BISPECIFIC BINDING MOLECULES BINDING TO VEGF AND ANG2
20170247475 · 2017-08-31
Inventors
- Andreas Gschwind (Ingelheim am Rhein, DE)
- Rene Georg Ott (Perchtoldsdorf, AT)
- Joachim Boucneau (De Pinte, BE)
- Marie-Ange Buyse (Merelbeke, BE)
- Erik Depla (Destelbergen, BE)
Cpc classification
C07K16/468
CHEMISTRY; METALLURGY
C07K2317/569
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/22
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/22
CHEMISTRY; METALLURGY
C07K2317/62
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
Bispecifc binding molecules binding to both VEGF and Ang2, preferably in the form of immunoglobulin single variable domains like VHHs and domain antibodies, pharmaceutical compositions containing the same and their use in the treatment of diseases that are associated with VEGF- and/or Ang2-mediated effects on angiogenesis are disclosed. Further, nucleic acids encoding bispecific binding molecules, host cells and methods for preparing same are also described.
Claims
1-26. (canceled)
27. An Ang2-binding VHH with an amino acid sequence selected from acid sequences shown in SEQ ID NOs: 214, 215, 216, 217, 218, 219, 220, 221, 222, or 223.
28. An immunoglobulin single variable domain which has been obtained by humanization of a VHH defined in claim 27.
29-37. (canceled)
38. A method of treating a disease that is associated with VEGF- and/or Ang2-mediated effects on angiogenesis comprising administering to a patient an effective amount of a pharmaceutical composition comprising at least one binding molecule as the active ingredient and a pharmaceutically acceptable carrier, diluent or excipient, wherein the said binding molecule comprises: at least one VEGF-binding component, at least one serum albumin binding component, and at least one Angiopoietin-2 (Ang2) binding component, wherein said VEGF-, serum albumin and Ang2-binding components are immunoglobulin single variable domains, each immunoglobulin single variable domain consisting of four framework regions and three complementarity determining regions (CDRs), and wherein: said VEGF binding immunoglobulin single variable domain, comprises the following CDR sequences: TABLE-US-00050 (SEQ ID NO: 450) CDR1: SYSMG (SEQ ID NO: 465) CDR2: AISKGGYKYDAVSLEG (SEQ ID NO: 4) CDR3: SRAYGSSRLRLADTYEY said serum albumin binding immunoglobulin single variable domain, comprises the following CDR sequences: TABLE-US-00051 (SEQ ID NO: 255) CDR1: SFGMS (SEQ ID NO: 256) CDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 257) CDR3: GGSLSR; and said Ang2-binding immunoglobulin single variable domain, comprises the following CDR sequences: TABLE-US-00052 (SEQ ID NO: 248) CDR1: DYAIG (SEQ ID NO: 49) CDR2: AIRSSGGSTYYADSVKG (SEQ ID NO: 250) CDR3: VPAGRLRYGEQWYPIYEYDA.
39. The method of claim 38 wherein the disease is selected from cancer and cancerous diseases.
40. The method of claim 38 wherein the disease is eye diseases.
41. An isolated nucleic acid molecule encoding a bispecific binding molecule, wherein said binding molecule comprises: at least one VEGF-binding component, at least one serum albumin binding component, and at least one Angiopoietin-2 (Ang2) binding component, wherein said VEGF-, serum albumin and Ang2-binding components are immunoglobulin single variable domains, each immunoglobulin single variable domain consisting of four framework regions and three complementarity determining regions (CDRs), and wherein: said VEGF binding immunoglobulin single variable domain, comprises the following CDR sequences: TABLE-US-00053 (SEQ ID NO: 450) CDR1: SYSMG (SEQ ID NO: 465) CDR2: AISKGGYKYDAVSLEG (SEQ ID NO: 4) CDR3: SRAYGSSRLRLADTYEY said serum albumin binding immunoglobulin single variable domain, comprises the following CDR sequences: TABLE-US-00054 (SEQ ID NO: 255) CDR1: SFGMS (SEQ ID NO: 256) CDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 257) CDR3: GGSLSR; and said Ang2-binding immunoglobulin single variable domain, comprises the following CDR sequences: TABLE-US-00055 (SEQ ID NO: 248) CDR1: DYAIG (SEQ ID NO: 249) CDR2: AIRSSGGSTYYADSVKG (SEQ ID NO: 250) CDR3: VPAGRLRYGEQWYPIYEYDA.
42. An isolated nucleic acid molecule encoding a bispecific binding molecule, wherein said bispecific binding molecule comprises: a VEGF-binding VHH, comprising the following CDR sequences: TABLE-US-00056 (SEQ ID NO: 450) CDR1: SYSMG (SEQ ID NO: 465) CDR2: AISKGGYKYDAVSLEG (SEQ ID NO: 4) CDR3: SRAYGSSRLRLADTYEY a serum albumin binding VHH, comprising the following CDR sequences: TABLE-US-00057 (SEQ ID NO: 255) CDR1: SFGMS (SEQ ID NO: 256) CDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 257) CDR3: GGSLSR; and an Ang2-binding VHH, comprising the following CDR sequences: TABLE-US-00058 (SEQ ID NO: 248) CDR1: DYAIG (SEQ ID NO: 249) CDR2: AIRSSGGSTYYADSVKG (SEQ ID NO: 250) CDR3: VPAGRLRYGEQWYPIYEYDA.
43. The isolated nucleic acid molecule of claim 42, wherein said VEGF-binding VHH comprised in said bispecific binding molecule has the amino acid sequence shown in SEQ ID NO: 57.
44. The isolated nucleic acid molecule of claim 42, wherein said serum albumin binding VHH comprised in said bispecific binding molecule has the amino acid sequence shown in SEQ ID NO: 254.
45. The isolated nucleic acid molecule of claim 42, wherein said Ang2-binding VHH comprised in said bispecific binding molecule has the amino acid sequence shown in SEQ ID NO: 222.
46. The isolated nucleic acid molecule of claim 42, wherein said serum albumin binding VHH has the amino acid sequence shown in SEQ ID NO: 254 and wherein the said Ang2-binding VHH has the amino acid sequence shown in SEQ ID NO: 222.
47. An isolated nucleic acid molecule encoding a bispecific binding molecule comprising the amino acid sequence shown in SEQ ID NO: 207.
48. An expression vector containing a nucleic acid molecule of claim 41.
49. A host cell containing an expression vector of claim 48.
50. A method of manufacturing a bispecific binding molecule comprising the steps of: culturing a host cell according to claim 49 under conditions that allow expression of said bispecific binding molecule; and recovering or isolating said bispecific binding molecule expressed by said host cell from the culture.
51. The method of claim 50, additionally comprising the step of: further purifying or modifying or formulating said bispecific binding molecule.
Description
BRIEF DESCRIPTION OF THE FIGURES
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MATERIALS AND METHODS
a) Production and Functionality Testing of VEGF109
[0335] A cDNA encoding the receptor binding domain of human vascular endothelial growth factor isoform VEGF165 (GenBank: AAM03108.1; AA residues 27-135) is cloned into pET28a vector (Novagen, Madison, Wis.) and overexpressed in E. coli (BL21 Star DE3) as a His-tagged insoluble protein. Expression is induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37° C. Cells are harvested by centrifugation and lysed by sonication of the cell pellet. Inclusion bodies are isolated by centrifugation. After a washing step with 1% Triton X 100 (Sigma-Aldrich), proteins are solubilized using 7.5M guanidine hydrochloride and refolded by consecutive rounds of overnight dialysis using buffers with decreasing urea concentrations from 6M till 0M. The refolded protein is purified by ion exchange chromatography using a MonoQ5/50GL (Amersham BioSciences) column followed by gel filtration with a Superdex75 10/300 GL column (Amersheim BioSciences). The purity and homogeneity of the protein is confirmed by SDS-PAGE and Western blot. In addition, binding activity to VEGFR1, VEGFR2 and Bevacizumab is monitored by ELISA. To this end, 1 μg/mL of recombinant human VEGF109 is immobilized overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). Serial dilutions of VEGFR1, VEGFR2 or Bevacizumab are added to the VEGF109 coated plate and binding is detected using alkaline phosphatase (AP) conjugated goat anti-human IgG, Fc specific (Jackson Immuno Research Laboratories Inc., West Grove, Pa., USA) and a subsequent enzymatic reaction in the presence of the substrate PNPP (p-nitrophenylphosphate) (Sigma-Aldrich). VEGF109 could bind to VEGFR1, VEGFR2 and Bevacizumab, indicating that the produced VEGF109 is active.
b) KLH Conjugation of VEGF165 and Functionality Testing of KLH-Conjugated VEGF165
[0336] Recombinant human VEGF165 (R&D Systems, Minneapolis, Minn., USA) is conjugated to mariculture keyhole limpet hemocyanin (mcKLH) using the Imject Immunogen EDC kit with mcKLH (Pierce, Rockford, Ill., USA) according to the manufacturer's instructions. Efficient conjugation of the polypeptide to mcKLH is confirmed by SDS-PAGE. Functionality of the conjugated protein is checked by ELISA: 2 μg/mL of KLH conjugated VEGF165 is immobilized overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). Serial dilutions of VEGFR1 or VEGFR2 are added and binding is detected using a horseradish peroxidase (HRP)-conjugated goat anti-human IgG, Fc specific (Jackson Immuno Research Laboratories Inc., West Grove, Pa., USA) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA). The KLH conjugated protein could still interact with VEGFR1, VEGFR2 and Bevacizumab, confirming that the relevant epitopes onVEGF165 are still accessible.
Example 1
[0337] Immunization with Different VEGF Formats Induces a Humoral Immune Response in Llama
1.1 Immunizations
[0338] After approval of the Ethical Committee of the faculty of Veterinary Medicine (University Ghent, Belgium), 4 llamas (designated No. 264, 265, 266, 267) are immunized according to standard protocols with 6 intramuscular injections (100 or 50 μg/dose at weekly intervals) of recombinant human VEGF109. The first injection at day 0 is formulated in Complete Freund's Adjuvant (Difco, Detroit, Mich., USA), while the subsequent injections are formulated in Incomplete Freund's Adjuvant (Difco, Detroit, Mich., USA). In addition, four llamas (designated No. 234, 235, 280 and 281) are immunized according to the following protocol: 5 intramuscular injections with KLH-conjugated human VEGH165 (100 or 50 μg/dose at biweekly intervals) followed by 4 intramuscular injections of human VEGF109 (first dose of 100 μg followed 2 weeks later with three 50 μg/dose at weekly interval).
1.2 Evaluation of VEGF-Induced Immune Responses in Llama
[0339] To monitor VEGF specific serum titers, an ELISA assay is set up in which 2 μg/mL of recombinant human VEGF165 or VEGF109 is immobilized overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). After addition of serum dilutions, bound total IgG is detected using 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′-tetramethylbenzidine) (Pierce, Rockford, Ill., USA). For llamas 264, 265, 266 and 267, an additional ELISA is performed in which the isotype-specific responses against VEGF165 and VEGF109 are evaluated. Isotype specific responses are detected using mouse mAbs specifically recognizing conventional llama IgG1 and the heavy-chain only llama IgG2 and IgG3 [Daley et al. (2005). Clin. Diagn. Lab. Imm. 12:380-386] followed by a rabbit anti-mouse-HRP conjugate (DAKO). ELISAs are developed using TMB as chromogenic substrate and absorbance is measured at 450 nm. The serum titers for each llama are depicted in Table 1.
TABLE-US-00004 TABLE 1 Antibody-mediated specific serum response against VEGF165 and VEGF109 ELISA (recombinant protein solid phase coated) Recombinant human Recombinant human EGF165 VEGF109 Llama Immunogen Total IgG IgG1 IgG2 IgG3 Total IgG IgG1 IgG2 IgG3 234 VEGF165-KLH + ++ n/d n/d n/d ++ n/d n/d n/d VEGF109 235 VEGF165-KLH + ++ n/d n/d n/d ++ n/d n/d n/d VEGF109 280 VEGF165-KLH + + n/d n/d n/d + n/d n/d n/d VEGF109 281 VEGF165-KLH + + n/d n/d n/d + n/d n/d n/d VEGF109 264 VEGF109 n/d ++ + + ++ ++ + + 265 VEGF109 n/d ++ + + + ++ + + 266 VEGF109 n/d ++ + +/− ++ ++ + +/− 267 VEGF109 n/d +/− − − +/− +/− − − n/d, not determined
Example 2
Cloning of the Heavy-Chain Only Antibody Fragment Repertoires and Preparation of Phage
[0340] Following the final immunogen injection, immune tissues as the source of B-cells that produce the heavy-chain antibodies are collected from the immunized llamas. Typically, two 150-ml blood samples, collected 4 and 8 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, total RNA is extracted, which is used as starting material for RT-PCR to amplify the VHH encoding DNA segments, as described in WO2005/044858. 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 gill protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multiple cloning site and a hybrid gIII-pelB leader sequence (pAX050). 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
Selection of VEGF-Specific VHHs Via Phage Display
[0341] VHH phage libraries are used in different selection strategies applying a multiplicity of selection conditions. Variables include i) the VEGF protein format (rhVEGF165, rhVEGF109 or rmVEGF164), ii) the antigen presentation method (solid phase: directly coated or via a biotin-tag onto Neutravidin-coated plates; solution phase: incubation in solution followed by capturing on Neutravidin-coated plates), iii) the antigen concentration and iv) the elution method (trypsin or competitive elution using VEGFR2). All selections are carried out in Maxisorp 96-well plates (Nunc, Wiesbaden, Germany).
[0342] Selections are performed as follows: Phage libraries are incubated at RT with variable concentrations of VEGF antigen, either in solution or immobilized on a solid support. After 2 hrs of incubation and extensive washing, bound phage are eluted. In case trypsin is used for phage elution, the protease activity is immediately neutralized by addition of 0.8 mM protease inhibitor AEBSF. 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 agar plates (LB+amp+glucose.sup.2%) for analysis of individual VHH clones. In order to screen a selection output for specific binders, single colonies are picked from the agar plates and grown in 1 mL 96-deep-well plates. The lacZ-controlled VHH expression is induced by adding IPTG (0.1-1 mM final). Periplasmic extracts (in a volume of ˜80 μL) are prepared according to standard methods.
Example 4
Identification of VEGF-Binding and VEGF Receptor-Blocking VHHs
[0343] Periplasmic extracts are tested for binding to human VEGF165 by ELISA. In brief, 2 μg/mL of recombinant human VEGF165 is immobilized overnight at 4° C. in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1%). After addition of typically a 10-fold dilution of the periplasmic extracts, VHH binding is detected using a mouse anti-myc (Roche) and an anti-mouse-HRP conjugate (DAKO). Clones showing ELISA signals of >3-fold above background are considered as VEGF binding VHHs.
[0344] In addition, periplasmic extracts are screened in a human VEGF165/human VEGFR2 AlphaScreen assay (Amplified Luminescent Proximity Homogeneous Assay) to assess the blocking capacity of the VHHs. Human VEGF165 is biotinylated using Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill., USA). Human VEGFR2/Fc chimera (R&D Systems, Minneapolis, Minn., USA) is captured using an anti-humanFc VHH which is coupled to acceptor beads according to the manufacturer's instructions (Perkin Elmer, Waltham, Mass., US). To evaluate the neutralizing capacity of the VHHs, periplasmic extracts are diluted 1/25 in PBS buffer containing 0.03% Tween 20 (Sigma-Aldrich) and preincubated with 0.4 nM biotinylated human VEGF165 for 15 minutes at room temperature (RT). To this mixture the acceptor beads (10 μg/ml) and 0.4 nM VEGFR2-huFc are added and further incubated for 1 hour at RT in the dark. Subsequently donor beads (10 μg/ml) are added followed by incubation of 1 hour at RT in the dark. Fluorescence is measured by reading plates on the Envision Multi label Plate reader (Perkin Elmer, Waltham, Mass., USA) using an excitation wavelength of 680 nm and an emission wavelength between 520 nm and 620 nm. Periplasmic extract containing irrelevant VHH is used as negative control. Periplasmic extracts containing anti-VEGF165 VHHs which are able to decrease the fluorescence signal with more than 60% relative to the signal of the negative control are identified as a hit. All hits identified in the AlphaScreen are confirmed in a competition ELISA. To this end, 1 μg/mL of human VEGFR2 chimera (R&D Systems, Minneapolis, Minn., USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Fivefold dilutions of the periplasmic extracts are incubated in the presence of a fixed concentration (4 nM) of biotinylated human VEGF165 in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma-Aldrich). Binding of these VHH/bio-VEGF165 complexes to the human VEGFR2 chimera coated plate is detected using horseradish peroxidase (HRP) conjugated extravidin (Sigma, St Louis, Mo., USA). VHH sequence IDs and the corresponding AA sequences of VEGF-binding (non-receptor-blocking) VHHs and inhibitory (receptor-blocking) VHHs are listed in Table 2 and Table 3, respectively.
TABLE-US-00005 TABLE 2 Sequence IDs and AA sequences of monovalent “non-receptor-blocking” anti-VEGF VHHs (FR, framework; CDR, complementary determining region) VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII01C02/ EVQLVESGGG SYGMG WFRQSPG AISEYSNTY RFTISRDNTKNTV SPTILLTTEQWYK WGQGTQ 58 LVQAGGSLRL KEREFVS CSDSVRG YLQMNSLTPDDTA Y VTVSS SCTASGGSFS IYYCAA VEGFBII01E07/ EVQLVESGGG ASDMG WFRQAPG AINWSGLST RFTISRDNDNGAL GRIPSSSRFSSPA WGQGTQ 59 LVQAGDSLRL KEREFVA FYTDSVKG YLQMNTLKPEDTA AYAS VTVSS SCVATGRTFR VYSCAA VEGFBII03D12/ EVQLVESGGG ITVMA WFRQAPG AITWSAPTT RFTISRDNAKNTV DRFKGRSIVTPSD WGQGTQ 60 LVQAGGSLRL KEREFVA YYADSVKG YLRMNSLKPEDSA YRY VTVSS SCTASTSIYT IYYCAA VEGFBII04B08/ EVQLVESGGG DITVA WYRQAPG TITPSGYTY RFTISRDNSKNIV QFY WGQGTQ 61 LVQPGGSLRL IQRQLVA YWDFVKG YLQMNSLKPEDTA VTVSS SCAASGSAVG AYYCNT VEGFBII05B02/ EVQLVESGGG TDDVG WFRQAPG VIRWSTGGT RFTLSRDNAKNTM RSRPLGAGAWYSG WGQGTQ 62 LVQAGGSLRL KEREFVA YTSDSVKG YLQMNSLKPEDTA EKHYNY VTVSS SCAASGRTFS VYYCAA VEGFBII05B03/ EVQLVESGGG HYNMG WFRQAPG SIRGGGGST RFTISRENAKNTV TAFYRGPYDYDY WGQGTQ 63 LAQAGDSLRL KEREFVA TYANSVKD YLQMNSLKPEDTA VTVSS SCAASGRSFS VYYCAA VEGFBII05B05/ EVQLVESGGG SMA WYRQAPG RISSGGTTA RFTISRDNSKNTV FSSRPNP WGAGTQ 64 LVQPGGSLRL KHRELVA YVDSVKG YLQMNSLKAEDTA VTVSS SCVASGIRFM VYYCNT VEGFBII06G02/ EVQLVESGGG NNAMA WYRQAPG RISSGGGFT RFTVSRDNAKNTV AYRTYNY WGQGTQ 65 LVQPGGSLRL KQRELVA YYLDSVKG YLQMNSLKPEDTA VTVSS SCAASGNIFS VYYCNA VEGFBII07A03/ EVQLVESGGG ITVMA WFRQAPG AITWSAPSS RFTISRDNAKNTV DRFKGRSIVTRSD WGQGTQ 66 LVQAGGSLRL KESEFVA YYADSVKG YLQMNSLKPEDSA YKY VTVSS SCAASTSIYS IYYCAA VEGFBII07A06/ EVQLVESGGG ISVMA WFRQAPG AITWSAPTT RFTISRDNAKNTV DRFKGRSIVTRSD WGQGTQ 67 LVQAGGSLRL KERAFVA YYADSVKG YLQTNSLKPEDSA YRY VTVSS SCAVSTSIYS IYYCAA VEGFBII07D08/ EVQLVESGGG NYAMA WFRQAPG AINQRGSNT RFTISRDSAKNSV STWYGYSTYARRE WGQGTQ 68 LVQTGGSLRL KEREFVS NYADSVKG FLQMNSLKPEDTA EYRY VTVSS SCAASGRTFS VYYCAA VEGFBII08D09/ EVQLVESGGG DNVMG WFRQAAG HISRGGSRT RFTISRDNTKKTM SRSVALATARPYD WGQGTQ 69 LVQAGGSLRL KEREFVA EYAESVKG YLQMNSLKPEDTA Y VTVSS SCAASGRSFS VYYCAA VEGFBII08E07/ EVQLVESGGG SYYMG WFRQAPG TISWNKIST RFTVSRDNNKNTV DASRPTLRIPQY WGQGTQ 70 LAQAGGSLRL KEREFVA IYTDSVKG YLQMNSLKPEDTA VTVSS SCTTSGLTFS VYYCAA VEGFBII08E06/ EVQLVESGGG SDVMG WYRQAPG FIRSLGSTY RFTISRDDAANTV RFSGESY WGQGTP 71 LVQPGGSLRL KQRELVA YAGSVKG YLQMNNLKPEDTA VTVSS SCAASGSIVR VYYCNA VEGFBII08F07/ EVQLVESGGG LYAMG WFRQAPG AITWSAGDT RFTISRDNARNTV RQWGGTYYYHGSY WGQGTQ 72 LVQAGGSLRL REREFLS QYADSVKG NLQMNGLKPEDTA AY VTVSS SCAVSGSTFG VYYCAG VEGFBII09A09/ EVQLVESGGG SMA WYRQAPG RISSEGTTA RFTISRDNSKNTV FSSRPNP WGAGTT 73 LVQPGGSLRL KHRELVA YVDSVKG YLQMNSLKAEDTA VTVSS SCVASGIRFM VYYCNT VEGFBII09A12/ EVQLVESGGG TDDVG WFRQAPG VIRWSTGGT RFTLSRDNAKNTM RSRPLGAGAWYTG WGQGTQ 74 LVQAGGSLRL KEREFVA YTSDSVAG YLQMNSLKPEDTA ETRYDS VTVSS SCAASGRTFS VYYCAA VEGFBII09D05/ EVQLVESGGG RYGMG WFRQAPG AISEYDNVY RFTISRDNSKSTV SPTILLSTDEWYK WGRGTQ 75 LVQPGDSLRL KEREFVI TADSVRG YLQMNSLKSEDTA Y VTVSS SCAASGLSFS VYYCAA VEGFBII09F05/ EVQLVESGGG TDDVG WFRQAPG VIRWSTGGT RFTLSRDNAKNTM RSRPLGAGAWYTG WGQGTQ 76 LVQAGGSLRL KEREFVA YTSDSVKG YLQMNSLKPEDTA ETRYNY VTVSS SCAASGRTFS VYYCAA VEGFBII10C07/ EVQLVESGGG NYAMG WFRQVPG VITRSPSNT RFTISRDNAKNIV HYWNSDSYTYTDS WGQGTQ 77 LVQAGGSLSL REREFVA YYTDSVKG YLQMNSLKPEDTA RWYNY VTVSS SCAASARAFS VYYCAA VEGFBII10E07/ EVQLVESGGG NYAMG WFRQAPG DISSSGINT RFTISRDNAKNTV SAWWYSQMARDNY WGQGTQ 78 LVQAGGSLRL KERVLVA YVADAVKG YLQMNSLKPEDTA RY VTVSS SCAASGRTFS VYYCAA VEGFBII10G04/ EVQLVESGGG RYAMG WFRQAPG SINTSGKRT RFAVSRDNAKNTG DRFFGSDSNEPRA WGQGTQ 79 LVQAGGSLRL KEREFVA SYADSMKG YLQMNSLKLEDTA YRY VTVSS SCAASGDTLS TYYCAA VEGFBII10G05/ EVQLVESGGG NYNMG WFRQAPG TIRHHGYDT RFTISRDNAKNTV KLFWDMDPKTGFS WGQGTQ 80 LVQAGESLRL KEREFVA YYAESVKG YLQMNSLKPEDTA S VTVSS SCVASGITFS LYSCAK VEGFBII11C08/ EVQLVESGGG SYGLG WFRQAPG AIGWSGSST RFTVSVDNAKNTV KVRNFNSDWDLLT WGQGTQ 81 LVQAGGSLRL KEREFVA YYADSVKG YLKMNSLEPEDTA SYNY VTVSS SCAASGRTLS VYYCAA VEGFBII11C11/ EVQLVESGGG SYAIG WFRQAPG RISWSGANT RFTISRGNAKNTV QTTSKYDNYDARA WGQGTQ 82 LVQAGGSLML REREFVA YYADSVKG YLQMNSLKPEDTA YGY VTVSS SCAASGRALS AYYCAA VEGFBII11D09/ EEQLVESGGG SYAIG WFRQAPG RISWSGANT RFTISRGNAKNTV QTTSKYDNYDARA WGQGTQ 83 LVQAGGSLML REREFVA YYADSVKG YLQMNSLKPEDTA YGY VTVSS SCAASGRALS AYYCAA VEGFBII11E04/ EVQLVESGGG SYAMG WFRQAPG TISQSGYST RFTISRDNAKNTV DPFYSYGSPSPYR WGQGTQ 84 LVQAGGSLRL KEREFVA YYADSVKG NLQMNSLKPEDTA Y VTVSS SCAASGRTFS VYYCAA VEGFBII11E05/ EVQLVESGGG FSAMG WFRQAPG AFKWSGSTT RFTISTDNAKNIL DRFYTGRYYSSDE WGQGTQ 85 LVQPGGSLRL KEREFVA YYADYVKG FLQMNSLKPEDTA YDY VTVSS SCASSGRLFS IYYCAV VEGFBII11F10/ EVQLVESGGG ITVMA WFRQAPG AITWSAPSS RFTISRDNAKNTV DRFKGRSIVTRSD WGQGTQ 86 LVQAGGSLRL KEREFVA YYADSVKG YLQVNSLKPEDSA YRY VTVSS SCAASTSIYS IYYCAA VEGFBII11F12/ EVQLVESGGG SLAMG WFRQVPG SISQSGITT RFTISRDSAKNTV SVFYSTALTRPVD WGQGTQ 87 LVQSGGSLRL KDREFVA SYADSVKS YLQMNLLKPEDTA YRY VTVSS SCAASGRSFS VYYCAT VEGFBII11G09/ EVQLVESGGG ITVMA WFRQAPG AITWSAPTT RFTISRDNAKNTV DRFKGRSIVTRSD WGQGTQ 88 LVQAGGSLRL KEREFVA YSADSVKG YLQMNSLKPEDSA YRY VTVSS SCAASTSIYS IYYCAA VEGFBII12A07/ EVQLVESGGG KYVMG WFRQAPG AITSRDGPT RFTISGDNTKNKI DEDLYHYSSYHFT WGQGTQ 89 LVQAGGSLRL NDREFVA YYADSVKG FLQMNSLMPEDTA RVDLYHY VTVSS SCSVTGRTFN VYYCAI VEGFBII12B01/ EVQLVESGGG SSWMY WVRQAPG RISPGGLFT RFSVSTDNANNTL GGAPNYTP RGRGTQ 90 LVQPGGSLRL KGLEWVS YYVDSVKG YLQMNSLKPEDTA VTVSS ACAASGFTLS LYSCAK VEGFBII12C04/ EVQLVESGGG SDVMG WYRQAPG FIRSLGSTY RFTISRDNAANTV RFSGESY WGQGTP 91 LVQPGGSLRL KQRELVA YAGSVKG YLQMNNLKPEDTA VTVSS SCAASGSIVR VYYCNA VEGFBII12E10/ EVQLVESGGG NYVMG WFRQAPG AITSTNGPT RFTISGDNTKNKV DEDLYHYSSYHYT WGQGTQ 92 LAQAGGSLRL NEREFVA YYADSVKG FLQMDSLRPEDTA RVALYHY VTVSS SCTASGRTFN VYYCAI VEGFBII12G04/ EVQLVESGGG LYAMG WFRQAPG AITWSAGDT RFTISRDNARNTV RQWGGTYYYHGSY WGQGTQ 93 LVQSGDSLRL REREFVS QYADSVKG NLQMNGLKPEDTA AW VTVSS SCAVSGNTFG VYYCAG VEGFBII16C03/ EVQLVESEGG TDDVG WFRQAPG VIRWSTGGT RFTLSRDNAKNTM RSRPLGAGAWYTG WGQGTQ 94 LVQAGGSLRL KEREFVA YTSDSVKG YLQMNSLKPEDTA ENYYNY VTVSS SCAASGRTFS VYYCAA VEGFBII16F11/ EVQLVESGGG GYDMG WFRQAPG AITWSGGST RFTISRDNAKNTV GRIWRSRDYDSEK WGHGTQ 95 LVQAGGSLRL KEREFVT YSPDSVKG YLQMNNLTPEDTA YYDI VTVSS SCAASGRTSS VYYCAS VEGFBII36C08/ EVQLVESGGG AYDMG WFRQAPG VISWTNSMT RFTISRDNAKNTV DRRRTYSRWRFYT WGQGTQ 96 LVQAGGSLRL KEREFVA YYADSVKG YLQMNSLKPEDTA GVNDYDY VTVSS SCAASGRTFS VYYCAV VEGFBII37F09/ EVQLVESGGG AYDMG WFRQAPG VISWSGGMT RFTISRDNAKSTV DRRRAYSRWRYYT WGQGTQ 97 LVQTGGSLRL KEREFVA YYADSVQG YLQMNSPKPEDTA GVNDYEF VTVSS SCAASGRTFS VYYCAV VEGFBII38A06/ EVQLVESGGG AYDMG WFRQAPG VISWSGGMT RFTISRDNAKNTV DRRRLYSRWRYYT WGQGTQ 98 LVQAGGSLRL KEREFVA YYADSVKG YLQMNSLKPEDTA GVNDYDY VTVSS SCAASGRTFS VYYCAV VEGFBII39H11/ EVQLVESGGG AYDMG WFRQAPG VISWTGGMT RFTISRDKAKNTV DRRRTYSRWRYYT WGQGTQ 99 LVQAGGSLRL KEREFVA YYADSVKG SLQMNSLKPEDTA GVNEYEY VTVSS SCAASGRTFS VYYCAV VEGFBII41B06/ EVQLVESGGG AYDMG WFRQAPG VISWTGDMT RFTISRDKAKNTV DRRRTYSRWRYYT WGQGTQ 100 LVQAGGSLRL KEREFVA YYADSVKG SLQMNSLKPEDTA GVNEYEY VTVSS SCAASGRTFS VYYCAA VEGFBII41C05/ EVQLVESGGG VYTMG WFRQAPG TISRTGDRT RFTISRENAKNTV GPIAPSPRPREYY WGQGTQ 101 LVQAGGSLRL KEREFVA SYANSVKG YLQMNSLKPEDTA Y VTVSS SCAASGRTFS VYSCAA VEGFBII41D11/ EVQLMESGGG AYDMG WFRQAPG VISWTGGMT RFTISRDKAKNTV DRRRTYSRWRYYT WGQGTQ 102 LVQAGGSLRL KEREFVA YYADSVKG SLQMNSLKPEDTA GVNEYEY VTVSS SCAASGRTFS VYYCAV VEGFBII42F10/ EVQLVESGGG AYDMG WFRQAPG VISWSGGMT RFTISRENAKNTQ GRRRAYSRWRYYT WGQGTQ 103 LVQAGGSLRL KEREFVA DYADSVKG FLQMNSLKPEDTA GVNEYDY VTVSS SCAASGRTFS VYYCAV VEGFBII86C11/ EVQLVESGGG SYAMG WFRQAPG HINRSGSST RFTISRDNAKNTV GRYYSSDGVPSAS WGQGTQ 104 LVQAGDSLRL KERESVA YYADSVKG YLQLNSLKPEDTA FNY VTVSS SCTASGRTFN VYYCAA VEGFBII86F11/ EVQLVESGGG TWAMA WFRQAPG AISWSGSMT RFIISRDNAQNTL KTVDYCSAYECYA WGRGAQ 105 LVQAGDSLRL KEREFIS YYTDSVKG FLQMNNTAPEDTA RLEYDY VTVSS SCFTSARTFD VYYCAA VEGFBII86G08/ EVQLVESGGG STNMG WFRQGPG AITLSGTTY RFTISRDNDKNTV DPSYYSTSRYTKA WGQGTQ 106 LMQTGDSLRL KEREFVA YAEAVKG ALQMNSLKPEDTA TEYDY VTVSS SCAASGLRFT VYYCGA VEGFBII86G10/ EVQLVESGGG TYTMG WFRQTPG AIRWTVNIT RFTISRDIVKNTV QTSAPRSLIRMSN WGQGTQ 107 LVQAGGSLRL TEREFVA YYADSVKG YLQMNSLKPEDTA EYPY VTVSS SCAASGRTFN VYYCAA VEGFBII86G11/ EVQLVESGGG LYTVG WFRQAPG YISRSGSNR RFTLSRDNAKNTV TSRGLSSLAGEYN WGRGTQ 108 LVQAGGSLRL KEREFVA YYVDSVKG DLQMNSLKTEDTA Y VTVSS SCAASGLTFS VYYCAA VEGFBII86H09/ EVQLVESGGG SYRMG WFRRTPG SISWTYGST RFTMSRDKAKNAG GAQSDRYNIRSYD WGQGTQ 109 LVQAGGSLRL KEDEFVA FYADSVKG YLQMNSLKPEDTA Y VTVSS SCTASGSAFK LYYCAA VEGFBII87B07/ EVQLVESGGG TSWMH WVRQAPG SIPPVGHFA RFTISRDNAKNTL DSAGRT KGQGTQ 110 LVQPGGSLKL KGLEWVS NYAPSVKG FLQMNSLKSEDTA VTVSS SCTASGFTFS VYYCAK VEGFBII88A01/ KVQLVESGGG NYAMD WFRQAPG AITRSGGGT RFTISRDNAKNTV TRSSTIVVGVGGM WGKGTL 111 LVQAGGSLRL KEREFVA YYADSVKG YLQMNSLKPEDTA EY VTVSS SCAASERTFS VYYCAA VEGFBII88A02/ EVQLVESGGG DYDIG WFRQAPG CITTDVGTT RFTISSDNAKNTV DTQDLGLDIFCRG WGQGTQ 112 LVQAGGSLRL NEREGVS YYADSVKG YLQINDLKPEDTA NGPFDG VTVSS SCAASGFTFG IYYCAV VEGFBII88B02/ EVQLVESGGG DYAIG WFRQAPG CISSYDSVT RFTISRDSAKNTL EREQLRRRESPHD SGKGTL 113 LVQPGGSLRL KEREGVS YYADHVKG YLQMNSLSIEDTG ELLRLCFYGMRY VTVSS SCTASGLNLD VYYCAA VEGFBII88E02/ EVQLVESGGG DYAIG WFRQAPG CISSSDTSI RFTFSRDNAKNTV AFRCSGYELRGFP WGQGTQ 114 LVQPGGSLRL KEREAVS DYTNSVKG YLQMNSLKPEDTA T VTVSS SCVASGFRLD VYYCAA VEGFBII88G03/ EVQLVESGGG SLAVG WFRQAPG RITWSGATT RFTISRDNAKNTM DRSPNIINVVTAY WGQGTQ 115 LVQAGGSLRL KEREFVA YYADAVKD YLQMNSLKPEDTA EYDY VTVSS SCAASGGTFS VYYCAA VEGFBII88G05/ EVQLVESGGG LYNMG WFRQAPG AITSSPMST RFSISINNDKTTG PEGSFRRQYADRA WGQGTQ 116 LVQPGASLRL KEREFVA YYADSVKG FLQMNVLKPEDTG MYDY VTVSS SCAASGDGFT VYFCAA VEGFBII88G11/ EVQLVESGGG GSDMG WFRQSPG AIRLSGSIT RFTISRDNAKNTV RSTYSYYLALADR WGQGTQ 117 LAQAGGSLRL KEREIVA YYPDSVKG YLQMNSLKPEDTA GGYDY VTVSS SCAASGRTFS VYYCAA VEGFBII88H01/ EVQLVESGGG TYAIG WFRQAPG CMSAGDSIP RFTTSTDNARNTV ARYHGDYCYYEGY WGQGTQ 118 LVQAGGSLRL KEREAVS WYTASVKG YLQMNSLKPEDTA YPF VTVSS SCVASGFTLG HYYCAA VEGFBII89B04/ EVQLVESGGG TNFMG WYRQAPG TITSSSITN RFTISRDNAKNTV RWRWSDVEY WGKGTL 119 LVQAGGSLRL KQRELVA YVDSVKG YLQMTSLKPEDTA VTVSS SCAASTSISS VYYCHA VEGFBII89B08/ EVQLVESGGG IFAMR WYRQAPG SITRSSITT RFTPSRDNAKNTV AIRPELYSVVNDY WGQGTQ 120 LVQPGGSLRL KQRELVA YADSVKG SLQMNSLKPEDTA VTVSS SCAASGTTSS VYYCNA VEGFBII89D04/ EVQLVESGGG DYNLG WFRQAPG VISWRDSFA RFTISRDNAKNTV DRVSSRLVLPNTS WGQGTQ 121 LVQPGGSLRL KERQFVA YYAEPVKG YLQMNSLKPEDTA PDFGS VTVSS SCATSGLTFS VYYCAA VEGFBII89F09/ EVQLVESGGG NAIMG WFRQAPG AMNWRGGPT RFTISGDNTKNTV DEDLYHYSSYHYS WGQGTQ 122 LVQAGDSLRL QEREFVA YYADSVKG FLQMNFLKPEDTA RVDLYHY VTVSS SCAASGRTFN VYYCAA VEGFBII89G09/ EVQLVESGGG IFAMR WYRQAPG SITRSSITT RFTLSRDNAKNTV AIRPELYSVVNDY WGQGTQ 123 LVQPGGSLRL KQRELVA YADSVKG SLQMNSLKPEDTA VTVSS SCAASGTTSS VYYCNA VEGFBII89H08/ EVQLVESGGG SYAPG WFRQAPG AFTRSSNIP RFTISRDNAHTVY NLGSTWSRDQRTY WGQGTQ 124 LVQAGGSLRL KEREFVA YYKDSVKG LQMNSLKPEDTAI DY VTVSS SCAASGGSFS YYCAV
TABLE-US-00006 TABLE 3 Sequence IDs and AA sequences of monovalent receptor-blocking anti-VEGF VHHs (FR, framework; CDR, complementary determining region) SEQ ID NO: 9-46 VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII22A10/9 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFS VEGFBII22A11/10 EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL A REFVV DAVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TAVYYCAA TFS VEGFBII22B06/11 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAASGR TAVYYCAA TFS VEGFBII22B07/12 EVQLVESGG SYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQAGDSL G REFVV YDSVSLEG VYLQINSLKPED GDTYDY TVSS RLSCAASGR TAVYYCAA TFS VEGFBII22E04/13 EVQLVESGG SYSM WFRQAQGKE AISSGGSIY RFTISRDNTKNT SRAYASSRLRL WGQGTQV GLVQPGDSL G REFVV DSVSLQG VYLQTPSLKPED ADTYDY TVSS KLSCVASGR TAVYYCAA TSS VEGFBII23A03/14 EVQLVESGG SYSM WFRQAQGKE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV DSVSLQG VYLQTPSLKPED ADTYDY TVSS KLSCVASGR TAVYYCAA TFS VEGFBII23A06/15 EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV DAVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TAVYYCAA TFS VEGFBII23A08/16 EVQLVESGG SYSM WFRQAQGKE AISNGGYKY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQTGDSL G REFVV DSVSLEG VYLQINSLKPED ADTYDY TVSS RLSCVASGR TAVYYCAA TFS VEGFBII23A09/17 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNSKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED PDTYDY TVSS KLSCAFSGR TAVYYCAA TFG VEGFBII23B04/18 EVQLVESGG SYSM WFRQAQGKE AISKGGYKY RFTISKDNAKNT SRAYGSSRLRL WGQGTQV GLVQTGDSL G REFVV DSVSLEG VYLQINSLKPED ADTYEY TVSS RLSCEVSGR TAVYYCAS TFS VEGFBII23D11/19 EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL A REFVV DAVSLEG VYLQTPSLKPED ADTYDY TVSS RLSCAFSGR TAVYYCAA TFS VEGFBII23E05/20 EVQLVESEG SYSM WFRQAQGKE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV DSVSLQG VYLQTPSLKPED ADTYDY TVSS KLSCVASGR TAVYYCAA TSS VEGFBII23F02/21 EMQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFS VEGFBII23F05/22 EVQLVESGG SYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQAGDSL G REFVV YDSVSLEG VYLQINSLKPKD GDTYDY TVSS RLSCAASGR TAVYYCAA TFS VEGFBII23F11/23 EVQLVESGG SYSM WFRQAQGKE AISSGGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFS VEGFBII23G03/24 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNSKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED PGTYDY TVSS KLSCAFSGR TAVYYCAA TFG VEGFBII24C04/25 EVQLVESGG SYSM WFRQAQGKE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV DSVSLQG VYLQTPSLKPED ADTYDY TVSS KLSCVASGR TAVYYCAA TSS VEGFBII27D08/26 EVQLVESGG SYSM WFRQAQGKE AISSGGYKY RFTISRDNTQNT SRAYGSGRLRL WGQGTQV GLVQTGDSL G REFVV DSVSLEG VYLQINSLKPED ADTYDY TVSS RLSCAASGR TAVYYCAA TFS VEGFBII27G07/27 EVQLVESGG SYSM WFRQAQGQE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV DSVSLQG VYLQTPSLKPED ADTYDY TVSS KLSCVASGR TAVYYCAA TSS VEGFBII30009/28 EVQLVESGG SYSM WFRQAQGQE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV DSVSLQG VYLQTPSLKPED ADTYDY TVSS KLSCIASGR TAVYYCAA TSS VEGFBII30E07/29 EVQLVESGG SYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTRV GLVQAGDSL G REFVV YDSVSLEG VYLQINSLKPED GDTYDY TVSS RLSCAASGR TAVYYCAA TFS VEGFBII31C07/30 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQTGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS RLSCAASGG TADYYCAA TFS VEGFBII39E02/31 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDPL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFS VEGFBII39G04/32 EVPLVESGG SYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQAGDSL G REFVV YDSASLEG VYLQINSLKPED GDTYDY TVSS RLSCAASGR TAVYYCAA TFS VEGFBII40F02/33 EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL A REFVV DAVSLEG VYLQTPSLKPEG ADTYDY TVSS KLSCAFSGR TAVYYCAA TFS VEGFBII40G07/34 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNA SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFS VEGFBII40H10/35 EVQLMESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFS VEGFBII41B05/36 EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGGSL G REFVV DAVSLEG VYLQTPSLKPED ADTYDY TVSS RLSCAFSGR TAVYYCAA TFS VEGFBII41G03/37 EVQLVESGG SYSM WFRQAQGKE AISSGGFIY RFTISRENTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL A REFVV DAVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TAVYYCAA TFS VEGFBII42A05/38 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQMPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFS VEGFBII42D05/39 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TAVYYCAA TFS VEGFBII42F11/40 EVQLVESGG SYSV WFRQAQGKE AISSGGYIY RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV DSVSLQG VYLQTPSLKPED ADTYDY TVSS KLSCVASGR TAVYYCAA TSS VEGFBII56E11/41 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR AADYYCAA TFS VEGFBII60A09/42 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTRNT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TADYYCAA TFS VEGFBII61A01/43 EVQLVESGG SYSM WFRQAQGKE AISSGGYKY RFTISRDNTKNT SRAYASSRLRL WGQGTQV GLVQAGGSL G REFVV DAVSLEG VYLQTPSLKPED ADTYDY TVSS RLSCAFSGR TAVYYCAA TFS VEGFBII62A09/44 EVQLVESGG SYSM WFRQAQGKE AISSSGGYI RFTISRDNTKNT SRAYGSSRLRL WGQGTQV DLVQPGDSL G REFVV YDSVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAASGR TAVYYCAA TFS VEGFBII62D10/45 EVQLVESEG SYSM WFRQAQGKE AISSSGNYK RFTISRDNTKNT SRAYGSSRLRL WGQGTQV GLVQAGDSL G REFVV YDSVSLEG VYLQINSLKPED GDTYDY TVSS RLSCAASGR TAVYYCAA TFS VEGFBII62F02/46 EVQLVESGG SYSM WFRQAQGKE AIASGGYIY RFTISRDNTKDT SRAYGSSRLRL WGQGTQV GLVQPGDSL G REFVV DAVSLEG VYLQTPSLKPED ADTYDY TVSS KLSCAFSGR TAVYYCAA TFS
[0345] Dissociation rates of inhibitory VHHs are analyzed on Biacore (Biacore T100 instrument, GE Healthcare). HBS-EP+ buffer is used as running buffer and experiments are performed at 25° C. Recombinant human VEGF165 is irreversibly captured on a CM5 sensor chip via amine coupling (using EDC and NHS) up to a target level of +/−1500RU. After immobilization, surfaces are deactivated with 10 min injection of 1M ethanolamine pH8.5. A reference surface is activated and deactivated with respectively EDC/NHS and ethanolamine. Periplasmic extracts of VHHs are injected at a 10-fold dilution in running buffer for 2 min at 45 μl/min and allowed to dissociate for 10 or 15 min. Between different samples, the surfaces are regenerated with regeneration buffer. Data are double referenced by subtraction of the curves on the reference channel and of a blank running buffer injection. The of the processed curves is evaluated by fitting a two phase decay model in the Biacore T100 Evaluation software v2.0.1. Values for k.sub.d-fast, k.sub.d-slow and % fast are listed in Table 4.
TABLE-US-00007 TABLE 4 Off-rate determination of anti-VEGF receptor-blocking VHHs with Biacore Unique B-cell sequence Representative Binding level lineage variant VHH ID k.sub.d (fast) k.sub.d (slow) % fast (RU) 1 1 VEGFBII22B07 1.50E−02 7.80E−05 31 328 1 2 VEGFBII23A08 1.30E−02 5.00E−05 19 502 1 3 VEGFBII23B04 8.80E−03 4.00E−05 12 768 1 4 VEGFBII27D08 2.40E−02 8.10E−05 13 225 1 5 VEGFBII24C04 1.30E−02 3.40E−05 17 456 1 6 VEGFBII27G07 1.30E−02 3.80E−05 18 471 1 7 VEGFBII22E04 1.80E−02 1.10E−04 14 520 1 8 VEGFBII23A03 1.50E−02 3.20E−05 15 487 1 9 VEGFBII22B06 3.80E−02 9.00E−05 23 168 1 10 VEGFBII23A09 2.70E−02 4.60E−05 20 247 1 11 VEGFBII23G03 2.80E−02 8.60E−05 28 141 1 12 VEGFBII22A11 2.20E−02 4.70E−05 12 461 1 13 VEGFBII23A06 1.70E−02 3.70E−05 13 547 1 14 VEGFBII23F11 2.70E−02 1.30E−04 22 134 1 15 VEGFBII22A10 3.70E−02 4.00E−05 19 229 1 16 VEGFBII23F05 1.60E−02 1.30E−04 29 198 1 17 VEGFBII23D11 1.90E−02 5.80E−05 13 510 1 18 VEGFBII23F02 n/d n/d n/d n/d 1 19 VEGFBII23E05 1.50E−02 6.90E−05 18 275 1 20 VEGFBII31C07 3.70E−02 1.50E−04 25 77 1 21 VEGFBII30C09 1.50E−02 7.60E−05 19 264 1 22 VEGFBII30E07 1.70E−02 1.30E−04 29 226 1 23 VEGFBII39G04 1.40E−02 7.40E−04 40 210 1 24 VEGFBII41G03 1.20E−02 2.70E−04 20 332 1 25 VEGFBII41B05 1.90E−02 1.20E−04 16 324 1 26 VEGFBII40F02 1.20E−02 9.80E−05 20 258 1 27 VEGFBII39E02 1.90E−02 2.40E−04 13 181 1 28 VEGFBII42D05 3.30E−02 1.50E−04 26 77 1 29 VEGFBII40G07 1.80E−02 3.20E−04 19 139 1 30 VEGFBII42A05 1.60E−02 3.40E−04 25 118 1 31 VEGFBII42F11 9.10E−03 5.00E−04 46 100 1 32 VEGFBII40H10 1.40E−02 2.90E−04 17 200 1 33 VEGFBII62A09 4.10E−02 1.10E−04 23 84 1 34 VEGFBII60A09 3.70E−02 9.30E−05 20 106 1 35 VEGFBII62F02 1.40E−02 8.50E−05 21 205 1 36 VEGFBII62D10 1.90E−02 1.60E−04 40 94 1 37 VEGFBII61A01 7.40E−03 1.70E−04 21 275 1 38 VEGFBII56E11 3.30E−02 1.40E−04 24 76 n/d, not determined
Example 5
Characterization of Purified Anti-VEGF VHHs
[0346] Three inhibitory anti-VEGF VHHs are selected for further characterization as purified protein: VEGFBII23B04, VEGFBII24C4 and VEGFBII23A6. These 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 for VHH purification via IMAC and size exclusion chromatography (SEC). Final VHH preparations show 95% purity as assessed via SDS-PAGE.
5.1 Evaluation of Human VEGF165/VEGFR2 Blocking VHHs in Human VEGF165/Human VEGFR2-Fc Blocking ELISA
[0347] The blocking capacity of the VHHs is evaluated in a human VEGF165/human VEGFR2-Fc blocking ELISA. In brief, 1 μg/mL of VEGFR2-Fc chimera (R&D Systems, Minneapolis, Minn., USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Dilution series (concentration range 1 mM-64 pM) of the purified VHHs in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) are incubated in the presence of 4 nM biotinlyated VEGF165. Residual binding of bio-VEGF165 to VEGFR2 is detected using horseradish peroxidase (HRP) conjugated extravidin (Sigma, St Louis, Mo., USA) and TMB as substrate. As controls Bevacizumab (Avastin®) and Ranibizumab (Lucentis®) are taken along. Dose inhibition curves are shown in
TABLE-US-00008 TABLE 5 IC.sub.50 (nM) values and % inhibition for monovalent VHHs in hVEGF165/hVEGFR2-Fc competition ELISA IC.sub.50 % VHH ID (nM) inhibition VEGFBII23B04 2.1 100 VEGFBII23A06 3.0 100 VEGFBII24C04 2.5 100 Ranibizumab 1.6 100 Bevacizumab 1.7 100
5.2 Evaluation of Human VEGF165/VEGFR2 Blocking VHHs in Human VEGF165/Human VEGFR1-Fc Blocking ELISA
[0348] VHHs are also evaluated in a human VEGF165/human VEGFR1-Fc blocking ELISA. In brief, 2 μg/mL of VEGFR1-Fc chimera (R&D Systems, Minneapolis, Minn., USA) is coated in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Dilution series (concentration range 1 mM-64 pM) of the purified VHHs in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) are incubated in the presence of 0.5 nM biotinlyated VEGF165. Residual binding of bio-VEGF165 to VEGFR1 is detected using horseradish peroxidase (HRP) conjugated extravidin (Sigma, St Louis, Mo., USA) and TMB as substrate. As controls Bevacizumab, Ranibizumab and an irrelevant VHH (2E6) are taken along. Dose inhibition curves are shown in
TABLE-US-00009 TABLE 6 IC.sub.50 (nM) values and % inhibition of monovalent VHHs in hVEGF165/hVEGFR1-Fc competition ELISA % VHH ID IC.sub.50 (nM) inhibition VEGFBII23B04 0.5 64 VEGFBII23A06 0.9 55 VEGFBII24C04 0.8 71 Ranibizumab 1.2 91 Bevacizumab 1.5 96
5.3 Evaluation of the Anti-VEGF165 VHHs in the Human VEGF165/Human VEGFR2-Fc Blocking AlphaScreen
[0349] The blocking capacity of the VHHs is also evaluated in a human VEGF165/human VEGFR2-Fc blocking AlphaScreen. Briefly, serial dilutions of purified VHHs (concentration range: 200 nM-0.7 pM) in PBS buffer containing 0.03% Tween 20 (Sigma) are added to 4 pM bio-VEGF165 and incubated for 15 min. Subsequently VEGFR2-Fc (0.4 nM) and anti-Fc VHH-coated acceptor beads (20 μg/ml) are added and this mixture is incubated for 1 hour in the dark. Finally, streptavidin donor beads (20 μg/ml) are added and after 1 hour of incubation in the dark, fluorescence is measured on the Envision microplate reader. Dose-response curves are shown in the
TABLE-US-00010 TABLE 7 IC.sub.50 (pM) values and % inhibition for VHHs in hVEGF165/hVEGFR2-Fc competition AlphaScreen % VHH ID IC.sub.50 (pM) inhibition VEGFBII23B04 160 100 VEGFBII23A06 250 100 VEGFBII24C04 250 100 Ranibizumab 860 100
5.4 Evaluation of the Anti-VEGF165 VHHs in the Human VEGF165/Human VEGFR1-Fc Blocking AlphaScreen
[0350] The blocking capacity of the VHHs is also evaluated in a human VEGF165/human VEGFR1-Fc blocking AlphaScreen. Briefly, serial dilutions of purified VHHs (concentration range: 500 nM-1.8 pM)) in PBS buffer containing 0.03% Tween 20 (Sigma) are added to 0.4 nM bio-VEGF165 and incubated for 15 min. Subsequently VEGFR1-Fc (1 nM) and anti-Fc VHH-coated acceptor beads (20 μg/ml) are added and this mixture is incubated for 1 hour in the dark. Finally, streptavidin donor beads (20 μg/ml) are added and after 1 hour of incubation in the dark, fluorescence is measured on the Envision microplate reader. Dose-response curves are shown in the
TABLE-US-00011 TABLE 8 IC.sub.50 (nM) values for VHHs in hVEGF165/hVEGFR1-Fc competition AlphaScreen VHH ID IC.sub.50 (nM) % inhibition VEGFBII23B04 0.9 41 VEGFBII23A06 0.4 46 VEGFBII24C04 0.2 53 Ranibizumab 3.3 79
5.5 Determination of the Affinity of the Human VEGF165-VHH Interaction
[0351] Binding kinetics of VHH VEGFBII23B04 with hVEGF165 is analyzed by SPR on a Biacore T100 instrument. Recombinant human VEGF165 is immobilized directly on a CM5 chip via amine coupling (using EDC and NHS). VHHs are analyzed at different concentrations between 10 and 360 nM. Samples are injected for 2 min and allowed to dissociate up to 20 min at a flow rate of 45 μI/min. In between sample injections, the chip surface is regenerated with 100 mM HCl. HBS-EP+(Hepes buffer pH7.4+EDTA) is used as running buffer. Binding curves are fitted using a Two State Reaction model by Biacore T100 Evaluation Software v2.0.1. The calculated affinities of the anti-VEGF VHHs are listed in Table 9.
TABLE-US-00012 TABLE 9 Affinity K.sub.D (nM) of purified VHHs for recombinant human VEGF165 VEGF165 k.sub.a k.sub.a1 k.sub.a2 k.sub.d k.sub.d1 k.sub.d2 K.sub.D VHH ID (M.sup.−1 .Math. s.sup.−1) (M.sup.−1 .Math. s.sup.−1) (M.sup.−1 .Math. s.sup.−1) (s.sup.−1) (s.sup.−1) (s.sup.−1) (nM) VEGFBII23B04.sup.(a) — 2.1E+05 1.4E−02 — 8.6E−03 2.4E−04 0.7 VEGFBII23A06.sup.(a) — 4.2E+05 2.0E−02 — 5.7E−02 1.0E−04 0.7 VEGFBII24C04.sup.(a) — 3.2E+05 1.8E−02 — 2.6E−02 9.6E−05 0.4 .sup.(a)Heterogeneous binding curve resulting in no 1:1 fit, curves are fitted using a Two State Reaction model by Biacore T100 Evaluation Software v2.0.1
5.6 Binding to Mouse VEGF164
[0352] Cross-reactivity to mouse VEGF164 is determined using a binding ELISA. In brief, recombinant mouse VEGF164 (R&D Systems, Minneapolis, Minn., USA) is coated overnight at 4° C. at 1 μg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1% in PBS). VHHs are applied as dilution series (concentration range: 500 nM-32 pM) in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-HRP conjugate (DAKO) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA) (
TABLE-US-00013 TABLE 10 EC.sub.50 (pM) values for VHHs in a recombinant human VEGF165 and mouse VEGF164 binding ELISA rhVEGF165 rmVEGF164 VHH ID EC.sub.50 (pM) EC.sub.50 (pM) VEGFBII23B04 297 NB VEGFBII24C04 453 NB VEGFBII23A06 531 NB NB, no binding
5.7 Binding to VEGF121
[0353] Binding to recombinant human VEGF121 is assessed via a solid phase binding ELISA. Briefly, recombinant human VEGF121 (R&D Systems, Minneapolis, Minn., USA) is coated overnight at 4° C. at 1 μg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1% in PBS). VHHs are applied as dilution series (concentration range: 500 nM-32 pM) in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-HRP conjugate (DAKO) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA) (
TABLE-US-00014 TABLE 11 EC.sub.50 (pM) values for monovalent VHHs in a recombinant human VEGF121 binding ELISA VHH ID EC.sub.50 (pM) VEGFBII23B04 510 VEGFBII24C04 792 VEGFBII23A06 928
5.8 Binding to VEGF Family Members VEGFB, VEGFC, VEGFD and PIGF
[0354] Binding to VEGFB, VEGFC, VEGFD and PIGF is assessed via a solid phase binding ELISA. In brief, VEGFB, VEGFC, VEGFD and PIGF (R&D Systems, Minneapolis, Minn., USA) are coated overnight at 4° C. at 1 μg/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells are blocked with a casein solution (1% in PBS). VHHs are applied as dilution series (concentration range: 500 nM-32 pM) and binding is detected using a mouse anti-myc (Roche) and an anti-mouse-AP conjugate (Sigma, St Louis, Mo., USA). As positive controls serial dilutions of the appropriate receptors are taken along and detected with horseradish peroxidase (HRP)-conjugated goat anti-human IgG, Fc specific antibody (Jackson Immuno Research Laboratories Inc., West Grove, Pa., USA) and a subsequent enzymatic reaction in the presence of the substrate TMB (3,3′,5,5′-tetramentylbenzidine) (Pierce, Rockford, Ill., USA). Dose-response curves of VHHs and controls are shown in
5.9 Epitope Binning
[0355] Biacore-based epitope binning experiments are performed to investigate which VEGF binders bind to a similar or overlapping epitope as VEGFBII23B04. To this end, VEGFBII23B04 is immobilized on a CM5 sensor chip. For each sample, human VEGF165 is passed over the chip surface and reversibly captured by VEGFBII23B4. Purified VHHs (100 nM) or periplasmic extracts (1/10 diluted) are then injected with a surface contact time of 240 seconds and a flow rate of 10 uL/minute. Between different samples, the surface is regenerated with regeneration buffer (100 mM HCl). Processed curves are evaluated with Biacore T100 Evaluation software. VHHs could be divided within two groups: group one which gave additional binding to VEGFBII23B04 captured VEGF165 and a second group which is not able to simultaneously bind to VEGFBII23B04 captured VEGF165. Table 12-A summarizes the binding epitopes of the tested VHHs.
[0356] The same assay set-up is used to assess whether VEGFR1, VEGFR2, Ranibizumab and Bevacizumab are able to bind to human VEGF-165 simultaneously with VEGFBII23B04. Table 12-B presents the additional binding responses to VEGFBII23B04-captured VEGF165. Only VEGFR2 is not able to bind to VEGFBII23B04-captured VEGF165, underscoring the blocking capacity of VEGFBII23B04 for the VEGF-VEGFR2 interaction. In addition, these data show that the VEGFBII23B04 epitope is different from the Bevacizumab and Ranibizumab epitope.
TABLE-US-00015 TABLE 12-A Epitope binning of anti-VEGF VHHs - simultaneous binding with VEGFBII23B04 No or low 1C02 1E07 4B08 8E07 8F07 12A07 12B01 86C11 86F11 86G08 additional 86G10 86G11 87B07 88A01 88A02 88B02 88E02 88G03 88G05 88G11 binding to 88H01 89B04 89D04 89F09 89G09 89H08 24C04 23A6 27G07 23B04 23B04- captured VEGF165* Additional 3D12 5B02 5B03 5B05 6G02 7D08 8D09 8F06 10C07 10E07 binding to 10G04 10G05 11C08 11D09 11E04 11E05 11F12 86H09 41C05 23B04- captured VEGF165 *indicating same or overlapping epitopes
TABLE-US-00016 TABLE 12-B Epitope binning of VEGFBII23B04 - binding of benchmark inhibitors or cognate receptors on VEGFBII23B04 captured VEGF165 Injection Binding step Binding [sample] level (RU) 1 VEGF165 100 nM 1727 2 VEGFBII23B04 100 nM — 3 Ranibizumab 100 nM 763 4 Bevacizumab 100 nM 1349 5 VEGFR1 100 nM 1011 6 VEGFR2 100 nM —
5.10 Characterization of the Anti-VEGF VHHs in the HUVEC Proliferation Assay
[0357] The potency of the selected VHHs is evaluated in a proliferation assay. In brief, primary HUVEC cells (Technoclone) are supplement-starved over night and then 4000 cells/well are seeded in quadruplicate in 96-well tissue culture plates. Cells are stimulated in the absence or presence of VHHs with 33 ng/mL VEGF. The proliferation rates are measured by [.sup.3H] Thymidine incorporation on day 4. The results of the HUVEC proliferation assay are shown in Table.
TABLE-US-00017 TABLE 13 IC.sub.50 (nM) values and % inhibition of monovalent VEGFBII23B04, VEGFBII23A06 and VEGFBII24C04 in VEGF HUVEC proliferation assay VHH ID IC.sub.50 (nM) % inhibition VEGFBII23B04 0.36 91 Bevacizumab 0.21 92 VEGFBII23A06 4.29 73 VEGFBII24C04 3.8 79 Bevacizumab 0.78 78
5.11 Characterization of the Anti-VEGF VHHs in the HUVEC Erk Phosphorylation Assay
[0358] The potency of the selected VHHs is assessed in the HUVEC Erk phosphorylation assay. In brief, primary HUVE cells are serum-starved over night and then stimulated in the absence or presence of VHHs with 10 ng/mL VEGF for 5 min. Cells are fixed with 4% Formaldehyde in PBS and ERK phosphorylation levels are measured by ELISA using phosphoERK-specific antibodies (anti-phosphoMAP Kinase pERK1&2, M8159, Sigma) and polyclonal Rabbit Anti-Mouse-Immunoglobulin-HRP conjugate (PO161, Dako). As shown in Table 14, VEGFBII23B04 and Bevacizumab inhibit the VEGF induced Erk phosphorylation by at least 90%, with IC.sub.50s<1 nM.
TABLE-US-00018 TABLE 14 IC.sub.50 (nM) values and % inhibition of monovalent VEGFBII23B04 in VEGF HUVEC Erk phosphorylation assay VHH ID IC.sub.50 (nM) % inhibition VEGFBII23B04 0.37 90 Bevacizumab 0.63 98
Example 6
Generation of Multivalent Anti-VEGF Blocking VHHs
[0359] VHH VEGFBII23B04 is genetically fused to either VEGFBII23B04 resulting in a homodimeric VHH (AA sequence see Table 15) or different VEGF binding VHHs resulting in heterodimeric VHHs. To generate the heterodimeric VHHs, a panel of 10 unique VEGF binding VHHs are linked via a 9 or 40 Gly-Ser flexible linker in two different orientations to VEGFBII23B04 (AA sequences see Table 15). Homodimeric VEGFBII23B04 (VEGFBII010) and the 40 heterodimeric bivalent' 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 desalting resulting in 90% purity as assessed via SDS-PAGE.
TABLE-US-00019 TABLE 15 Sequence ID, VHH ID and AA sequence of bivalent anti-VEGF VHHs (each of the used linkers is highlighted in one relevant sequence) Sequence ID/ SEQ ID NO: VHH ID AA sequence VEGFBII23B04- 35GS-23B04/128 VEGFBII010
[0360] The panel of 40 bivalent VHHs is tested in the VEGFR2 and VEGFR1 blocking AlphaScreen assay, as described in Example 5.3 and 5.4, respectively. Based on potency and maximum level of inhibition, the 5 best bivalent VHHs (VEGFBII021, VEGFBII022, VEGFBI023, VEGFBI024 and VEGFBII025) are chosen for further characterization. An overview of the screening results for the 5 selected bivalent VHHs in the competitive VEGFR2 and VEGFR1 AlphaScreen is shown in Table 16.
TABLE-US-00020 TABLE 16 Potency and efficacy of 5 best bivalent VHHs in the VEGF/VEGFR1 and VEGF/VEGFR2 competition AlphaScreen assay VEGFR1 VEGFR2 IC.sub.50 % VHH ID IC.sub.50 (pM) (pM) inhibition VEGFBII021 9 16 100 VEGFBII022 7 8 100 VEGFBII023 38 44 91 VEGFBII024 12 46 100 VEGFBII025 51 39 82
Example 7
Characterization of Formatted Anti-VEGF VHHs
[0361] VHHs VEGFBII010, VEGFBII021, VEGFBII022, VEGFBII023, VEGFBII024 and VEGFBII025 are compared side-by-side in the VEGFR2 and VEGFR1 blocking ELISA (
TABLE-US-00021 TABLE 17 IC.sub.50 (pM) values and % inhibition for formatted VHHs in hVEGF165/hVEGFR2-Fc competition ELISA IC.sub.50 VHH ID (pM) % inhibition VEGFBII010 49 100 VEGFBII021 204 100 VEGFBII022 164 100 VEGFBII023 213 100 VEGFBII024 292 100 VEGFBII025 577 100 Bevacizumab 315 100 Ranibizumab 349 100
TABLE-US-00022 TABLE 18 IC.sub.50 (pM) values and % inhibition of formatted VHHs in VEGF165/hVEGFR1-Fc competition ELISA IC.sub.50 VHH ID (pM) % inhibition VEGFBII010 73.5 67 VEGFBII021 254 97 VEGFBII022 225 89 VEGFBII023 279 91 VEGFBII024 326 92 VEGFBII025 735 91 Bevacizumab 484 91 Ranibizumab 594 96
TABLE-US-00023 TABLE 19 IC.sub.50 (pM) values and % inhibition for formatted VHHs in hVEGF165/hVEGFR2-Fc competition AlphaScreen IC.sub.50 VHH ID (pM) % inhibition VEGFBII010 16 100 VEGFBII021 7 100 VEGFBII022 7 100 VEGFBII023 46 100 VEGFBII024 50 100 VEGFBII025 51 100 Ranibizumab 600 100
TABLE-US-00024 TABLE 20 IC.sub.50 (pM) values and % inhibition of formatted VHHs in VEGF165/hVEGFR1-Fc competition AlphaScreen IC.sub.50 VHH ID (pM) % inhibition VEGFBII010 21 70 VEGFBII021 12 100 VEGFBII022 9 98 VEGFBII023 48 87 VEGFBII024 69 98 VEGFBII025 71 82 Ranibizumab 1300 87
[0362] In addition, formatted VHHs are also tested for their capacity to block the mVEGF164/mVEGFR2-huFc interaction. In brief, serial dilutions of purified VHHs (concentration range: 4 μM-14.5 pM) in PBS buffer containing 0.03% Tween 20 (Sigma) are added to 0.1 nM biotinylated mVEGF164 and incubated for 15 min. Subsequently mouse VEGFR2-huFc (0.1 nM) and anti-huFc VHH-coated acceptor beads (20 μg/ml) are added and this mixture is incubated for 1 hour. Finally, streptavidin donor beads (20 μg/ml) are added and after 1 hour of incubation fluorescence is measured on the Envision microplate reader. Dose-response curves are shown in
TABLE-US-00025 TABLE 21 IC.sub.50 (pM) values and % inhibition for formatted VHHs in mVEGF164/mVEGFR2-hFc competition AlphaScreen IC.sub.50 VHH ID (nM) % inhibition VEGFBII022 108 100 VEGFBII024 — — mVEGF164 0.05 100 Ranibizumab — —
[0363] The formatted VHHs are also tested in ELISA for their ability to bind mVEGF164 and human VEGF165 (Example 5.6;
TABLE-US-00026 TABLE 22 EC.sub.50 (pM) values for formatted VHHs in a recombinant human VEGF165 and mouse VEGF164 binding ELISA rhVEGF165 rmVEGF164 VHH ID EC.sub.50 (pM) EC.sub.50 (pM) VEGFBII010 428 — VEGFBII021 334 502 VEGFBII022 224 464 VEGFBII023 221 — VEGFBII024 320 — VEGFBII025 668 —
TABLE-US-00027 TABLE 23 EC.sub.50 (pM) values for formatted VHHs in a recombinant human VEGF121 binding ELISA rhVEGF121 VHH ID EC.sub.50 (pM) VEGFBII010 920 VEGFBII022 540 VEGFBII024 325 VEGFBII025 475
TABLE-US-00028 TABLE 24 Affinity K.sub.D (nM) of purified formatted VHHs for recombinant human VEGF165 k.sub.a1 K.sub.D VHH ID (1/Ms) k.sub.d1 (1/s) k.sub.a2 (1/s) k.sub.d2 (1/s) (nM).sup.(a) VEGFBII010.sup.(b) 4.5E+05 1.7E−02 2.9E−02 1.3E−04 0.16 VEGFBII021.sup.(b) 1.2E+06 1.1E−02 2.3E−02 1.9E−04 0.07 VEGFBII022.sup.(b) 1.2E+06 9.1E−03 1.4E−02 2.6E−04 0.14 VEGFBII023.sup.(b) 3.0E+05 1.8E−02 2.4E−02 2.7E−04 0.69 VEGFBII024.sup.(b) 3.0E+05 1.3E−02 2.6E−02 2.8E−04 0.47 VEGFBII025.sup.(b) 3.3E+05 1.7E−02 1.8E−02 3.7E−04 1.1 .sup.(a)K.sub.D = k.sub.d1/k.sub.a1 * (k.sub.d2/(k.sub.d2 + k.sub.a2)) .sup.(b)Curves are fitted using a Two State Reaction model by Biacore T100 Evaluation Software v2.0.1
[0364] VHHs VEGFBII010, VEGFBII022, VEGFBII024 and VEGFBII025 are also tested in the VEGF-mediated HUVEC proliferation and Erk phosphorylation assay.
[0365] The potency of the selected formatted VHHs is evaluated in a proliferation assay. In brief, primary HUVEC cells (Technoclone) are supplement-starved over night and then 4000 cells/well are seeded in quadruplicate in 96-well tissue culture plates. Cells are stimulated in the absence or presence of VHHs with 33 ng/mL VEGF. The proliferation rates are measured by [.sup.3H] Thymidine incorporation on day 4. The results shown in Table 25 demonstrate that the formatted VHHs and Bevacizumab inhibit the VEGF-induced HUVEC proliferation by more than 90%, with IC.sub.50s<1 nM.
TABLE-US-00029 TABLE 25 IC.sub.50 (nM) values and % inhibition of formatted VHHs in VEGF HUVEC proliferation assay VHH ID IC.sub.50 (nM) % inhibition VEGFBII010 0.22 95 VEGFBII021 0.40 98 VEGFBII022 0.34 100 VEGFBII023 0.52 98 VEGFBII024 0.38 96 VEGFBII025 0.41 104 Bevacizumab 0.21 92
[0366] The potency of the selected formatted VHHs is assessed in the HUVEC Erk phosphorylation assay. In brief, primary HUVE cells are serum-starved over night and then stimulated in the absence or presence of VHHs with 10 ng/mL VEGF for 5 min. Cells are fixed with 4% Formaldehyde in PBS and ERK phosphorylation levels are measured by ELISA using phosphoERK-specific antibodies (anti-phosphoMAP Kinase pERK1&2, M8159, Sigma) and polyclonal Rabbit Anti-Mouse-Immunoglobulin-HRP conjugate (PO161, Dako). As shown in Table 26, the formatted VHHs and Bevacizumab inhibit the VEGF induced Erk phosphorylation by more than 90%, with IC.sub.50s<1 nM.
TABLE-US-00030 TABLE 26 IC.sub.50 (nM) values and % inhibition of formatted VHHs in VEGF HUVEC Erk phosphorylation assay VHH ID IC.sub.50 (nM) % inhibition VEGFBII010 0.19 92 VEGFBII021 0.21 103 VEGFBII022 0.18 94 VEGFBII023 0.25 100 VEGFBII024 0.23 94 VEGFBII025 0.23 99 Bevacizumab 0.63 98
Example 8
Sequence Optimization
8.1 Sequence Optimization of VEGFBII23B04
[0367] The amino acid sequence of VEGFBII23B04 is aligned to the human germline sequence VH3-23/JH5, see
[0368] The alignment shows that VEGFBII23B04 contains 19 framework mutations relative to the reference germline sequence. Non-human residues at positions 14, 16, 23, 24, 41, 71, 82, 83 and 108 are selected for substitution with their human germline counterparts. A set of 8 VEGFBII23B04 variants is generated carrying different combinations of human residues at these positions (AA sequences are listed in Table 27). One additional variant is constructed in which the potential isomerization site at position D59S60 (CDR2 region, see
TABLE-US-00031 TABLE 27 AA sequence of sequence-optimized variants of VHH VEGFBII23B04 (FR, framework; CDR, complementary determining region) VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII111D05/ EVQLVESGG SYSM WFRQAPGKEREF AISKGGY RFTISRDNAKNTVYLQI SRAYGSS WGQGTLVTV 47 GLVQTGGSLR G VV KYDSVSL NSLRPEDTAVYYCAS RLRLADT SS LSCEASGRTF EG YEY S VEGFBII111G06/ EVQLVESGG SYSM WFRQAPGKEREF AISKGGY RFTISRDNAKNTVYLQM SRAYGSS WGQGTLVTV 48 GLVQPGGSL G VV KYDSVSL NSLRPEDTAVYYCAS RLRLADT SS RLSCAASGRT EG YEY FS VEGFBII112D11/ EVQLVESGG SYSM WFRQAPGKEREF AISKGGY RFTISRDNAKNTVYLQI SRAYGSS WGQGTLVTV 49 GLVQPGGSL G VV KYDSVSL NSLRPEDTAVYYCAS RLRLADT SS RLSCEASGRT EG YEY FS VEGFBII113A08/ EVQLVESGG SYSM WFRQAPGKEREF AISKGGY RFTISKDNAKNTVYLQIN SRAYGSS WGQGTLVTV 50 GLVQTGGSLR G VV KYDSVSL SLRPEDTAVYYCAS RLRLADT SS LSCEVSGRTF EG YEY S VEGFBII113E03/ EVQLVESGG SYSM WFRQAQGKERE AISKGGY RFTISKDNAKNTVYLQM SRAYGSS WGQGTLVTV 51 GLVQTGDSLR G FVV KYDSVSL NSLRPEDTAVYYCAS RLRLADT SS LSCEVSGRTF EG YEY S VEGFBII114C09/ EVQLVESGG SYSM WFRQAPGKEREF AISKGGY RFTISKDNAKNTVYLQIN SRAYGSS WGQGTLVTV 52 GLVQPGDSLR G VV KYDSVSL SLRPEDTAVYYCAS RLRLADT SS LSCEVSGRTF EG YEY S VEGFBII114D02/ EVQLVESGG SYSM WFRQAPGKEREF AISKGGY RFTISRDNAKNTVYLQI SRAYGSS WGQGTLVTV 53 GLVQTGGSLR G VV KYDSVSL NSLRPEDTAVYYCAS RLRLADT SS LSCEVSGRTF EG YEY S VEGFBII114D03/ EVQLVESGG SYSM WFRQAQGKERE AISKGGY RFTISKDNAKNTVYLQIN SRAYGSS WGQGTLVTV 54 GLVQTGDSLR G FVV KYDSVSL SLRPEDTAVYYCAS RLRLADT SS LSCAVSGRTF EG YEY S VEGFBII118E10/ EVQLVESGG SYSM WFRQAQGKERE AISKGGY RFTISKDNAKNTVYLQIN SRAYGSS WGQGTQVTV 55 GLVQTGDSLR G FVV KYDAVSL SLKPEDTAVYYCAS RLRLADT SS LSCEVSGRTF EG YEY S
[0369] These variants are characterized as purified proteins in the VEGF165/VEGFR2 AlphaScreen (Example 5.3,
TABLE-US-00032 TABLE 28 IC.sub.50 (pM) values, % inhibition and melting temperature (@pH 7) of sequence-optimized variants of VEGFBII23B04 VHH ID IC.sub.50 (pM) % inhibition T.sub.m @ pH 7 (° C.) VEGFBII23B04 (wt) 169 100 63 VEGFBII111D05 209 100 68 VEGFBII111G06 366 100 71 VEGFBII112D11 221 100 70 VEGFBII113A08 253 100 69 VEGFBII113E03 290 100 68 VEGFBII114C09 215 100 71 VEGFBII114D02 199 100 74 VEGFBII114D03 227 100 64 VEGFBII118E10 189 100 62
[0370] In a second cycle, tolerated mutations from the humanization effort (VEGFBII111G06) and mutations to avoid potential posttranslational modification at selected sites (the D16G, the S60A substitution and an E1D mutation) are combined resulting in a sequence-optimized clone derived from VEGFBII23B04: VEGFBII0037. One extra sequence-optimized variant (VEGFBII038) is anticipated which contains the same substitutions as VEGFBII0037, with the exception of the 182M mutation, as this mutation may be associated with a minor drop in potency. The sequences from both sequence-optimized clones are listed in Table 29. VEGFBII0037 and VEGFBII0038 are characterized in the VEGF165/VEGFR2 blocking AlphaScreen (Example 5.3,
TABLE-US-00033 TABLE 29 AA sequences of sequence-optimized variants of VHH VEGFBII23B04 VHH ID/ CDR CDR FR SEQ ID NO: FR 1 1 FR2 2 FR3 CDR 3 4 VEGFBII037 DVQL SYSM WFRQA AISK RFTIS SRAYG WGQ 56 VESG G PGKER GGYK RDNAK SSRLR GTL GGLV EFVV YDAV NTVYL LADTY VTV QPGG SLEG QMNSL EY SS SLRL RPEDT SCAA AVYYC SGRT AS FS VEGFBII038 DVQL SYSM WFRQA AISK RFTIS SRAYG WGQ 57 VESG G PGKER GGYK RDNAK SSRLR GTL GGLV EFVV YDAV NTVYL LADTY VTV QPGG SLEG QINSL EY SS SLRL RPEDT SCAA AVYYC SGRT AS FS
TABLE-US-00034 TABLE 30 IC.sub.50 (pM) values, % inhibition, melting temperature (@pH 7) and affinity (pM) of sequence-optimized clones VEGFBII037 and VEGFBII038 % T.sub.m (° C.) VHH ID IC.sub.50 (pM) inhibition @ pH 7 K.sub.D (pM) VEGFBII23B04 152 100 63 560 VEGFBII037 300 100 72 270 VEGFBII038 143 100 71 360
8.2 Sequence Optimization of VEGFBII5B05
[0371] The amino acid sequence of VEGFBII5B05 is aligned to the human germline sequence VH3-23/JH5, see
TABLE-US-00035 TABLE 31 AA sequences of sequence-optimized variants of VHH VEGFBII5B05 (FR, framework; CDR, complementary determining region) VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII119G11/ EVQ SMA WYR RISSG RFT FSSRP WGQ 125 LVE QAP GTTA ISR NP GTL SGG GKQ YADS DNS VTV GLV REL VKG KNT SS QPG VA VYL GSL QMN RLS SLR CAA AED TAV SGI YY RFM CNT VEGFBII120E10/ EVQ SMA WYR RISSG RFT FSSRP WGA 126 LVE QAP GTTA ISR NP GTQ SGG GKH YVDS DNS VTV GLV REL VKG KNT SS QPG VA VYL GSL QMN RLS SLK CVA AED SGI TAV RFI YYC NT
[0372] One additional variant is constructed in which the potential oxidation site at position M30 (CDR1 region, see
TABLE-US-00036 TABLE 32 Off-rates sequence-optimized variants VEGFBII5B05 VHH ID binding level (RU) k.sub.d (1/s) VEGFBII5B05 242 6.15E−02 VEGFBII119G11 234 7.75E−02 VEGFBII120E10 257 4.68E−02
[0373] In a second cycle, mutations from the humanization effort and the M30I substitution are combined resulting in a sequence-optimized clone of VEGFBII5B05, designated VEGFBII032. The sequence is listed in Table 33. Affinity of VEGFBII032 is determined by Biacore (see Example 5.5) and the melting temperature is determined in the thermal shift assay as described above. An overview of the characteristics of the sequence-optimized VHH VEGFBII032 is presented in Table 34.
TABLE-US-00037 TABLE 33 A sequence of sequence-optimized clone AVEGFBII032 (FR, framework; CDR, complementary determining region) VHH ID/ SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 VEGFBII032/ EVQ SMA WYRQAP RISSG RFT FSSR WGQ 127 LVE GKQREL GTTA ISR PNP GTL SGG VA YADS DNS VTV GLV VKG KNT SS QPG VYL GSL QMN RLS SLR CAA AED SGI TAV RFI YYCNT
TABLE-US-00038 TABLE 34 Melting temperature (@pH 7) and affinity (nM) of sequence-optimized clone VEGFBII032 T.sub.m (° C.) VHH ID @ pH 7 K.sub.D (nM) VEGFBII5B05(wt) 69 32 VEGFBII0032 71 44
[0374] The potency of the sequence-optimized clones VEGFBII037 and VEGFBII038 is evaluated in a proliferation assay. In brief, primary HUVEC cells (Technoclone) are supplement-starved over night and then 4000 cells/well are seeded in quadruplicate in 96-well tissue culture plates. Cells are stimulated in the absence or presence of VHHs with 33 ng/mL VEGF. The proliferation rates are measured by [.sup.3H] Thymidine incorporation on day 4. The results shown in Table 35, demonstrate that the activity (potency and degree of inhibition) of the parental VHH VEGFBII23B04 is conserved in the sequence optimized clone VEGFBII038.
TABLE-US-00039 TABLE 35 IC.sub.50 (nM) values and % inhibition of the sequence optimized clones VEGFBII037 and VEGFBII038 in VEGF HUVEC proliferation assay VHH ID IC.sub.50 (nM) % inhibition VEGFBII23B04 0.68 92 VEGFBII037 1.54 78 VEGFBII038 0.60 92 Bevacizumab 0.29 94
Example 9
Construction, Production and Characterization of Bivalent VHHs Targeting Ang2
[0375] VHHs 1 D01 (SEQ ID No:214), 11B07, 00908 and 00027 (SEQ ID No:216) are genetically fused to 1 D01 (SEQ ID No: 214), 11B07, 00908 and 00027 (SEQ ID No:216), respectively, resulting in homodimeric VHHs. The bivalent VHHs are linked via a 9-GlySer or 40-GlySer flexible linker. The encoding DNA sequences of the formatted VHHs are cloned in the expression vector pAX172. VHHs are expressed in Pichia pastoris as c-terminally myc-His6 tagged proteins. In brief, BGCM cultures are started from a single colony streak incubated over weekend at 30° C. (250 rpm). After medium switch to BMCM, cultures are incubated until evening at 30° C. (250 rpm) and followed by an induction with 100% methanol. The next day the cultures are induced an additional 3 times (morning, afternoon, evening). The next day cultures are centrifuged for 20 min at 4° C. (1,500×g). The His6-tagged VHHs present in the supernatant are purified through immobilized metal affinity chromatography (IMAC) followed by desalting (DS) and finally gel filtration (GF) to remove any endotoxins/impurities. An overview of the format and sequence of all bivalent VHHs is depicted in
[0376] To explore the anti-Ang2 blocking properties in comparison with the monovalent building blocks, bivalent VHHs are analyzed in a human Ang2/hTie2 (
TABLE-US-00040 TABLE 36-A Sequences of bivalent VHH targeting Ang2 VHH ID AA sequence ANGBII00001 EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGR FTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRF TISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 180) ANGBII00002 EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGR FTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWF RQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRS KLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 181) ANGBII00003 EVQLVESGGGLVQVGDSLRLSCAASGRTFSTYLMVGWFRQAPGKEREFAAGIWSSGDTAYADSVRGR FTISRDNAKNTVYLQMNSLKTEDTAVYYCAGSYDGNYYIPGFYKDWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQVGDSLRLSCAASGRTFSTYLMVGWFRQAPGKEREFAAGIWSSGDTAYADSVRGRFT ISRDNAKNTVYLQMNSLKTEDTAVYYCAGSYDGNYYIPGFYKDWGQGTLVTVSS (SEQ ID NO: 182) ANGBII00004 EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADS VKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 183) ANGBII00005 EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDY AIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAV PAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 184) ANGBII00006 EVQLVESGGGLVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSSGGGGS GGGSEVQLLESGGGLVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADS VKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS (SEQ ID NO: 185)
Example 10
Construction, Production and Characterization of Trivalent Bispecific VHHs Targeting VEGF and Ang2 Using Anti-Serum Abumin as Half-Life Extension
[0377] The anti-VEGF VHH VEGFBII00038 (US 2011/0172398 A1) and the anti-Ang2 VHH 00027 (SEQ ID No:216) are used as building blocks to generate bispecific VHHs VEGFANGBII00001-00004. A genetic fusion to a serum albumin binding VHH is used as half-life extension methodology. Building blocks are linked via a triple Ala or 9 Gly-Ser flexible linker. VHHs are produced and purified as described in Example 9. An overview of the format and sequence of all four bispecific VHHs is depicted in
TABLE-US-00041 TABLE 38-A Sequences of bispecific VHH targeting VEGF and Ang2 VHH ID AA sequence VEGFANGBII00001 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKN TVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 186) VEGFANGBII00002 EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSDVQLVE SGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISRDN AKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSS (SEQ ID NO: 187) VEGFANGBII00003 EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSDVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSL EGRFTISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 188) VEGFANGBII00004 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSAAAEVQLVE SGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRD NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSAAAEVQLVESGGGLVQAGGSLRLSC AASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKNTVYLQMNSLKPE DTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 189)
[0378] To explore the anti-VEGF blocking properties in comparison with the monovalent building block VEGFBII00038, all four bispecific VHHs are analyzed in the VEGF/VEGFR2-Fc (
[0379] To explore the anti-Ang2 blocking properties in comparison with the monovalent building block 00027 (SEQ ID No:216), all four bispecific VHHs are analyzed in a human Ang2/hTie2-Fc (
Example 11
Construction, Production and Characterization of Trivalent and Tetravalent Bispecific VHHs Targeting VEGF and Ang2 Using Anti-Serum Albumin Binding as Half-Life Extension
[0380] Ten bispecific VHHs targeting VEGF and Ang2 are constructed (VEGFANGBII00005-00015). In these constructs monovalent and bivalent 1D01 (SEQ ID NO:214), monovalent and bivalent 7G08 (SEQ ID NO:215) and bivalent 00027 (SEQ ID NO:216) anti-Ang2 building blocks are included. A genetic fusion to a serum albumin binding VHH is used as half-life extension methodology. Building blocks are linked via a 9 Gly-Ser flexible linker. VHHs are produced and purified as described in Example 8. An overview of the format and sequence of all ten bispecific VHHs is depicted in
TABLE-US-00042 TABLE 41-A Sequences of bispecific VHH targeting VEGF and Ang2 VHH ID AA sequence VEGFANGBII00005 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKG RFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 190) VEGFANGBII00006 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGRFTISRDNAKN TVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSS (SEQ ID NO: 191) VEGFANGBII00007 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSSGGGGSGGG SEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKG RFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGGSLRLSCAASGFALDYYAIGWFRQVPGKEREGVSCISSSDGITYYVDSVKGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCATDSGGYIDYDCMGLGYDYWGQGTLVTVSS (SEQ ID NO: 192) VEGFANGBII00008 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADS VKGRFTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSG GGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 193) VEGFANGBII00009 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSDNDKN TVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVE SGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSD NDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 194) VEGFANGBII00010 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGR FTISSDNDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVE SGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSSIRDNDGSTYYADSVKGRFTISSD NDKNTVYLQMNSLKPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS (SEQ ID NO: 195) VEGFANGBII00011 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGR FTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRF TISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFT ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 196) VEGFANGBII00012 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKN TVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGL VQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNT VYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 197) VEGFANGBII00013 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGR FTISSDNAKNTVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGL VQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKNT VYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 198) VEGFANGBII00014 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQAGGSLRLSCAASGFTFDDYALGWFRQAAGKEREGVSCIRCSDGSTYYADSVKGRFTISSDNAKN TVYLQMNSLKPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS (SEQ ID NO: 199)
[0381] To explore the anti-VEGF blocking properties in comparison with the monovalent building block VEGFBII00038, all ten bispecific VHHs are analyzed in the VEGF/VEGFR2-Fc (Example 10;
[0382] To explore the anti-Ang2 blocking properties in comparison with their respective monovalent building block 7G08 (SEQ ID No:215), 1D01 (SEQ ID No:214) and 00027 (SEQ ID No:216), all ten bispecific VHHs are analyzed in the human Ang2/hTie2-Fc (see Example 5.1;
[0383] Affinities of for human serum albumin have been determined and are shown in Table 44. Briefly, human serum albumin (Sigma, St Louis, Mo., USA) is immobilized on a CM5 chip via amine coupling. A multicycle kinetic approach is used: increasing concentrations of VHH (2-8-31-125-500 nM) are injected and allowed to associate for 2 min and to dissociate for 10 min at a flow rate of 100 μL/min. Between VHH injections, the surfaces are regenerated with a 10 sec pulse of 10 mM Glycine-HCl pH 1.5 and 60 sec stabilization period. Association/dissociation data are evaluated by fitting a 1:1 interaction model (Langmuir binding) or Heterogeneous Ligand model. The affinity constant K.sub.D is calculated from resulting association and dissociation rate constants k.sub.a and k.sub.d (Table 44).
TABLE-US-00043 TABLE 44 Affinity K.sub.D of purified VHHs for human (HSA), cyno (CSA) and mouse serum albumin (MSA) HSA CSA MSA 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 (1/MS) (1/s) (nM) (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) ALB11 4.5E+05 1.8E−03 4 4.3E+05 1.6E−03 4 6.6E+05 3.2E−02 49 VEGFANGBII00001 2.3E+05 4.8E−03 22 1.8E+05 4.3E−03 24 n.d. n.d. n.d. VEGFANGBII00005 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII00006 2.0E+05 4.6E−03 22 1.5E+05 4.5E−03 30 1.7E+05 6.0E−02 360 VEGFANGBII00007 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII00008 1.3E+05 4.3E−03 34 n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII00009 1.5E+05 4.6E−03 30 1.1E+05 4.2E−03 39 1.2E+05 4.0E−02 340 VEGFANGBII00010 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII00011 1.3E+05 4.0E−03 31 n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII00012 1.5E+05 4.3E−03 31 1.2E+05 4.2E−03 24 1.0E+05 2.5E−02 240 VEGFANGBII0013 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. VEGFANGBII0014 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d., not determined
Example 12
Construction, Production and Characterization of Sequence Optimized and Affinity Matured Bispecific VHHs Targeting VEGF and Ang2 Using Anti-Serum Albumin Binding as Half-Life Extension
[0384] 14 bispecific VHHs targeting VEGF and Ang2 are constructed (VEGFANGBII00015-00028). In these constructs bivalent 00921 (a sequence optimized 1D01 variant) (SEQ ID No:220), monovalent VHHs 00908-00932-00933-00934-00935-00936-00937-00938 (sequence optimized/affinity matured 28D10 variants) (SEQ ID No:222), bivalent 00956 (SEQ ID NO:223) (sequence optimized 28D10 variant) and monovalent 00928 (SEQ ID NO:221) (sequence optimized 37F02 variant) anti-Ang2 building blocks are included. A genetic fusion to a serum albumin binding VHH is used as half-life extension methodology. Building blocks are linked via a 9 Gly-Ser flexible linker. An overview of the format and sequence of all 14 bispecific VHHs is depicted in
[0385] Expression levels are indicated in Table 45-B.
TABLE-US-00044 TABLE 45-A Sequences of bispecific VHH targeting VEGF and Ang2 VHH ID AA sequence VEGFANGBII00015 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS (SEQ ID NO: 200) VEGFANGBII00016 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSSIRDNGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 201) VEGFANGBII00017 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 202) VEGFANGBII00018 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSAIRESGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 203) VEGFANGBII00019 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGITLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 204) VEGFANGBII00020 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 205) VEGFANGBII00021 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSAIRESGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 206) VEGFANGBII00022 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAVSGITLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS SEQ ID NO: 207) VEGFANGBII00023 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVE SGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGRFTISSD NSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS SEQ ID NO: 208) VEGFANGBII00024 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGS GGGSEVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRDNGGSTYYADS VKGRFTISSDNSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSG GGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS SEQ ID NO: 209) VEGFANGBII00025 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGL VQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRFTISSDNSKNT VYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSS SEQ ID NO: 210) VEGFANGBII00026 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGR FTISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDYALGWFRQAPGKEREGVSCIRCSGGSTYYADSVKGRF TISSDNSKNTVYLQMNSLRPEDTAVYYCAASIVPRSKLEPYEYDAWGQGTLVTVSSGGGGSGGGSEV QLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFT ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS SEQ ID NO: 211) VEGFANGBII00027 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGFALDYYAIGWFRQAPGKEREGVSCISSSGGITYYADSVKGRFTISRDNSKN TVYLQMNSLRPEDTAVYYCATDSGGYIDYDCSGLGYDYWGQGTLVTVSS SEQ ID NO: 212) VEGFANGBII00028 DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRF TISRDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGR FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKN TVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSSGGGGSGGGSEVQLVE SGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSD NSKNTVYLQMNSLRPEDTAVYYCAAVPAGRLRFGEQWYPLYEYDAWGQGTLVTVSS SEQ ID NO: 213)
[0386] To explore the anti-VEGF blocking properties in comparison with the monovalent building block VEGFBII00038, the bispecific VHHs are analyzed in the VEGF/VEGFR2-Fc (Example 10;
[0387] Binding kinetics of the bispecific VHHs on human VEGF165 is analyzed by SPR on a Biacore T100 instrument (see Example 12.5 described in patent US 2011/0172398 A1). Monovalent Nanobody VEGFBII00038 is taken along as reference (Table 46-B).
TABLE-US-00045 TABLE 46-B Overview of kinetic parameters in hVEGF165 Biacore assay. ka1 kd1 ka2 kd2 KD1 (1/Ms) (1/s) (1/s) (1/s) (M) VEGFBII00038 2.6E+05 1.3E−02 1.3E−02 1.9E−04 7.5E−10 VEGFANGBII00022 1.6E+05 1.4E−02 1.4E−02 2.2E−04 1.4E−09 VEGFANGBII00025 1.1E+05 1.4E−02 1.4E−02 2.1E−04 1.9E−09 VEGFANGBII00028 1.7E+05 1.3E−02 1.3E−02 2.1E−04 1.1E−09
[0388] The ability of the VHHs to bind to human isoform VEGF121 is determined in a binding ELISA. Binding of a dilution series of VHH to 1 μg/mL directly coated human VEGF121 (R&D) (human VEGF165 as reference) is detected using biotinylated anti-VHH 1A4 followed by extravidin-HRP. 1A4 is a anti-VHH VHH (generated in-house by Ablynx NV). The benchmark Avastin serves as positive control and is detected using a HRP conjugated anti-human Fc antibody. An irrelevant VHH serves as negative control. Representative binding response curves on VEGF165 and VEGF121 are shown in
TABLE-US-00046 TABLE 46-C Overview of EC.sub.50 values in hVEGF165 and hVEGF121 binding ELISA. hVEGF165 hVEGF121 EC.sub.50 (M) EC.sub.50 (M) VEGFANGBII00022 1.4E−09 2.3E−09 VEGFANGBII00025 1.5E−09 2.5E−09 VEGFANGBII00028 1.2E−09 2.1E−09
[0389] Binding to rat and mouse VEGF164 is assessed in a binding ELISA. VHHs binding to 1 μg/mL directly coated murine or rat VEGF164 (R&D) are detected using biotinylated anti-VHH 1A4 followed by extravidin-HRP. As positive control the mouse/rat cross-reactive monoclonal antibody B20-4.1 (Genentech) is titrated and detected with an HRP conjugated anti-human Fc antibody. An irrelevant VHH serves as negative control. Results are shown in
[0390] Binding to human VEGF-B, VEGF-C, VEGF-D and PIGF is assessed via a binding ELISA. Binding of VHHs to 1 μg/mL directly coated VEGF-B (R&D), VEGF-C(R&D), VEGF-D (R&D) and PIGF (R&D) was detected using biotinylated anti-VHH 1A4 followed by extravidin-HRP. As positive controls a series of dilutions of the appropriate receptors (hVEGFR1-Fc for hVEGF-B and hPIGF, hVEGFR2-Fc for hVEGF-C, anti-hVEGF-D mAb (R&D) for hVEGF-D) are taken along. An irrelevant VHH serves as negative control. Results are shown in
[0391] To explore the anti-Ang2 blocking properties in comparison with their respective monovalent building block 00921 (SEQ ID NO:220) and 00938 (SEQ ID NO:222), all 3 bispecific VHHs are analyzed in the human Ang2/hTie2-Fc (see Example 5.1;
[0392] Affinities of VEGFANGBII00022-25-28 for human, mouse, cyno and rat Ang2 (see Example 5.4) have been determined and are shown in Table 47-B.
TABLE-US-00047 TABLE 47-B Affinity KD of purified VHHs for recombinant human, cyno, mouse and rat Ang2 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) human Ang2-FLD cyno Ang2-FLD VEGFANGBII00022 9.7E+05 1.5E−05 1.6E−11 1.5E+06 1.3E−05 8.1E−12 VEGFANGBII00025 2.7E+06 1.2E−02 4.5E−09 4.3E+06 1.1E−02 2.7E−09 VEGFANGBII00028 5.9E+05 9.6E−04 1.6E−09 8.4E+05 8.7E−04 1.0E−09 mouse Ang2-FLD rat Ang2-FLD VEGFANGBII00022 5.5E+05 2.8E−05 5.1E−11 3.9E+05 3.8E−05 9.9E−11 VEGFANGBII00025 1.3E+06 1.4E−02 1.1E−08 8.7E+05 2.9E−02 3.3E−08 VEGFANGBII00028 3.6E+05 2.0E−03 5.6E−09 2.5E+05 3.1E−03 1.2E−08
[0393] Affinities of VEGFANGBII00022-25-28 for human, mouse and cyno serum albumin have been determined (Example 11) and are shown in Table 48. The affinity constant K.sub.D is calculated from resulting association and dissociation rate constants k.sub.a and k.sub.d (Table 48).
TABLE-US-00048 TABLE 48 Affinity KD (nM) of purified VHHs for recombinant human, mouse and cyno serum albumin using (A) 1:1 interaction model or (B) heterogeneous ligand model (A) HSA CSA k.sub.a k.sub.d K.sub.D k.sub.a k.sub.d K.sub.D (1/MS) (1/s) (nM) (1/Ms) (1/s) (nM) ALB11 5.6E+05 1.9E−03 4 4.5E+05 1.7E−03 4 VEGFANGBII00022 6.7E+05 6.0E−03 9 6.2E+05 5.4E−03 9 VEGFANGBII00025 5.6E+05 5.6E−03 12 4.3E+05 5.1E−03 12 VEGFANGBII00028 5.6E+05 5.8E−03 10 5.2E+05 5.3E−03 10 MSA k.sub.a k.sub.d K.sub.D (1/Ms) (1/s) (nM) ALB11 5.9E+05 3.0E−02 51 VEGFANGBII00022 5.2E+05 5.4E−03 150 VEGFANGBII00025 — — — VEGFANGBII00028 — — — (B) MSA k.sub.a1 k.sub.d1 k.sub.a2 k.sub.d2 K.sub.D1 K.sub.D2 (1/MS) (1/s) (1/s) (1/s) (nM) (nM) VEGFANGBII00025 6.2E+05 9.9E−02 4.7E+04 5.7E−04 160* 12 VEGFANGBII00028 5.9E+04 6.9E−04 5.7E+05 9.4E−02 12 160* *describes 70% or more of the interaction
TABLE-US-00049 Ang2-binding components (Table 49) FR1 CDR1 FR2 CDR2 1D01 EVQLVESGGGLVQAGGSLRLSCAASGFTFD DYALG WFRQAAGKEREGVS CIRCSDGSTYYADSVKG 7G08 EVQLVESGGGLVQPGGSLRLSCAASGFALD YYAIG WFRQVPGKEREGVS CISSSDGITYYVDSVKG 027 EVQLVESGGGLVQAGGSLRLSCAASGFTLD DYAIG WFRQAPGKEREGVS CIRDSDGSTYYADSVKG FR3 CDR3 FR4 1D01 RFTISSDNAKNTVYLQMNSLKPEDTAVYYCAA SIVPRSKLEPYEYDA WGQGTQVTVSS 7G08 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAT DSGGYIDYDCMGLGYDY WGQGTQVTVSS 027 RFTI SDNDKNTVYLQMNSLKPEDTAVYYCAA VPAGRLRFGEQWYPLYEYDA WGQGTQVTVSS FR1 CDR1 FR2 CDR2 00042 EVQLVESGGGLVQPGGSLRLSCAASGFTLD DYAIG WFRQAPGKEREGVS SIRDNDGSTYYADSVKG 00050 EVQLVESGGGLVQPGGSLRLSCAASGFTFD DYALG WFRQAPGKEREGVS CIRCSDGSTYYADSVKG 00045 EVQLVESGGGLVQPGGSLRLSCAASGFALD YYAIG WFRQAPGKEREGVS CISSSDGITYYADSVKG FR3 CDR3 FR4 00042 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA VPAGRLRFGEQWYPLYEYDA WGQGTLVTVSS 00050 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA SIVPRSKLEPYEYDA WGQGTLVTVSS 00045 RFTISRDNSKNTVYLQMNSLRPEDTAVYYCAT DSGGYIDYDCMGLGYDY WGQGTLVTVSS FR1 CDR1 FR2 CDR2 00921 EVQLVESGGGLVQPGGSLRLSCAASGFTFD DYALG WFRQAPGKEREGVS CIRCSGGSTYYADSVKG 00928 EVQLVESGGGLVQPGGSLRLSCAASGFALD YYAIG WFRQAPGKEREGVS CISSSGGITYYADSVKG 00938 EVQLVESGGGLVQPGGSLRLSCAVSGITLD DYAIG WFRQAPGKEREGVS AIRSSGGSTYYADSVKG 00956 EVQLVESGGGLVQPGGSLRLSCAASGFTLD DYAIG WFRQAPGKEREGVS AIRSSGGSTYYADSVKG FR3 CDR3 FR4 00921 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA SIVPRSKLEPYEYDA WGQGTLVTVSS 00928 RFTISRDNSKNTVYLQMNSLRPEDTAVYYCAT DSGGYIDYDCSGLGYDY WGQGTLVTVSS 00938 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA VPAGRLRYGEQWYPIYEYDA WGQGTLVTVSS 00956 RFTISSDNSKNTVYLQMNSLRPEDTAVYYCAA VPAGRLRFGEQWYPLYEYDA WGQGTLVTVSS (1D01 (SEQ ID No: 214); 7G08 (SEQ ID No: 215); 027 (SEQ ID No: 216); 00042 (SEQ ID No: 217); 00050 (SEQ DI No: 218); 00045 (SEQ ID No: 219); 00921 (SEQ ID No: 220); 00928 (SEQ ID No: 221); 00938 (SEQ ID No: 222); 00956 (SEQ ID No: 223)