FLAG-cAng2 is detected using HRP conjugated anti-FLAG M2 mAb (Sigma, St. Louis, Mo., USA). Reference molecule is the Fab fragment of Ab536 (mouse:
TABLE-US-00011 TABLE 6-1 IC.sub.50 (nM) values of purified VHHs blocking the interaction of mAng2 to mTie2 (competition ELISA; VHH: n = 2-3; Fab Ab536: n = 5) mAng2 VHH ID IC.sub.50 (nM) 1D01 6.3 2F04 57.4 3A07 99.3 3F02 32.7 6H05 7.7 7G08 0.09 8A11 442.1 10C06 45.2 10H02 5.2 11B07 21.0 12B03 6.7 13A02 6.1 14H02 143.2 15H04 124.6 16G09 19.4 21G10 16.8 22C07 13.6 24B05 1.5 25F01 13.3 Fab Ab536 15.3
TABLE-US-00012 TABLE 6-2 IC.sub.50 (nM) values of purified VHHs blocking the interaction of mAng2 and cAng2 to mTie2 and cTie2, respectively (competition ELISA; VHH: n = 1-3; AMG386 peptide: n = 3; n.d., not determined) mAng2 cAng2 VHH ID IC.sub.50 (nM) IC.sub.50 (nM) 1D01 10.0 16.4 7G08 0.07 0.14 10H02 21.4 23.7 11B07 39.7 24.8 13A02 26.6 33.1 24B05 1.1 2.1 28D10 6.1 2.0 32H10 13.0 n.d. 37A09 0.1 0.2 37F02 0.09 0.1 AMG386 peptide 5.2 6.6
[0294] 5.3. Evaluation of Selectivity of Human Ang2 Blocking VHHs Towards Human Ang1 in ELISA
[0295] In order to determine whether the anti-Ang2 blocking VHHs are selective over human Ang1 binding to human Tie2, a competition ELISA is performed. In brief, 2 μg/mL of recombinant human Tie2-Fc (R&D Systems, Minneapolis, Minn., USA) is coated overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Coated wells are blocked with a 1% casein solution. A fixed concentration (0.02 nM) of FLAG-tagged recombinant human Ang1 (Alexis Biochemicals, San Diego, Calif., USA) and a dilution series of VHH (diluted in PBS+0.1% casein+0.05% Tween-20) are incubated on the coated receptor Tie2-Fc for 2 hours at room temperature to reach binding equilibrium. Residual binding of FLAG-hAng1 is detected using HRP conjugated anti-FLAG M2 mAb. Reference molecule is the peptide moiety of AMG386 peptibody (
TABLE-US-00013 TABLE 7 IC.sub.50 (nM) values of purified VHHs blocking the interaction of human Ang1 to human Tie2 (competition ELISA; VHH: n = 2-3; AMG386 peptide: n = 3) hAng1/hAng2 VHH ID IC.sub.50 (nM) ratio 1D01 >67,000 >10,800 7G08 >84,000 >2,333,333 10H02 2,000 230 11B07 120,000 8,570 13A02 17,000 739 24B05 120,000 109,090 28D10 >4,000 >3,076 37A09 >10,000 >100,000 37F02 >10,000 >100,000 AMG386 peptide 4,000 1,176
[0296] 5.4. Determining the Affinity of the Human, Mouse, Cyno Ang2-VHH Interaction
[0297] Affinities of the VHH for binding to human, mouse and cyno Ang2 are determined using surface plasmon resonance (SPR) analysis (Biacore T100). In brief, VHH and benchmark compounds are immobilized on a CM5 chip via amine coupling. A multi-cycle kinetic approach is used: different concentrations of human, mouse and cyno Ang2-FLD (0.4-1-2.6-6.4-16-40-100 nM) are injected. Ang2-FLD species are allowed to associate for 2 min and to dissociate for 20 min at a flow rate of 45 μL/min. In between injections, the surfaces are regenerated with a 10 sec pulse of 25 mM NaOH and 60 sec stabilization period. Association/dissociation data are evaluated by fitting a 1:1 interaction model (Langmuir binding). The affinity constant K.sub.D is calculated from resulting association and dissociation rate constants k.sub.a and k.sub.d and are depicted in Table 8.
TABLE-US-00014 TABLE 8 Affinity K.sub.D (nM) of purified VHHs for human, mouse and cyno Ang2 human Ang2-FLD mouse Ang2-FLD cyno Ang2-FLD VHH k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D ID (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) 1D01 7.7E+06 1.5E−02 2.0E−09 3.3E+06 1.5E−02 4.6E−09 9.9E+06 1.4E−02 1.4E−09 7G08 1.0E+06 1.0E−04 9.7E−11 8.1E+05 1.4E−04 1.8E−10 1.5E+06 1.1E−04 7.2E−11 10H02 5.7E+06 2.1E−02 3.6E−09 2.5E+06 2.6E−02 1.1E−08 6.9E+06 2.3E−02 3.4E−09 11B07 9.2E+06 6.2E−02 6.7E−09 4.8E+06 1.4E−01 2.9E−08 1.2E+07 7.3E−02 6.1E−09 13A02 9.2E+06 9.1E−02 9.9E−09 1.9E+06 3.4E−02 1.8E−08 1.1E+07 9.4E−02 8.7E−09 24B05 2.6E+06 2.5E−03 9.6E−10 1.7E+06 2.9E−03 1.7E−09 4.1E+06 2.4E−03 5.9E−10 28D10 4.9E+06 6.2E−03 1.3E−09 1.9E+06 1.1E−02 5.6E−09 1.8E+07 2.5E−02 1.4E−09 mAb 5.0E+07 5.5E−02 1.1E−09 1.1E+07 6.1E−02 5.5E−09 n.d. n.d. n.d. 3.19.3 Fab 3.1E+06 3.7E−02 1.2E−08 1.6E+06 1.7E−02 1.1E−08 4.1E+06 4.5E−02 1.1E−08 Ab536
Example 6
[0298] Affinity Maturation of Selected VHH
[0299] A variant of VHH 28D10 (00027 carrying C.sub.50S/S.sub.53N and Q.sub.108L substitution—Example 7.3) is subjected to affinity maturation.
[0300] In a first cycle, amino acid substitutions are introduced randomly in both framework (FW) and complementary determining regions (CDR) using the error-prone PCR method. Mutagenesis is performed in a two-round PCR-based approach using the Genemorph II Random Mutagenesis kit (Stratagene, La Jolla, Calif., USA) using 1 ng of VHH 00027 cDNA template, followed by a second error-prone PCR using 0.1 ng of product of round 1. After a polish step, PCR products are inserted via unique restriction sites into a vector designed to facilitate phage display of the VHH library. Consecutive rounds of in-solution selections are performed using decreasing concentrations of biotinylated recombinant human Ang2 (R&D Systems, Minneapolis, Minn., USA) and trypsin elutions. Periplasmic extracts (in a volume of ˜80 uL) are prepared according to standard methods and screened for binding to recombinant human Ang2-FLD in a ProteOn (BioRad, Hercules, Calif., USA) off-rate assay. In brief, a GLC ProteOn Sensor chip is coated with recombinant human Ang2-FLD on the “ligand channels” L3, L4, L5 and L6 (with L1/L2 as reference channel). Periplasmic extract of affinity matured clones is diluted 1:10 and injected across the “analyte channels” A1-A6. An average off-rate is calculated of the reference VHH 00027 which is prepared and tested in the same way as the affinity matured VHHs and serves as a reference to calculate off-rate improvements. The top 25 affinity matured variants are shown in Table 9. VHH are sequenced (Table 10-A) to identify amino acid mutations beneficial for improving the off-rate (Table 10-B).
TABLE-US-00015 TABLE 9 Off-rate and fold improvement of affinity matured variants of VHH 00027. VHH ID k.sub.d (1/s) fold improvement 64G03 7.4E−05 15.7 64F03 1.1E−04 10.8 64D11 1.1E−04 10.3 64G11 1.2E−04 9.6 55D06 1.2E−04 9.4 64F07 1.2E−04 9.4 64G02 1.2E−04 9.4 55A06 1.3E−04 9.2 64C03 1.3E−04 9.2 64G12 1.3E−04 8.9 65F03 1.4E−04 8.9 55F02 1.4E−04 8.7 64E12 1.3E−04 8.7 60C09 1.6E−04 8.5 64B02 1.4E−04 8.5 64A03 1.4E−04 8.3 64C07 1.4E−04 8.3 60A06 1.6E−04 8.3 64B01 1.4E−04 8.2 64G01 1.4E−04 8.2 56A07 1.5E−04 8.1 58D10 1.3E−04 8.1 65F01 1.6E−04 8.1 53A06 1.6E−04 8.0 55G03 1.5E−04 8.0
[0301] Initially 12 VHH variants containing combinations of mutations on Kabat position 27, 29, 100b and 100i (Table 11;
TABLE-US-00016 TABLE 10-A Amino acid sequence of affinity matured anti-Ang2 VHHs VHH ID/ SEQ ID NOs: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 64G03/87 EVQLVES DYAI WFRQA SIRDND RSTISSDNDK VPAGRLRY WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMDSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD 64F03/88 EVQLVES DYAV WFRQA TIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPKDTAVYYC DA TVSS CAVSGITL AA D 64D11/89 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGQAQ G PGKERE GSTYYT NTVYLQMNSL EQWYPIYEY GTLV AGGSLRL GVS DSVKG KPEDTAVYYC DA TVSS SCAVSGF AA TLD 64G11/90 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERV GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGITL AA D 55D06/91 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGLT AA LD 64F07/92 QVQLVES DYAI WFRQA SIRDND RFTISSDNDQ VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KSEDTAVYYC DA TVSS CAVSGFT AA LD 64G02/93 EVQLVES DYAI WFRQA SIRDND RFIISSDNDKN VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA TVYLQMNSLK EQWYPIYEY GTLV GGSLRLS GVS DSVKG PEDTAVYYCA DA TVSS CAISGFTL A V 55A06/94 EVQLMES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVNLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGITL AA D 64C03/95 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRY WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD 64G12/96 EVQLVES DYAI WFRQA SIREND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLRMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVQG KPEDTAVYYC DA TVSS CAVSGFT AA LD 65F03/97 EVQMVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNGL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGLT AA LD 55F02/98 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQS G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGLT AA LD 64E12/99 EVQLVES DYAI WFRQA SIRDND RSTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAVSGFT AA LD 60C09/100 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNS EQWYPIYEY GILVT GGSLRLS GVS DSVKW MKPEDTAVYY DA VSS CAVSGITL CAA D 64B02/101 EVQLVES DYAI WFRQA SIRDND RFTISSDNVK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGFLRLT GVS DSVKG KPEDTAVYYC DA TVSS CAVSGFT AA LD 64A03/102 EVQLLES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRIG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGFT AA LD 64C07/103 EVHLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGVERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGLT AA LD 60A06/104 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRY WGQ GGGLVQA G PGKDRE GSTYYA NTVYLQMNSL GEQWYPIYE GTVV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD 64B01/105 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GILVT GGTLRLS GVS DSVKG KPEDTAVYYC DA VSS CAVSGFT AA LD 64G01/106 EVQLVES DYAI WFRQA SIRDND RFIISSDNDKN VPAGRLRY WGQ GGGLVQA G PGKERE GSTYYA TVYLQMNNLK GEQWYPIYE GTLV GGSLRLS GVS DSVKG PEDTAVYYCA YDA TVSS CAASGFT A LD 56A07/107 EVQLVES DYAI WFRQA SIRDND RSTISSDNAR VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVFLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGFT AA LD 58D10/108 EELLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGSVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLKLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGLT AA LD 65F01/109 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSRYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGLT AA LD 53A06/110 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGSLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGLT AA LD 55G03/111 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGSLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG KPEDTAVYYC DA TVSS CAASGLT AA LD
TABLE-US-00017 TABLE 10-B Single mutations or combination thereof yielding improvements in off-rate Fold improvement Mutation(s) off-rate A24V 2 F27I 1.9-2.2 F27L; L100il 9.4 L29I; L100il 5.6-6.0 F100bY; L100il 5.1-9.2 L100il 3.1-7.0
TABLE-US-00018 TABLE 11 Amino acid sequence of affinity matured anti-Ang2 VHHs VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 00903/112 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC DA TVSS CAASGFT AA LD 00904/113 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGFT AA LD 00905/114 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC DA TVSS CAASGFTI AA D 00906/115 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGFTI AA D 00907/116 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC DA TVSS CAASGITL AA D 00908/117 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGITL AA D 00909/118 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC DA TVSS CAASGITI AA D 00910/119 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGITI AA D 00911/120 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC DA TVSS CAASGLT AA LD 00912/121 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGLT AA LD 00913/122 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPIYEY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC DA TVSS CAASGLTI AA D 00914/123 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGLTI AA D
TABLE-US-00019 TABLE 12 Overview of T.sub.m, IC.sub.50 (pM) in human, mouse and cyno Ang2 competition ELISA and hAng1/hAng2 IC.sub.50 ratios of affinity matured variants of VHH 00027 TSA Tm @ pH IC.sub.50 in Ang2/Tie2 ELISA 7.0 hAng2 mAng2 cAng2 hAng1/hAng2 VHH ID (° C.) (pM) (pM) (pM) IC.sub.50 ratio 00027 61.1 672 1975 728 >14,878 00042 61.9 646 1910 753 >15,476 00903 64.0 89 n.d. n.d. >112,202 00904 64.4 62 n.d. n.d. >162,181 00905 64.0 79 n.d. n.d. >125,893 00906 64.4 45 n.d. n.d. >223,872 00907 67.3 62 n.d. n.d. >162,181 00908 67.3 45 85 79 >192,014 00909 66.1 53 n.d. n.d. >190,546 00910 66.1 42 n.d. n.d. >239,883 00911 66.1 59 n.d. n.d. >169,824 00912 66.1 62 n.d. n.d. >162,181 00913 64.0 42 n.d. n.d. >239,883 00914 64.4 37 n.d. n.d. >269,153
[0302] From potency, T.sub.m and sequence perspective VHH 00908 is taken forward into a second cycle of combined affinity maturation and sequence optimization (Example 7.3).
Example 7
[0303] Sequence Optimization of Selected VHH 1D01, 28D10 and 37F02
[0304] 7.1 VHH 1D01
[0305] The amino acid sequence of anti-Ang2 VHH 1D01 (see
[0306] These variants are characterized as purified protein in the human (
TABLE-US-00020 TABLE 13 Overview of T.sub.m, IC.sub.50 (nM) in human, mouse and cyno Ang2 competition ELISA and hAng1/hAng2 IC.sub.50 ratios of sequence optimized variants of VHH 1D01 TSA IC.sub.50 in Ang2-Tie2 ELISA hAng1/ % FR VHH T.sub.m (° C.) hAng2 mAng2 cAng2 hAng2 identity ID @ pH7 (nM) (nM) (nM) IC.sub.50 ratio AbM 1D01 65.0 6.6 12.4 9.1 >1,511 85.4 00039 61.5 11.9 28.1 6.4 n.d 91.0 00040 64.0 6.4 29.0 5.8 n.d 92.1 00049 66.1 6.9 20.3 4.5 n.d 89.9 00050 67.7 6.0 18.6 4.8 n.d 91.0 00051 64.8 16.1 37.9 15 n.d 89.9 00921 67.3 30.4 27.0 37 >329 91.0 00925 66.5 22.1 28.4 33 >453 89.9 n.d., not determined
TABLE-US-00021 TABLE 14 Affinity K.sub.D of purified VHH 00921 for recombinant human, cyno, mouse and rat Ang2 human Ang2-FLD cyno Ang2-FLD k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) 00921 4.0E+06 2.7E−02 6.6E−09 1.1E+06 2.5E−02 2.3E−09 mouse Ang2-FLD rat Ang2-FLD k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) 00921 3.3E+06 2.5E−02 7.5E−09 1.2E+06 4.9E−02 4.2E−08
TABLE-US-00022 TABLE 15 Amino acid sequence of sequence optimized variants of anti-Ang2 VHH 1D01 VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 00039/124 EVQLVES DYAL WFRQA CIRCSD RFTISRDNSK SIVPRSKLE WGQ GGGLVQP G AGKERE GSTYYA NTVYLQMNSL PYEYDA GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFT AA FD 00040/125 EVQLVES DYAL WFRQA CIRCSD RFTISRDNSK SIVPRSKLE WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL PYEYDA GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFT AA FD 00049/126 EVQLVES DYAL WFRQA CIRCSD RFTISSDNSK SIVPRSKLE WGQ GGGLVQP G AGKERE GSTYYA NTVYLQMNSL PYEYDA GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFT AA FD 00050/127 EVQLVES DYAL WFRQA CIRCSD RFTISSDNSK SIVPRSKLE WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL PYEYDA GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFT AA FD 00051/128 EVQLVES DYAL WFRQA CIRCSG RFTISSDNSK SIVPRSKLE WGQ GGGLVQP G AGKERE GSTYYA NTVYLQMNSL PYEYDA GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFT AA FD 00921/129 EVQLVES DYAL WFRQA CIRCSG RFTISSDNSK SIVPRSKLE WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL PYEYDA GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFT AA FD 00925/130 DVQLVES DYAL WFRQA CIRCSG RFTISSDNSK SIVPRSKLE WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL PYEYDA GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFT AA FD
[0307] 7.2 VHH 37F02
[0308] The amino acid sequence of anti-Ang2 VHH 37F02 (see
[0309] These variants are characterized as purified protein in the human (
TABLE-US-00023 TABLE 16 Overview of T.sub.m, IC.sub.50 (pM) in human, mouse and cyno Ang2 competition ELISA and hAng1/hAng2 IC.sub.50 ratios of cycle 1 sequence optimized variants of VHH 37F02 TSA IC.sub.50 in Ang2-Tie2 ELISA % FR VHH Tm (° C.) hAng2 mAng2 cAng2 hAng1/hAng2 identity ID @ pH7 (pM) (pM) (pM) IC.sub.50 ratio AbM 37F02 66.1 77 110 150 >130,317 87.6 00044 66.9 69 91 130 n.d. 91.0 00045 71.1 120 110 160 n.d. 92.1 00046 69.8 95 83 160 n.d. 91.0
[0310] A NNK library approach is used to knock out two potential post-translational modifications sites in CDR3: i) oxidation sensitive Met on position 100e and ii) Asp isomerization site on position D.sub.95S.sub.96. Since D.sub.54G is tolerated (VHH 00046 and 00920; Table 16 and Table 17) no NNK approach was used to knock out this potential Asp isomerization site.
[0311] In the end, 3 NNK libraries containing VHH clones carrying substitutions at positions D.sub.95, S.sub.96 and M.sub.100e to all other amino acids are screened in a hAng2/hTie2 competition AlphaScreen assay (Example 2). Briefly, periplasmic extracts containing expressed VHH are screened at 3 different dilutions (corresponding roughly to EC.sub.20, EC.sub.50 and EC.sub.80 of the parental VHH 37F02) and changes in % inhibition at the different dilution points are compared to parental 37F02. Based on the screening results and the data shown in Table 17, 8 additional cycle 2 VHH variants are constructed (based on VHH 00920 backbone) carrying different knock-out combinations of D.sub.95S.sub.96 and M.sub.100e (
[0312] All these variants are characterized as purified protein in the human (
TABLE-US-00024 TABLE 17 Overview of T.sub.m, IC.sub.50 (pM) in human, mouse and cyno Ang2 competition ELISA and hAng1/hAng2 IC.sub.50 ratios of cycle 2 sequence optimized variants of VHH 37F02 TSA IC50 in Ang2-Tie2 ELISA hAng1/ % FR Tm (° C.) hAng2 mAng2 cAng2 hAng2 identity VHH ID @ pH7 (pM) (pM) (pM) ratio AbM 37F02 66.1 77 110 150 >130,317 87.6 00920 73.6 130 150 230 >79,159 92.1 00924 73.1 110 n.d. n.d. n.d. 91.0 00926 71.5 250 200 290 >39,446 92.1 00927 73.6 220 190 330 >44,668 92.1 00928 72.7 220 200 390 >45,604 92.1 00929 69.0 150 150 250 >65,013 92.1 00930 69.8 190 170 310 >53,580 92.1 00931 69.4 170 150 290 >57,677 92.1 n.d., not determined
TABLE-US-00025 TABLE 18 Affinity K.sub.D of purified VHH 00928 for recombinant human, cyno, mouse and rat Ang2 human Ang2-FLD cyno Ang2-FLD k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) 00928 6.2+05 4.0E−05 6.4E−11 9.7E+05 4.9E−05 5.0E−11 mouse Ang2-FLD rat Ang2-FLD k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) 00928 4.2E+05 7.3E−05 1.7E−10 1.8E+05 5.4E−05 2.9E−10
TABLE-US-00026 TABLE 19-1 VHH ID/ SEQ ID NOs: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 00044/131 EVQLVES YYAI WFRQA CISSSD RFTISRDNSK DSGGYIDYD WGQ GGGLVQP G PGKERE GITYYV NTVYLQMNSL CMGLGYDY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFA AT LD 00045/132 EVQLVES YYAI WFRQA CISSSD RFTISRDNSK DSGGYIDYD WGQ GGGLVQP G PGKERE GITYYA NTVYLQMNSL CMGLGYDY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFA AT LD 00046/133 EVQLVES YYAI WFRQA CISSSG RFTISRDNSK DSGGYIDYD WGQ GGGLVQP G PGKERE GITYYV NTVYLQMNSL CMGLGYDY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFA AT LD
TABLE-US-00027 TABLE 19-2 VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 00920/134 EVQLVES YYAI WFRQA CISSSG RFTISRDNSK DSGGYIDYD WGQ GGGLVQP G PGKERE GITYYA NTVYLQMNSL CMGLGYDY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFA AT LD 00924/135 DVQLVES YYAI WFRQA CISSSG RFTISRDNSK DSGGYIDYD WGQ GGGLVQP G PGKERE GITYYA NTVYLQMNSL CMGLGYDY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFA AT LD 00926/136 EVQLVES YYAI WFRQA CISSSG RFTISRDNSK DSGGYIDYD WGQ GGGLVQP G PGKERE GITYYA NTVYLQMNSL CQGLGYDY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFA AT LD 00927/137 EVQLVES YYAI WFRQA CISSSG RFTISRDNSK DSGGYIDYD WGQ GGGLVQP G PGKERE GITYYA NTVYLQMNSL CRGLGYDY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFA AT LD 00928/138 EVQLVES YYAI WFRQA CISSSG RFTISRDNSK DSGGYIDYD WGQ GGGLVQP G PGKERE GITYYA NTVYLQMNSL CSGLGYDY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFA AT LD 00929/139 EVQLVES YYAI WFRQA CISSSG RFTISRDNSK ESGGYIDYD WGQ GGGLVQP G PGKERE GITYYA NTVYLQMNSL CQGLGYDY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFA AT LD 00930/140 EVQLVES YYAI WFRQA CISSSG RFTISRDNSK ESGGYIDYD WGQ GGGLVQP G PGKERE GITYYA NTVYLQMNSL CRGLGYDY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFA AT LD 00931/141 EVQLVES YYAI WFRQA CISSSG RFTISRDNSK ESGGYIDYD WGQ GGGLVQP G PGKERE GITYYA NTVYLQMNSL CSGLGYDY GTLV GGSLRLS GVS DSVKG RPEDTAVYYC TVSS CAASGFA AT LD
[0313] 7.3 VHH 28D 10
[0314] The amino acid sequence of anti-Ang2 VHH 28D10 (see
[0315] These variants are characterized as purified protein in the human (
TABLE-US-00028 TABLE 20 Overview of T.sub.m, IC.sub.50 (nM) in human, mouse and cyno Ang2 competition ELISA and hAng1/hAng2 IC.sub.50 ratios of cycle 1 sequence optimized variants of VHH 28D10 TSA IC50 in Ang2-Tie2 ELISA hAng1/ % FR VHH ID Tm (° C.) hAng2 mAng2 cAng2 hAng2 identity @ pH7 (nM) (nM) (nM) ratio AbM 28D10 66.1 1.8 5.7 2.1 >5,675 86.5 00025 63.2 2.1 4.9 1.7 n.d. 87.6 00026 68.2 1.0 2.6 1.1 n.d. 87.6 00027 61.1 0.7 2.0 0.7 >14,878 87.6 00041 62.3 0.6 1.7 0.9 n.d. 89.9 00042 61.9 0.7 1.9 0.8 >15,476 91.0 00043 65.7 9.5 29.2 12.6 n.d. 91.0 00048 63.2 0.6 2.1 1.0 n.d. 87.6 00052 61.1 1.0 2.1 0.8 n.d. 89.9 00053 64.8 10.7 36.7 13.8 n.d. 91.0 00054 65.7 9.5 22.9 11.7 n.d. 92.1 00055 63.6 1.8 4.6 2.0 n.d. 87.6 n.d., not determined
[0316] An additional set of variants (cycle 2) is created to explore the affinity maturation substitution on position A.sub.24V and to further explore C.sub.50X-S.sub.53X variants (Table 21;
[0317] Additionally, melting temperature (Tm) of each variant is determined in thermal shift assay (Example 6).
TABLE-US-00029 TABLE 21 Overview of T.sub.m, IC.sub.50 (nM) in human, mouse and cyno Ang2 competition ELISA and hAng1/hAng2 IC.sub.50 ratios of cycle 2 sequence optimized variants of VHH 28D10 TSA IC50 in Ang2-Tie2 ELISA hAng1/ % FR Tm (° C.) hAng2 mAng2 cAng2 hAng2 identity VHH ID @ pH7 (nM) (nM) (nM) ratio AbM 28D10 66.1 1.8 5.7 2.1 >5,675 86.5 00898 66.1 1.2 n.d. n.d. >8,279 87.6 00899 61.9 0.9 n.d. n.d. >11,749 87.6 00900 64.3 0.2 n.d. n.d. >42,855 86.5 00901 67.3 1.2 n.d. n.d. >8,054 87.6 00902 66.1 1.3 n.d. n.d. >7,907 87.6 00919 67.3 0.9 1.8 0.7 >11,212 91.0 00923 67.7 0.7 n.d. n.d. n.d. 89.9 n.d., not determined
[0318] In parallel, a NNK library approach is used to knock out two post-translational modifications sites in CDR2: two sequential Asp isomerization sites on position D.sub.52aS.sub.53 and D.sub.54G.sub.55. Since D.sub.54G is tolerated (Example 6; Table 20) no NNK approach was used to knock out this potential Asp isomerization site. In the end, 2 NNK libraries containing VHH clones carrying substitutions at positions D.sub.52a and S.sub.53 to all other amino acids are screened in a hAng2/hTie2 competition AlphaScreen assay (Example 2). Briefly, periplasmic extracts containing expressed VHH are screened at 3 different dilutions (corresponding roughly to EC.sub.20, EC.sub.50 and EC.sub.80 of the reference 00902) and changes in % inhibition at the different dilution points are compared to reference 00902. Based on the screening results and the data shown in Table 21, 7 additional VHH cycle 3 variants are constructed (based on 00908 backbone) (see
TABLE-US-00030 TABLE 22 Overview of T.sub.m, IC.sub.50 (nM) in human, mouse and cyno Ang2 competition ELISA and hAng1/hAng2 IC.sub.50 ratios of cycle 3 sequence optimized variants of VHH 28D10 TSA IC50 in Ang2/Tie2 HUVEC Tm @ ELISA hAng1/ survival % FR pH 7.0 hAng2 mAng2 cAng2 hAng2 IC.sub.50 identity VHH ID (° C.) (pM) (pM) (pM) IC.sub.50 ratio (nM) AbM 00027 61.1 672 1,975 728 >14,878 n.d. 87.6 00908 67.3 45 85 79 >192,014 n.d. 91.0 00932 70.5 49 38 54 >205,747 n.d. 89.9 00933 70.5 56 62 71 >179,887 n.d. 91.0 00934 67.1 64 74 71 >156,675 n.d. 91.0 00935 68.6 68 78 69 >146,218 n.d. 91.0 00936 73.9 45 42 60 >223,872 n.d. 89.9 00937 72.0 54 52 85 >186,209 n.d. 89.9 00938 73.9 50 55 91 >201,064 4.3 89.9 00956 68.0 1,300 2,200 830 >7,727 6.8 91.0 n.d. not determined
TABLE-US-00031 TABLE 23 Affinity K.sub.D of purified VHHs 00919, 00938 and 00956 for recombinant human, cyno, mouse and rat Ang2 human Ang2-FLD cyno Ang2-FLD k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) 00919 9.5E+05 1.5E−03 1.6E−09 2.3E+06 1.2E−03 5.4E−10 00938 1.6E+06 2.8E−05 1.7E−11 2.6E+06 2.2E−05 8.7E−12 00956 1.3E+06 1.5E−03 1.2E−09 1.7E+06 1.3E−03 7.2E−10 mouse Ang2-FLD rat Ang2-FLD k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) 00919 1.3E+06 3.9E−03 3.0E−09 5.0E+05 5.0E−03 1.0E−08 00938 1.1E+06 5.6E−05 5.0E−11 5.1E+05 7.2E−05 1.4E−10 00956 9.8E+05 3.9E−03 4.0E−09 4.2E+05 5.2E−03 1.3E−08
TABLE-US-00032 TABLE 24-1 VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 00025/142 EVQLVES DYAI WFRQA TIRDSD RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD 00026/143 EVQLVES DYAI WFRQA AIRDSD RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD 00027/144 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD 00041/145 EVQLVES DYAI WFRQA SIRDND RFTISSDNAK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGFT AA LD 00042/146 EVQLVES DYAI WFRQA SIRDND RFTISSDNSK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGFT AA LD 00043/147 EVQLVES DYAI WFRQA SIRDND RFTISRDNAK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGFT AA LD 00048/148 EVQLVES DYAI WFRQA SIRDSD RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD 00052/149 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGFT AA LD 00053/150 EVQLVES DYAI WFRQA SIRDND RFTISRDNDK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGFT AA LD 00054/151 EVQLVES DYAI WFRQA SIRDND RFTISRDNSK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGFT AA LD 00055/152 EVQLVES DYAI WFRQA SIRDNG RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD
TABLE-US-00033 TABLE 24-2 VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 00898/153 EVQLVES DYAI WFRQA AIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD 00899/154 EVQLVES DYAI WFRQA SIRDIDG RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE STYYAD NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS SVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD 00900/155 EVQLVES DYAI WFRQA SIRDND RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAVSGFT AA LD 00901/156 EVQLVES DYAI WFRQA AIRDSD RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD 00902/157 EVQLVES DYAI WFRQA AIRDSG RFTISSDNDK VPAGRLRFG WGQ GGGLVQA G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG KPEDTAVYYC YDA TVSS CAASGFT AA LD
TABLE-US-00034 TABLE 24-3 VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 00919/158 EVQLVES DYAI WFRQA AIRDNG RFTISSDNSK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGFT AA LD 00923/159 DVQLVES DYAI WFRQA AIRDNG RFTISSDNSK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGFT AA LD 00932/160 EVQLVES DYAI WFRQA SIRDNG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAVSGITL AA D 00933/161 EVQLVES DYAI WFRQA AIRDNG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGITL AA D 00934/162 EVQLVES DYAI WFRQA AIRESG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGITL AA D 00935/163 EVQLVES DYAI WFRQA AIRSSG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGITL AA D 00936/164 EVQLVES DYAI WFRQA AIRDNG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAVSGITL AA D 00937/165 EVQLVES DYAI WFRQA AIRESG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAVSGITL AA D 00938/166 EVQLVES DYAI WFRQA AIRSSG RFTISSDNSK VPAGRLRY WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL GEQWYPIYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAVSGITL AA D 00956/167 EVQLVES DYAI WFRQA AIRSSG RFTISSDNSK VPAGRLRFG WGQ GGGLVQP G PGKERE GSTYYA NTVYLQMNSL EQWYPLYE GTLV GGSLRLS GVS DSVKG RPEDTAVYYC YDA TVSS CAASGFT AA LD