METHOD FOR MEASUREMENT AND CONTROL OF INTRACULAR VEGF CONCENTRATION

Abstract

The invention describes a new method for in vivo measurement and control of intraocular VEGF concentration using bioluminescence resonance energy transfer (BRET) of a VEGF-binding biosensor. Furthermore, the method is suitable for highly sensitive in vitro determination of VEGF concentration from a small sample volume.

Claims

1. A method for measurement and control of intraocular vascular endothelial growth factor (VEGF) concentration, which comprises the steps of: adding a Renilla luciferase substrate to VEGF-binding biosensor molecules each comprising an anti-VEGF single chain variable fragment (anti-VEGF-scFv) with Renilla luciferase fused to its N-terminus and a fluorescent protein or peptide fused to its C-terminus in a liquid containing VEGF, measuring bioluminescence resonance energy transfer (BRET) signal depending on binding of prevalent VEGF to the biosensor molecules as an indicator for VEGF concentration, and inducing expression of anti-VEGF molecules by addition of doxycycline to a vector encoding anti-VEGF molecules that is transduced into eukaryotic cells.

2. The method according to claim 1, wherein the fluorescent protein is GFP2, YFP, eYFP, TurboYFP, or peptides or derivatives or mutants thereof.

3. The method according to claim 1, wherein measurement and control of intraocular VEGF concentration are performed in vivo.

4. The method according to claim 1, wherein the VEGF-binding biosensor and the vector encoding anti-VEGF molecules that is transduced into eukaryotic cells are encapsulated in an eye-implantable, permeable microcapsule, microparticle, microbead, or gel.

5. The method according to claim 4, wherein the eye-implantable, permeable microcapsule, microparticle, microbead, or gel is permeable for VEGF, Renilla luciferase substrate, doxycycline, and anti-VEGF molecules, but is not permeable for VEGF-anti-VEGF antigen-antibody complexes and VEGF bound to VEGF-binding biosensor.

6. The method according to claim 5, wherein the eye-implantable, permeable microcapsule, microparticle, microbead, or gel is made from alginate.

7. The method according to claim 1, wherein measurement of intraocular VEGF concentration is performed in vitro and which comprises the following steps: addition of a Renilla luciferase substrate to VEGF-binding biosensor molecules each comprising an anti-VEGF single chain variable fragment (anti-VEGF-scFv) with Renilla luciferase fused to its N-terminus and a fluorescent protein or peptide fused to its C-terminus in a liquid containing VEGF, measurement of bioluminescence resonance energy transfer (BRET) signal depending on binding of VEGF to the biosensor molecules as an indicator for VEGF concentration.

8. The method according to claim 7, wherein the measurement is performed with a sample volume of 1 to 10 l.

9. The method according to claim 7, wherein the lower detection limit of VEGF concentration is 100 fg/ml and the upper detection limit is 10 ng/ml.

10. A method for diagnosis and/or therapy of VEGF-concentration-related retinal neovascular disorders comprising the steps of: adding a Renilla luciferase substrate to VEGF-binding biosensor molecules each comprising an anti-VEGF single chain variable fragment (anti-VEGF-scFv) with Renilla luciferase fused to its N-terminus and a fluorescent protein or peptide fused to its C-terminus in a liquid containing VEGF, measuring bioluminescence resonance energy transfer (BRET) signal depending on binding of prevalent VEGF to the biosensor molecules as an indicator for VEGF concentration, and inducing expression of anti-VEGF molecules by addition of doxycycline to a vector encoding anti-VEGF molecules that is transduced into eukaryotic cells.

11. A method for diagnosis of VEGF-concentration-related retinal neovascular disorders comprising the steps of: adding a Renilla luciferase substrate to VEGF-binding biosensor molecules each comprising an anti-VEGF single chain variable fragment (anti-VEGF-scFv) with Renilla luciferase fused to its N-terminus and a fluorescent protein or peptide fused to its C-terminus in a liquid containing VEGF, and measuring bioluminescence resonance energy transfer (BRET) signal depending on binding of prevalent VEGF to the biosensor molecules as an indicator for VEGF concentration, wherein measurement of intraocular VEGF concentration is performed in vitro and which comprises the steps of: adding a Renilla luciferase substrate to VEGF-binding biosensor molecules each comprising an anti-VEGF single chain variable fragment (anti-VEGF-scFv) with Renilla luciferase fused to its N-terminus and a fluorescent protein or peptide fused to its C-terminus in a liquid containing VEGF, and measuring bioluminescence resonance energy transfer (BRET) signal depending on binding of VEGF to the biosensor molecules as an indicator for VEGF concentration.

12. The method of claim 10 wherein the VEGF concentration-related retinal neovascular disorder is selected from the group consisting of age-related macular degeneration, diabetic macular edema, diabetic retinopathy, retinopathy of prematurity, and retinal vein occlusion.

13. The method of claim 11 wherein the VEGF-concentration-related retinal neovascular disorder is selected from the group consisting of age-related macular degeneration, diabetic macular edema, diabetic retinopathy, retinopathy of prematurity, and retinal vein occlusion.

Description

DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1. Antigen-induced conformational change of the VEGF-binding biosensor according to the present invention and BRET assay. If VEGF molecules are present, VEGF binds to the Ra02 parts (Ra02-L and Ra02-H) of the biosensor molecule and therefore induces a conformational change of the biosensor molecule. Thus, in the presence of Renilla luciferase substrate Coelenterazine, the Renilla luciferase RLuc8 at the N-terminus of the biosensor molecule emits radiation as a donor (BRET signal 1) that is then accepted by a fluorescent protein or peptide, e.g. GFP2, eGFP, eYFP, or TurboYFP, at the C-terminus of the biosensor molecule. The acceptor molecule then emits itself radiation at another wavelength than the donor (BRET signal 2). Both BRET signals are measured using a device using appropriate filters (e.g. magenta for BRET signal 1 and green for BRET signal 2) and a software that determines the BRET ratio. Then the BRET ratio is used for determination of the VEGF concentration by linear regression methods. The different parts of biosensor molecule (RLuc8, Ra02, and the fluorescent protein or peptide) are either fused directly to each other or are separated by proline (Pro) or 4 glycine (4Gly) linkers.

[0030] FIG. 2. Insert for implantation into the eye of a patient whose VEGF concentration in the eye should be measured in vivo. The insert is a microcapsule, microparticle, microbead, or gel, for example made from alginate, that is permeable for VEGF, Renilla luciferase substrate Coelenterazine, doxycycline, and anti-VEGF molecules, but is not permeable for VEGF-anti-VEGF antigen-antibody complexes and VEGF bound to the VEGF-binding biosensor. The insert encapsulates VEGF-binding biosensor molecules, like e.g. RLuc8-Ra02-GFP2 (SEQ ID No. 1), and additionally a doxycycline-inducible vector, TetOn-Ra02, that encodes anti-VEGF molecules and which is transduced into eukaryotic cells. A In the absence of free VEGF molecules, RLuc8 may convert its substrate Colenterazine and emit radiation, but this radiation can be transferred via BRET to the acceptor GFP only at a very low level. B In the presence of free VEGF molecules, the biosensor molecules undergoes a conformational change upon binding of VEGF to Ra02 of the biosensor. As a consequence, in the presence of Coelenterazine, the radiation of RLuc8 is transferred to the acceptor GFP, which then itself emits radiation. Both signals can be measured with an appropriate device and the BRET ratio as well as the VEGF concentration can be determined as described herein. If the VEGF concentration is too high, synthesis of anti-VEGF molecules can be triggered by administration of doxycycline to the patient. Doxycycline then induces expression of anti-VEGF molecules from the vector TetOn-Ra02 in the eukaryotic cells that are also encapsulated in the insert. Newly synthesized anti-VEGF molecules are small enough to leave the insert and to bind to free VEGF that is present in the eye of the patient.

[0031] FIG. 3. Change of BRET ratio in dependence of the VEGF concentration. The VEGF concentration is displayed on the x-axis (c(VEGF)) at ng/ml. On the y-axis the change of the BRET ratio dependent on the VEGF concentration is shown in delta milli BRET Units (mBU). Exponential growth is measured in the range of 0.0001 ng/ml up to 0.01 ng/ml, which corresponds to 2 log units of the VEGF concentration.

[0032] FIG. 4. Change of BRET ratio in dependence of the VEGF concentration. The VEGF concentration is displayed on the x-axis (c(VEGF)) at pg/ml. On the y-axis the change of the BRET ratio (BR) dependent on the VEGF concentration is shown in delta milli BRET Units (mBU). Exponential growth is measured in the range of 0.0001 ng/ml up to 0.01 ng/ml, which corresponds to 2 log units of the VEGF concentration. In one case, the concentration of the biosensor RLuc8-Ra02-GFP2 was 90,000 RLU (relative luciferase units; displayed as black dots), in another case the biosensor concentration was 180,000 RLU (displayed as white triangles).

[0033] The following sequences referred to herein are shown in the accompanying sequence listing.

SEQUENCE LISTING

[0034] SEQ ID No. 1: RLuc8-Ra02-GFP2 biosensor molecule

[0035] SEQ ID No. 2: Vector with TetOn-Ra02 expression cassette