US20220119536A1 - Agonistic trkb binding molecules for the treatment of eye diseases - Google Patents

Agonistic trkb binding molecules for the treatment of eye diseases Download PDF

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US20220119536A1
US20220119536A1 US17/505,763 US202117505763A US2022119536A1 US 20220119536 A1 US20220119536 A1 US 20220119536A1 US 202117505763 A US202117505763 A US 202117505763A US 2022119536 A1 US2022119536 A1 US 2022119536A1
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trkb
scfv
binding molecule
molecule
agonistic
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Peter Michael Benz
Remko Alexander BAKKER
Holger Fuchs
Fei Han
Sandeep Kumar
Sarah Low
Justin M. Scheer
Leo Thomas
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Boehringer Ingelheim International GmbH
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    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This invention relates to agonistic TrkB binding molecules, methods for improving agonistic TrkB binders, methods for producing or generating agonistic TrkB binding molecuels, their use in medicine, pharmaceutical compositions comprising the same, and methods of using the same as agents for treatment and/or prevention of diseases e.g. of the eye.
  • TrkB Tropomyosin receptor kinase B
  • TrkB also known as tyrosine receptor kinase B, or BDNF/NT-3 growth factors receptor or neurotrophic tyrosine kinase, receptor, type 2
  • TrkB is a protein that in humans is encoded by the NTRK2 gene (Genbank ID: 4915).
  • TrkB is a receptor for brain-derived neurotrophic factor (BDNF).
  • TrkB The neurotrophic tyrosine kinase receptor B (TrkB; gene symbol: NTRK2) is expressed by retinal neurons and glial cells.
  • TrkB signaling counteracts cell stress and promotes cell survival.
  • loss and functional impairments of retinal neurons and glial cells occur which cause visual impairments and vision loss.
  • Activating TrkB signaling above the basal level can counteract the loss and functional impairments of neurons and glial cells, thus improving visual function.
  • TrkB activation has the potential to regenerate lost synaptic connections in the diseased eye, thereby promoting the regain of visual function.
  • TrkB Upon ligand binding, TrkB undergoes homodimerization followed by autophosphorylation.
  • Dependent on the phosphorylation sites (Y516, Y702, Y706, Y707 or Y817) different signal transduction pathways are activated, including the activity of PLC ⁇ 1 or different subforms of AKT and ERK which regulate distinct overlapping signalling cascades inducing axonal/neurite outgrowth, increasing synaptic plasticity, or increasing cell survival.
  • Agonistic anti-TrkB antibodies have been described in the US20100196390 and US20100150914 as well as their proposed use in the treatment of e.g. Charcot-Marie-Tooth disease or diabetes.
  • the present invention is based on the surprising finding that the design of the TrkB binding sites (two scFv's) in the TrkB binding molecules of the invention apparently supports an optimal sterical formation of TrkB binding and activation.
  • the invention is directed to a TrkB binding molecule comprising or consisting of two scFv's, wherein each scFv binds specifically to TrkB and both together act as a TrkB agonist.
  • TrkB binding molecule comprising or consisting of two scFv's, wherein each scFv binds specifically to TrkB and both together act as a TrkB agonist.
  • the invention relates to a TrkB binding molecule comprising or consisting of two scFv's, wherein each scFv binds specifically to TrkB.
  • the scFv's are connected to each other via a hinge region or a linker.
  • the TrkB binding molecule is in the format of an Fc-scFv, scFv-Fc, (scFv′)2 or scFv-CH 3 .
  • the TrkB binding molecule further comprises an Ig molecule.
  • the Ig molecule is a monoclonal antibody, a human monoclonal antibody, a humanized monoclonal antibody, a chimeric antibody, a fragment of an antibody, such as a Fc, Fv, Fab, Fab′, or F(ab′)2 fragment, a single chain antibody, such as a single chain variable fragment (scFv), a Small Modular Immunopharmaceutical (SMIP), a domain antibody, a nanobody, or a diabody.
  • scFv single chain variable fragment
  • SMIP Small Modular Immunopharmaceutical
  • each scFv is fused to the C- terminus of the heavy chain of the Ig molecule. Further relating to these embodiments, each scFv is fused to the N- terminus of the heavy chain of the Ig molecule. Further relating to these embodiments, the Ig molecule is an IgG, F(ab), or F(ab′)2. Further relating to these embodiments, the Ig molecule comprises or consists of an Fc region. In a further related embodiment, each scFv is fused to the Ig molecule by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids.
  • the two scFv's bind to the same epitope or bind to a different epitope.
  • the two scFv's are each TrkB agonists, preferably partial agonists.
  • the TrkB binding molecule is a TrkB agonist.
  • the TrkB binding molecule is bispecific and tetravalent.
  • the invention relates to a method for improving the efficacy of an agonistic TrkB binder, wherein the agonistic TrkB binder contains a light chain variable domain (VL) and a heavy chain variable domain (VH),
  • VL light chain variable domain
  • VH heavy chain variable domain
  • the agonistic TrkB binder is a partial agonist.
  • the efficacy of the TrkB binding molecule compared to the efficacy of the agonistic TrkB binder is higher by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%.
  • the TrkB binding molecule is about at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% as efficacious as compared to BDNF.
  • the two agonistic TrkB binding scFv's are identical.
  • the scFv's are connected to each other via a hinge region or a linker.
  • the TrkB binding molecule is in the format of an Fc-scFv, scFv-Fc, (scFv′)2 or scFv-CH 3 .
  • the TrkB binding molecule further comprises an Ig molecule.
  • the Ig molecule is a monoclonal antibody, a human monoclonal antibody, a humanized monoclonal antibody, a chimeric antibody, a fragment of an antibody, such as a Fv, Fab, Fab′, or F(ab′)2 fragment, a single chain antibody, such as a single chain variable fragment (scFv), a Small Modular Immunopharmaceutical (SMIP), a domain antibody, a nanobody, or a diabody.
  • scFv single chain variable fragment
  • SMIP Small Modular Immunopharmaceutical
  • each scFv is fused to the C-terminus of the heavy chain of the Ig molecule.
  • each scFv is fused to the N-terminus of the heavy chain of the Ig molecule.
  • the Ig molecule is an IgG, F(ab), or F(ab′)2.
  • the Ig molecule comprises or consists of an Fc region.
  • each scFv is fused to the Ig molecule by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids.
  • the TrkB binding molecule is a TrkB agonist.
  • the agonistic TrkB binder is bivalent.
  • the agonistic TrkB binder is a bivalent partial agonist.
  • the invention relates to a method for improving the efficacy of a bivalent partial agonistic TrkB binder, wherein the bivalent partial agonistic TrkB binder contains a light chain variable domain (VL) and a heavy chain variable domain (VH),
  • VL light chain variable domain
  • VH heavy chain variable domain
  • the bivalent partial agonistic TrkB binder is an IgG.
  • the TrkB binding molecule is in the format of an an Fc-scFv, scFv-Fc, (scFv′)2 or scFv-CH 3 .
  • the TrkB binding molecule is bispecific and tetravalent.
  • the invention is directed to a method to produce or generate a TrkB binding molecule having improved efficacy compared to an agonistic TrkB binder it is based upon, wherein the agonistic TrkB binder contains a light chain variable domain (VL) and a heavy chain variable domain (VH),
  • TrkB binding molecule comprises at least the two agonistic TrkB binding scFv's from step (i) and (ii),
  • TrkB binding molecule has a higher efficacy compared to the efficacy of the agonistic TrkB binder, and wherein the efficacy is the maximum response as determined by incubating CHO cells stably expressing a TrkB receptor with the agonistic TrkB binder or the TrkB binding molecule and measuring the TrkB phosphorylation at Y706/707 in the cell lysate of the treated CHO cells.
  • the invention relates to an isolated nucleic acid molecule encoding (i) the heavy chain or heavy chain variable domain, and/or (ii) the light chain or light chain variable domain of the TrkB binding molecule according to the first aspect or any embodiments related to the first aspect.
  • the invention relates to a viral vector comprising the isolated nucleic acid molecule of the fourth aspect.
  • the invention relates to an expression vector comprising a nucleic acid molecule according to the fifth aspect.
  • the invention relates to a host cell transfected with an expression vector according to the sixth aspect.
  • the invention relates to a method of manufacturing a TrkB binding molecule according to the first aspect or any embodiments related to the first aspect comprising
  • the invention relates to the TrkB binding molecule according to the first aspect or any embodiments related to the first aspect for use in medicine, wherein the use is the treatment of eye or retinal or neurodegenerative diseases.
  • the TrkB binding molecule is for the use according to the ninth aspect, wherein the use is for the treatment and/or prevention of macular degeneration, age-related macular degeneration, wet age-related macular degeneration (wAMD), retinal vein occlusion (RVO), diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, inherited retinal dystrophy, inherited macular dystrophy, myopic degeneration, geographic atrophy, retinal artery occlusions, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane secondary to any retinal diseases, optic neuropathies, glaucoma, retinal detachment, toxic retinopathy, radiation
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TrkB binding molecule according to the first aspect or any embodiments related to the first aspect.
  • FIG. 1 Illustration of the design of VEGF-TrkB single binding molecules from series 1 to series 4 .
  • FIG. 2 A-B (A) TrkB phosphorylation (Y706/707) or (B) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of the indicated molecules. Data represent mean+/ ⁇ SEM.
  • FIG. 3 A-D TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing (A) cyno TrkB, (B) rabbit TrkB, (C) rat TrkB or (D) mouse TrkB after incubation with growing concentrations of the indicated molecules. Data represent mean+/ ⁇ SEM.
  • FIG. 4 A-C Trk phosphorylation (Y706/707) was measured in CHO cells stably expressing (A) human TrkA, (B) human TrkB, or (C) human TrkC after incubation with growing concentrations of the indicated molecules.
  • the natural ligands NGF for TrkA, BDNF for TrkB, and NT-3 for TrkC were used as controls. Data represent mean+/ ⁇ SEM.
  • FIG. 5 A-B (A) CHO cells stably expressing human TrkB were incubated with the indicated concentrations of the natural TrkB ligand BDNF (in duplicate), 1 nM BDNF with the indicated concentrations of the agonistic TrkB antibody C2 (in triplicate) or 1 nM BDNF with the indicated concentrations of the Doppelmab TPP-11736 (in triplicate). (B) TrkB internalization was assessed by immunofluorescence staining of surface TrkB receptors followed by confocal microscopy analysis. Dark and light fields of the heatmap represent high and low percentage of cells above fluorescence threshold, respectively.
  • FIG. 6 ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of the indicated molecules, with or without pre-incubation of 200 ng/mL human VEGF-A. Incubation only with VEGF-A served as control. Data represent the mean. For clarity error bars are omitted.
  • FIG. 7 Neuroprotective function of TrkB activation in a rat model of diabetes-induced retinal neurodegeneration. Animals were treated with STZ to induce hyperglycemia. The retinal function was then assessed by electroretinography (ERG) and rod-driven B-wave implicit time delays immediately before and two weeks after intravitreal application of the agonistic TrkB antibody C2 or Doppelmab TPP-11736; mean+/ ⁇ SEM; n.s. p>0.05, non-significant; **p ⁇ 0.01; ***p ⁇ 0.001; one-way Anova with Tukey multi-comparison test. Anti-TNP served as isotype control antibody.
  • FIG. 8 A-B VEGF-A scavenging was assessed by measuring VEGF receptor 2 phosphorylation (Y1175-VEGFR2).
  • HRMECs human retinal microvascular endothelial cells
  • A Comparison of Doppelmabs TPP-11735, -736, -737 and -738.
  • B Comparison of TPP-11736 and TPP-11738 with EYLEA® (aflibercept).
  • Basal Non-stimulated cells
  • 50 ng/ml human VEGF without antibody treatment served as control. Data represent mean+/ ⁇ SEM.
  • FIG. 9 A-B VEGF-A scavenging was assessed by ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2).
  • HRMECs human retinal microvascular endothelial cells
  • A Comparison of Doppelmabs TPP-11735, -736, -737 and -738.
  • B Comparison of TPP-11736, -738 with EYLEA® (aflibercept).
  • FIG. 10 VEGF-A scavenging was assessed by measuring p38 MAPK phosphorylation (T180/Y182).
  • HRMECs human retinal microvascular endothelial cells
  • Non-stimulated cells (Basal) and 50 ng/ml human VEGF without antibody treatment served as control.
  • Data represent mean+/ ⁇ SEM.
  • HRMECs Human retinal microvascular endothelial cells
  • FIG. 12 TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of the indicated molecules. Data represent mean+/ ⁇ SEM.
  • FIG. 13 ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of the indicated molecules. Data represent mean+/ ⁇ SEM.
  • FIG. 14 A-B VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 phosphorylation (Y1175) or (B) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2).
  • HRMECs human retinal microvascular endothelial cells
  • Non-stimulated cells (Basal) and 50 ng/ml human VEGF without molecule treatment served as control.
  • Data represent mean+/ ⁇ SEM.
  • FIG. 15 VEGF-A scavenging was assessed by image-based quantification of HRMEC cell numbers.
  • HRMECs human retinal microvascular endothelial cells
  • FIG. 16 Endothelial sprouting was assessed by confocal microscopy and displayed spheroid perimeter obtained from maximum projections of Z-stacks.
  • HRMECs human retinal microvascular endothelial cells
  • Endothelial sprouting was induced for 24 hours by incubation with 50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of the indicated Doppelmabs or 5 nM EYLEA® (aflibercept) for 24 hours.
  • Non-stimulated cells served as control (Basal). Data represent mean+/ ⁇ SEM. n
  • FIG. 17 A-C VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 phosphorylation (Y1175), (B) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2), or (C) p38 MAPK phosphorylation (T180/Y182).
  • HRMECs human retinal microvascular endothelial cells
  • 50 ng/mL human VEGF with or without pre-incubation with growing concentrations of Doppelmab TPP-11736 or TPP-13788 (B20 IgG). 50 ng/ml human VEGF without antibody treatment served as control.
  • Data represent mean+/ ⁇ SEM.
  • FIG. 18 Endothelial sprouting was assessed by confocal microscopy and displayed spheroid perimeter obtained from maximum projections of Z-stacks.
  • HRMECs human retinal microvascular endothelial cells
  • Endothelial sprouting was induced for 24 hours by incubation with 50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of Doppelmab TPP-11736 or 2.5 nM TPP-13788 (B20 IgG).
  • Non-stimulated cells served as control (Basal). Data represent mean+/ ⁇ SEM.
  • FIG. 19 A-C (A) TrkB phosphorylation (Y706/707) or (B & C) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of the indicated molecules. Data represent mean+/ ⁇ SEM.
  • FIG. 20 A-B TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing (A) cyno TrkB or (B) rat TrkB after incubation with growing concentrations of the indicated molecules. Data represent mean+/ ⁇ SEM.
  • FIG. 21 A-C VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 phosphorylation (Y1175), (B) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) or (C) Src phosphorylation (Y419).
  • HRMECs human retinal microvascular endothelial cells
  • HRMECs human retinal microvascular endothelial cells
  • FIG. 22 Endothelial sprouting was assessed by confocal microscopy and displayed spheroid perimeter obtained from maximum projections of Z-stacks.
  • HRMECs retinal microvascular endothelial cells
  • Endothelial sprouting was induced for 24 hours by incubation with 50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of the indicated molecules.
  • Non-stimulated cells served as control (Basal).
  • Data represent mean+/ ⁇ SEM. n.s. p>0.05 non-significant, ****p ⁇ 0.0001.
  • FIG. 23 A-D VEGF-A scavenging was assessed by image-based quantification of HRMEC cell numbers.
  • HRMECs human retinal microvascular endothelial cells
  • FIG. 24 A-B (A) TrkB phosphorylation (Y706/707) or (B) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of the indicated molecules of the second series. Data represent mean+/ ⁇ SEM.
  • FIG. 25 Cartoon depicting the proposed underlying effect of VEGF induced clustering with the single binding molecules resulting in increased efficacy and potency of TrkB activation.
  • FIG. 26 A-B TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing (A) cyno TrkB or (B) rat TrkB after incubation with growing concentrations of the indicated molecules of the second series. Data represent mean+/ ⁇ SEM.
  • FIG. 27 A-C TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of Doppelmabs (A) TPP-14940, (B) TPP-14941 or (C) C2 antibody with or without pre-incubation with 200 ng/mL human VEGF-A. Data represent the mean+/ ⁇ SEM.
  • FIG. 28 A-C ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of Doppelmabs (A) TPP-14940, (B) TPP-14941 or (C) C2 antibody with or without pre-incubation with 200 ng/mL human VEGF-A. Data represent the mean+/ ⁇ SEM.
  • FIG. 29 A-B (A) TrkB phosphorylation (Y706/707) or (B) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of either human VEGF-A or with growing concentrations of BDNF alone, or growing concentrations of BDNF with a fixed concentration of 200 ng/mL hVEGF. Data represent the mean+/ ⁇ SEM.
  • FIG. 30 A-C TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing cyno TrkB after incubation with growing concentrations of Doppelmabs (A) TPP-14940, (B) TPP-14941 or (C) C2 antibody with or without pre-incubation with 200 ng/mL human VEGF-A. Data represent the mean+/ ⁇ SEM.
  • FIG. 31 A-C ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing cyno TrkB after incubation with growing concentrations of Doppelmabs (A) TPP-14940, (B) TPP-14941 or (C) C2 antibody with or without pre-incubation with 200 ng/mL human VEGF-A. Data represent the mean+/ ⁇ SEM.
  • FIG. 32 A-C VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 phosphorylation (Y1175), (B) ERK1/2 phosphorylation (T202/Y204 -ERK1) (T185/Y187-ERK2) or (C) Src phosphorylation (Y419).
  • HRMECs human retinal microvascular endothelial cells
  • HRMECs human retinal microvascular endothelial cells
  • HRMECs Human retinal microvascular endothelial cells
  • FIG. 34 A-C VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 phosphorylation (Y1175), (B) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) or (C) Src phosphorylation (Y419).
  • HRMECs human retinal microvascular endothelial cells
  • HRMECs human retinal microvascular endothelial cells
  • FIG. 35 A-D VEGF-A scavenging was assessed by image-based quantification of HRMEC cell numbers.
  • HRMECs human retinal microvascular endothelial cells
  • FIG. 36 A-C VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 phosphorylation (Y1175), (B) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) or (C) Src phosphorylation (Y419).
  • HRMECs human retinal microvascular endothelial cells
  • HRMECs human retinal microvascular endothelial cells
  • FIG. 37 A-D VEGF-A scavenging was assessed by image-based quantification of HRMEC cell numbers.
  • HRMECs human retinal microvascular endothelial cells
  • FIG. 38 Endothelial sprouting was assessed by confocal microscopy and displayed spheroid perimeter obtained from maximum projections of Z-stacks.
  • HRMECs retinal microvascular endothelial cells
  • Endothelial sprouting was induced for 24 hours by incubation with 50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of the indicated molecules.
  • Non-stimulated cells served as control (Basal). Data represent mean+/ ⁇ SEM. n.s.
  • FIG. 39 A-B (A) Time protocol showing the experimental procedure. Fifteen minutes after intravitreal (ivt) administration of the anti-VEGF compound (13 or 26 pmol per eye of EYLEA® (aflibercept) or TPP-14940) or the control (26 pmol TPP-11737), 13 pmol human VEGF-A per eye was administered by ivt injection. PBS injection served as control. Twenty-four hours later 1 mL/kg of an Evans Blue (EB) solution (45 mg/mL in 0.9% saline) were administered by intravenous (iv) injection for 30 minutes before the eyes were isolated and fixed. Plasma samples were collected at the same point in time to confirm equal systemic EB exposure.
  • EB Evans Blue
  • the tissue was covered with mounting medium (Vectashield® antifade mounting media H-1200 containing the DNA stain DAPI) and a coverslip was put on top to obtain a retinal flatmount.
  • the samples were excited at a wavelength of 639 nm and emission of Evans Blue at 669 nm was recorded with a LSM 700 confocal laser scanning-microscope (Carl Zeiss, Jena; gain 800, laser strength 2%, 5 stacks of 60 ⁇ m) and images of the retinal flatmounts with maximum intensity projection were obtained. Analysis of fluorescence intensity sum was done after opening the images in the program ImageJ with a threshold of 30. ***p ⁇ 0.001; *p ⁇ 0.05; n.s. p>0.05; #p>0.05 non-significant vs. TPP-11737+PBS.
  • One-way Anova with Tukey multi comparison test, n 9 ⁇ 17.
  • FIG. 40 A-D (A & B) TrkB phosphorylation (Y706/707) or (C & D) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of BDNF or growing concentrations of the indicated molecules.
  • FIG. 41 A-B (A) CHO cells stably expressing human TrkB were incubated with the indicated concentrations of the natural TrkB ligand BDNF (in duplicate), or 1 nM BDNF with the indicated concentrations of the Doppelmabs (each in triplicate). (B) TrkB internalization was assessed by immunofluorescence staining of surface TrkB receptors followed by confocal microscopy analysis. Dark and light fields of the heatmap represent high and low percentage of cells above fluorescence threshold, respectively.
  • FIG. 42 VEGF-A scavenging was assessed by measuring VEGF receptor 2 phosphorylation (Y1175).
  • HRMECs human retinal microvascular endothelial cells
  • 50 ng/ml human VEGF without molecule treatment served as control.
  • Data represent mean+/ ⁇ SEM.
  • FIG. 43 Endothelial sprouting was assessed by confocal microscopy and displayed spheroid perimeter obtained from maximum projections of Z-stacks.
  • HRMECs retinal microvascular endothelial cells
  • Endothelial sprouting was induced for 24 hours by incubation with 50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of the indicated molecules.
  • Non-stimulated cells served as control (Basal). Data represent mean+/ ⁇ SEM. n.s.
  • FIG. 44 A-D TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of Doppelmabs (A) TPP-22180 vs. TPP-22204; (B) TPP-22192 vs. TPP-22216; (C) TPP-22190 vs. TPP-22214; (D) TPP-22191 vs. TPP-22215. Data represent mean+/ ⁇ SEM.
  • FIG. 45 A-B TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing rat TrkB after incubation with growing concentrations of Doppelmabs (A) TPP-22180 vs. TPP-22204; (B) TPP-22192 vs. TPP-22216. Data represent mean+/ ⁇ SEM.
  • FIG. 46 A-C Trk phosphorylation (Y706/707) was measured in CHO cells stably expressing (A) human TrkA, (B) human TrkB or (C) human TrkC after incubation with growing concentrations the C2 antibody or the Doppelmabs TPP-22204 or TPP-22214.
  • the natural ligands NGF for TrkA, BDNF for TrkB, and NT-3 for TrkC were used as controls. Data represent mean+/ ⁇ SEM.
  • FIG. 47 A-B TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing human TrkB receptor after incubation with (A) growing concentrations of the C2 antibody with or without constant concentrations of 0.3 nM, 1 nM or 3 nM BDNF or (B) growing concentrations of the Doppelmab TPP-22214 with or without constant concentrations of 0.3 nM, 1 nM or 3 nM BDNF. Data represent the mean+/ ⁇ SEM.
  • FIG. 48 A-D TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing human TrkB receptor after incubation with growing concentrations of Doppelmab TPP-22214 with or without pre-incubation with (A) 200 ng/mL human VEGF-A (hVEGF), (B) 50 ng/mL hVEGF, (C) 10 ng/mL hVEGF or (D) 2 ng/mL hVEGF. Data represent the mean+/ ⁇ SEM.
  • FIG. 49 A-D ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of Doppelmab TPP-22214 with or without pre-incubation with (A) 200 ng/mL human VEGF-A (hVEGF), (B) 50 ng/mL hVEGF, (C) 10 ng/mL hVEGF or (D) 2 ng/mL hVEGF. Data represent the mean+/ ⁇ SEM.
  • FIG. 50 Size of Dopplemab complex was assessed using size exclusion chromatography combined with a multi-angle light scattering detector.
  • the differential refractive index (black, dark grey, light grey and dashed lines) and light scattering were monitored over the time it takes for the proteins to elute from the size exclusion column.
  • the light scattering data is used to determine the molar mass at each point.
  • the molar mass at the midpoint of each peak is denoted by a star and measured using the right axis.
  • a thru D represent possible complex schematics based on the measured molar masses.
  • FIG. 51 A-C TrkB internalization was assessed by immunofluorescence staining the surface TrkB receptors without permeabilization of the cells followed by confocal microscopy analysis.
  • CHO cells with stable expression of human TrkB were incubated with (A) growing concentrations of the natural TrkB ligand BDNF, (B) growing concentrations of TPP-22214 or (C) 1 nM BDNF with growing concentrations of TPP-22214.
  • Data represent the percent of cells with surface TrkB staining intensity above threshold; mean+/ ⁇ SEM.
  • FIG. 52 Neuroprotective function of TrkB activation in a rat model of diabetes-induced retinal neurodegeneration. Animals were treated with STZ to induce hyperglycemia. The retinal function was then assessed by electroretinography (ERG) and rod-driven B-wave implicit time delays immediately before and two weeks after intravitreal application of the agonistic TrkB antibody C2 or Doppelmab TPP-22214; mean+/ ⁇ SEM; n.s. p>0.05; non-significant (n.s.), ****p ⁇ 0.0001; one-way Anova with Tukey multi-comparison test. Anti-TNP served as isotype control antibody.
  • FIG. 53 A-B VEGF-A scavenging was assessed by measuring VEGF receptor 2 phosphorylation (Y1175).
  • HRMECs human retinal microvascular endothelial cells
  • A Comparison of Doppelmabs TPP-14941, TPP-22216, TPP-22192, TPP-22204, and TPP-22180.
  • B Comparison of Doppelmabs TPP-14940, TPP-14941, TPP-22190, TPP-22214, TPP-22191 and TPP-22215.
  • Non-stimulated cells (Basal) and 50 ng/ml human VEGF without molecule treatment served as control. Data represent mean+/ ⁇ SEM.
  • FIG. 54 A-B VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation (Y1175) or (B) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2).
  • VEGFR2 VEGF receptor 2
  • ERK1/2 phosphorylation T202/Y204-ERK1
  • HRMECs human retinal microvascular endothelial cells
  • HRMECs Human retinal micro
  • FIG. 56 A-B VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 phosphorylation (Y1214) or (B) p38-MAPK phosphorylation (T180/Y182).
  • HRMECs human retinal microvascular endothelial cells
  • 50 ng/mL human VEGF with or without pre-incubation with growing concentrations of TPP-22214 or EYLEA® (aflibercept).
  • 50 ng/ml human VEGF without molecule treatment served as control.
  • Data represent mean+/ ⁇ SEM.
  • FIG. 57 A-B Spheroids of human retinal microvascular endothelial cells (HRMECs) were embedded in a collagen matrix. Endothelial sprouting was induced for 24 hours by incubation with 50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of TPP-22204, TPP-22214, TPP-22215, TPP-22216 or 5 nM EYLEA® (aflibercept).
  • HRMECs retinal microvascular endothelial cells
  • FIG. 58 A-B VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 phosphorylation (Y951) or (B) Src phosphorylation (Y419).
  • HRMECs human retinal microvascular endothelial cells
  • 50 ng/mL human VEGF with or without pre-incubation with growing concentrations of TPP-22214 or EYLEA® (aflibercept).
  • 50 ng/ml human VEGF without antibody treatment served as control.
  • Data represent mean+/ ⁇ SEM.
  • FIG. 59 A-B TPP-22214 prevented human VEGF-A-induced hyperpermeability in the rat retina.
  • A Time protocol showing the experimental procedure. Fifteen minutes after intravitreal (ivt) administration of the anti-VEGF compound (13 or 26 pmol per eye of EYLEA® (aflibercept) or TPP-14940) or the control (26 pmol TPP-11737), 13 pmol human VEGF-A per eye was administered by ivt injection. PBS injection served as control. Twenty-four hours later 1 mL/kg of an Evans Blue (EB) solution (45 mg/mL in 0.9% saline) were administered by intravenous (iv) injection for 30 minutes before the eyes were isolated and fixed.
  • EB Evans Blue
  • the tissue was covered with mounting medium (Vectashield® antifade mounting media H-1200 containing the DNA stain DAPI) and a coverslip was put on top to obtain a retinal flatmount.
  • the samples were excited at a wavelength of 639 nm and emission of Evans Blue at 669 nm was recorded with a LSM 700 confocal laser scanning-microscope (Carl Zeiss, Jena; gain 800, laser strength 2%, 5 stacks of 60 ⁇ m) and images of the retinal flatmounts with maximum intensity projection were obtained. Analysis of fluorescence intensity sum was done after opening the images in the program ImageJ with a threshold of 30. ***p ⁇ 0.001; *p ⁇ 0.05; n.s. p>0.05.
  • FIG. 60 A-B (A) Functional characterization of the TkrB extracellular domain (TrkB-ECD). CHO cells with stable expression of human TrkB were incubated with growing concentrations of the natural ligand BDNF or 10 nM BDNF with growing concentrations of TrkB-ECD. TrkB activation was assessed by measuring TrkB phosphorylation (Y706/707). (B) Impact of TrkB-ECD binding of TPP-22214 on inhibition of VEGF-induced VEGFR2 phosphorylation.
  • HRMECs Human retinal microvascular endothelial cells
  • 50 ng/mL human VEGF 50 ng/mL human VEGF with growing concentrations of TPP-22214 or 50 ng/mL human VEGF with growing concentrations of TPP-22214 and 100 nM TrkB-ECD.
  • VEGF-A scavenging was assessed by measuring VEGF receptor 2 phosphorylation (Y1175). Data represent mean+/ ⁇ SEM.
  • FIG. 61 A-B (A) TrkB phosphorylation (Y706/707) or (B) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of the indicated molecules. Data represent mean+/ ⁇ SEM.
  • FIG. 62 A-C TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of Doppelmabs (A) TPP-23457, (B) TPP-23459 or (C) TPP-6830 antibody with or without pre-incubation with 200 ng/mL human VEGF-A. Data represent the mean+/ ⁇ SEM.
  • FIG. 63 A-C ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with growing concentrations of Doppelmabs (A) TPP-23457, (B) TPP-23459 or (C) TPP-6830 antibody with or without pre-incubation with 200 ng/mL human VEGF-A. Data represent the mean+/ ⁇ SEM.
  • FIG. 64 A-B TrkB phosphorylation (Y706/707) was measured in CHO cells stably expressing human TrkB receptor after incubation with (A) growing concentrations of the C2 antibody with or without constant concentrations of 0.3 nM, 1 nM or 3 nM BDNF or (B) growing concentrations of the Doppelmab TPP-23457 with or without constant concentrations of 0.3 nM, 1 nM or 3 nM BDNF. Data represent the mean+/ ⁇ SEM.
  • FIG. 65 A-B ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was measured in CHO cells stably expressing human TrkB after incubation with (A) growing concentrations of the C2 antibody with or without constant concentrations of 0.3 nM, 1 nM or 3 nM BDNF or (B) growing concentrations of the Doppelmab TPP-23457 with or without constant concentrations of 0.3 nM, 1 nM or 3 nM BDNF. Data represent the mean+/ ⁇ SEM.
  • FIG. 66 A-B VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation (Y1175) or (B) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2).
  • VEGFR2 VEGF receptor 2
  • ERK1/2 phosphorylation T202/Y204-ERK1
  • HRMECs human retinal microvascular endothelial cells
  • 50 ng/mL human VEGF 50 ng/mL human VEGF with or without pre-incubation with growing concentrations of TPP-22215, TPP-23457, TPP-23459 or EYLEA® (aflibercept). 50 ng/ml human VEGF without molecule treatment served as control.
  • Data represent mean+/ ⁇ SEM.
  • FIG. 67 A-C TrkB binders identified via na ⁇ ve Phage display or via B-cell to Phage were formatted either as IgG, scFv-Fc or Fc-scFv.
  • the potency (EC 50 ) for each hit and each of the three molecule formats was determined by measuring the TrkB phosphorylation (Y706/707) in CHO cells stably expressing human TrkB receptor after incubation with growing concentrations of the molecules.
  • Figures A-C show the pairwise alignment (scatter plots with line of equality) of the EC 50 of the molecules with Figure (A) comparing the EC 50 of IgG (x-axis) against scFv-Fc (y-axis) for each hit, Figure (B) comparing the EC 50 of IgG (x-axis) against Fc-scFv (y-axis) for each hit, and Figure (C) comparing the EC 50 of scFv-Fc (x-axis) against Fc-scFv (y-axis) for each hit.
  • FIG. 68 A-C TrkB binders identified via na ⁇ ve Phage display or via B-cell to Phage were formatted either as IgG, scFv-Fc or Fc-scFv. The efficacy for each hit and each of the three molecule formats was determined by measuring the TrkB phosphorylation (Y706/707) in CHO cells stably expressing human TrkB receptor after incubation with growing concentrations of the molecules. BDNF treated cells were measured as well and the efficacy of BDNF was set to 100% as control.
  • Figures A-C show the pairwise alignment (scatter plots with line of equality) of the efficacy of the molecules.
  • the efficacy of each molecule is expressed as percentage in comparison to the natural ligand BDNF.
  • Figure (A) compares the efficacy of IgG (x-axis) against scFv-Fc (y-axis) for each hit
  • Figure (B) compares the efficacy of IgG (x-axis) against Fc-scFv (y-axis) for each hit
  • Figure (C) compares the efficacy of scFv-Fc (x-axis) against Fc-scFv (y-axis) foreach hit.
  • FIG. 69 A-H Selected hits identified via na ⁇ ve Phage display or via B-cell to Phage were formatted either as IgG, scFv-Fc or Fc-scFv.
  • the efficacy for each hit and each of the three molecule formats was determined by measuring the TrkB phosphorylation (Y706/707) in CHO cells stably expressing human TrkB receptor after incubation with growing concentrations of the molecules.
  • BDNF treated cells were measured as well and the efficacy of BDNF was set to 1.0 as control.
  • the molecule concentration is plotted on the x-axis and the y-axis shows the TrkB phosphorylation relative to BDNF.
  • Figures A-H show in total 8 selected hits and for each hit the three different formats IgG, scFv-Fc and Fc-scFv in direct comparison. The lowest compound concentration was solvent alone. Data represent mean ⁇ SEM.
  • FIG. 70 A-C Schematic of the three different molecule formats: (A) IgG, (B) scFv-Fc and (C) Fc-scFv.
  • FIG. 71 A selected TrkB binder was formatted either as IgG, scFv-Fc or as two scFvs connected to each other via two different peptide linkers (scFv-linker1-scFv and scFv-linker2-scFv).
  • the resulting molecules were tested for their ability to activate TrkB by measuring the TrkB phosphorylation (Y706/707) in CHO cells stably expressing human TrkB receptor after incubation with growing concentrations of the molecules.
  • BDNF treated cells were measured as well and served as reference.
  • the molecule concentration is plotted on the x-axis and the y-axis shows the TrkB phosphorylation. The lowest compound concentration was solvent alone. Data represent mean ⁇ SEM.
  • FIG. 72 A selected TrkB binder was formatted either as IgG or scFv-Fc.
  • the scFv-Fc was cleaved by a cysteine protease below the hinge to remove the Fc portion, creating two scFvs connected to each other via the hinge region (scFv-hinge-scFv). All three molecules were tested for their ability to activate TrkB by measuring the TrkB phosphorylation (Y706/707) in CHO cells stably expressing human TrkB receptor after incubation with growing concentrations of the molecules. The molecule concentration is plotted on the x-axis and the y-axis shows the TrkB phosphorylation. The lowest compound concentration was solvent alone. Data represent mean ⁇ SEM.
  • the present invention is based on the concept of combining an antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) with an antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB) within a single binding molecule.
  • VEGF Vascular Endothelial Growth Factor
  • TrkB Tropomyosin receptor kinase B
  • binding molecules targeting these two antigens are prepared binding molecules including at least one antigen binding site that binds specifically to VEGF and at least one antigen binding site that binds specifically to TrkB.
  • the initial goal of the inventors was to design a single binding molecule which had comparable efficacy and/or potency when compared to their respective individual binders, i.e. the individual VEGF or TrkB binder which only bind to their respective target. This was already considered challenging in itself, as it was not fully understood but expected that formatting of the individual binders into a single binding molecule would negatively impact the original efficacy and/or potency of the individual binders within the single binding molecule.
  • TrkB activation with VEGF scavenging in a single binding molecule resulted in an unexpected increase of both efficacy as wells as potency of TrkB activation.
  • the single binding molecules according to the invention show full TrkB agonist activity—opposed to partial agonist activity of the individual TrkB binder.
  • potency of TrkB activation is further enhanced after binding of VEGF to the single binding molecule.
  • VEGF induced clustering with the single binding molecules of the invention may be responsible for the observed increase in potency of TrkB activation.
  • the design of the TrkB binding sites in the binding molecules of the invention support an optimal sterical formation of TrkB binding which resulted in the observed full TrkB agonist activity.
  • VEGF-scavenging resulting in inhibition of vascular dysfunction and leakage
  • the activation of the neuroprotective TrkB-receptor resulting in reduction of neuronal death
  • the binding molecules according to the invention are useful for treatment and/or prevention of loss in visual function and thereby improvement in quality of life.
  • the present invention provides a binding molecule, in particular a molecule having at least one antigen binding site that binds specifically to vascular endothelial growth factor VEGF, preferably VEGF-A and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB) having one or more of the properties described herein below.
  • VEGF vascular endothelial growth factor
  • TrkB Tropomyosin receptor kinase B
  • a binding molecule of the present invention binds with high affinity to human TrkB. In an embodiment relating to this aspect, a binding molecule of the present invention binds to human TrkB at a K D ⁇ 500 nM. In another embodiment, a binding molecule of the present invention binds to human TrkB at a KD ⁇ 450 nM. In another embodiment, a binding molecule of the present invention binds to human TrkB at a K D ⁇ 400 nM. In another embodiment, a binding molecule of the present invention binds to human TrkB at a K D ⁇ 300 nM.
  • a binding molecule of the present invention binds to human TrkB at a KD ⁇ 250 nM. In an embodiment relating to this aspect, a binding molecule of the present invention binds to human TrkB at a K D ⁇ 200 nM. In another embodiment, a binding molecule of the present invention binds to human TrkB at a K D ⁇ 150 nM. In another embodiment, a binding molecule of the present invention binds to human TrkB at a K D ⁇ 100 nM. In another embodiment, a binding molecule of the present invention binds to human TrkB at a K D ⁇ 50 nM.
  • a binding molecule of the present invention under conditions comprising pre-incubation with 200 ng/mL human VEGF-A—activates TrkB with high potency.
  • a binding molecule of the present invention activates human TrkB with an EC 50 ⁇ 100 nM.
  • a binding molecule of the present invention activates human TrkB with an EC 50 ⁇ 90 nm.
  • a binding molecule of the present invention activates human TrkB with an EC 50 ⁇ 80 nm.
  • a binding molecule of the present invention activates human TrkB with an EC 50 ⁇ 70 nm.
  • a binding molecule of the present invention activates human TrkB with an EC 50 ⁇ 60 nm. In a further embodiment, a binding molecule of the present invention activates human TrkB with an EC 50 ⁇ 50 nm. In a further embodiment, a binding molecule of the present invention activates human TrkB with an EC 50 ⁇ 40 nm. In a further embodiment, a binding molecule of the present invention activates human TrkB with an EC 50 ⁇ 30 nm. In a further embodiment, a binding molecule of the present invention activates human TrkB with an EC 50 ⁇ 20 nm. In a further embodiment, a binding molecule of the present invention activates human TrkB with an EC 50 ⁇ 10 nm.
  • a binding molecule of the present invention binds with high affinity to human VEGF, preferably VEGF-A. In an embodiment relating to this aspect, a binding molecule of the present invention binds to human VEGF-A at a K D ⁇ 1 nM. In another embodiment, a binding molecule of the present invention binds to human VEGF-A at a K D ⁇ 900 pM. In another embodiment, a binding molecule of the present invention binds to human VEGF-A at a K D ⁇ 800 pM. In another embodiment, a binding molecule of the present invention binds to human VEGF-A at a K D ⁇ 700 pM.
  • a binding molecule of the present invention binds to human VEGF-A at a K D ⁇ 600 pM. In another embodiment, a binding molecule of the present invention binds to human VEGF-A at a K D ⁇ 500 pM. In another embodiment, a binding molecule of the present invention binds to human VEGF-A at a K D ⁇ 400 pM.
  • a binding molecule of the present invention inhibits VEGF-A phosphorylation (Tyr1175) with high potency. In an embodiment relating to this aspect, a binding molecule of the present invention inhibits human VEGF-A phosphorylation (Tyr1175) with an IC 50 ⁇ 1 nM. In an embodiment relating to this aspect, a binding molecule of the present invention inhibits human VEGF-A phosphorylation (Tyr1175) with an IC 50 ⁇ 900 pM. In an embodiment relating to this aspect, a binding molecule of the present invention inhibits human VEGF-A phosphorylation (Tyr1175) with an IC 50 ⁇ 800 pM.
  • a binding molecule of the present invention inhibits human VEGF-A phosphorylation (Tyr1175) with an IC 50 ⁇ 700 pM. In an embodiment relating to this aspect, a binding molecule of the present invention inhibits human VEGF-A phosphorylation (Tyr1175) with an IC 50 ⁇ 600 pM. In an embodiment relating to this aspect, a binding molecule of the present invention inhibits human VEGF-A phosphorylation (Tyr1175) with an IC 50 ⁇ 500 pM. In an embodiment relating to this aspect, a binding molecule of the present invention inhibits human VEGF-A phosphorylation (Tyr1175) with an IC 50 ⁇ 400 pM. In an embodiment relating to this aspect, a binding molecule of the present invention inhibits human VEGF-A phosphorylation (Tyr1175) with an IC 50 ⁇ 300 pM.
  • a binding molecule of the present invention is more potent in inducing activation of TrkB downstream signaling pathways than the natural TrkB ligand, BDNF.
  • a binding molecule of the present invention regulates gene expression through TrkB-mediated signaling pathways in a comparable pattern to that of BDNF.
  • a binding molecule of the present invention does not reduce BDNF induced ERK phosphorylation.
  • a binding molecule of the present invention is specific for TrkB phosphorylation and/or activation and does not unspecifically phosphorylate/activate TrkA or TrkC.
  • the binding molecules according to the invention are useful to prevent neurodegeneration and loss of retinal function in a disease-related animal model.
  • a binding molecule of the present invention protects neurons, glial cells and/or the neurovascular unit in the retina of patients with e.g. macular degeneration, age-related macular degeneration, geographic atrophy or diabetic retinopathy by stimulating TrkB-dependent survival signaling pathways and thereby providing neuroprotection.
  • a binding molecule of the present invention regenerates axons/dendrites and/or synapses in the retina after disease onset in e.g. macular degeneration, age-related macular degeneration, geographic atrophy or diabetic retinopathy and thereby resulting in neuroregeneration.
  • a binding molecule of the present invention can be formulated to high concentrations for intravitreal injections into the eye.
  • the binding molecules according to the invention show superior VEGF-A scavenging compared to the current anti-VEGF standard of care compound aflibercept (EYLEA® (aflibercept)) in terms of inhibition of VEGF-induced signaling, endothelial cell proliferation/sprouting and/or inhibition of VEGF-induced hyperpermeability in the rat retina.
  • EYLEA® aflibercept
  • the binding molecules according to the invention show a substantially longer vitreal half-life in rabbit PK studies compared to e.g. EYLEA® (aflibercept) or other IgG antibodies. Accordingly, the binding molecules according to the invention are suitable for a quarterly injection or even less frequent injections.
  • the binding molecules according to the invention are useful to target the high unmet need of patients with wAMD and at risk of developing geographic atrophy.
  • the binding molecules according to the invention are uniquely useful for treating patients with wAMD and preventing the development of geographic atrophy.
  • the development of geographic atrophy is delayed or the severity of the disease is reduced, i.e. a reduction of the onset and/or the rate of progression of geographic atrophy.
  • treatment of patients with the binding molecules according to the invention may already be initiated before the definite diagnosis of geographic atrophy, i.e. for patients being at risk of developing geographic atrophy.
  • binding molecules according to the invention are useful to target the high unmet need of patients with retinal vein occlusion (CRVO).
  • CRVO retinal vein occlusion
  • binding molecules according to the invention improve the ERG-deficit implicit time when compared to the current standard of care (EYLEA® (aflibercept)).
  • binding of the binding molecules according to the invention to TrkB does not induce receptor internalization.
  • binding of the binding molecules according to the invention to TrkB will result in less receptor internalization when compared to the natural ligand BDNF.
  • VEGF-A binding to the binding molecules according to the invention increases the potency of TrkB activation.
  • Said increase in potency may be measured e.g. in vitro by determining the EC 50 for TrkB or Erk1/2 phosphorylation in an appropriate cell model after treatment with the single binding molecules, e.g such as in CHO cells overexpressing (human) TrkB (cf. examples).
  • the single binding molecules will be tested with or without pre-incubating the cells with appropriate concentrations of human VEGF-A (hVEGF), such as 200 ng/mL, 50 ng/mL, 10 ng/mL or 2 ng/mL.
  • the single binding molecules of the invention will show an increase in potency, as measured by EC 50 for TrkB or Erk1/2 phosphorylation of at least approximately 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or at least approximately 10-fold increase in potency when compared to cells not pre-incubated with VEGF-A.
  • the binding molecules according to the invention show full TrkB agonist activity.
  • the binding molecules according to the invention are as efficacious in activating TrkB as the natural ligand BDNF.
  • the binding molecules according to the invention can simultaneously activate the TrkB receptor and scavenge VEGF, preferably VEGF-A.
  • an antigen binding site comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) derived from an antibody.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • each variable domain comprises 3 CDRs.
  • an antigen binding site according to the present invention or certain portions of the protein is generally derived from an antibody.
  • the generalized structure of antibodies or immunoglobulin molecules is well known to those of skill in the art.
  • Antibodies or “immunoglobulin molecules” (also known as immunoglobulins, abbreviated Ig) are gamma globulin proteins that can be found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. They are typically made of basic structural units - each with two large heavy chains and two small light chains - to form, for example, monomers with one unit, dimers with two units or pentamers with five units. Antibodies can bind, by non-covalent interaction, to other molecules or structures known as antigens. This binding is specific in the sense that an antibody will only bind to a specific structure with high affinity.
  • variable domain The unique part of the antigen recognized by an antibody is called an epitope, or antigenic determinant.
  • the part of the antibody binding to the epitope is sometimes called paratope and resides in the so-called variable domain, or variable region (Fv) of the antibody.
  • the variable domain comprises three so-called complementary-determining region (CDR's) spaced apart by framework regions (FR's).
  • CDR's is based on different definitions, such as e.g. CCG, also referred to as IMGT (Lefranc MP, Pommié C, Ruiz M, Giudicelli V, Foulquier E, Truong L, Thouvenin-Contet V, Lefranc G. “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.” Dev Comp Immunol. 2003 Jan.; 27(1):55-77; Giudicelli V,
  • IMGT/V-QUEST IMGT standardized analysis of the immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences”.
  • IG immunoglobulin
  • TR T cell receptor
  • An alternative definition of CDRs is based on Chothia (Chothia and Lesk, J. Mol. Biol. 1987, 196: 901-917), together with Kabat (E. A. Kabat, T. T. Wu, H. Bilofsky, M. Reid-Miller and H. Perry, Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda (1983)).
  • variable domains or “variable region” or Fv as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen.
  • the variable domain of a light chain is abbreviated as “VL” and the variable domain of a heavy chain is abbreviated as “VH”.
  • the variable light and heavy chain domains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three HVRs (or CDRs).
  • the framework regions adopt a beta-sheet conformation and the CDRs may form loops connecting the beta-sheet structure.
  • the CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site.
  • the antibody's heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.
  • the term “constant domains” or “constant region” as used within the current application denotes the sum of the domains of an antibody other than the variable region. Such constant domains and regions are well known in the state of the art and e.g. described by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91).
  • the “Fc part” of an antibody is not involved directly in binding of an antibody to an antigen,but exhibit various effector functions.
  • An “Fc part of an antibody” is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies.
  • antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG and IgM. According to the heavy chain constant regions the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ respectively. Several of these may be further divided into subclasses (isotypes), e.g.
  • the Fc part of an antibody is directly involved in ADCC (antibody dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, Clq binding and Fc receptor binding.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Complement activation (CDC) is initiated by binding of complement factor Clq to the Fc part of most IgG antibody subclasses. While the influence of an antibody on the complement system is dependent on certain conditions, binding to Clq is caused by defined binding sites in the Fc part. Such binding sites are known in the state of the art and described e.g.
  • antibody molecule or “antibody” or “Ig molecule” (used synonymously herein) do not only include antibodies as they may be found in nature, comprising e.g. two light chains and two heavy chains, or just two heavy chains as in camelid species, but furthermore encompasses all molecules comprising at least one paratope with binding specificity to an antigen and structural similarity to a variable domain of an immunoglobulin.
  • an antibody or immunoglobulin or Ig molecule may comprise a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a fragment of an antibody, in particular a Fv, Fab, Fab′, or F(ab′)2 fragment, a single chain antibody, in particular a single chain variable fragment (scFv), a Small Modular Immunopharmaceutical (SMIP), a domain antibody, a nanobody, a diabody.
  • the antibody may have an effector function, such as ADCC or CDC, that is usually mediated by the Fc part (antibody constant region) of the antibody, or it may have no effector function, e.g.
  • Monoclonal antibodies are monospecific antibodies that are identical in amino acid sequence. They may be produced by hybridoma technology from a hybrid cell line (called hybridoma) representing a clone of a fusion of a specific antibody-producing B cell with a myeloma (B cell cancer) cell (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256:495-7.).
  • monoclonal antibodies may be produced by recombinant expression in host cells (Norderhaug L, Olafsen T, Michaelsen T E, Sandlie I. (May 1997). “Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells.” J Immunol Methods 204 (1): 77-87; see also below).
  • a “recombinant antibody” or “recombinant binding molecule” is an antibody or binding molecule which has been produced by a recombinantly engineered host cell. It is optionally isolated or purified.
  • a “chimeric antibody” is understood to be antibody comprising a sequence part (e.g. a variable domain) derived from one species (e.g. mouse) fused to a sequence part (e.g. the constant domains) derived from a different species (e.g. human).
  • a “humanized antibody” is an antibody comprising a variable domain originally derived from a non-human species, wherein certain amino acids have been mutated to make the overall sequence of that variable domain more closely resemble to a sequence of a human variable domain.
  • a “humanized” antibody refers to an antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g. complementary determining regions (CDRs)) correspond to those of a non-human antibody, and all or substantially the entire framework regions (FRs) correspond to those of a human antibody.
  • HVRs complementary determining regions
  • FRs framework regions
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g. a non-human antibody refers to an antibody that has undergone humanization.
  • Antibody can also include fragments of immunoglobulins which retain antigen binding properties, like Fab, Fab′, or F(ab′)2 fragments. Such fragments may be obtained by fragmentation of immunoglobulins e.g. by proteolytic digestion, or by recombinant expression of such fragments. For example, immunoglobulin digestion can be accomplished by means of routine techniques, e.g. using papain or pepsin (WO 94/29348). Papain digestion of antibodies typically produces two identical antigen binding fragments, so-called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields an F(ab′)2.
  • variable domains are each fused to an immunoglobulin constant domain, preferably of human origin.
  • the heavy chain variable domain may be fused to a CH1 domain (a so-called Fd fragment), and the light chain variable domain may be fused to a CL domain.
  • Fab molecules may be produced by recombinant expression of respective nucleic acids in host cells, see below.
  • variable domains of immunoglobulins or molecules derived from such variable domains, in a different molecular context.
  • these antibody molecules are smaller in size compared to immunoglobulins,and may comprise a single amino acid chain or several amino acid chains.
  • a single-chain variable fragment scFv
  • S serine
  • G glycine
  • Single domain antibodies or “nanobodies” harbour an antigen-binding site in a single Ig-like domain (WO 94/04678; WO 03/050531, Ward et al., Nature. 1989 Oct. 12; 341(6242): 544-6; Revets et al., Expert Opin Biol Ther. 5(1): 111-24, 2005).
  • One or more single domain antibodies with binding specificity for the same or a different antigen may be linked together.
  • Diabodies are bivalent antibody molecules consisting of two amino acid chains comprising two variable domains (WO 94/13804, Holliger et al., Proc Natl Acad Sci U S A. 1993 Jul. 15; 90(14): 6444-8).
  • SMIP Small Modular Immunopharmaceutical
  • binding or “specifically binding” refers to the binding of an antibody or antigen binding site (e.g., in the binding molecule described herein) to an epitope of the antigen in an in-vitro assay.
  • binding affinity of an antibody molecule may be enhanced by a process known as affinity maturation (Marks et al., 1992, Biotechnology 10:779-783; Barbas, et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169: 147-155). Affinity matured antibodies are therefore also embraced in the present invention.
  • epitope is a region of an antigen that is bound by an antibody or antigen binding moiety.
  • epitope includes any polypeptide determinant capable of specific binding to an antibody or antigen binding moiety.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, glycan side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • binding and “specific binding” refer to the binding of the antibody or antigen binding moiety to an epitope of the antigen in an in vitro assay, preferably in a plasmon resonance assay (BlAcore®, GE-Healthcare Uppsala, Sweden) with purified wild-type antigen.
  • BlAcore® plasmon resonance assay
  • a “single chain Fv fragment” is a polypeptide comprising an antibody heavy chain variable domain (VH), a linker, and an antibody light chain variable domain (VL), wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-linker-VL, b) VL-linker-VH; and wherein said linker is a polypeptide of 15 to 25 amino acids, preferably 20 amino acids, in length.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences that are compared are the same length after gaps are introduced within the sequences, as appropriate (e.g., excluding additional sequence extending beyond the sequences being compared). For example, when variable region sequences are compared, the leader and/or constant domain sequences are not considered.
  • a “corresponding” CDR refers to a CDR in the same location in both sequences (e.g., CDR-H1 of each sequence).
  • the determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410.
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • BLAST Gapped BLAST
  • PSI-Blast programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, 1994, Comput. Appl.
  • the “therapeutically effective amount” of the molecule to be administered is the minimum amount necessary to prevent, ameliorate, or treat clinical symptoms of e.g. eye or retinal or neurodegenerative diseases, in particular the minimum amount which is effective to these disorders.
  • the terms “monovalent”, “bivalent”, “tetravalent” refer to the number (one, two or four, respectively) of antigen binding elements in a binding molecule.
  • the terms “monospecific”, “bispecific” refer to the number (one, two) of different antigens or epitopes a binding molecule specifically binds.
  • a typical monoclonal antibody is bivalent and monospecific, with two antigen-binding arms that both recognize the same epitope.
  • Bispecific antibodies have two antigen-binding sites, which are capable of recognizing and binding two different antigens or epitopes.
  • TrkB Tropomyosin receptor kinase B
  • TrkB also known as tyrosine receptor kinase B, or BDNF/NT-3 growth factors receptor or neurotrophic tyrosine kinase, receptor, type 2
  • TrkB is a protein that in humans is encoded by the NTRK2 gene (Genbank ID: 4915).
  • TrkB is a receptor for brain-derived neurotrophic factor (BDNF).
  • TrkB The neurotrophic tyrosine kinase receptor B (TrkB; gene symbol: NTRK2) is expressed by retinal neurons and glial cells.
  • TrkB signaling counteracts cell stress and promotes cell survival.
  • loss and functional impairments of retinal neurons and glial cells occur which cause visual impairments and vision loss.
  • Activating TrkB signaling above the basal level can counteract the loss and functional impairments of neurons and glial cells, thus improving visual function.
  • TrkB activation has the potential to regenerate lost synaptic connections in the diseased eye, thereby promoting the regain of visual function.
  • TrkB Upon ligand binding, TrkB undergoes homodimerization followed by autophosphorylation.
  • Dependent on the phosphorylation sites (Y516, Y702, Y706, Y707 or Y817) different signal transduction pathways are activated, including the activity of PLC ⁇ 1 or different subforms of AKT and ERK which regulate distinct overlapping signalling cascades inducing axonal/neurite outgrowth, increasing synaptic plasticity, or increasing cell survival.
  • TrkB-binding components of the binding molecules according to the invention e.g. scFv or scFv2 specifically bind to native or recombinant human TrkB.
  • the binding molecules of the invention By binding to TrkB the binding molecules of the invention thereby activate TrkB signaling, hence the binding molecules of the invention act as TrkB agonists.
  • the binding molecules of the present invention may recognize specific “TrkB antigen epitope” and “TrkB epitope”.
  • the binding molecules of the invention bind to an epitope in the extracellular domain of human TrkB.
  • the TrkB-binding components of the binding molecule is a TrkB agonist and more preferably a full agonist.
  • the extracellular domain of human TrkB essentially comprises the following sequence (SEQ ID NO.226):
  • TrkB antigen epitope and “TrkB epitope” refer to a molecule (e.g., a peptide) or a fragment of a molecule capable of binding to an antigen binding site of the binding molecule or binding molecules fragments according to the invention that binds specifically to Tropomyosin receptor kinase B. These terms further include, for example, a TrkB antigenic determinant recognized by any of the binding molecules or binding molecules fragments of the present invention, which has a light and heavy chain CDR combination selected from:
  • Light chain CDRs comprising the amino acid sequences of SEQ ID NO: 201 (CDR1), SEQ ID NO: 202 (CDR2) and SEQ ID NO: 203 (CDR3), and Heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 204 (CDR1), SEQ ID NO: 205 (CDR2) and SEQ ID NO: 206 (CDR3); or Heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 207 (CDR1), SEQ ID NO: 208 (CDR2) and SEQ ID NO: 209 (CDR3); or Heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 210 (CDR1), SEQ ID NO: 211 (CDR2) and SEQ ID NO: 212 (CDR3).
  • TrkB antigen epitopes can be included in proteins, protein fragments, peptides or the like.
  • the epitopes are most commonly proteins, short oligopeptides, oligopeptide mimics (i.e., organic compounds that mimic antibody binding properties of the TrkB antigen), or combinations thereof.
  • VEGF Vascular endothelial growth factor
  • VEGF-A vascular permeability factor
  • VEGF-B vascular permeability factor
  • VEGF-D vascular permeability factor
  • VEGF-E vascular permeability factor
  • Alternative splicing of mRNA of a single gene of human VEGF results in at least six isoforms (VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, and VEGF206), VEGF165 being the most abundant isoform.
  • VEGFR-1 also known as Flt-1
  • VEGFR-2 also known as KDR or FIK-1
  • VEGFR-1 has the highest affinity for VEGF
  • VEGFR- 2 has a somewhat lower affinity for VEGF.
  • Ferrara Endocrine Rev. 2004, 25: 581-611
  • VEGF has been reported to be a pivotal regulator of both normal and abnormal angiogenesis (Ferrara and Davis-Smyth, Endocrine Rev. 1997, 18: 4-25; Ferrara J. MoL Med. 1999, 77: 527-543). Compared to other growth factors that contribute to the processes of vascular formation, VEGF is unique in its high specificity for endothelial cells within the vascular system.
  • VEGF is in particular involved in eye diseases.
  • concentration of VEGF in eye fluids is highly correlated with the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies.
  • studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by age-related macular degeneration (AMD).
  • AMD age-related macular degeneration
  • the VEGF-binding components of the binding molecule according to the invention e.g. immunoglobulin (Ig) molecules, have specificity for VEGF in that they bind specifically to one or more epitopes within the VEGF molecule.
  • Ig immunoglobulin
  • the binding molecules of the invention act as VEGF antagonists.
  • the binding molecules of the invention bind specifically to VEGF-A.
  • Specific binding of a VEGF-binding component to its antigen VEGF can be determined in any suitable manner known per se, including, for example, the assays described herein, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA and ELISA) and sandwich competition assays, and the different variants thereof known per se in the art.
  • RIA radioimmunoassays
  • EIA and ELISA enzyme immunoassays
  • sandwich competition assays sandwich competition assays
  • a VEGF-binding component of the invention e.g. an immunoglobulin (Ig) molecule
  • the immunoglobulin (Ig) molecule preferably binds to human VEGF, if intended for therapeutic purposes in humans.
  • immunoglobulin (Ig) molecules that bind to VEGF from another mammalian species are also within the scope of the invention.
  • An immunoglobulin (Ig) molecule binding to one species form of VEGF may cross-react with VEGF, which has a different sequence than the human one, from one or more other species.
  • immunoglobulin (Ig) molecules binding to human VEGF may exhibit cross reactivity with VEGF from one or more other species of primates and/or with VEGF from one or more species of animals that are used in animal models for diseases, for example monkey, mouse, rat, rabbit, pig, dog, and in particular in animal models for diseases and disorders associated with VEGF-mediated effects on angiogenesis (such as the species and animal models mentioned herein).
  • Immunoglobulin (Ig) molecules that show such cross-reactivity are advantageous in a research and/or drug development, since it allows the immunoglobulin single variable domains of the invention to be tested in acknowledged disease models such as monkeys, in particular Cynomolgus or Rhesus, or mice and rats.
  • a VEGF-binding component recognizes an epitope in a region of the VEGF of interest that has a high degree of identity with human VEGF.
  • the VEGF-binding components of the binding molecule according to the invention e.g. an immunoglobulin (Ig) molecule recognizes an epitope which is, totally or in part, located in a region of VEGF that is relevant for binding to its receptor.
  • the VEGF-binding components of the binding molecule according to the invention block VEGF receptor activation, preferably substantially and most preferably totally.
  • VEGF-binding component As described above, the ability of a VEGF-binding component to block the interaction between VEGF and its receptors can be determined by an Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen®), a competition ELISA, or a plasmon resonance (SPR) based assay (Biacore®), as described in the Examples.
  • AlphaScreen® Amplified Luminescent Proximity Homogeneous Assay
  • Biacore® plasmon resonance
  • the present invention relates to binding molecules that have binding specificities for at least two different targets.
  • the binding molecules are derived from antibodies.
  • Techniques for making binding molecules include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain- light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731, 168).
  • Binding molecules of the invention may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross- linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., Immunol., 148(5): 1547-1553 (1992)); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci.
  • the binding molecule comprises or consists of at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF), preferably VEGF-A and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB).
  • VEGF Vascular Endothelial Growth Factor
  • TrkB Tropomyosin receptor kinase B
  • the binding molecule is bispecific and tetravalent.
  • the binding molecule comprises or consists of at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the at least one antigen binding site that binds specifically to VEGF is an immunoglobulin (Ig) molecule, preferably an IgG molecule.
  • VEGF Vascular Endothelial Growth Factor
  • TrkB Tropomyosin receptor kinase B
  • the binding molecule is bispecific and tetravalent.
  • the binding molecule comprises or consists of at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the at least one antigen binding site that binds specifically to TrkB comprises one or more scFv(s), preferably in a VL-VH orientation from N-to C-terminus.
  • VEGF Vascular Endothelial Growth Factor
  • TrkB Tropomyosin receptor kinase B
  • the binding molecule is bispecific and tetravalent.
  • the binding molecule comprises or consists of at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the at least one antigen binding site that binds specifically to VEGF is an immunoglobulin (Ig) molecule, preferably an IgG molecule, and wherein the at least one antigen binding site that binds specifically to TrkB is fused to the C- terminus of the heavy chain of the Ig molecule.
  • the binding molecule is bispecific and tetravalent.
  • the binding molecule comprises or consists of at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the at least one antigen binding site that binds specifically to VEGF is an immunoglobulin (Ig) molecule, preferably an IgG molecule, and wherein the at least one antigen binding site that binds specifically to TrkB comprises one or more scFv(s), preferably in a VL-VH orientation from N-to C-terminus.
  • the binding molecule is bispecific and tetravalent.
  • the binding molecule comprises or consists of at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the at least one antigen binding site that binds specifically to VEGF is an immunoglobulin (Ig) molecule, preferably an IgG molecule, and wherein the at least one antigen binding site that binds specifically to TrkB comprises one or more scFv(s), preferably in a VL-VH orientation from N-to C-terminus, and is fused to the C- terminus of the heavy chain of the Ig molecule.
  • the binding molecule is bispecific and tetravalent.
  • the binding molecules of the invention comprise or consists of (i) two heavy chains each comprising or consisting of a heavy chain variable region specific for VEGF, constant IgG domains and an scFv specific for TrkB and (ii) two light chains each comprising or consisting of a light chain variable region specific for VEGF.
  • these single chain Fv fragments might be further stabilized by incorporation of disulfide bonds between the VH and VL domains, within the VH domain, or within the VL domain, via incorporation of cysteine residues.
  • N-terminus denotes the first amino acid of the polypeptide chain while the term C-terminus denotes the last amino acid of the C-terminus of the polypeptide chain.
  • the one or more scFv(s) comprises additional cysteine residues to form disulfide bonds.
  • the present invention provides a binding molecule which is a multi-specific binding protein comprising (i) an Ig molecule specifically binding to VEGF with two heavy and two light chains, and (ii) two scFv molecules (scFv(s)) each specifically binding to TrkB.
  • each heavy chain of the Ig molecule has one scFv fused to its C-terminus, thereby forming a bispecific tetravalent binding protein.
  • the present invention provides a binding molecule (also referred to herein multi-specific binding protein or a modified Ig molecule) with:
  • the present invention provides a binding molecule having at least one antigen binding site that binds specifically to vascular endothelial growth factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB).
  • VEGF vascular endothelial growth factor
  • TrkB Tropomyosin receptor kinase B
  • antibodies may be generated via any one of several methods which employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al 1991, Nature 349:293-299) or generation of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique (Kohler et al 1975.
  • EBV Epstein-Barr virus
  • the present inventors conceived binding molecules against VEGF/TrkB of the invention as shown in Table 1 and a selection of those molecules were prepared and are discussed in the accompanying examples.
  • the binding molecule of the invention or the TrkB binding molecule may be based on any of the below disclosed TrkB binders which are also disclosed in WO2018/224630.
  • the binding molecule comprises at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the antigen binding site that binds specifically to TrkB comprises light chain CDRs comprising or consisting of the amino acid sequences of SEQ ID NO: 201 (CDR1), SEQ ID NO: 202 (CDR2) and SEQ ID NO: 203 (CDR3) and heavy chain CDRs comprising or consisting of the amino acid sequences of SEQ ID NO: 204 (CDR1), SEQ ID NO: 205 (CDR2) and SEQ ID NO: 206 (CDR3); or heavy chain CDRs comprising or consisting of the amino acid sequences of SEQ ID NO: 207 (CDR1), SEQ ID NO: 208 (CDR2) and SEQ ID NO: 209 (CDR3); or heavy chain CDRs comprising VEGF
  • the binding molecule comprises at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the antigen binding site that binds specifically to TrkB comprises a light chain variable domain comprising or consisting of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 213 or 215 and a heavy chain variable domain comprising or consisting of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 9
  • the binding molecule comprises at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the antigen binding site that binds specifically to TrkB comprises a light chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 213 or 215 and a heavy chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 214 or 216; and wherein the antigen binding site that binds specifically to VEGF comprises a light chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 189 or 193 and a heavy chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 190, 191, 192 or 194.
  • VEGF Vascular Endothelial Growth Factor
  • TrkB Tropomyosin receptor kinase B
  • the binding molecule comprises at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the antigen binding site that binds specifically to TrkB comprises a light chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 213 and a heavy chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 214; and wherein the antigen binding site that binds specifically to VEGF comprises a light chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 193 and a heavy chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 191.
  • VEGF Vascular Endothelial Growth Factor
  • TrkB Tropomyosin receptor kinase B
  • the binding molecule comprises at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the antigen binding site that binds specifically to TrkB comprises a light chain variable domain comprising or consisting of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 213 or 215 and a heavy chain variable domain comprising or consisting of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 9
  • the binding molecule comprises at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the antigen binding site that binds specifically to TrkB comprises light chain CDRs comprising or consisting of the amino acid sequences of SEQ ID NO: 228 (CDR1), SEQ ID NO: 229 (CDR2) and SEQ ID NO: 230 (CDR3) and heavy chain CDRs comprising or consisting of the amino acid sequences of SEQ ID NO: 231 (CDR1), SEQ ID NO: 232 (CDR2) and SEQ ID NO: 23 (CDR3); or heavy chain CDRs comprising or consisting of the amino acid sequences of SEQ ID NO: 234 (CDR1), SEQ ID NO: 235 (CDR2) and SEQ ID NO: 236 (CDR3); or heavy chain CDRs comprising or consisting or consist
  • the binding molecule comprises at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB), wherein the antigen binding site that binds specifically to TrkB comprises a light chain variable domain and a heavy chain variable domain, each comprising or consisting of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequences of SEQ ID NO: 240 and 241, 242 and 243, 244 and 245, 246 and 247, 248 and 249, 250 and 251, 252 and 253, 254 and 255, or 256 and 257; and wherein the antigen binding site that binds specifically to VEGF comprises
  • the binding molecule is bispecific and tetravalent.
  • the binding molecules of the inventions act as VEGF antagonists or TrkB agonists, respectively.
  • the antigen binding site that binds specifically to VEGF is an immunoglobulin (Ig) molecule and the at least one antigen binding site that binds specifically to TrkB comprises one or more scFv(s).
  • the one or more scFv(s) is fused to the C- terminus of the heavy chain of the Ig molecule.
  • the at least one antigen binding site that binds specifically to VEGF is an immunoglobulin (Ig) molecule, more preferably an IgG, and the at least one antigen binding site that binds specifically to TrkB comprises one or more scFv(s), more preferably two scFv.
  • Ig immunoglobulin
  • the antibody molecule or binding molecule described herein may be fused (as a fusion protein) or otherwise linked (by covalent or non-covalent bonds) to other molecular entities having a desired impact on the properties of the antibody molecule.
  • it may be desirable to improve pharmacokinetic properties of antibody or binding molecules described herein, stability e.g. in body fluids such as blood, in particular in the case of single chain antibodies or domain antibodies.
  • the antibody is, in certain embodiments, a full length antibody or an antibody that contains a portion of the Fc region, the latter as long as the antibody exhibits specific binding both to the relevant portion of the antigen and to Fc receptors and complements.
  • effector functions like complement fixation or antibody-dependent cell-mediated cytotoxicity are desirable features, and on the desired pharmacological properties of the antibody protein.
  • stability of the scFv moiety can be increased by incorporation of two cysteine residues in close 3-dimensional proximity to form a disulfide bond within the scFv.
  • residues at these positions are preferably substituted with cysteine residues.
  • TrkB scFv having a VL-VH orientation from N-to C-terminus can function in the binding molecules of the invention to activate TrkB signalling. While a TrkB scFv having a VH-VL orientation from N-to C-terminus can also function, the activity may be reduced in this orientation. Hence a preferred embodiment of the invention is where the order is VL-VH from N-to C-terminus.
  • a further preferred embodiment of the invention is wherein the one or more scFv(s) specifically binding to TrkB is fused to the Ig molecule (e.g., human IgG1, IgG1(KO), IgG1FcRnmut, IgG4Pro) specifically binding to VEGF by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids (e.g., anyone of 6, 9, 10, 12, or 15).
  • the scFv is fused to the C- terminus of the heavy chain of the Ig molecule.
  • the Ig molecule is an IgG.
  • scFv molecules to the C- terminus of the heavy chain of the IgG molecule or linking the variable domains within scFv molecules are well known in the art.
  • a small linker sequence of glycine and serine (termed a GS mini-linker) amino acids is used.
  • the number of amino acids in the linker can vary, from 4 (GGGS) (SEQ ID NO: 227), 6 (GGSGGS) (SEQ ID NO: 217), 10 (GGGGSGGGGS) (SEQ ID NO: 218), 15 (GGGGSGGGGSGGGGS) (SEQ ID NO: 219), 20 (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 220) or more.
  • the linker is formed by combining the nucleic acid molecule encoding the IgG of interest (which in the present case would include the nucleic acid encoding the variable domain of the heavy chain for the VEGF binding site and constant domains of the IgG type) with the nucleic acid encoding the desired scFv (which in the present case would include the nucleic acid encoding the variable domain of the heavy and light chain, either in VL-VH or VH-VL orientation for the TrkB binding site) interspaced by the nucleic acid molecule encoding the linker sequence (e.g. a GS mini linker of any one of 5, 10, 15, or 20 amino acids, preferably a linker of SEQ ID NO: 218).
  • this complete HC-scFv encoding nucleic acid molecule is placed within an expression vector and introduced to appropriate host cells such that the complete IgG heavy chain-scFv single polypeptide is formed.
  • the GS mini-linker between the scFV molecule and the C-terminus of the heavy chain of the IgG molecule is 10L1 (SEQ ID NO: 218).
  • binding to complement product C1q or Fc gamma receptor by the binding molecule in this invention is ablated by utilization of the IgG4 constant region or of the IgG1 constant region with directed L to A mutagenesis at positions 234 and 235.
  • the binding molecule of the invention may have an Fc region, or the relevant section thereof, that has been engineered to avoid unintended cross-linking by soluble Fc gamma receptors or complement Cl q.
  • such binding molecule or antibody variant has much lower affinities to Fc gamma receptors and complement C1q than the parent antibody.
  • parent in the context of an antibody molecule, or in the context of IgG or the Fc region, refers to the non-engineered antibody molecule, Fc region or IgG, respectively, from which the mutated (engineered) molecule is derived.
  • the Ig molecule comprises a Fc variant having a reduced affinity to Fc gamma receptors or complement receptors or both compared to a wildtype Fc region.
  • IgG1(KO) Such Ig molecule is referred to herein as IgG1(KO).
  • a binding molecule comprising an Fc region, or the relevant section thereof, that has been engineered to modify serum levels (half-life) by optimizing its interaction with the neonatal Fc receptor (FcRn), e.g. by a point mutation in the CH2 domain at position H310A or a point mutation at position H435A).
  • FcRn neonatal Fc receptor
  • Such Ig molecule is referred to herein as IgG1 FcRnmut.
  • binding molecule comprising an Ig molecule which comprises a hinge region variant of IgG4 that ablates swapping of the heavy chains with other IgG4 molecules.
  • IgG4Pro Such Ig molecule is referred to herein as IgG4Pro.
  • a further aspect of the invention provides isolated nucleic acid molecules that encode the binding molecule of the invention or the antibody molecule of the invention, or an expression vector comprising such a nucleic acid molecule(s).
  • binding molecules of the invention or antibody molecule of the invention comprise antibody heavy chain and/or light chain polypeptides.
  • nucleic acid molecules can be readily prepared which encode the heavy chain polypeptides, light chain polypeptides, or heavy chain polypeptides and light chain polypeptides.
  • Nucleic acid molecules coding for the light chain and the heavy chain may be synthesized chemically and enzymatically by Polymerase Chain Reaction (PCR) using standard methods.
  • PCR Polymerase Chain Reaction
  • suitable oligonucleotides can be synthesized with methods known in the art (e.g. Gait, 1984), which can be used to produce a synthetic gene. Methods to generate synthetic genes from oligonucleotides are known in the art (e.g. Stemmer et al., 1995; Ye et al., 1992; Hayden et Mandecki, 1988; Frank et al., 1987).
  • the nucleic acid molecules of the invention include, but are not limited to, the DNA molecules encoding the polypeptide sequences shown in the sequence listing. Also, the present invention also relates to nucleic acid molecules that hybridize to the DNA molecules encoding the polypeptide sequences shown in the sequence listing under high stringency binding and washing conditions, as defined in WO 2007/042309. Preferred molecules (from an mRNA perspective) are those that have at least 75% or 80% (preferably at least 85%, more preferably at least 90% and most preferably at least 95%) homology or sequence identity with one of the DNA molecules described herein.
  • the DNA sequences shown in the sequence listing have been designed to match codon usage in eukaryotic cells. If it is desired to express the antibodies in E. coli , these sequences can be changed to match E. coli codon usage.
  • Variants of DNA molecules of the invention can be constructed in several different ways, as described e.g. in WO 2007/042309.
  • a further aspect of the invention provides a method of production of a binding or antibody molecule described herein, comprising:
  • An embodiment of this aspect of the invention is wherein the method of production further comprises step (c) further purifying and/or modifying and/or formulating the binding molecule of the invention.
  • the DNA molecules encoding full-length light and/or heavy chains or fragments thereof are inserted into an expression vector such that the sequences are operatively linked to transcriptional and translational control sequences.
  • binding molecules or antibodies of the invention For manufacturing the binding molecules or antibodies of the invention, the skilled artisan may choose from a great variety of expression systems well known in the art, e.g. those reviewed by Kipriyanov and Le Gall, Curr Opin Drug Discov Devel. 2004 Mar.; 7(2):233-42.
  • Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes, and the like.
  • the expression vector and expression control sequences are selected to be compatible with the host cell.
  • the antibody light chain gene and the antibody heavy chain gene or the gene of the heavy chain of the binding molecule described herein e.g. the gene comprising an immunoglobulin heavy chain sequence attached with its C-terminus to a scFv sequence
  • both DNA sequences, light and heavy chain sequences are inserted into the same expression vector.
  • Convenient vectors are those that encode a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed, as described above.
  • the constant chain is usually kappa or lambda for the antibody light chain, for the antibody heavy chain, it can be, without limitation, any IgG isotype (IgG 1 , IgG2, IgG3, IgG4) or other immunoglobulins, including allelic variants.
  • the recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain (e.g., the heavy and light chains of the binding molecules or antibodies described herein) from a host cell.
  • the DNA encoding the antibody chain may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the mature antibody chain DNA.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous peptide from a non-immunoglobulin protein.
  • the DNA sequence encoding the antibody chain (e.g., the heavy and light chains of the binding molecules or antibodies described herein) may already contain a signal peptide sequence.
  • the recombinant expression vectors carry regulatory sequences including promoters, enhancers, termination and polyadenylation signals and other expression control elements that control the expression of the antibody chains in a host cell.
  • promoter sequences are promoters and/or enhancers derived from (CMV) (such as the CMV Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.
  • AdMLP adenovirus major late promoter
  • polyoma g., the adenovirus major late promoter (AdMLP)
  • AdMLP adenovirus major late promoter
  • polyoma g., the adenovirus major late promoter (AdMLP)
  • AdMLP adenovirus major late promoter
  • polyoma g., the adenovirus major late promoter (AdMLP)
  • strong mammalian promoters such as native immunoglobulin and actin promoters.
  • polyadenylation signals are BGH polyA, SV40 late or early polyA; alternatively, 3′UTRs of immunoglobulin genes etc. can be used.
  • the recombinant expression vectors may also carry sequences that regulate replication of the vector in host cells (e. g. origins of replication) and selectable marker genes.
  • Nucleic acid molecules encoding the heavy chain or an antigen-binding portion thereof and/or the light chain or an antigen-binding portion thereof of a binding molecule or antibody described herein, and vectors comprising these DNA molecules can be introduced into host cells, e.g. bacterial cells or higher eukaryotic cells, e.g. mammalian cells, according to transfection methods well known in the art, including liposome-mediated transfection, polycation-mediated transfection, protoplast fusion, microinjections, calcium phosphate precipitation, electroporation or transfer by viral vectors.
  • the nucleic acid molecules encoding the heavy chain and the light chain of the binding molecules or antibodies described herein are present on two vectors which are co-transfected into the host cell, preferably a mammalian cell.
  • a further aspect provides a host cell comprising an expression vector comprising a nucleic acid molecule encoding the heavy chain and an expression vector comprising a nucleic acid molecule encoding the light chain of the binding molecules or antibodies described herein.
  • Mammalian cell lines available as hosts for expression are well known in the art and include, inter alia, Chinese hamster ovary (CHO, CHO-DG44) cells, NSO, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human carcinoma cells (e. g., Hep G2), A549 cells, 3T3 cells or the derivatives/progenies of any such cell line.
  • Other mammalian cells including but not limited to human, mice, rat, monkey and rodent cells lines, or other eukaryotic cells, including but not limited to yeast, insect and plant cells, or prokaryotic cells such as bacteria may be used.
  • the binding molecules of the invention are produced by culturing the host cells for a period of time sufficient to allow for expression of the binding molecule in the host cells.
  • Binding molecules and antibody molecules as described herein are preferably recovered from the culture medium as a secreted polypeptide or it can be recovered from host cell lysates if for example expressed without a secretory signal. It is necessary to purify the binding molecules or antibody molecules described herein using standard protein purification methods used for recombinant proteins and host cell proteins in a way that substantially homogenous preparations of the binding molecule or antibody as described herein are obtained.
  • state-of-the art purification methods useful for obtaining the binding molecules and antibodies of the invention include, as a first step, removal of cells and/or particulate cell debris from the culture medium or lysate.
  • the binding molecule or antibody is then purified from contaminant soluble proteins, polypeptides and nucleic acids, for example, by fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, chromatography on silica or on a cation exchange resin.
  • the purified binding molecule may be dried, e.g. lyophilized, as described below for therapeutic applications.
  • a further aspect of the invention provides the binding molecules of the invention for use in medicine. It will be understood that this use in medicine and the uses for treatment of diseases as described in the following also includes the TrkB binding molecules, scFv and antibodies according to the invention.
  • the binding molecules of the invention are indicated e.g. for use in the therapy/treatment of eye or retinal or neurodegenerative diseases, preferably for the treatment of neural/neuronal eye or retinal diseases.
  • the present invention relates to methods for the treatment and/or prevention of eye or retinal or neurodegenerative diseases, which method comprises the administration of an effective amount of the binding molecule of the invention to a human being (e.g. an individual suffering from wAMD or being at risk of developing geographic atrophy), thereby ameliorating one or more symptoms of the eye or retinal or neurodegenerative diseases.
  • treatment and “therapy” and the like, as used herein, are meant to include therapeutic as well as prophylactic, or suppressive measures for a disease or disorder leading to any clinically desirable or beneficial effect, including but not limited to alleviation or relief of one or more symptoms, regression, slowing or cessation of progression of the disease or disorder.
  • treatment includes the administration of a binding molecule prior to or following the onset of a symptom of a disease or disorder thereby preventing or removing one or more signs of the disease or disorder.
  • the term includes the administration of a binding molecule after clinical manifestation of the disease to combat the symptoms of the disease.
  • administration of a binding molecule after onset and after clinical symptoms have developed where administration affects clinical parameters of the disease or disorder, such as the degree of tissue injury or the amount or extent of metastasis, whether or not the treatment leads to amelioration of the disease, comprises “treatment” or “therapy” as used herein.
  • treatment or “therapy” as used herein.
  • compositions of the invention either alone or in combination with another therapeutic agent alleviate or ameliorate at least one symptom of a disorder being treated as compared to that symptom in the absence of use of the binding molecule, the result should be considered an effective treatment of the underlying disorder regardless of whether all the symptoms of the disorder are alleviated or not.
  • a binding molecule of the invention can be administered to a subject having or at risk of having an eye or retinal disease.
  • the invention further provides for the use of a binding molecule in the manufacture of a medicament for prevention and/or treatment of an eye or retinal disease.
  • subject as used herein means any mammalian patient to which a binding molecule can be administered, including, e.g., humans and non-human mammals, such as primates, rodents, and dogs. Subjects specifically intended for treatment using the methods described herein include humans.
  • the binding molecule can be administered either alone or in combination with other compositions.
  • the binding molecules of the invention are used for the treatment of macular degeneration, age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, inherited retinal dystrophy, inherited macular dystrophy, myopic degeneration, geographic atrophy, geographic atrophy secondary to age-related macular degeneration, retinal artery occlusions, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane secondary to any retinal diseases, optic neuropathies, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, and traumatic retinopathy, prodromal and mild-to-moderate alzheimer's diseases, delaying disease progression of patients with Alzheimer's disease, Huntington's disease, Parkinson's disease, major depressive disorder, schizophrenia, cognitive impairment associated with schizophrenia, prevention of first-episode psychosis in individuals with attenuated
  • the binding molecules of the invention have utility in the treatment of wAMD. Further preferred, the binding molecules of the invention have utility in the treatment of wAMD and treatment of geographic atrophy. In a yet further preferred embodiment the binding molecules of the invention have utility in the treatment of geographic atrophy secondary to age-related macular degeneration. In a further preferred embodiment the binding molecules of the invention have utility in the treatment of geographic atrophy or the treatment of patients at risk for developing geographic atrophy. In a further preferred embodiment the binding molecules of the invention have utility in the prevention of geographic atrophy. In a most preferred embodiment the binding molecules of the invention have utility in the treatment of wAMD in patients at risk for developing geographic atrophy.
  • binding molecules of the invention may be useful for treatment of hearing loss, in particular for cis platin induced hearing loss as well as noise and age-related hearing loss.
  • the binding molecule of the invention is administered by any suitable means, including intravitreal, oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the binding molecule of the invention is suitably administered by pulse infusion, particularly with declining doses.
  • the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • the binding molecule of the invention is given through an intravitreal injection into the eye.
  • a binding molecule of the invention is used in combination with a device useful for the administration of the binding molecule, such as a syringe, injector pen, or other device.
  • a binding molecule of the invention is comprised in a kit of parts, for example also including a package insert with instructions for the use of the binding molecule.
  • the efficacy of the binding molecules of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease involved.
  • suitable assays and animal models will be clear to the skilled person, and for example include the assays and animal models used in the Examples below.
  • the actual pharmaceutically effective amount or therapeutic dosage will of course depend on factors known by those skilled in the art such as age and weight of the patient, route of administration and severity of disease.
  • the binding molecule of the invention will be administered at dosages and in a manner which allows a pharmaceutically effective amount to be delivered based upon patient's unique condition.
  • binding molecules of the invention may be used on their own or in combination with other pharmacologically active ingredients, such as state-of-the-art or standard-of-care compounds.
  • a further aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a binding molecule of the invention, together with a pharmaceutically acceptable carrier and optionally one or more further active ingredients.
  • the binding molecule of the invention is administered every 6 weeks, preferably every 7 weeks, also preferred every 8 weeks, further preferred every 9 weeks, more preferred every 10 weeks, further preferred every 11 weeks, and more preferred every 12 weeks.
  • the TrkB-antibody is administered once every 3 months.
  • the frequency of administration may be even longer, such as every 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks or 20 weeks.
  • dosage regimens may be applied including e.g. a loading dose, wherein the binding molecules of the invention may be injected monthly for 3 loading doses and then every 12 weeks.
  • the frequency of administration may also be extended in such cases and may be even longer, such as every 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks or 20 weeks.
  • the predicted estimated human dose of approximately 2.5 mg/eye corresponds to a 50 mg/mL formulation in which 50 ⁇ L will be injected into the eye.
  • the binding molecule of the invention is formulated into pharmaceutical compositions appropriate to facilitate administration to animals or humans.
  • Typical formulations of the binding molecule or antibody molecule described herein can be prepared by mixing the binding with physiologically acceptable carriers, excipients or stabilizers, in the form of lyophilized or otherwise dried formulations or aqueous solutions or aqueous or non-aqueous suspensions. Carriers, excipients, modifiers or stabilizers are nontoxic at the dosages and concentrations employed.
  • buffer systems such as phosphate, citrate, acetate and other inorganic or organic acids and their salts; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone or polyethylene glycol (PEG); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, oligosaccharides,
  • suitable formulations for therapeutic proteins such as the binding molecules of the invention are buffered protein solutions, such as solutions including the protein in a suitable concentration (such as from 0.001 to 400 mg/ml, preferably from 0.005 to 200 mg/ml, more preferably 0.01 to 200 mg/ml, more preferably 1.0 -100 mg/ml.
  • TrkB binding sites two scFv's
  • the invention is further directed to a TrkB binding molecule comprising or consisting of two scFv's, wherein each scFv binds specifically to TrkB and both together act as a TrkB agonist.
  • TrkB binding molecules are full TrkB agonists, i.e. the TrkB binding molecules are as efficacious in activating TrkB as the natural ligand BDNF.
  • the invention relates to a TrkB binding molecule comprising or consisting of two scFv's, wherein each scFv binds specifically to TrkB.
  • Specific binding of a an scFv and in particular a TrkB binding molecule to its antigen TrkB can be determined in any suitable manner known per se, including, for example, the assays described herein, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA and ELISA) and sandwich competition assays, and the different variants thereof known per se in the art.
  • RIA radioimmunoassays
  • EIA and ELISA enzyme immunoassays
  • sandwich competition assays sandwich competition assays
  • a TrkB binding molecule is considered as efficacious as BDNF if it shows at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the efficacy of BDNF.
  • both BDNF as well as the TrkB binding molecule will be tested by measuring/determing the efficacy, which is the maximum response as determined by incubating CHO cells stably expressing a TrkB receptor with BDNF or the TrkB binding molecule and measuring the TrkB phosphorylation at Y706/707 in the cell lysate of the treated CHO cells. Further details are described in the Examples.
  • the TrkB binding molecules may be about at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as efficacious as compared to BDNF as determined by incubating CHO cells stably expressing a TrkB receptor with BDNF or the TrkB binding molecule and measuring the TrkB phosphorylation at Y706/707 in the cell lysate of the treated CHO cells. Further details are described in the Examples.
  • the two scFv's in the TrkB binding molecule are connected to each other to form a TrkB binding molecule, in particular a bivalent TrkB binding molecule.
  • the two scFv's in the TrkB binding molecule may be fused or otherwise covalently attached to each other.
  • the two scFv's may be linked via a peptide linker, preferably a peptide linker e.g. having a length of about 4 to 20 amino acids and more preferably a flexible peptide linker.
  • other linkers may be used having lengths ranging from 4 to 100 amino acids and being flexible, helical or rigid.
  • the scFv's in the TrkB binding molecule may also be connected to each other via a hinge region. It is well understood by the skilled artisan that the hinge region is a short sequence of the heavy chains of antibodies linking the Fab (Fragment antigen binding) region to the Fc (Fragment crystallisable) region.
  • TrkB binding molecule comprises or consists of two scFv's that are linked via a peptide linker. In another alternative the TrkB binding molecule comprises or consists of two scFv's that are linked via a hinge region. In another alternative the TrkB binding molecule consists of two scFv's linked via a hinge region.
  • Each scFv in the TrkB binding molecule may bind to the same or a different epitope within the TrkB protein.
  • both scFv bind to the same TrkB epitope.
  • the two scFv's are identical.
  • both scFv will be based on the same VL and VH sequence.
  • the two scFv's are substantially the same, i.e. both scFv's have the same CDR regions but may have some variability in the framework region.
  • the two scFv's have the same CDR regions and the remaining framework regions are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each other.
  • both scFv's are different from each other, i.e. have a different VL and/or VH sequence.
  • the scFv's in the TrkB binding molecule may comprise from N to C terminus a VH domain (e.g. murine, humanized or human VH domain) a linker and a VL domain (e.g. murine, humanized or human VL domain), or vice versa a VL domain a linker and a VH domain).
  • a VH domain e.g. murine, humanized or human VH domain
  • VL domain e.g. murine, humanized or human VL domain
  • the scFv's in the TrkB binding molecule have a VL-VH orientation from N-to C-terminus.
  • the single chain Fv fragments might be further stabilized by incorporation of disulfide bonds between the VH and VL domains, within the VH domain, or within the VL domain, via incorporation of cysteine residues.
  • the term N-terminus denotes the first amino acid of the polypeptide chain while the term C-terminus denotes the last amino acid of the C-terminus of the polypeptide chain.
  • the one or both scFv(s) comprise additional cysteine residues to form disulfide bonds.
  • the TrkB binding molecule further comprises an Ig molecule.
  • the Ig molecule may be a monoclonal antibody, a human monoclonal antibody, a humanized monoclonal antibody, a chimeric antibody, a fragment of an antibody, such as a Fv, Fab, Fab′, or F(ab′)2 fragment, a single chain antibody, such as a single chain variable fragment (scFv), a Small Modular Immunopharmaceutical (SMIP), a domain antibody, a nanobody, or a diabody.
  • each scFv may be fused to the C-terminus of the heavy chain of the Ig molecule.
  • each scFv may be fused to the N-terminus of the heavy chain of the Ig molecule.
  • the Ig molecule is an IgG, F(ab), or F(ab′)2.
  • the Ig molecule comprises or consists of an Fc region.
  • each scFv may be fused to the Ig molecule by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids. In any case also other linkers may be used having lengths ranging from 4 to 100 amino acids and being flexible, helical or rigid. In some instances a longer linker and/or rigid linker may be preferable.
  • the TrkB binding molecule is in the format of an Fc-scFv, scFv-Fc, (scFv′)2 or scFv-CH 3 .
  • the scFv-Fc format is depicted in FIG. 70B and comprises or consists of two polypeptide chains, each polypeptide chain containing a scFv, a CH2 and a CH3 domain of an IgG, wherein both polypeptide chains form together a dimer through the disulfide bonds in the hinge region and each scFv is fused to the hinge region.
  • the Fc-scFv format is depicted in FIG.
  • each polypeptide chain containing a CH2 and a CH3 domain of an IgG and a scFv, wherein both polypeptide chains form together a dimer through the disulfide bonds in the hinge region and each scFv is fused to the C-temrinus of the CH3 domain of one polypeptide chain.
  • the Fc-scFv format as depicted in FIG. 70C may further comprise one or more Fab domains.
  • the TrkB binding molecule is a TrkB agonist.
  • the TrkB binding molecule is about at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% as efficacious as compared to BDNF.
  • the TrkB binding molecule is bispecific and tetravalent, more preferably a Doppelmab.
  • the Ig molecule may be designed to bind to any target. Since, it is believed that the observed full agonist effect is mainly based on the sterical formation of the scFv's within the TrkB binding molecule, it follows that the Ig molecule may also not bind to any target specifically or does not contain any target specific binding domain at all.
  • the Ig molecule comprises or consists of an Fc region.
  • each scFv is fused to the C- terminus of the heavy chain of the Fc region.
  • each scFv is fused to the Fc region by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids.
  • the two scFv's and the Ig molecule in the TrkB binding molecule are connected to each other to form a TrkB binding molecule.
  • the scFv's may be fused or otherwise covalently attached directly to the Ig molecule or they may be linked to the Ig molecule via a linker, preferably a peptide linker e.g. having a length of about 4 to 20 amino acids and more preferably a flexible peptide linker.
  • the scFv are fused to the C-terminus of the heavy chain of the Ig molecule.
  • the Ig molecule is an IgG, F(ab), or F(ab')2.
  • the TrkB binding molecule comprises or consists of two scFv's and an IgG, F(ab), or F(ab′)2 wherein each scFv binds specifically to TrkB.
  • the TrkB binding molecule is bispecific and tetravalent and comprises or consists of two scFv's and an IgG or F(ab′)2 wherein each scFv binds specifically to TrkB.
  • the Ig molecule and more preferably the IgG, F(ab), or F(ab′)2 binds specifically to VEGF.
  • scFv molecules to the C-terminus of the heavy chain of the Ig molecule e.g. such as an IgG molecule or linking the variable domains within scFv molecules are well known in the art.
  • a small linker sequence of glycine and serine (termed a GS mini-linker) amino acids is used.
  • the number of amino acids in the linker can vary, from 4 (GGGS) (SEQ ID NO: 227), 6 (GGSGGS) (SEQ ID NO: 217), 10 (GGGGSGGGGS) (SEQ ID NO: 218), 15 (GGGGSGGGGSGGGGS) (SEQ ID NO: 219), 20 (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 220) or more.
  • the linker is formed by combining the nucleic acid molecule encoding the Ig of interest with the nucleic acid encoding the desired scFv (which in the present case would include the nucleic acid encoding the variable domain of the heavy and light chain, either in VL-VH or VH-VL orientation for the TrkB binding site) interspaced by the nucleic acid molecule encoding the linker sequence (e.g. a GS mini linker of any one of 5, 10, 15, or 20 amino acids, preferably a linker of SEQ ID NO: 218). Then as explained beforehand this complete HC-scFv encoding nucleic acid molecule is placed within an expression vector and introduced to appropriate host cells such that the complete Ig heavy chain-scFv single polypeptide is formed.
  • the linker sequence e.g. a GS mini linker of any one of 5, 10, 15, or 20 amino acids, preferably a linker of SEQ ID NO: 218
  • the GS mini-linker between the scFv molecule and the C-terminus of the heavy chain of the Ig molecule is 10L1 (SEQ ID NO: 218).
  • a TrkB binding molecule is more potent in inducing activation of TrkB downstream signaling pathways than the natural TrkB ligand, BDNF.
  • a TrkB binding molecule regulates gene expression through TrkB-mediated signaling pathways in a comparable pattern to that of BDNF.
  • TrkB binding molecule is specific for TrkB phosphorylation and/or activation and does not unspecifically phosphorylate/activate TrkA or TrkC.
  • the TrkB binding molecule comprises or consists of:
  • the TrkB binding molecule comprises or consists of:
  • the TrkB binding molecule comprises or consists of:
  • the invention provides for a method to improve the efficacy of an agonistic TrkB binder.
  • the inventors have surprisingly found that agonistic TrkB binders and in particular partial agonistic TrkB binders, such as IgG could be improved in their efficacy when applying the inventive method.
  • the invention provides for a method to produce or generate a TrkB binding molecule having improved efficacy compared to the agonistic TrkB binder it is based upon.
  • the method comprises generating a first scFv using the variable light and heavy chain domains of an agonistic TrkB binder. Subsequently generating a second scFv, which is either identical or different to the first scFv and linking or attaching both scFv in one molecule to yield a bivalent TrkB binding molecule comprising both scFv's.
  • the method can be used to improve the efficacy of any agonistic TrkB binder and to produce or generate a TrkB binding molecule having improved efficacy compared to the agonistic TrkB binder it is based upon.
  • “Agonistic TrkB binder(s)” as used herein refers to binding molecules characterized by comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) that form together the antigen binding part of the binding molecule and specifically bind to the TrkB receptor and act as agonists.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH heavy chain variable domain
  • monoclonal antibody human monoclonal antibody
  • humanized monoclonal antibody chimeric antibody
  • fragment of an antibody such as Fv, Fab, Fab′, or F(ab')2 fragment
  • single chain antibody such as a single chain variable fragment (scFv)
  • scFv single chain variable fragment
  • SMIP Small Modular Immunopharmaceutical
  • the method can be used to improve the efficacy of agonistic TrkB binders that were identified and characterized e.g. via screenings and appropriate assays.
  • the method is especially useful to improve the efficacy of agonistic TrkB binding antibodies, such as IgG.
  • agonistic TrkB binder may also be a scFv.
  • TrkB binders In the art numerous agonistic TrkB binders are described and the skilled artisan will be able to choose freely among those to improve their efficacy according to the method as described herein.
  • TrkB binding molecule generated according to the method of the invention will be more efficacious, i.e. will show a higher efficacy to activate TrkB compared to the agonistic TrkB binder that served as the basis to generate the TrkB binding molecule.
  • the efficacy as used herein describes the maximum response that can be achieved with a drug.
  • the effect of the drug is plotted against the concentraion in a graph, to give the concentration-response curve.
  • the increasing concentrations used are displayed by the X axis and the half maximal and maximal responses are displayed by the Y axis.
  • the highest point on the curve shows the maximum response (efficacy) and is referred to as the Emax.
  • Efficacy (Emax) is the maximum effect which can be expected from this drug (i.e. when this magnitude of effect is reached, increasing the dose will not produce a greater magnitude of effect).
  • it is not the absolute increase in efficacy that is important but the relative increase that is achieved by generating the TrkB binding molecule from the VL and VH of the agonistic TrkB binder.
  • the invention provides for a method for improving the efficacy of an agonistic TrkB binder, wherein the agonistic TrkB binder contains a light chain variable domain (VL) and a heavy chain variable domain (VH), comprising
  • the agonistic TrkB binder is a partial agonist.
  • the efficacy of the TrkB binding molecule compared to the efficacy of the agonistic TrkB binder is higher by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.
  • both the agonistic TrkB binder as well as the TrkB binding molecule will be tested by measuring/determing the efficacy, which is the maximum response as determined by incubating CHO cells stably expressing a TrkB receptor with the agonistic TrkB binder or the TrkB binding molecule and measuring the TrkB phosphorylation at Y706/707 in the cell lysate of the treated CHO cells. Further details are described in the Examples.
  • the TrkB binding molecule is about at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% as efficacious as compared to BDNF.
  • both BDNF as well as the TrkB binding molecule will be tested by measuring/determing the efficacy, which is the maximum response as determined by incubating CHO cells stably expressing a TrkB receptor with BDNF or the TrkB binding molecule and measuring the TrkB phosphorylation at Y706/707 in the cell lysate of the treated CHO cells. Further details are described in the Examples.
  • the two scFv's are identical. In this embodiment both scFv will be based on the same VL and VH of the same agonistic TrkB binder. In another embodiment the two scFv's are substantially the same, i.e. both scFv's have the same CDR regions but may have some variability in the framework region. In a related embodiment the two scFv's have the same CDR regions and the remaining framework regions are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each other.
  • both scFv's are different from each other, i.e. are based on two different agonistic TrkB binders.
  • the efficacy of the TrkB binding molecule will be both improved compared to both agonistic TrkB binders it is based upon.
  • the two agonistic TrkB binding scFv's bind to the same epitope or bind to a different epitope.
  • TrkB binding molecule is formed by the first and the second scFv, i.e. both are included to form the TrkB binding.
  • both scFv's may be fused or otherwise covalently attached directly to each other to form the TrkB binding molecule (scFv-scFv).
  • the scFv's may also be linked to each other via a linker (scFv-linker-scFv).
  • the scFv's may be linked to each other via a hinge region (scFv-hinge-scFv).
  • the scFv's are not directly linked to each other but are part of an Ig molecule, i.e. both scFv are attached to the Ig molecule.
  • the TrkB binding molecule further comprises an Ig molecule.
  • the Ig molecule may be a monoclonal antibody, a human monoclonal antibody, a humanized monoclonal antibody, a chimeric antibody, a fragment of an antibody, such as a Fc, Fv, Fab, Fab′, or F(ab′)2 fragment, a single chain antibody, such as a single chain variable fragment (scFv), a Small Modular Immunopharmaceutical (SMIP), a domain antibody, a nanobody, or a diabody.
  • scFv single chain variable fragment
  • SMIP Small Modular Immunopharmaceutical
  • each scFv is fused to the C-terminus of the heavy chain of the Ig molecule.
  • each scFv is fused to the N-terminus of the heavy chain of the Ig molecule or replaces an antigen binding domain at the N-terminus.
  • the Ig molecule is an IgG, F(ab), or F(ab′)2. In a further preferred embodiment the Ig molecule comprises or consists of an Fc region. In a more preferred embodiment the TrkB binding molecule is in the format of an Fc-scFv, scFv-Fc, (scFv′) 2 or scFv-CH 3 .
  • each scFv is fused to the Ig molecule by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids.
  • the scFv's may be connected to each other via a hinge region or a linker only.
  • the two scFv's in the TrkB binding molecule are connected to each other to form the TrkB binding molecule.
  • the scFv's may be fused or otherwise covalently attached directly to each other or they may be linked via a linker, preferably a peptide linker e.g. having a length of about 4 to 20 amino acids and more preferably a flexible peptide linker.
  • the TrkB binding molecule comprises or consists of two scFv's that are connected via a linker.
  • Methods of linking scFv molecules are well known in the art.
  • a small linker sequence of glycine and serine (termed a GS mini-linker) amino acids is used.
  • the number of amino acids in the linker can vary, from 4 (GGGS) (SEQ ID NO: 227), 6 (GGSGGS) (SEQ ID NO: 217), 10 (GGGGSGGGGS) (SEQ ID NO: 218), 15 (GGGGSGGGGSGGGGS) (SEQ ID NO: 219), 20 (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 220) or more.
  • the TrkB binding molecule comprises or consists of two scFv's that are connected via a hinge region only.
  • Such molecule can be generated for example by subjecting a scFv-Fc molecule to pepsin treatment.
  • Other enzymes that may be used are known to the skilled artisan, such as Fabricator, which is a cysteine protease that cleaves IgGs and Fc-fusions at one specific single site just below the hinge region. It will be understood that other methods, substances and/or enzymes can be used equally to generate two scFv's that are connected via a hinge region only. Such methods, substances and/or enzymes may result in hinge regions with different lengths depending on the specific cleavage site.
  • the TrkB binding molecule consists of two scFv's and a hinge region connecting the two scFv's.
  • the TrkB binding molecule is a TrkB agonist.
  • the agonistic TrkB binder is bivalent and the resulting TrkB binding molecule is either bivalent or tetravalent.
  • the agonistic TrkB binder is a bivalent partial agonist.
  • the invention provides for a method for improving the efficacy of a bivalent partial agonistic TrkB binder, wherein the bivalent partial agonistic TrkB binder contains a light chain variable domain (VL) and a heavy chain variable domain (VH),
  • the bivalent partial agonistic TrkB binder is an IgG.
  • the TrkB binding molecule is in the format of an an Fc-scFv, scFv-Fc, (scFv′)2 or scFv-CH 3 .
  • the TrkB binding molecule is bispecific and tetravalent.
  • the TrkB binding molecule is as potent as the IgG molecule it is based upon. In another aspect the TrkB binding molecule is as potent and more efficacious than the IgG molecule it is based upon. In another aspect the TrkB binding molecule is more efficacious than the IgG molecule it is based upon. In another aspect the TrkB binding molecule is more efficacious and slightly less potent than the IgG molecule it is based upon.
  • the invention also relates to an isolated nucleic acid molecule encoding (i) the heavy chain or heavy chain variable domain, and/or (ii) the light chain or light chain variable domain of the TrkB binding molecule according any of the aforementioned embodiments.
  • the invention also relates to a viral vector comprising the isolated nucleic acid molecule of the TrkB binding molecules.
  • the invention further relates to an expression vector comprising a nucleic acid molecule of the TrkB binding molecules.
  • the invention relates further to a host cell transfected with an expression vector comprising a nucleic acid molecule of the TrkB binding molecules.
  • the invention relates to the TrkB binding molecule according to any of the aforementioned embodiments for use in medicine, wherein the use is the treatment of eye or retinal or neurodegenerative diseases.
  • the TrkB binding molecule is used for the treatment and/or prevention of macular degeneration, age-related macular degeneration, wet age-related macular degeneration (wAMD), retinal vein occlusion (RVO), diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, inherited retinal dystrophy, inherited macular dystrophy, myopic degeneration, geographic atrophy, retinal artery occlusions, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane secondary to any retinal diseases, optic neuropathies, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, and traumatic retinopathy, prodromal
  • TrkB binding molecules may be useful for treatment of hearing loss, in particular for cis platin induced hearing loss as well as noise and age-related hearing loss.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TrkB binding molecule according to any of the aforementioned embodiment.
  • a first item relates to a binding molecule comprising at least one antigen binding site that binds specifically to Vascular Endothelial Growth Factor (VEGF) and at least one antigen binding site that binds specifically to Tropomyosin receptor kinase B (TrkB).
  • VEGF Vascular Endothelial Growth Factor
  • TrkB Tropomyosin receptor kinase B
  • the binding molecule is bispecific and tetravalent.
  • the at least one antigen binding site that binds specifically to VEGF is an immunoglobulin (Ig) molecule.
  • the at least one antigen binding site that binds specifically to TrkB is fused to the C- terminus of the heavy chain of the Ig molecule.
  • the at least one antigen binding site that binds specifically to TrkB comprises one or more scFv(s).
  • the at least one antigen binding site that binds specifically to VEGF is an immunoglobulin (Ig) molecule and the at least one antigen binding site that binds specifically to TrkB comprises one or more scFv(s).
  • the one or more scFv(s) have a VL-VH orientation from N-to C-terminus.
  • the one or more scFv(s) is fused to the C- terminus of the heavy chain of the Ig molecule.
  • the Ig molecule is a monoclonal antibody, a human monoclonal antibody, a humanized monoclonal antibody, a chimeric antibody, an (scFv) 2 or a fragment of an antibody such as a F(ab′)2 fragment.
  • the Ig molecule is an IgG or F(ab′)2.
  • the one or more scFv(s) is fused to the Ig molecule by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids.
  • the antigen binding site that binds specifically to TrkB is selected from:
  • the antigen binding site that binds specifically to TrkB is selected from:
  • the binding molecule comprises the amino acid sequence of SEQ ID NO: 222, or the amino acid sequence of SEQ ID NO: 223, or the amino acid sequence of SEQ ID NO: 224, or the amino acid sequence of SEQ ID NO: 225.
  • the antigen binding site that binds specifically to VEGF is selected from the group consisting of antigen binding sites a) to c):
  • the antigen binding site that binds specifically to VEGF is selected from the group consisting of antigen binding sites a) to d):
  • the antigen binding site that binds specifically to VEGF is selected from the group consisting of antigen binding sites a) to d):
  • a second item relates to a binding molecule comprising a light chain comprising an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,
  • a third item relates to a binding molecule comprising: (i) a light chain comprising the amino acid sequence of SEQ ID NO: 41 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 42, or (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 43 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 44, or (iii) a light chain comprising the amino acid sequence of SEQ ID NO: 45 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 46, or (iv) a light chain comprising the amino acid sequence of SEQ ID NO: 47 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 48, or (v) a light chain comprising the amino acid sequence of SEQ ID NO: 49 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 50, or (vi) a light chain comprising the amino acid sequence of SEQ ID NO: 51 and a heavy chain comprising the amino acid sequence of SEQ ID NO
  • a fourth item relates to a TrkB binding molecule comprising or consisting of two scFv's, wherein each scFv binds specifically to TrkB.
  • TrkB binding molecule further comprises an Ig molecule.
  • the Ig molecule is a monoclonal antibody, a human monoclonal antibody, a humanized monoclonal antibody, a chimeric antibody, a fragment of an antibody, such as a Fv, Fab, Fab′, or F(ab′)2 fragment, a single chain antibody, such as a single chain variable fragment (scFv), a Small Modular Immunopharmaceutical (SMIP), a domain antibody, a nanobody, or a diabody.
  • scFv single chain variable fragment
  • SMIP Small Modular Immunopharmaceutical
  • each scFv is fused to the C-terminus of the heavy chain of the Ig molecule.
  • the Ig molecule is an IgG, F(ab), F(ab′)2.
  • the Ig molecule comprises or consists of an Fc region.
  • the TrkB binding molecule is bispecific and tetravalent.
  • a fifth item relates to an scFv binding specifically to TrkB comprising (i) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 213 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 214 or (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 215 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 216.
  • the scFv comprises the amino acid sequence of SEQ ID NO: 222, or the amino acid sequence of SEQ ID NO: 223, or the amino acid sequence of SEQ ID NO: 224, or the amino acid sequence of SEQ ID NO: 225.
  • a sixth item relates to an antibody molecule binding specifically to VEGF comprising:
  • the antibody molecule comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 195 or 199, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 196, 197, 198 or 200.
  • a seventh item relates to an isolated nucleic acid molecule encoding (i) the heavy chain or heavy chain variable domain, and/or (ii) the light chain or light chain variable domain of a binding molecule of any one of the aforementioned items.
  • An eigth item relates to an isolated nucleic acid molecule encoding (i) the heavy chain or heavy chain variable domain, and/or (ii) the light chain or light chain variable domain of a TrkB binding molecule according of any one of the aforementioned items.
  • a ninth item relates to an isolated nucleic acid molecule encoding (i) the heavy chain variable domain, and/or (ii) the light chain variable domain of an scFv according to any one of the aforementioned items.
  • a tenth item relates to an isolated nucleic acid molecule encoding (i) the heavy chain or heavy chain variable domain, and/or (ii) the light chain or light chain variable domain of an antibody molecule according to any one of the aforementioned items.
  • An eleventh item relates to a viral vector comprising the isolated nucleic acid molecule according to seventh, eighth, ninth or tenth item.
  • a twelfth item relates to an expression vector comprising a nucleic acid molecule according to the eleventh item.
  • a thirteenth item relates to a host cell transfected with an expression vector according to the twelfth item.
  • a fourteenth item relates to a method of manufacturing a binding molecule, a TrkB binding molecule, a scFv, or an antibody molecule according to any of the aforementioned items, comprising
  • a fifteenth item relates to the binding molecule, a TrkB binding molecule, the scFv, or the antibody molecule according to any of the aforementioned items for use in medicine.
  • the binding molecule, the TrkB binding molecule, the scFv, or the antibody molecule for the use according to the fifteenth item wherein the use is for the treatment of neural/neuronal eye or retinal diseases.
  • the binding molecule, the TrkB binding molecule, the scFv, or the antibody molecule for the use according to the fifteenth aspect wherein the use is for the treatment of macular degeneration, age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinitis pigmentosa, inherited retinal dystrophy, inherited macular dystrophy, myopic degeneration, geographic atrophy, retinal artery occlusions, endophthalmitis, uveitis, cystoid macular edema, choroidal neovascular membrane secondary to any retinal diseases, optic neuropathies, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, and traumatic retinopathy, prodromal and mild-to-moderate alzheimer's diseases, delaying disease progression of patients with Alzheimer's disease, Huntington's disease, Parkinson's disease, major depressive disorder, schizophrenia, cognitive dysfunction
  • the binding molecule, the TrkB binding molecule, the scFv, or the antibody molecule for the use according to the fifteenth item wherein the use is for the treatment of macular degeneration and in particular wet age-related macular degeneration (wAMD). Further relating to the fifteenth item, the binding molecule, the TrkB binding molecule, the scFv, or the antibody molecule for the use according to the fifteenth item, wherein the use is for the treatment of retinal vein occlusion (RVO). Further relating to the fifteenth item, the binding molecule, the TrkB binding molecule, the scFv, or the antibody molecule for the use according to the fifteenth item, wherein the use is for the treatment and/or prevention of geographic atrophy.
  • RVO retinal vein occlusion
  • a sixteenth item relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the binding molecule, the TrkB binding molecule, the scFv, or the antibody molecule according to any of the aforementioned items.
  • a seventeemth item relates to a method of treating or preventing an eye or retinal or neurodegenerative disease comprising administering to a patient in need thereof a therapeutically effective amount of the binding molecule, the TrkB binding molecule, the scFv, or the antibody molecule according to any of the aforementioned items.
  • An eightteenth item relates to the use of the binding molecule, the TrkB binding molecule, the scFv or the antibody molecule according to any of the aforementioned items for preparing a pharmaceutical composition for treating or preventing an eye or retinal or neurodegenerative diseases.
  • CHO cells expressing the human TrkB receptor (ThermoFisher Scientific, #K1491), and customised CHO cells expressing the human TrkA or TrkC receptor were cultured in DMEM (Lonza, #BE12-604F) supplemented with 10% fetal bovine serum, glutamax, non-essential amino acids, 20 mM HEPES, 5 ⁇ g/mL blasticidin and 200 ⁇ g/mL zeocin.
  • Customized CHO cells expressing cyno, rabbit or rat TrkB receptor were cultured in a 1:1 mixture of Hams F12 (Lonza #BE12-615F) and DMEM (Lonza #BE12-604F) with 5% fetal bovine serum, 8 mM Glutamine, 0.5 mg/mL G418.
  • Customised CHO cells expressing mouse TrkB receptor were cultured DMEM (Lonza #BE12-604F) supplemented with 10% fetal bovine serum, glutamax, 10 mM HEPES and 0.8 mg/mL G418.
  • TrkA/B/C and ERK1/2 Phosphorylation in CHO Cells Expressing Human, Cyno, Rabbit, Rat, or MouseTrk Receptors
  • CHO cells expressing the respective Trk receptor were seeded in each cavity of a 384 well clear tissue culture plate (BD Falcon, # 353963) and incubated in a humidified incubator at 37° C. and 5% CO 2 . Twenty-four hours after seeding, the supernatant of the cells was replaced with room-temperature starvation medium (DMEM with 0.1% BSA (Sigma, #A-3059) but without other supplements).
  • DMEM room-temperature starvation medium
  • starvation medium with increasing concentrations of human BDNF (R&D #248-BD or Bachem #H-5594), human NGF (Biovision #4303R-20), human NT-3 (Sigma #N1905), agonistic antibodies, or isotype controls was added in triplicate for 45 minutes at room temperature to stimulate TrkB and ERK1/2 phosphorylation. Starvation medium alone served as control.
  • BDNF or the agonistic antibodies were pre-incubated for one hour without or with 2, 10, 50, or 200 ng/mL human VEGF (R&D Systems #293-VE-050), prior to stimulation of the cells. In these experiments, incubation with human VEGF alone served as control.
  • TrkB a agonistic TrkB antibody limits the BDNF-induced phosphorylation of TrkB and/or downstream ERK1/2 phosphorylation
  • CHO cells expressing the human TrkB receptor were incubated with growing concentrations of the antibody without or with a constant concentration of 0.3 nM, 1 nM or 3 nM BDNF.
  • TrkA phosphorylation at Y680/681, TrkB phosphorylation at Y706/707, or TrkC phosphorylation at Y709/710 using a commercially available assay (Perkin Elmer #ALSU-PTRKAB-A10K), according to the manufacturer's instructions.
  • Quantification of ERK1/2 phosphorylation at T202/Y204 (ERK1) and T185/Y187 (ERK2) was done similarly using another commercially available assay (Perkin Elmer #TGRES10K or ALSU-PERK-A10K), according to the manufacturer's instructions.
  • TkrB Extracellular Domain TrkB-ECD
  • TrkB activation was assessed by measuring TrkB phosphorylation on Y706/707 as outlined above. Data represent mean+/ ⁇ SEM.
  • CHO cells expressing human TrkB receptor were seeded in each cavity of a 96 well black clear bottom tissue culture plate (PerkinElmer, #6055300) and incubated in a humidified incubator at 37° C. and 5% CO 2 .
  • the supernatant of the CHO/hTrkB cells was replaced with starvation medium (DMEM with 0.1% BSA (Sigma, #A-3059) and 20 mM Hepes (Lonza, # BE17-737F)) and the cells were incubate for 30 minutes at 37° C.
  • BDNF R&D #248-BD or Bachem #H-5594
  • agonistic TrkB antibodies alone, or a combination of 1 nM BDNF with increasing concentrations of the agonistic antibodies in starvation medium for 50 minutes at 37° C.
  • HCS-Cellmask® green stain (ThermoFisher Scientific, #H32714) in PBS/0.05% Tween-20.
  • Cell surface receptors were imaged on a PerkinElmer Opera PhenixTM High Content Screening System equipped with a 20 ⁇ water objective and analyzed with PerkinElmer Harmony High-Content Imaging and Analysis Software. Data are either presented as heatmap, with dark and light fields of the heatmap representing high and low percentage of cells above fluorescence threshold, respectively, or as diagram representing the percent of cells with surface TrkB staining intensity above threshold. In the latter case, data were prepared for presentation with GraphPad Prism (version 8), including the raw data (mean ⁇ SEM) and a non-linear regression (log(agonist) vs.
  • HRMEC Human retinal microvascular endothelial cells
  • HRMEC Human retinal microvascular endothelial cells
  • gelatine- (Millipore #ES-006B) coated plates in endothelial cell basal medium (Promocell #C-22210) with supplements (Promocell #C-39210) and 10 U/mL penicillin/streptomycin, each.
  • 12000 cells were seeded in each cavity of a 96 well clear tissue culture plate using normal growth medium and incubated in a humidified incubator at 37° C. and 5% CO 2 .
  • the medium was replaced with starvation medium (Promocell #C-22210 supplemented with 0.1% BSA (Sigma, #A-3059) and 10 U/mL penicillin/streptomycin) and cells were starved for 20 hours in a humidified incubator at 37° C. and 5% CO 2 .
  • starvation medium Promocell #C-22210 supplemented with 0.1% BSA (Sigma, #A-3059) and 10 U/mL penicillin/streptomycin
  • 50 ng/mL human VEGF R&D Systems #293-VE-050
  • EYLEA® aflibercept
  • VEGF-antibody/EYLEA® aflibercept
  • Starvation medium alone served as control.
  • Cells were lysed in lysis buffer (Perkin Elmer #ALSU-PVGFR-A500) for 10 minutes at room temperature and then incubated for additional 10 minutes on ice.
  • VEGFR2 phosphorylation at Y1175 Perkin Elmer #ALSU-PVGFR-A500 or #ALSU-PVGFR-A10K
  • VEGFR2 phosphorylation at Y1214 Perkin Elmer #ALSU-PVGFR-C500
  • VEGFR2 phosphorylation at Y951 Perkin Elmer #ALSU-PVGFR-B500
  • ERK1/2 phosphorylation at T202/Y204 (ERK1) and T185/Y187 (ERK2) Perkin Elmer #TGRES10K or ALSU-PERK-A10K
  • Src phosphorylation at Y419 Perkin Elmer #ALSU-PSRC-A10K
  • p38-MAPK phosphorylation at Thr180/Tyr182 Perkin Elmer #ALSU-PP38-B500.
  • TrkB-binding inhibition of VEGF-induced VEGFR2 phosphorylation was assessed in starved HRMEC in the absence or presence of the TrkB-ECD.
  • Prior to stimulation growing concentrations of the respective Doppelmab were incubated with 100 nM TrkB-ECD (R&D Systems #1494-TB) in starvation medium at room temperature for one hour, followed by incubation with 50 ng/mL human VEGF for another hour.
  • HRMEC were incubated with (i) starvation medium alone, (ii) 50 ng/mL human VEGF, (iii) pre-formed complexes (one hour at room temperature) of 50 ng/mL human VEGF with growing concentrations of TrkB-ECD, (iv) pre-formed complexes (one hour at room temperature) of 50 ng/mL human VEGF with growing concentrations of the respective Doppelmab, and (v) growing concentrations of the TrkB-ECD alone.
  • VEGF-A scavenging was assessed by measuring VEGF receptor 2 (VEGFR2) phosphorylation at Y1175 as outlined above.
  • VEGF human VEGF
  • R&D Systems #293-VE-050 human VEGF
  • EYLEA® aflibercept
  • VEGF vascular endothelial growth factor
  • Starvation medium alone served as control (basal proliferation).
  • Cell proliferation was assessed by automated, phase contrast image-based quantification of the total HRMEC nuclear areas (Essen Bioscience, IncuCyte S3), which was considered to be proportional to the HRMEC numbers. Four images per well were recorded with a 10 ⁇ objective every four hours for a total period of 96 hours.
  • the area between each growth curve and the basal growth curve (proliferation in starvation medium) was plotted against the decadic logarithm of the respective compound concentration.
  • Data were prepared for presentation with GraphPad Prism (version 8), including the raw data (mean ⁇ SEM) and a non-linear regression (log(agonist) vs. response (three parameters)) if applicable.
  • HRMEC-based spheroid assay HRMEC were resuspended in normal growth medium containing 20% MethocelTM modified cellulose media (1.2% methylcellulose and 10% fetal bovine serum in endothelial basal medium) and 25 ⁇ l drops containing 500 HRMEC were applied on square petri dishes. The plates were turned upside down to cultivate the cells in hanging drops, which allows the spontaneous formation of spheroids.
  • spheroids were harvested and embedded in a MethocelTM modified cellulose media—collagen mixture (80% Methocoel and 20% FCS 1:1 mixed with 3 mg/mL rat tail collagen I (Corning #354236) in M199 medium) in 48 well plates and incubated in a humidified incubator at 37° C. and 5% CO 2 for 30 minutes to accomplish collagen polymerization.
  • human VEGF R&D Systems #293-VE-050
  • EYLEA® aflibercept
  • VEGF-antibody/EYLEA® aflibercept
  • Basal endothelial growth medium supplemented with 2% fetal bovine serum without VEGF served as control.
  • cells were extensively washed and stained over night with 2 pg/mL HCS-CellmaskTM green stain (ThermoFisher Scientific, #H32714) in PBS containing 0.2% Triton X-100.
  • Spheroids were analyzed on a ZEISS LSM 780 confocal microscope.
  • the three-dimensional sprouting of HRMEC was quantified from maximum projection images of Z-stacks either manually and expressed as accumulated sprout length per spheroid (mm), or semi-automated with the Zeiss ZEN-imaging software and expressed as spheroid perimeter (pixel). Data were prepared for presentation with GraphPad Prism (version 8) and represent mean ⁇ SEM.
  • Electroretinography is a non-invasive electrophysiological technique to assess light- induced electrical activity of different retinal neurons,and allows for quantifying different aspects of retinal function such as dim light or color vision.
  • ERGs were measured as the potential change between a corneal and a reference electrode using the Espion E3 ERG recording system (Diagnosys LLC). Prior to ERG recordings, animals were dark adapted for at least 2 h, and anesthetized by i.p. injection of ketamine (Ketanest®, ca. 100 mg/kg) and xylazin (RompunTM, ca. 7 mg/kg). The animals were placed on a heated stage to maintain the body temperature constant at 37° C.
  • Pupils were dilated with topical Tropicamid and 10% phenylephrine.
  • a drop of MethocelTM 2% solution (Omni Vision) was placed on the cornea to prevent corneas eyes from drying during recordings. Recordings were performed simultaneously from both eyes with gold loop electrodes.
  • the reference electrode was a toothless alligator clip wetted with MethocelTM media and attached to the cheek of the animal. For electrical grounding, a clip was attached to the tail of the animal. ERG signals were sampled at 1 kHz and recorded with 0.15 Hz low-frequency and 500 Hz high-frequency cutoffs.
  • the light stimuli consisted of full-field flashes (duration ⁇ 4 ms) delivered by a set of light-emitting diodes or a Xenon light bulb (for flashes ⁇ 1 cd ⁇ s/m 2 ). All flashes were produced by a Ganzfeld stimulator (ColorDome; Diagnosys), either in darkness or on background light.
  • ERG responses were first recorded from dark-adapted animals (for isolating rod-driven responses), followed by recordings from animals adapted to red background light (50 cd/m 2 , for isolating UV cone-driven ERG responses) and finally adapted to green-blue background light (25.5 cd/m 2 , for isolating M cone-driven ERG responses).
  • responses were evoked by a series of flashes ranging from 1 ⁇ 10 ⁇ 5 to 100 cd ⁇ s/m 2 .
  • For the flashes between 1 ⁇ 10 ⁇ 4 up to 0.05 cd ⁇ s/m 2 responses of 10 trials were averaged, for the flash of 0.1 cd ⁇ s/m 2 , responses of 8 trials and for the flash of 1 cd ⁇ s/m 2 , 5 trials were averaged.
  • the last flashes of 10 cd ⁇ s/m 2 we recorded responses of 3 trials and for the final flash 100 cd ⁇ s/m 2 , a single flash was recorded.
  • Intervals between individual flashes were chosen to ensure that the retina recovered completely from each flash (no indications of flash-induced reduction of response amplitudes or shortening of implicit times). Based on these criteria, the inter-flash intervals were 2 s for the 1 ⁇ 10 ⁇ 5 and 3 ⁇ 10 ⁇ 5 cd ⁇ s/m 2 flashes, 5 s for flashes between 1 ⁇ 10 ⁇ 4 up to 0.05 cd ⁇ s/m 2 , 10 s for the flash of 0.1 cd ⁇ s/m 2 , and 20 s for the flash of 1 cd ⁇ s/m 2 . After the single flashes of 10 cd ⁇ s/m 2 and 100 cd ⁇ s/m 2 , there was a recovery time of 30 s and 120 s, respectively.
  • UV cone-driven and M cone-driven responses Animal were light-adapted for 2 min first to a red background light and afterwards to a green background light. Light responses were evoked by UV flashes of 0.02, 0.04, 0.08, 0.17, 0.35, 0.83, 1.66, 2.90, and 4.15 ⁇ W/m 2 and respectively by M cone flashes from 0.1 up to 110 cd ⁇ s/m 2 flashes. All responses of 10 trials were averaged with inter-flash intervals of 3 s.
  • BN rats Male Brown Norway rats (BN rats) were obtained from Charles River (Germany). Hyperglycemia was induced by i.p. injections of STZ (65 mg/kg body weight). Non- or poorly responding animals were not included into the study, i.e. animals with blood glucose concentrations ⁇ 20 mM at day 7 post STZ application. Body weight and blood glucose levels were monitored regularly. STZ was administered ca. 3 weeks before intravitreal dosing.
  • rats were anesthetized with 2.5-3% isoflurane (Forene; Abbvie).
  • 5 ⁇ L were injected via a 34-gauge needle (fitted on a 10 ⁇ l Hamilton glass syringe) into the vitreous just behind the limbus in each eye.
  • ERGs were measured as the potential change between a corneal and a reference electrode using the Espion E3 ERG recording system (Diagnosys LLC).
  • ERG data were processed and analyzed using the MATLABTM software (version R2014a; MathWorks). In our case we sorted the data with an individual macro and prepared a file for our Matlab routines.
  • the b-wave amplitude was calculated from the bottom of the a-wave response to the peak of the b-wave peak.
  • the b-wave implicit time was measured as the time after the flash stimulus needed to reach the peak of the b-wave.
  • the amplitudes of b-waves as a function of the stimulus intensity were fitted by using a least-square fitting procedure (GraphPad Prism, Version 6.01 and later on an upper Version of GraphPad Prism).
  • the a-wave amplitude was calculated from baseline (zero line) to the negative a-wave response.
  • FIG. 1 A first figure.
  • the present inventors have developed binding molecules that bind VEGF and TrkB and act as VEGF antagonists and TrkB agonists respectively.
  • the molecular design used has an IgG antibody (termed the “master antibody”) which has specificity for one target antigen, with scFvs of a different specificity coupled to the C terminus of the heavy chain.
  • a schematic of the design is shown in FIG. 1 .
  • the binding molecule is bispecific and tetravalent.
  • the bispecific molecule contains flexible peptide sequences between the variable heavy (VH) and variable light (VL) domains of the scFv, and the scFv domains are linked to the master IgG antibody via further series of linkers.
  • the scFv is oriented such that the VL domain forms the “N-terminal” end of the scFv and is thus fused to the C-terminus of the heavy chain of the master antibody while the VH forms the C-terminus of the scFv and indeed the whole heavy chain polypeptide.
  • this “N-VL-VH-C” structure can be reversed, i.e. “N-VH-VL-C”.
  • FIG. 1 A first figure.
  • Immunoglobulin (Ig) VH and VL genes were obtained from different VEGF and TrkB binders and formatted into the bispecific molecules of the invention.
  • three different VEGF binding domains were obtained from the individual VEGF binders termed B20, G6, and Ranibizumab.
  • the binding domains were obtained from the individual TrkB binder termed C2 (WO2010086828).
  • VL and VH genes encoding the Fab ( 2) part were then formatted into the bispecific format outlined in Example 1.
  • the VH genes were cloned into pTT5 expression vector as an in-frame fusion at the 5′ end of a gene encoding human Ig ⁇ .
  • a gene encoding the scFv binder was cloned in frame at the 3′ end of the same Ig ⁇ encoding segment.
  • the VL genes were cloned into pTT5 expression vector as an in-frame fusion with a gene encoding human IgG kappa light chain.
  • In-Fusion® HD Cloning Kit (Clonetech, U.S.A.) was used in the above procedure for directional cloning of VH and VL genes.
  • PCR primers for VL/VH with 15 bp extensions complementary to the ends of the linearized vector were synthesized.
  • PCR was performed using the manufacturer's standard protocol and the amplicons were purified or treated with Cloning Enhancer, then cloned into the appropriate vector.
  • E. coli were then transformed according to manufacturer's instructions (Clonetech, U.S.A.). DNA mini-preps were sequenced.
  • Each expression vector contains eukaryotic promoter elements for the chain-encoding gene, the gene encoding the signal sequence and the heavy or light chain, an expression cassette for a prokaryotic selection marker gene such as ampicillin, and an origin of replication. These DNA plasmids were propagated in ampicillin resistant E. coli colonies and purified.
  • the expression vectors were transfected into CHO-E cells.
  • Transfected CHO-E cells growing in suspension in serum-free media were cultivated in shake flasks under agitation at 140 rpm, 37° C. and 5% CO 2 and kept at conditions of exponential growth.
  • cells were chemically transfected with 1 mg of light chain plasmid and 0.5 mg of heavy chain plasmid. They were then seeded at 1 to 2 ⁇ 10 6 cells/ml in 1 L of Gibco® FreeStyleTM CHO expression medium (LifeTechnologies, NY, US). Cells were then incubated under orbital shaking for 10 to 12 days with one-time feeding of 150 ml commercial feed solution to allow expression of the proteins.
  • Binding molecule titers in the cell culture supernatants were determined using an Octet® instrument (Pall ForteBio, CA, US) and protA biosensor tips according to manufacturer's instructions.
  • Recombinant binding molecules were purified from culture supernatant by Protein A affinity chromatography using MabSelectTM SuReTM (Cytiva) followed by either size exclusion chromatography (Superdex® 200 column, Cytiva) or cation exchange chromatography (POROSTM50 HS, ThermoFisher) and stored in 60 mM NaOAc buffer, 100 mM NaCI (pH 5.0). Purity and degree of heterogeneity of the samples were assessed by mass spectrometry and analytical size exclusion chromatography. All samples were confirmed to have a monomer content of ⁇ 90% and contain ⁇ 10% impurities prior to functional testing.
  • VEGF binding part was formatted as scFv(2) and different VEGF binders were evaluated, e.g.
  • TrkB activation was assessed by measuring (A) TrkB phosphorylation on Y706/707 or (B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), respectively, downstream of TrkB. The lowest compound concentration is solvent alone.
  • TrkB activation by DMabs was virtually identical to the parental C2 molecule. Also, the DMabs showed the same properties as the parental C2 molecule and acted as partial TrkB agonists having an efficacy of TrkB activation at around -40-50% of BDNF (pTrkB). Although, the DMab did incorporate the entire (Fab) 2 portion of the parental C2 antibody without changing the sequence or the orientation/layout this was a very encouraging result, as it showed that formatting into this specific format did not negatively affect the ability of the TrkB binder to activate TrkB and TrkB downstream signalling. As expected the isotype control did not activate TrkB.
  • TrkB activation was assessed by measuring TrkB phosphorylation on Y706/707. The lowest compound concentration is solvent alone. Data represent mean+/ ⁇ SEM.
  • TrkB activation by DMabs was virtually identical to the parental C2 molecule and activation was comparable to activation of human TrkB receptor. Also in this assay the DMabs showed the same properties as the parental C2 molecule and acted as partial TrkB agonists having an efficacy of TrkB activation at around ⁇ 40-50% of BDNF (pTrkB).
  • TrkA human TrkA
  • TrkB human TrkB
  • TrkC human TrkC
  • TrkB TrkB internalization was assessed by immunofluorescence staining of surface TrkB receptors without permeabilization of the cells, followed by confocal microscopy analysis. Dark and light fields of the heatmap represent high and low percentage of cells above fluorescence threshold, respectively.
  • the antibody C2 and TPP-11736 decreased the BDNF-induced internalization of the TrkB receptor (lanes 3-5 and 6-8; note that heatmap fields are getting darker from bottom to top).
  • TrkB activation was assessed by measuring ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), downstream of TrkB. Incubation with growing concentrations of human VEGF-A without antibody served as control. The lowest compound concentration is solvent alone. Data represent the mean. For clarity, error bars are omitted.
  • TrkB activation in a rat model of diabetes-induced retinal neurodegeneration using IVT injection of an agonistic TrkB tool antibody (C2) as well as Doppelmab TPP-11736.
  • Animals were treated with STZ to induce hyperglycemia.
  • the retinal function was assessed by electroretinography (ERG) before and after treatment. Diabetes induction led to delayed implicit times within 3 weeks after STZ treatment.
  • animals were intravitreally dosed with an isotype control antibody (anti-TNP) or C2 (19 ⁇ g/5 ⁇ l, each), or an equimolar amount of TPP-11736 (25 ⁇ g/5 ⁇ l). After two weeks of treatment, ERG recordings were repeated and analyzed.
  • Rod-driven B-wave implicit time delays immediately before and two weeks after intravitreal application of the antibodies are shown in FIG. 7 ; mean+/ ⁇ SEM; n.s. p>0.05, non-significant; **p ⁇ 0.01; ***p ⁇ 0.001; one-way Anova with Tukey multi-comparison test.
  • HRMECs Human retinal microvascular endothelial cells
  • VEGF-A scavenging was assessed by measuring VEGF receptor 2 (VEGFR2) phosphorylation on Y1175.
  • VEGFR2 VEGF receptor 2
  • A Comparison of Doppelmabs TPP-11735, -736, -737 and -738.
  • B Comparison of TPP-11736 and TPP-11738 with EYLEA® (aflibercept). Non-stimulated cells (Basal) and 50 ng/ml human VEGF without antibody treatment served as control . Data represent mean+/ ⁇ SEM.
  • TPP-11737 vs. TPP-11738 These molecules had both the G6 anti-VEGF molecule as scFv. 737 is connected in a VH-VL orientation, whereas 738 has the VL-VH orientation. It turned out that the VL-VH orientation worked a lot better based on the G6 anti-VEGF antibody.
  • TPP-11735 vs. TPP-11736 These molecules had both the B20 anti-VEGF molecule as scFv. For the B20 VEGF antibody in this assay, there was not much of a performance difference between the two orientations.
  • TPP-11736 vs. TPP-11738 These molecules had either the B20 (VL-VH) or the G6 (VL-VH) anti-VEGF molecule as scFv. Both binding molecules showed similar potency (1050 ⁇ 4 nM), but TPP-11736 was more efficacious.
  • HRMECs Human retinal microvascular endothelial cells
  • VEGF-A scavenging was assessed by TrkB activation was assessed by measuring ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2).
  • ERK1 T202/Y204
  • ERK2 T185/Y187
  • A Comparison of Doppelmabs TPP-11735, -736, -737 and -738.
  • B Comparison of TPP-11736, -738 with EYLEA® (aflibercept).
  • Non-stimulated cells (Basal) and 50 ng/ml human VEGF without antibody treatment served as control. Data represent mean+/ ⁇ SEM.
  • TPP-11737 vs. TPP-11738 These molecules had both the G6 anti-VEGF molecule as scFv. 737 is connected in a VH-VL orientation, whereas 738 has the VL-VH orientation. It turned out that the VL-VH orientation worked a lot better based on the G6 anti-VEGF antibody.
  • TPP-11735 vs. TPP-11736 These molecules had both the B20 anti-VEGF molecule as scFv. For the B20 VEGF antibody in this assay, there was not much of a performance difference between the two orientations.
  • TPP-11736 vs. TPP-11738 These molecules had either the B20 (VL-VH) or the G6 (VL-VH) anti-VEGF molecule as scFv. Both binding molecules showed similar potency (IC 50 ⁇ 4 nM), but TPP-11736 was more efficacious.
  • HRMECs Human retinal microvascular endothelial cells
  • HRMECs Human retinal microvascular endothelial cells
  • 50 ng/mL human VEGF with or without pre-incubation with growing concentrations of TPP-11736 or EYLEA® (aflibercept).
  • VEGF-A scavenging was assessed by measuring p38 MAPK phosphorylation on T180/Y182.
  • Non-stimulated cells (Basal) and 50 ng/ml human VEGF without antibody treatment served as control. Data represent mean+/ ⁇ SEM.
  • HRMECs Human retinal microvascular endothelial cells
  • EYLEA® aflibercept
  • Molecule concentrations are given in mol/L.
  • TPP-11737 vs. TPP-11738 These molecules have both the G6 anti-VEGF molecule as scFv. TPP-11737 is connected in a VH-VL orientation, whereas TPP-11738 has a VL-VH orientation. Obviously, the VL-VH orientation worked a lot better in the case of G6. There is virtually no effect of TPP-11737.
  • TPP-11735 VH-VL vs.
  • TPP-11736 VL-VH: These molecules have both the B20 anti-VEGF molecule as scFv. Again, the VL-VH orientation performed better.
  • EYLEA® (aflibercept) was more efficacious than TPP-11736.
  • TrkB activation by DMabs was virtually identical to the parental C2 molecule. This was not unexpected since the entire (Fab) 2 portion of the C2 TrkB binder was incorporated into the DMabs without changing the sequence or the orientation/layout. Also, all Doppelmabs (and C2) were only partial TrkB agonists.
  • VL-VH VEGF binders and B20
  • VL-VH was more efficacious than G6
  • both Doppelmabs (B20 and G6) were inferior to EYLEA® (aflibercept) in vitro.
  • TrkB activation was assessed by measuring TrkB phosphorylation on Y706/707. The lowest compound concentration was solvent alone. Data represent mean. For clarity, error bars were omitted.
  • TrkB activation by DMabs TPP-16061 to 16064 was virtually identical to the parental C2 molecule and TPP-11736. TrkB activation appeared to be largely independent of the linker variations between the Fc and the scFv (anti-VEGF) portion of the protein. Although this was expected - because the linkers were located distant to the TrkB (Fab) 2 fragment between the Fc and the scFv (anti-VEGF) portion of the protein—this was nonetheless a good confirmation that different linkers can be utilized without impacting the activity of the binding molecule. Finally, all displayed binding molecules only showed partial TrkB agonist activity.
  • TrkB activation was assessed by measuring ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2).
  • TrkB activation by DMabs TPP-16061 and 16062 was virtually identical to TPP-11736. Also here it showed that TrkB activation was largely independent of the linker variations and the displayed binding molecules showed only partial TrkB agonist activity.
  • Human retinal microvascular endothelial cells were starved and then incubated with 50 ng/mL human VEGF with or without pre-incubation with growing concentrations of the indicated antibodies; TPP-11736 (B20, scFv, 20L3, VL-VH), or four Doppelmabs with different linkers TPP-16061 (B20, scFv, 20L1, VL-VH), TPP-16062 (B20, scFv, 15L1, VL-VH), TPP-16063 (B20, scFv, 10L1, VL-VH), TPP-16064 (B20, scFv, 6GS, VL-VH).
  • TPP-11736 B20, scFv, 20L3, VL-VH
  • TPP-16062 B20, scFv, 15L1, VL-VH
  • TPP-16063 B20, scFv, 10L1, VL-VH
  • VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y1175 or (B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), respectively.
  • A VEGF receptor 2
  • B ERK1/2 phosphorylation on T202/Y204
  • ERK2 ERK1
  • T185/Y187 ERK2
  • linkers had virtually no impact on the potency or efficacy of VEGF-scavenging. This was somewhat unexpected—given that the linkers were located in proximity to the VEGF binding sites—but further confirmed that different linkers can be utilized without impacting the activity of the binding molecule.
  • HRMECs Human retinal microvascular endothelial cells
  • 10 ng/mL human VEGF with or without pre-incubation with 0.5 nM Doppelmabs or 1 nM EYLEA® (aflibercept).
  • EYLEA® (aflibercept) was used at 1 nM because EYLEA® (Aflibercept) Was supposed to be a mono-valent molecule, whereas the Doppelmabs are bi-valent. Under these conditions, EYLEA® (aflibercept) performed better in inhibition of VEGF-induced proliferation of HRMEC.
  • Spheroids of human retinal microvascular endothelial cells were embedded in a collagen matrix. Endothelial sprouting was induced for 24 hours by incubation with 50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of the indicated Doppelmabs or 5 nM EYLEA® (aflibercept) for 24 hours. Endothelial sprouting was assessed by confocal microscopy and displayed spheroid perimeter obtained from maximum projections of Z-stacks. Non-stimulated cells served as control (Basal). Data represent mean+/ ⁇ SEM. n.s. p>0.05 non-significant vs. 50 ng/mL hVEGF+2.5 nM TPP-11736.
  • EYLEA® (aflibercept) was used at 1 nM because EYLEA® (aflibercept) is supposed to be a mono-valent molecule, whereas the Doppelmabs are bi-valent. Under these conditions, EYLEA® (aflibercept) performed better in inhibition of VEGF-induced proliferation of HRMEC.
  • TrkB activation No difference/improvement compared to the parental molecule TPP-11736 was observed.
  • VEGF-scavenging No difference/improvement compared to the parental molecule TPP-11736.
  • the inventors set out to compare the VEGF-scavenging of TPP-11736 (B20 as scFv) with TPP-13788 (B20 IgG) to test if re-formatting of the B20 as a Fab would improve VEGF-scavenging.
  • VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y1175, (B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), or (C) p38 MAPK phosphorylation on T180/Y182. 50 ng/ml human VEGF without molecule treatment served as control. Data represent mean+/ ⁇ SEM. The Table 4 below reports the corresponding best-fit IC 50 values (non-linear regression; log(agonist) vs. response (three parameters).
  • TPP-11736 TPP-13788 IC 50 (VEGFR2 phosphorylation) 2.6 nM 1.0 nM IC 50 (ERK1/2 phosphorylation) 9.1 nM 3.0 nM IC 50 (p38 MAPK phosphorylation) 7.7 nM 2.4 nM
  • the tested B20 IgG TPP-13788 displays an approximately 3-fold increased VEGF-A scavenging potency vs. TPP-11736
  • Spheroids of human retinal microvascular endothelial cells were embedded in a collagen matrix. Endothelial sprouting was induced for 24 hours by incubation with 50 ng/mL human VEGF without or with pre-incubation with 2.5 nM of Doppelmab TPP-11736 or 2.5 nM TPP-13788 (B20 IgG). Endothelial sprouting was assessed by confocal microscopy and displayed spheroid perimeter obtained from maximum projections of Z-stacks. Non-stimulated cells served as control (Basal). Data represent mean+/ ⁇ SEM. n.s. p>0.05 non-significant vs. 50 ng/mL hVEGF+2.5 nM TPP-11736.
  • TPP-13788 B20 IgG mediated inhibition of VEGF-induced sprouting was not better than that of TPP-11736.
  • TrkB activation was assessed by measuring (A) TrkB phosphorylation on Y706/707, (B & C) ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), respectively. The lowest compound concentration was solvent alone. Data represent mean+/ ⁇ SEM.
  • TrkB activation by DMabs TPP-14936 and TPP-14937 was virtually identical to TPP-11736. This was expected since the TrkB-binding component of the binding molecules was always the C2 as Fab fragment.
  • TrkB activation was assessed by measuring TrkB phosphorylation on Y706/707. The lowest compound concentration was solvent alone. Data represent mean+/ ⁇ SEM.
  • TrkB activation by DMab TPP-14936 was virtually identical to TPP-11736. Again, this is was expected since the TrkB-binding component of the two molecules is C2 as Fab fragment in both cases.
  • HRMECs Human retinal microvascular endothelial cells
  • TPP-11736 B20, scFv, 20L3, VL-VH
  • TPP-14936 Ranibizumab, scFv, 20L3, VH-VL
  • TPP-14937 TPP-14937
  • VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y1175, (B) ERK1/2 phosphorylation on T202/Y204 and T185/Y187, or (C) Src phosphorylation on Y419. 50 ng/ml human VEGF without antibody treatment served as control. Data represent mean+/ ⁇ SEM. The Table 5 below reports the corresponding best-fit IC 50 values and the absolute efficacy values (bottom plateau), respectively (non-linear regression; log(agonist) vs. response (three parameters).
  • Potency of VEGF-scavenging (IC 50 ) is similar between TPP-11736 and TPP-14936/TPP-14937.
  • TPP-14936 Potency of VEGF-scavenging (IC 50 ) by TPP-14936 is somewhat better than TPP-14937 ⁇ VH-VL better than VL-VH orientation of Ranibizumab scFv.
  • TPP-14936 and TPP-14937 were more efficacious than TPP-11736 (bottom values were smaller).
  • Spheroids of human retinal microvascular endothelial cells were embedded in a collagen matrix. Endothelial sprouting was induced for 24 hours by incubation with 50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of Doppelmab TPP-11736 (B20, scFv, 20L3, VL-VH), TPP-14936 (Ranibizumab, scFv, 20L3, VH-VL) or TPP-14937 (Ranibizumab, scFv, 20L3, VL-VH).
  • Endothelial sprouting was assessed by confocal microscopy and displayed spheroid perimeter obtained from maximum projections of Z-stacks. Non-stimulated cells served as control (Basal). Data represent mean+/ ⁇ SEM. n.s. p>0.05 non-significant, ****p ⁇ 0.0001.
  • TPP-14936 and TPP-14937 were similar in the VEGFR2/ERK1_2/Src phosphorylation assay, there were dramatic differences in the inhibition of VEGF-induced sprouting. No significant difference between TPP-11736 and TPP-14937, however, inhibition of VEGF-induced HRMEC sprouting by TPP-14936 was a lot more efficacious than TPP-14937 or TPP-11736.
  • VH-VL orientation of the anti-VEGF scFv is critical for the performance in this assay: VH-VL is much better than VL-VH. Interestingly, the VL-VH orientation was better than VH-VL in case of TPP-11735 vs. 736 (B20) and also in case of TPP-11737 vs. 738 (G6).
  • HRMECs Human retinal microvascular endothelial cells
  • 10 ng/mL human VEGF with or without pre-incubation with growing concentrations of the indicated binding molecules or EYLEA® (aflibercept).
  • Binding molecule/EYLEA® (aflibercept) concentrations are given in mol/L.
  • TPP-14936 and TPP-14937 displayed a better potency and especially efficacy of inhibition of VEGF-induced HRMEC proliferation compared to TPP-11736.
  • TPP-14936 was more potent than TPP-14937.
  • TPP-14936 and TPP-14937 were more efficacious than EYLEA® (aflibercept). Both Doppelmabs reduced the proliferation even below baseline. In contrast to EYLEA® (aflibercept), they showed full inhibition of VEGF-induced proliferation. However, EYLEA® (aflibercept) was still more potent than TPP-14936 and 14937.
  • TrkB activation No difference/improvement compared to the TPP-11736 were observed. Since both binding molecules had the C2 binder as Fab fragment this was kind of expected.
  • VEGF-scavenging Potency of VEGF-scavenging (IC 50 ) was similar between TPP-11736 and TPP-14936/TPP-14937. VEGF scavenging by TPP-14936 and TPP-14937 was more efficacious than TPP-11736 (bottom values).
  • TPP-14936 and TPP-14937 displayed a better potency and especially efficacy of inhibition of VEGF-induced HRMEC proliferation compared to TPP-11736.
  • TPP-14936 was more potent than TPP-14937 and TPP-14936 and TPP-14937 were both more efficacious than EYLEA® (aflibercept).
  • TrkB ligand BDNF TPP-11736
  • TPP-14938 C2 as scFv, 20L3, VH-VL
  • TPP-14939 C2 as scFv, 20L3, VL-VH
  • TPP-14940 C2 as scFv, 20L3, VH-VL
  • TPP-14941 C2 as scFv, 20L3, VL-VH
  • TrkB activation was assessed by (A) measuring TrkB phosphorylation on Y706/707 or by (B) measuring ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), respectively. The lowest compound concentration was solvent alone. Data represent mean+/ ⁇ SEM.
  • TPP-11736 contained the C2 CDRs as Fabs.
  • the four Doppelmabs TPP-14938, TPP-14939, TPP-14940 and TPP-14941 contained C2 as scFv.
  • TrkB activation by those Doppelmabs was now as efficacious as the natural ligand BDNF. This was totally unexpected since the original C2 antibody only showed partial TrkB receptor agonist. Without wishing to be bound by theory it appears that VEGF induced clustering with the single binding molecules, as well as the sterical formation of the binding molecules, may be responsible for the observed increase in efficacy and potency of TrkB activation (see also FIG. 25 ).
  • TrkB activation was also somewhat better with the Doppelmabs TPP-14938 & TPP-14940 as compared to TPP-14939 & TPP-14941.
  • the “VH-VL” orientation of the C2 scFv showed better potency of TrkB activation.
  • TrkB activation was assessed by measuring TrkB phosphorylation on Y706/707. The lowest compound concentration wa solvent alone. Data represent mean+/ ⁇ SEM.
  • TrkB activation was somewhat better with the Doppelmab TPP-14940 as compared to TPP-14941.
  • the VH-VL orientation of the C2 scFv showed better potency of TrkB activation.
  • TrkB activation was assessed by measuring TrkB phosphorylation on Y706/707. The lowest compound concentration was solvent alone. Data represent the mean+/ ⁇ SEM.
  • TrkB activation TrkB activation
  • TrkB activation The impact of VEGF on the potency of TrkB phosphorylation (TrkB activation) was greater for TPP-14941 compared to TPP-14940.
  • both molecules showed synergistic effects on potency of TrkB phosphorylation, it appears that the orientation/geometry of the TrkB activating part of the Doppelmabs (scFv VL-VH vs. scFv VH-VL vs. Fab) seems to have an impact on the synergistic effect, with scFv having a VL-VH orientation being preferred.
  • TrkB activation TrkB activation
  • TrkB activation was assessed by measuring ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), downstream of TrkB. The lowest compound concentration is solvent alone. Data represent the mean+/ ⁇ SEM.
  • TrkB activation the impact of VEGF on the potency of ERK1/2 phosphorylation (TrkB activation) was greater for TPP-14941 compared to TPP-14940.
  • both molecules showed synergistic effects on potency of TrkB phosphorylation, but it appears that the orientation/geometry of the TrkB activating part of the Doppelmabs (scFv VL-VH vs. scFv VH-VL vs. Fab) seems to have an impact on the synergistic effect with scFv having a VL-VH orientation being preferred.
  • VEGF pre-incubation did also not impact on the potency of ERK1/2 phosphorylation (TrkB activation) by TPP-11736 (series 1, C2 as Fab). Finally, VEGF pre-incubation did not impact on the potency of ERK1/2 phosphorylation (TrkB activation) by C2 (control).
  • TrkB activation was assessed by measuring (A) TrkB phosphorylation on Y706/707 or (B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), downstream of TrkB, respectively. The lowest compound concentration was solvent alone. Data represent the mean+/ ⁇ SEM.
  • hVEGF alone did not induce TrkB or ERK1/2 phosphorylation.
  • the BDNF dose-response-curve is largely independent of hVEGF.
  • CHO cells with stable expression of cyno TrkB were incubated with growing concentrations of Doppelmabs (A) TPP-14940, (B) TPP-14941, or (C) C2 with or witout pre-incubation with 200 ng/mL human VEGF-A (hVEGF). TrkB activation was assessed by measuring TrkB phosphorylation on Y706/707. The lowest compound concentration was solvent alone. Data represent the mean+/ ⁇ SEM.
  • TrkB activation phosphorylation by TPP-14941
  • TPP-14940 C2 as scFv, 20L3, HL
  • TrkB activation was assessed by measuring ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), downstream of TrkB. The lowest compound concentration was solvent alone. Data represent the mean+/ ⁇ SEM.
  • TPP-11736 B20 as scFv
  • TPP-14938 B20 as Fab
  • TPP-14939 B20 as Fab
  • VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y1175, (B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), or (C) Src phosphorylation on Y419.
  • TPP-14938 and TPP-14939 were more efficacious than TPP-11736 (bottom values are smaller).
  • HRMECs Human retinal microvascular endothelial cells
  • 10 ng/mL human VEGF with or without pre-incubation with growing concentrations of the indicated binding molecules. Molecule concentrations are given in mol/L.
  • HRMECs Human retinal microvascular endothelial cells
  • TPP-11736 B20 as scFv
  • TPP-14936 Ranibizumab as scFv, VH-VL
  • TPP-14937 Ranibizumab as scFv, VL-VH
  • TPP-14940 Ranibizumab as Fab
  • TPP-14941 TPP-14941
  • VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y1175, (B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), or (C) Src phosphorylation on Y419. 50 ng/ml human VEGF without antibody treatment served as control. Data represent mean+/ ⁇ SEM. Below Table 7 reports the corresponding best-fit 1050 values and the absolute efficacy values (bottom plateaus), respectively (non-linear regression; log(agonist) vs. response (three parameters).
  • HRMECs Human retinal microvascular endothelial cells
  • 10 ng/mL human VEGF with or without pre-incubation with growing concentrations of the indicated binding molecules. Molecule concentrations are given in mol/L.
  • TPP-14940 & TPP-14941 full VEGF-inhibition was already seen at a concentration of 0.25 nM, whereas 1 nM TPP-14936 and 4 nM TPP-14937 were needed for full inhibition, respectively.
  • VEGF-A-inhibition was similar among all four molecules (all full inhibitors).
  • HRMECs Human retinal microvascular endothelial cells
  • TPP-11736 B20 as scFv
  • TPP-13938 B20 as Fab
  • TPP-13939 B20 as Fab
  • TPP-14940 Ranibizumab as Fab
  • TPP-14941 TPP-14941
  • VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y1175, (B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), or (C) Src phosphorylation on Y419. 50 ng/ml human VEGF without molecule treatment served as control. Data represent mean+/ ⁇ SEM. Below Table 8 reports the corresponding best-fit IC 50 values and the absolute efficacy values (bottom plateaus), respectively (non-linear regression; log(agonist) vs. response (three parameters).
  • Doppelmabs with Ranibizumab Fab showed clearly better potency of VEGF-scavenging than Doppelmabs based on B20 Fab in all three assays.
  • HRMECs Human retinal microvascular endothelial cells
  • 10 ng/mL human VEGF with or without pre-incubation with growing concentrations of the indicated binding molecules. Molecule concentrations are given in mol/L.
  • TPP-14938 & TPP-14939 failed to fully inhibit VEGF-induced sprouting.
  • TPP-14940 & TPP-14941 fully inhibited VEGF-induced proliferation already at a 64-fold lower concentration of 0.25 nM.
  • Spheroids of human retinal microvascular endothelial cells were embedded in a collagen matrix. Endothelial sprouting was induced for 24 hours by incubation with 50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of Doppelmab TPP-11736 (B20 scFv), TPP-14936 (Ranibizumab scFv, VH-VL), TPP-14937 (Ranibizumab scFv, VL-VH), TPP-14938 (B20 Fab), TPP-14939 (B20 Fab), TPP-14940 (Ranibizumab Fab), TPP-14941 (Ranibizumab Fab) or 5nM EYLEA® (aflibercept).
  • Endothelial sprouting was assessed by confocal microscopy and displayed spheroid perimeter obtained from maximum projections of Z-stacks.
  • Non-stimulated cells served as control (Basal).
  • Data represent mean+/ ⁇ SEM.
  • n.s. p>0.05 no significant difference; ⁇ p ⁇ 0.0001 compared to 50 ng/mL hVEGF; #p>0.05 no significant difference compared to 50 ng/mL hVEGF.
  • TPP-14940 & TPP-14941 were clearly the best VEGF-scavengers in this assay.
  • TPP-14940 & TPP-14941 fully inhibited VEGF-induced sprouting; no statistically significant difference was seen between the endothelial sprouting in the absence of VEGF (Basal) and 50 ng/mL hVEGF pre-incubated with 2.5 nM TPP-14940 or TPP-14941.
  • TPP-14940 (but not EYLEA® (aflibercept)) prevented human VEGF-A-induced hyperpermeability in the rat retina.
  • A Time protocol showing the experimental procedure. Fifteen minutes after intravitreal (ivt) administration of the anti-VEGF compound (13 or 26 pmol per eye of EYLEA® (aflibercept) or TPP-14940) or the control (26 pmol TPP-11737), 13 pmol human VEGF-A per eye was administered by ivt injection. PBS injection served as control.
  • the retinae were separated from the outer segments (sclera and choroidea) and transferred to a glass slide and cut four times to achieve a flat cloverleaf-like structure.
  • the tissue was covered with mounting medium (Vectashield® antifade mounting media H-1200 containing the DNA stain DAPI) and a coverslip was put on top to obtain a retinal flatmount.
  • the samples were excited at a wavelength of 639 nm and emission of Evans Blue at 669 nm was recorded with a
  • TPP-11737 was used as control antibody because it has the same molecular format (Doppelmab) as TPP-14940 but in earlier in vitro assays the compound did not scavange VEGF.
  • TPP-14940 completely blocked vascular hyperpermeability.
  • EYLEA® aflibercept
  • TrkB activation was somewhat better in Doppelmabs with “VH-VL” orientation TPP-14938/40 vs. TPP-14939 & TPP-14941.
  • TrkB activation TrkB activation
  • TPP-14940 In contrast to TPP-14941, the impact of VEGF on the potency of TrkB phosphorylation (TrkB activation) by TPP-14940 [C2, scFv, 20L3, VH-VL] was comparatively smaller.
  • TPP-14940 TPP-19986 (VH-VL, 10L1), TPP-19987 (VH-VL, 20L1); and on the basis of TPP-14941: TPP-19988 (VL-VH, 10L1), TPP-19989 (VL-VH, 20L1), which were all based on Ranibizumab as Fab and C2 as scFv.
  • TrkB ligand BDNF TPP-14940 [C2, scFv, 20L3, VH-VL] or its linker variants TPP-19986 [C2, scFv, 10L1, VH-VL] or TPP-19987 [C2, scFv, 20L1, VH-VL], or TPP-14941 [C2, scFv, 20L3, V-VH] and its linker variants TPP-19988 [C2, scFv, 10L1, VL-VH] or TPP-19989 [C2, scFv, 20L1, VL-VH].
  • TrkB activation was assessed by (A & B) measuring TrkB phosphorylation on Y706/707 or by (C & D) measuring ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), respectively. The lowest compound concentration was solvent alone. Data represent mean+/ ⁇ SEM.
  • the Tables 9 and 10 below report the corresponding best-fit EC 50 values of TrkB and ERK1/2 phosphorylation, respectively (non-linear regression; log(agonist) vs. response (three parameters).
  • TrkB/ERK phosphorylation was better for TPP-14940 molecule and its linker variants (VH-VL orientation) compared to the TPP-14941 molecule and its linker variants (VL-VH orientation).
  • TPP-14940 [C2, scFv, 20L3, VH-VL] or TPP-14941 [C2, scFv, 20L3, VL-VH] and the linker variants TPP-19988 [C2, scFv, 10L1, VL-VH] or TPP-19989 [C2, scFv, 20L1, VL-VH]-Series 3
  • CHO cells with stable expression of cyno TrkB were incubated with growing concentrations of the natural TrkB ligand BDNF, or 1 nM BDNF with growing concentrations of the Doppelmabs of the first series TPP-14940 [C2, scFv, 20L3, VH-VL] or TPP-14941 [C2, scFv, 20L3, VL-VH] and the linker variants TPP-19988 [C2, scFv, 10L1, VL-VH] or TPP-19989 [C2, scFv, 20L1, VL-VH].
  • TrkB internalization was assessed by immunofluorescence staining the surface TrkB receptors without permeabilization of the cells followed by confocal microscopy analysis. Dark and light fields of the heatmap represent high and low percentage of cells above florescence threshold, respectively.
  • TPP-14940, TPP-14941, TPP-19988 and TPP-19989 were full TrkB receptor agonists, none of the Doppelmabs increased the BDNF-induced receptor internalization but rather decreased the BDNF-induced internalization of the TrkB receptor (note: heatmap fields are getting darker from bottom to top).
  • Human retinal microvascular endothelial cells were starved and then incubated with 50 ng/mL human VEGF with or without pre-incubation with growing concentrations of the Doppelmabs TPP-14940 and its linker variant TPP-19986, and TPP-14941 and its linker variant TPP-19988.
  • VEGF-A scavenging was assessed by measuring VEGF receptor 2 (VEGFR2) phosphorylation on Y1175. 50 ng/ml human VEGF without molecule treatment served as control. Data represent mean+/ ⁇ SEM.
  • the Table 11 below reports the corresponding best-fit IC 50 values of the inhibition of VEGFR2 phosphorylation (non-linear regression; log(agonist) vs. response (three parameters).
  • Spheroids of human retinal microvascular endothelial cells were embedded in a collagen matrix. Endothelial sprouting was induced for 24 hours by incubation with 50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of Doppelmabs TPP-11736 (B20 scFv), TPP-14940 [C2, scFv, 20L3, VH-VL] or its linker variants TPP-19986 [C2, scFv, 10L1, VH-VL] or TPP-19987 [C2, scFv, 20L1, VH-VL], or TPP-14941 [C2, scFv, 20L3, VL-VH] and its linker variants TPP-19988 [C2, scFv, 10L1, VL-VH] or TPP-19989 [C2, scFv, 20L1, VL-VH].
  • TPP-11736 B20 scFv
  • Endothelial sprouting was assessed by confocal microscopy and displayed spheroid perimeter obtained from maximum projections of Z-stacks.
  • Non-stimulated cells served as control (Basal).
  • Data represent mean+/ ⁇ SEM.
  • n.s. p>0.05 no significant difference; ⁇ p ⁇ 0.0001 compared to 50 ng/mL hVEGF; #p>0.05 no significant difference compared to 50 ng/mL hVEGF.
  • TrkB activation was assessed by measuring TrkB phosphorylation on Y706/707. The lowest compound concentration was solvent alone. Data represent mean+/ ⁇ SEM.
  • Table 12 below reports the corresponding best-fit EC 50 values, and the absolute or relative efficacy values (top plateaus) of TrkB activation, respectively (non-linear regression; log(agonist) vs. response (three parameters).
  • TrkB-phosphorylation efficacy relative to TrkB-phosphorylation TrkB-phosphorylation parental clone potency efficacy without CC bridge
  • TPP Figure Description (EC 50 , nM) (AlphaLISA counts) (%) TPP-22180 A (1Q6Q70G_TrkB-C2-VH-VL_10L1) 8.09 6642 100 TPP-22204 A (1Q6Q70G_TrkB-C2-VH-VL_10L1, CC) 3.88 7613 115 TPP-22192 B (1Q6Q70G_TrkB-C2-VL-VH_10L1) 12.2 5642 100 TPP-22216 B (1Q6Q70G_TrkB-C2-VL-VH_10L1, CC) 4.33 8407 149 TPP-22190 C (1Q70G_TrkB-C2-VL-VH_10L1) 9.95 4225 100 TPP-22214 C (1Q70G_TrkB-C
  • the disulfide bridge was introduced mainly to improve the CMC properties of the binding molecules. Surprisingly, by including the disulfide bridge in the scFv anti-TrkB part further improved both potency and efficacy of TrkB activation. A greater impact was observed in the binding molecule having the VL-VH orientation.
  • TrkB activation was assessed by measuring TrkB phosphorylation on Y706/707. The lowest compound concentration was solvent alone. Data represent mean+/ ⁇ SEM.
  • Table 13 below reports the corresponding best-fit EC 50 values and the absolute or relative efficacy values (top plateaus) of TrkB activation, respectively (non-linear regression; log(agonist) vs. response (three parameters).
  • TrkB-phosphorylation efficacy relative TrkB-phosphorylation TrkB-phosphorylation to parental clone potency efficacy without CC bridge
  • TPP Figure Description (EC 50 , nM) (AlphaLISA counts) (%) TPP-22180 A (1Q6Q70G_TrkB-C2-VH-VL_10L1) 4.21 19676 100 TPP-22204 A (1Q6Q70G_TrkB-C2-VH-VL_10L1, CC) 2.01 21787 111 TPP-22192 B (1Q6Q70G_TrkB-C2-VL-VH_10L1) 18.9 21213 100 TPP-22216 B (1Q6Q70G_TrkB-C2-VL-VH_10L1, CC) 3.31 25808 122
  • TrkA human TrkA
  • TrkB human TrkB
  • TrkC TrkC
  • TrkB CHO cells with stable expression of human TrkB receptor were incubated with growing concentrations of (A) C2 antibody or (B) Doppelmab TPP-22214 with or without a constant concentration of 0.3 nM, 1 nM or 3 nM BDNF. Activation of TrkB was assessed by measuring receptor phosphorylation on Y706/707. Data represent the mean+/ ⁇ SEM.
  • TPP-22214 did not limit BDNF-induced TrkB activation.
  • TrkB Phosphorylation Comparison of Human TrkB Activation (TrkB Phosphorylation) by TPP-22214 in the Presence or Absence of Human VEGF
  • TrkB activation was assessed by measuring TrkB phosphorylation on Y706/707. The lowest compound concentration was solvent alone. Data represent the mean+/ ⁇ SEM.
  • TrkB activation As shown before, pre-incubation with 200 ng/mL human VEGF improved potency of TrkB phosphorylation (TrkB activation) by TPP-22214. More importantly, this synergistic effect was largely retained at 50, 10 and even 2 ng/mL hVEGF. Similar effects were also obtained in other species such as cyno (data not shown).
  • TrkB activation was assessed by measuring ERKI /2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), downstream of TrkB. The lowest compound concentration was solvent alone. Data represent the mean+/ ⁇ SEM.
  • TPP-22214 and hVEGF were made by mixing together the samples at the described molar ratio (1:1, 4:1 or 20:1) in 1 ⁇ Dulbecco's PBS. Samples were incubated for 1 hour at room temperature prior to analysis. An Agilent 1200 was used in series with mini-DAWN® TREOS® compact SEC-MALs detector and Optilab® T-REX detector (Wyatt Technologies Corp). The samples were run in 1 ⁇ Dulbecco's PBS as the mobile phase at a flow rate of 0.6 ml/min.
  • TPP-22214 forms complexes in the presence of VEGF-A with largest complexes formed at 1:1 molar ratio. These complexes suggest complexing of the Doppelmab and VEGF beyond 1:1 ratio. These larger complexes can lead to clustering of the TrkB receptors on the cell surface. Experimental data support the proposed mechanism.
  • TrkB internalization was assessed by immunofluorescence staining the surface TrkB receptors without permeabilization of the cells followed by confocal microscopy analysis. Data represent the percent of cells with surface TrkB staining intensity above threshold; mean+/ ⁇ SEM.
  • TrkB activation in a rat model of diabetes-induced retinal neurodegeneration using IVT injection of an agonistic TrkB antibody (C2) as well as Doppelmab TPP-22214.
  • Animals were treated with STZ to induce hyperglycemia.
  • the retinal function was assessed by electroretinography (ERG) before and after treatment. Diabetes induction led to delayed implicit times within 3 weeks after STZ treatment.
  • animals were intravitreally dosed with an isotype control antibody (anti-TNP) or C2 (19 ⁇ g/5 ⁇ l, each), or an equimolar amount of TPP-22214 (25 ⁇ g/5 ⁇ l).
  • Rod-driven B-wave implicit time delays immediately before and two weeks after intravitreal application of the molecules are depicted; mean+/ ⁇ SEM; n.s. p>0.05; non-significant (n.s.), ****p ⁇ 0.0001; one-way Anova with Tukey multi-comparison test.
  • C2 antibody treatment blocked a further increase of the implicit time delay.
  • VEGF-A scavenging was assessed by measuring VEGF receptor 2 (VEGFR2) phosphorylation on Y1175. 50 ng/ml human VEGF without molecule treatment served as control. Data represent mean+/ ⁇ SEM. Below Table 14 reports the corresponding best-fit IC 50 values (non-linear regression; log(agonist) vs. response (three parameters).
  • Human retinal microvascular endothelial cells were starved and then incubated with 50 ng/mL human VEGF with or without pre-incubation with growing concentrations of TPP-22214 or EYLEA® (aflibercept).
  • VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y1175 or (B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2). 50 ng/ml human VEGF without molecule treatment served as control. Data represent mean+/ ⁇ SEM. Table 15 below reports the corresponding best-fit values form the non-linear regression (log(agonist) vs. response (three parameters). Best-fit values that significantly differ between EYLEA® (aflibercept) and TPP-22214 (P ⁇ 0.05) are shown in bold face. P-values are also reportet in Table 15.
  • TPP-22214 Compared to EYLEA® (aflibercept), TPP-22214 fully inhibited VEGF-induced phosphorylation of VEGFR2 on Y1175 and of ERK1/2. The improved efficacy in VEGF-scavenging of TPP-22214 can also be seen on the highly significant differences in the values of the bottom plateaus (see Table 15). Finally, TPP-22214 was significantly more potent in VEGF-scavenging than EYLEA® (aflibercept).
  • HRMECs Human retinal microvascular endothelial cells
  • 10 ng/mL human VEGF with or without pre-incubation with growing concentrations of the indicated binding molecules: (A) TPPP-22204, (B) TPP-22214; (C) TPP-22216 or (D) EYLEA® (aflibercept).
  • E Plot of the difference of the area under the growth curves and the basal curves vs. the concentration of EYLEA® (aflibercept) or TPP-22214.
  • Table 16 reports the corresponding best-fit values form the non-linear regression (log(agonist) vs. response (three parameters). Best-fit values that significantly differ between EYLEA® (aflibercept) and TPP-22214 (P ⁇ 0.05) are shown in bold face. P-values are also reportet in Table 16.
  • EYLEA® aflibercept
  • Human retinal microvascular endothelial cells were starved and then incubated with 50 ng/mL human VEGF with or without pre-incubation with growing concentrations of TPP-22214 or EYLEA® (aflibercept).
  • VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y1214 or (B) p38-MAPK phosphorylation on T180/Y182. 50 ng/ml human VEGF without molecule treatment served as control. Data represent mean+/ ⁇ SEM.
  • Table 17 reports the corresponding best-fit values form the non-linear regression (log(agonist) vs. response (three parameters). Best-fit values that significantly differ between EYLEA® (aflibercept) and TPP-22214 (P ⁇ 0.05) are shown in bold face. P-values are also reportet in Table 17.
  • TPP-22214 In strong contrast to EYLEA® (aflibercept), TPP-22214 fully inhibited VEGF-induced phosphorylation of VEGFR2 on Y1214 and p38-MAPK. The more efficacious VEGF-scavenging of TPP-22214 can be seen from the highly significant difference in the values of the bottom plateaus in Table 17. Moreover, a trend was observed that the potency of VEGF-scavenging by TPP-22214 is better than that of EYLEA® (aflibercept).
  • Spheroids of human retinal microvascular endothelial cells were embedded in a collagen matrix. Endothelial sprouting was induced for 24 hoursvby incubation with 50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of TPP-22204, TPP-22214, TPP-22215 or TPP-22216, or 5 nM EYLEA® (aflibercept).
  • A Endothelial sprouting was assessed by confocal microscopy and displayed spheroid perimeter obtained from maximum projections of Z-stacks. Non-stimulated cells served as control (Basal). Data represent mean+/ ⁇ SEM. n.s. p>0.05 non-significant, ****p ⁇ 0.0001.
  • the Doppelmabs were a lot more efficacious in inhibiting VEGF-induced HRMEC sprouting. Moreover, the Doppelmabs were able to fully inhibit VEGF-induced endothelial cell sprouting. Noteworthy, EYLEA® (aflibercept) was used at the double molecular concentration as compared to the Doppelmabs and still was not able to fully inhibit cell sprouting.
  • Human retinal microvascular endothelial cells were starved and then incubated with 50 ng/mL human VEGF with or without pre-incubation with growing concentrations of TPP-22214 or EYLEA® (aflibercept).
  • VEGF-A scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y951 or (B) Src phosphorylation on Y419. 50 ng/ml human VEGF without antibody treatment served as control. Data represent mean+/ ⁇ SEM.
  • Table 18 reports the corresponding best-fit values form the non-linear regression (log(agonist) vs. response (three parameters). Best-fit values that significantly differ between EYLEA® (aflibercept) and TPP-22214 (P ⁇ 0.05) are shown in bold face. P-values are also reported in Table 18.
  • TPP-22214 In strong contrast to EYLEA® (aflibercept), TPP-22214 fully inhibited VEGF-induced phosphorylation of VEGFR2 on Y951 and of Src on Y419. The more efficacious VEGF-scavenging of TPP-22214 can be seen from the highly significant difference in the values of the bottom plateaus (Table 18).
  • TPP-22214 prevented human VEGF-A-induced hyperpermeability in the rat retina.
  • A Time protocol showing the experimental procedure. Fifteen minutes after intravitreal (ivt) administration of the anti-VEGF compound (13 or 26 pmol per eye of EYLEA® (aflibercept) or TPP-14940) or the control (26 pmol TPP-11737), 13 pmol human VEGF-A per eye was administered by ivt injection. PBS injection served as control. Twenty-four hours later 1 mL/kg of an Evans Blue (EB) solution (45 mg/mL in 0.9% saline) were administered by intravenous (iv) injection for 30 minutes before the eyes were isolated and fixed.
  • EB Evans Blue
  • the tissue was covered with mounting medium (Vectashield® H-1200 antifade mounting media containing the DNA stain DAPI) and a coverslip was put on top to obtain a retinal flatmount.
  • the samples were excited at a wavelength of 639 nm and emission of Evans Blue at 669 nm was recorded with a LSM 700 confocal laser scanning-microscope (Carl Zeiss, Jena; gain 800, laser strength 2%, 5 stacks of 60 ⁇ m) and images of the retinal flatmounts with maximum intensity projection were obtained. Analysis of fluorescence intensity sum was done after opening the images in the program ImageJ with a threshold of 30. ***p ⁇ 0.001; *p ⁇ 0.05; n.s. p>0.05.
  • One-way Anova with Tukey multi comparison test, n 9-17. Incubation with 67: 1 molar ratio of EYLEA® (aflibercept): VEGF is shown for comparison.
  • TPP-11737 was used as control molecule because it had the same molecular format (Doppelmab) as TPP-22214 but in earlier in vitro assays did not scavange VEGF (see above).
  • TrkB-Binding on VEGF-Scavenging by TPP-22214-Comparison of Human VEGF-A Scavenging by TPP-22214 in the Presence or Absence of the TrkB Extracellular Domain
  • TrkB-ECD Functional characterization of the TkrB extracellular domain
  • TrkB-ECD binding of TPP-22214 on inhibition of VEGF-induced VEGFR2 phosphorylation.
  • Human retinal microvascular endothelial cells HRMECs
  • HRMEC incubation with growing concentrations of TrkB-ECD with or without 50 ng/mL VEGF and unstimulated cells (Basal) served as control.
  • VEGF-A scavenging was assessed by measuring VEGF receptor 2 (VEGFR2) phosphorylation on Y1175. Data represent mean+/ ⁇ SEM.
  • VEGF-scavenging by TPP-22214 was independent of the presence or absence of the TrkB-ECD. Both curves were nearly identical. This indicated that TrkB-binding of TPP-22214 did per se not limit VEGF-scavenging of the Doppelmab suggesting that the Doppelmab can simultaneously activate the TrkB receptor and scavenge VEGF.
  • Biophysical properties of molecules from series 3 and series 4 were assessed. During production of the molecules the quality after protein A measured by the percentage of monomer was tested using analytical size exclusion chromatography. Additionally, the thermal stability and aggregation onset of each molecule was assessed in 10 mM Histidine pH 6.0. The thermal stability was measured using a thermal shift assay which measures the unfolding of the protein with temperature using Sypro-orange dye. Each Tm was calculated as the peak maxima of the first derivative of fluorescence signal across temperature. Aggregation onset was measured using dynamic light scattering to measure the hydrodynamic radius as a function of temperature. Finally, the storage stability of various Doppelmabs was tested by measuring aggregation after 2 weeks at either 40° C. or 5° C. Aggregation was measured using analytical size exclusion chromatography.
  • Table 20 shows the results of the experimentally determined vitreous half-life of TPP-22214 and Bevacizumab and compares the inhouse experimentally determined values with the known literature values of Bevacizumab, Ranibizumab and Aflibercept.
  • the reported half-life of Bevacizumab is identical to the in-house/experimentally derived value confirming the accuracy of the applied method and the comparability of the data sets.
  • TPP-22214 was shown to have an in vivo half-life in rabbits which was approximately 50% higher compared to e.g Bevacizumab (6.7 days vs 4.3 days).
  • Ocular VEGF (aqueous humor, wet AMD patients) Hata M, IOVS Cabral T, Int J Hsu, M., Sci Rep Sato T, Sci Rep 2017, 58 (1): Retin Vitr 2017, Publication 2016, 6:34631 2018; 8:1098 292-298 3:6 VEGF pg/mL pM pg/mL pM pg/mL pM pg/mL pM concentration 546 28 228 12 90.0 4.7 180 9.4
  • Table 21 reports on the literature values of ocular VEGF concentrations in wet age-related macular degeneration (wAMD) patients.
  • TrkB ligand BDNF a further monoclonal TrkB antibody
  • TPP-23457 TPP-6380 as scFv, 10L1, VH-VL; Ranibizumab as Fab
  • TPP-23459 TPP6380 as scFv, 10L1, VL-VH; Ranibizumab as Fab
  • the new Doppelmab molecules were now based on a different TrkB binder (TPP-6830) to evaluate whether the observed effects with the Doppelmabs based on the C2 TrkB binder could be also reproduced with a different TrkB binder.
  • TrkB Phosphorylation Comparison of Human TrkB Activation (TrkB Phosphorylation) by TPP-6830 (TrkB Monoclonal Antibody) and the Two Doppelmabs TPP-23457 and TPP-23459 in the Presence or Absence of Human VEGF
  • TrkB activation was assessed by measuring TrkB phosphorylation on Y706/707. The lowest compound concentration was solvent alone. Data represent the mean+/ ⁇ SEM.
  • TrkB activation TrkB activation
  • TPP-23457 TPP-6830 as scFv, 10L1, VH-VL; Ranibizumab as Fab
  • TPP-23459 TPP-6380 as scFv, 10L1, VL-VH; Ranibizumab as Fab
  • TrkB activation TrkB activation
  • TrkB activation was assessed by measuring ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), downstream of TrkB. The lowest compound concentration is solvent alone. Data represent the mean+/ ⁇ SEM.
  • TPP-23457 TPP-6830 as scFv, 10L1, VH-VL; Ranibizumab as Fab
  • TPP-23459 TPP-6380 as scFv, 10L1, VL-VH; Ranibizumab as Fab
  • TrkB activation ERK1/2 phosphorylation
  • TrkB CHO cells with stable expression of human TrkB receptor were incubated with growing concentrations of (A) C2 antibody or (B) Doppelmab TPP-23457 with or without a constant concentration of 0.3 nM, 1 nM or 3 nM BDNF. Activation of TrkB was assessed by measuring receptor phosphorylation on Y706/707. Data represent the mean+/ ⁇ SEM.
  • TPP-23457 did not limit BDNF-induced TrkB activation.

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