Fusion proteins for ophthalmology with increased eye retention

Abstract

The combination of a first binding site specifically binding to a target associated with an eye disease and a second binding site specifically binding to a target influencing the retention in the eye a multispecific binder provides for improved intravitreal retention compared to a monospecific binder. The second binding site specifically binds to a compound/molecules found in the extracellular matrix (ECM) in vitreous humor/retina. This compound of the extracellular matrix has to be present in amounts allowing a sufficient loading/dose of the drug to be bound. It has been found that collagen, especially collagen II, is a suitable compound in the ECM in the vitreous humor for this purpose. Thus, herein is reported a multispecific binder comprising a first binding site specifically binding to a therapeutic ocular target, and a second binding site specifically binding to collagen II.

Claims

1. A method of treating an ocular vascular disease in an individual, the method comprising administering to the individual a multispecific binder comprising: (a) an anti-human collagen II antibody, or an antibody fragment thereof, comprising six CDRs determined according to Kabat from SEQ ID NO: 09 and SEQ ID NO: 10; and (b) an antibody, or an antibody fragment thereof, binding to a therapeutic ocular target wherein the therapeutic ocular target is selected from the group consisting of ANG2, VEGF, PDGF-B, and IL-1β.

2. The method of claim 1, wherein the anti-human collagen II antibody, or the antibody fragment thereof, comprises a heavy chain variable domain with the amino acid sequence of SEQ ID NO: 09 and a light chain variable domain with the amino acid sequence of SEQ ID NO: 10.

3. The method of claim 1, wherein the anti-human collagen II antibody, or the antibody fragment thereof, comprises a scFv.

4. A method of treating an ocular vascular disease in an individual, the method comprising administering to the individual a fusion protein comprising: (a) a Fab specifically binding to a therapeutic ocular target wherein the therapeutic ocular target is selected from the group consisting of ANG2, VEGF, PDGF-B, and IL-1β; and (b) a scFv specifically binding to collagen II, wherein the scFv specifically binding to collagen II comprises six CDRs determined according to Kabat from SEQ ID NO: 09 and SEQ ID NO: 10, wherein the Fab is conjugated by a peptide bond at one of its C-termini to the N-terminus of a peptidic linker and the scFv is conjugated by a peptide bond at its N-terminus to the C-terminus of the peptidic linker.

5. The method of claim 3, wherein the scFv comprises a peptidic linker.

6. The method of claim 2, wherein the anti-human collagen II antibody, or the fragment thereof, comprises the amino acid sequence of SEQ ID NO: 11.

7. The method of claim 4, wherein the scFv specifically binding to collagen II comprises the amino acid sequence of SEQ ID NO: 11.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 Half-life of the different constructs in the different compartments of the eye; 1: FAB-PEG, 2: FAB-HBD, 3: FAB, 4: FAB-COLL-1, 5: FAB-COLL-II, 6: FAB-COLL-III; upper bar: vitreous, middle bar: retina, lower bar: choroid.

(2) FIG. 2 Exposure of different compartments (tissues) of the eye to the different constructs; 1: FAB-COLL-I, 2: FAB-COLL-II, 3: FAB-COLL-III, 4: FAB, 5: FAB-HBD, 6: FAB-PEG; left bar: vitreous, middle bar: retina, right bar: choroid.

MATERIALS AND METHODS

(3) Recombinant DNA Techniques

(4) Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The molecular biological reagents were used according to the manufacturer's instructions.

(5) Gene Synthesis

(6) Desired gene segments were ordered according to given specifications at Geneart (Regensburg, Germany).

(7) DNA Sequence Determination

(8) DNA sequences were determined by double strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).

(9) DNA and Protein Sequence Analysis and Sequence Data Management

(10) The GCG's (Genetics Computer Group, Madison, Wis.) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation and illustration.

(11) Expression Vectors

(12) For the expression of the described antibodies expression plasmids for transient expression (e.g. in HEK293-F cells) based either on a cDNA organization with or without a CMV-Intron A promoter or on a genomic organization with a CMV promoter were used.

(13) The transcription unit of the antibody gene was composed of the following elements: unique restriction site(s) at the 5′ end, the immediate early enhancer and promoter from the human cytomegalovirus, in the case of the cDNA organization the Intron A sequence, a 5′-untranslated region of a human immunoglobulin gene, a nucleic acid encoding an immunoglobulin heavy chain signal sequence, a nucleic acid encoding the human antibody chain (wild-type or with domain exchange) either as cDNA or in genomic organization with the immunoglobulin exon-intron organization, a 3′ non-translated region with a polyadenylation signal sequence, and unique restriction site(s) at the 3′ end.

(14) Beside the antibody expression cassette the plasmids contained: an origin of replication which allows replication of this plasmid in E. coli, a β-lactamase gene which confers ampicillin resistance in E. coli., and the dihydrofolate reductase gene from Mus musculus as a selectable marker in eukaryotic cells.

(15) The nucleic acids encoding the antibody chains were generated by PCR and/or gene synthesis and assembled by known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective vectors. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfections larger quantities of the plasmids were prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).

(16) Cell Culture Techniques

(17) Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons, Inc.

(18) The bispecific antibodies were expressed by transient co-transfection of the respective expression plasmids in in HEK293-F cells growing in suspension as described below.

Example 1

(19) Expression and Purification

(20) Transient transfections in HEK293-F system

(21) The fusion constructs were generated by transient transfection with the respective plasmids using the HEK293-F system (Invitrogen) according to the manufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle™ 293 expression medium (Invitrogen) were transfected with a mix of the respective expression plasmids and 293fectin™ or fectin (Invitrogen). For 2 L shake flask (Corning) HEK293-F cells were seeded at a density of 1*10.sup.6 cells/mL in 600 mL and incubated at 120 rpm, 8% CO.sub.2. The day after the cells were transfected at a cell density of approx. 1.5*10.sup.6 cells/mL with approx. 42 mL of a mixture of A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmid DNA (1 μg/mL) encoding the heavy or modified heavy chain, respectively and the corresponding light chain in an equimolar ratio and B) 20 ml Opti-MEM with 1.2 mL 293 fectin or fectin (2 μL/mL). According to the glucose consumption glucose solution was added during the course of the fermentation. The supernatant containing the secreted antibody was harvested after 5-10 days and antibodies were either directly purified from the supernatant or the supernatant was frozen and stored.

(22) Purification

(23) The polypeptide-containing culture supernatants were filtered and purified by two chromatographic steps. The antibodies were captured by affinity chromatography using HiTrap KappaSelect (GE Healthcare) equilibrated with PBS (1 mM KH.sub.2PO.sub.4, 10 mM Na.sub.2HPO.sub.4, 137 mM NaCl, 2.7 mM KCl), pH 7.4. Unbound proteins were removed by washing with equilibration buffer, and the fusion polypeptide was recovered with 100 mM citrate buffer, pH 2.9, and immediately after elution neutralized to pH 6.0 with 1 M Tris-base, pH 9.0. Size exclusion chromatography on HiLoad 26/60 Superdex 75™ (GE Healthcare) was used as second purification step. The size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The polypeptide containing solutions were concentrated with an Ultra free -CL centrifugal filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, Mass.) and stored at −80° C.

(24) The protein concentrations of the polypeptides were determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.

(25) Purity and integrity of the polypeptides molecules were analyzed by CE-SDS using a LabChip GX II (PerkinElmer) with Protein Express Chip and HT Protein Express Reagents Kit.

(26) Aggregate content was determined by high-performance SEC using a Biosuite High Resolution SEC, 250 Å, 5 μm analytical size-exclusion column (Waters GmbH) using 200 mM K.sub.2HPO.sub.4/KH.sub.2PO.sub.4, 250 mM KCl, pH 7.0 as running buffer.

(27) The integrity of the amino acid backbone of reduced polypeptides was verified by Nano Electrospray QTOF mass spectrometry after removal of N-glycans by enzymatic treatment with a combination of neuraminidase, 0-glycanase and peptide-N-glycosidase F (Roche Applied Science).

Example 2

(28) Binding to Human and Porcine Collagen II

(29) Binding kinetics of anti-collagen antibodies to human Collage type II (Millipore CC052) and porcine Collagen type H (USBiological C7510-31) was investigated by surface plasmon resonance using a BIAcore T200 instrument (GE Healthcare). All experiments were performed at 25° C. using HBS-P (10 mM His, 140 mM NaCl, 0.05% Tween 20 pH 7.4) as running and dilution buffer. Collagen type II was immobilized on a Series S CM5 Sensor Chip (GE Healthcare) using standard amine coupling chemistry. Anti-Collagen antibodies were injected for 180 s with concentrations from 1.23 up to 900 nM (1:3 dilution series) onto the surface (association phase). The dissociation phase was monitored for 600 sec by washing with running buffer. The surface was regenerated by injecting 0.85% H3PO4 for 60 sec. Bulk refractive index differences were corrected by subtracting the response obtained from a mock surface. Blank injections were subtracted (double referencing). The derived curves were fitted to a 1:1 Langmuir binding model using the BIAevaluation software.

Example 3

(30) Minipig Pharmacokinetic Study

(31) Female minipigs, 7-8 kg each, were administered 1.25 nmol of each drug by IVT injection. The aimed initial concentration was 500 nM in the eye for each molecule. Vitreous, retina and choroid samples were collected at three termination time points 168, 336 and 672 hours after application.

Example 4

(32) Pharmacokinetic Parameter Determination

(33) Minipig serum, aqueous humor, vitreous humor and ocular tissue (retina, choroid, sclera, iris, lens, ciliary body) were analyzed with an ECLIA method using an ELECSYS instrument (Roche Diagnostics GmbH).

(34) Briefly, test sample (calibrator, quality control or study sample), first detection antibody mAb<H-Fab(kappa)>M-1.7.10-IgG-Bi, second detection antibody mAb<Fab(CH1)>M-1.19.31-IgG-Ru, and SA-beads are added stepwise to a detection vessel and incubated for 9 minutes in each step. Finally, the SA-beads-bound complex is detected by a measuring cell, which numbers the counts of SA-beads in repeat. The counts are proportional to the analyte concentration in the test sample.

(35) Bi=biotin, Ru=ruthenium label, SA=streptavidin

(36) Prior to analysis, vitreous humor and ocular tissue samples were mechanically lysed in tissue extraction buffer (10 mM Tris, 137 mM NaCl, 1% Triton, 10% Glycerin) containing protease inhibitors using the Magana Lyser Homogenisator (Roche Diagnostics GmbH).

(37) The assay calibration range for the three collagen binder conjugates FAB-COLL-I, -II, and -III was between 4.92 ng/mL and 3000 ng/mL (assay concentration).

(38) Serum samples were diluted 1:10 to 1:20 to obtain valid results. Standard curve, quality control and sample dilutions were done in assay buffer incl. minipig serum resulting in 10% matrix concentration. Experimental serum samples below 49.2 ng/mL were annotated as “BLQ”.

(39) Aqueous humor, vitreous humor and ocular tissue samples were measured undiluted and diluted up to 1:50 to obtain valid results. Standard curve, quality control and sample dilutions were done in assay buffer without matrix. Experimental aqueous humor, vitreous humor and ocular tissue samples below 4.92 ng/mL were annotated as “BLQ”.

Example 5

(40) Diffusion Parameter Determination

(41) The test solutions—vitreous fluid of minipigs—was stored at −80° C.

(42) Dig-3-cme-eda-Cy5 was dissolved in DMF and adjusted to 1 mM Dig-Cy5 in 30% DMF/dilution buffer). A working stock was prepared as a 50 μM Dig-Cy5 solution in PBS/0.2% BSA/1.5% DMF. PBS was purchased at LONZA (#17-516F), pH 7.3-pH 7.5 and was supplemented with 0.2% BSA (fraction V). Measurements are done in 384-well glass bottom assay plates (MMI, #60200).

(43) One sample was thawed on ice. The fluid is highly viscous and transparent. The sample was cautiously pipetted up and down ten times with a cropped 1000 μL tip. It does foam mildly. Aliquots of 100 μl (using a cropped 200 μl tip) are frozen on dry ice and stored at −80° C.

(44) The other samples were thawed and liquefied alike. The bulk amount of all three samples is pooled, aliquoted and stored at −80° C. with sample name “all”. Some original aliquots are stored as reference sample.

(45) FCS measurements were performed with a ConfoCor2 FCS unit connected to an Axiovert 100M equipped with a C-Apochromat 40× N. A. 1.2 water immersion lens (Carl Zeiss, Jena, Germany). At this instrument Cy5 was excited with a 633 helium-neon laser. The red fluorescence emitted by Cy5 was detected with an LP 650 long pass filter. Measurements were performed typically with acquisition settings of 10 times for 10 seconds. The fluorescence fluctuations were auto-correlated with appropriate fitting formalisms. Data analysis allows determining the brightness, behavior and diffusion time of fluorescent particles in homogenous solution.