Treatment of ophthalmologic diseases

Abstract

To provide an antibody which binds to VEGF and ANG2 for treatment of ophthalmologic diseases.SOLUTION: A bispecific antibody which binds to human vascular endothelial growth factor (VEGF) and to human angiopoietin-2 (ANG-2) is for use in treatment of an ocular vascular disease or a patient suffering from an ocular vascular disease. The bispecific antibody is administered (is to be administered) intravitreally every 8 weeks or less frequently. The bispecific antibody which binds to VEGF and to human ANG-2 may be a VEGF antagonist / inhibitor and an ANG2 antagonist / inhibitor, or inhibit binding of VEGF to its receptor VEGFR and inhibit binding of ANG2 to its receptor TIE2.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the use of antibodies that bind to VEGF and ANG2 for the treatment of ophthalmic diseases.

[0002] Background of the Invention Angiogenesis is involved in the pathogenesis of a variety of disorders, including solid tumors, intraocular neovascular syndromes such as proliferative retinopathies or age-related macular degeneration (AMD), rheumatoid arthritis, and psoriasis (Folkman, J., et al., J. Biol. Chem. 267 (1992) 10931-10934; Klagsbrun, M., et al., Annu. Rev. Physiol. 53 (1991) 217-239; and Garner, A., Vascular diseases, Pathobiology of ocular disease, A dynamic approach, Garner, A., and Klintworth, GK (eds.), 2nd edition, Marcel Dekker, New York (1994), pp. 1625-1710).

[0003] Ranibizumab (trade name Lucentis®) is a monoclonal antibody fragment derived from the same parent murine antibody as bevacizumab (Avastin®). However, it has been affinity matured to confer stronger binding to VEGF-A (WO 98 / 45331). Systemic blockade of VEGF-A is known to be associated with an increased risk of certain adverse events; therefore, ranibizumab lacks the Fc portion to reduce systemic exposure and the risk of systemic toxicity. It is an antiangiogenic agent approved for treating the "wet" form of age-related macular degeneration (neovascular age-related macular degeneration), a common form of age-related vision loss.

[0004] Corneal angiogenesis assays showed that both ANG-1 and ANG-2 have similar effects, promoting the growth of new blood vessels by acting synergistically with VEGF (Asahara, T., et al., Circ. Res. 83 (1998) 233-40). The possibility of a dose-dependent endothelial response was raised by the observation that ANG-2 can also be proangiogenic at high concentrations in vitro (Kim, I., et al., Oncogene 19 (2000) 4549-52). At high concentrations, ANG-2 acts as an apoptotic survival factor for endothelial cells during serum deprivation-induced apoptosis through activation of Tie2 via the PI-3 kinase and Akt pathways (Kim, I., et al., Oncogene 19 (2000) 4549-52).

[0005] Ocular vascular diseases such as "wet" age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR) result from abnormal choroidal or retinal neovascularization, respectively. Bleeding and leakage from these vessels can cause retinal dysfunction and vision loss. Other retinal vascular diseases, such as diabetic macular edema (DME) and macular edema secondary to retinal vein occlusion (RVO), result from abnormal retinal leakage, leading to retinal swelling and impaired visual function. These conditions are the leading cause of vision loss in industrialized countries. The retina is composed of well-defined layers of neuronal, glial, and vascular components, and relatively minor impairments, such as those seen in vascular proliferation or edema, can result in significant visual impairment. Inherited retinal degenerations, such as retinitis pigmentosa (RP), are also associated with vascular abnormalities, such as arteriolar narrowing and vascular atrophy. They affect as many as 1 in 3500 individuals and are characterized by progressive night blindness, visual field loss, optic nerve atrophy, arteriolar weakening, and central visual field loss that often progresses to total blindness.

[0006] Ischemic retinopathy is characterized by a decrease or dysfunction of the retinal vasculature, which leads to reduced blood flow and hypoxia. The retina responds to hypoxia by generating signals to grow new blood vessels, but these new vessels are usually fragile and disorganized. It is the growth of these abnormal new blood vessels that creates the majority of the threat to vision, as they may leak, bleed, or scar, which can ultimately lead to retinal detachment. Current treatments for ischemic retinopathy seek to stop the growth of pathological blood vessels but do not address the underlying ischemia that drives their growth. Furthermore, the standard treatment for diabetic retinopathy, a type of ischemic retinopathy that affects millions of people, involves the destruction of part of the retina with a laser in an attempt to stop the growth of new blood vessels and destroy the ischemic tissue to preserve central vision. Strategies have been used to block the function of vascular endothelial growth factor (VEGF), a major promoter of abnormal blood vessel growth and leakage. While anti-VEGF antibody therapy can improve vision in the short term, it does not address the underlying ischemia and may in fact worsen the condition by inhibiting the growth of all blood vessels, including beneficial collateral vessels. Furthermore, there are serious concerns about systemic exposure to these drugs in elderly and / or diabetic patients, whose ischemic brains, hearts, or limbs may require new blood vessel growth.

[0007] Summary of the Invention According to one aspect of the present invention, a method, use, bispecific antibody (for use), medicament, or pharmaceutical formulation is provided for treating a patient suffering from an ocular vascular disease, the method comprising administering to the patient an effective amount of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), wherein the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently; in one embodiment, every 16 weeks or less frequently).

[0008] One aspect of the invention is such a method, use, bispecific antibody (for use), medicament, or pharmaceutical formulation (for use) of / for the treatment of a patient suffering from ocular vascular disease, the method, use, bispecific antibody (for use), medicament, or pharmaceutical formulation (for use) comprising the step of intravitreally administering to the patient an effective amount of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), wherein the patient experiences an increase of 12 letters or more (in one embodiment, 13 letters or more, in one embodiment, 14 letters or more, and in one embodiment, 15 letters or more) in best corrected visual acuity (BCVA) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally every 8 weeks or less. One embodiment of the invention is a method of treating a patient suffering from ocular vascular disease, the method comprising the step of administering (intravitreally) to the patient an effective amount of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), wherein the patient exhibits improved visual acuity after administration of the bispecific VEGF / ANG2 antibody, as measured by an increase in best corrected visual acuity (BCVA) of 12 letters or more (in one embodiment, 13 letters or more, in one embodiment, 14 letters or more, and in one embodiment, 15 letters or more) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart, compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally every 8 weeks or less.

[0009] In one embodiment of the invention, the letter increase in the BCVA / ETDRS letter score is measured at 4 weeks, and / or 8 weeks, and / or 12 weeks, and / or 16 weeks, and / or 20 weeks, and / or 24 weeks, respectively, after initiation of treatment.

[0010] In one embodiment of the invention, the letter increase in the BCVA / ETDRS letter score is measured at 24 weeks, and / or 25 weeks, and / or 26 weeks, and / or 27 weeks, and / or 28 weeks, and / or 29 weeks, and / or 30 weeks, and / or 31 weeks, and / or 32 weeks, and / or 33 weeks, and / or 34 weeks, and / or 35 weeks, and / or 36 weeks, and / or 37 weeks, and / or 38 weeks, and / or 39 weeks, and / or 40 weeks after the start of treatment, respectively. and / or at 41st week, and / or 42nd week, and / or 43rd week, and / or 44th week, and / or 45th week, and / or 46th week, and / or 47th week, and / or 48th week, and / or 49th week, and / or 50th week, and / or 51st week, and / or 52nd week, and / or 53rd week, and / or 54th week, and / or 55th week, and / or 56th week, and / or 57th week, and / or 58th week, and / or 59th week, and / or 60th week. In one embodiment of the invention, the ocular vascular disease is selected from the group consisting of wet age-related macular degeneration (wet AMD), neovascular age-related macular degeneration, diabetic macular edema (DME), cystoid macular edema (CME), nonproliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), macular edema secondary to central retinal vein occlusion, secondary to hemiretinal vein occlusion, or secondary to branch retinal vein occlusion, retinitis, conjunctivitis, uveitis, choroiditis, choroidal neovascularization (CNV) secondary to intraocular inflammation (including secondary to ocular histoplasmosis or presumed histoplasmosis or choroiditis); myopic choroidal neovascularization (mCNV), and choroidal neovascularization secondary to trauma, retinopathy of prematurity, and rubeosis iridis / neovascular glaucoma.

[0011] In one embodiment of the invention, the ocular vascular disease is diabetic macular edema (DME).

[0012] In one embodiment of the invention, the ocular vascular disease is diabetic macular edema (DME), and the letter increase in the BCVA / ETDRS letter score is measured at about 9-15 months (in one embodiment, 9-14 months; in one embodiment, 9-12 months) after initiation of treatment.

[0013] In one embodiment of the invention, the ocular vascular disease is diabetic macular edema (DME) and the letter increase in the BCVA / ETDRS letter score is measured at 36 weeks, and / or 37 weeks, and / or 38 weeks, and / or 39 weeks, and / or 40 weeks, and / or 41 weeks, and / or 42 weeks, and / or 43 weeks, and / or 44 weeks, and / or 45 weeks, and / or 46 weeks, and / or 47 weeks, and / or 48 weeks, and / or 49 weeks, and / or 50 weeks, and / or 51 weeks, and / or 52 weeks, and / or 53 weeks, and / or 54 weeks, and / or 55 weeks, and / or 56 weeks, and / or 57 weeks, and / or 58 weeks, and / or 59 weeks, and / or 60 weeks, respectively, after initiation of treatment.

[0014] These time points are quite early, with maximum increases not typically reached until approximately 6-9 months in neovascular age-related macular degeneration and 9-12 months in diabetic macular edema.

[0015] In one embodiment of the invention, the ocular vascular disease is wet age-related macular degeneration (wet AMD) or neovascular age-related macular degeneration (nAMD).

[0016] In one embodiment of the invention, the ocular vascular disease is wet age-related macular degeneration (wet AMD) or neovascular age-related macular degeneration (nAMD), and the letter increase in the BCVA / ETDRS letter score is measured at about 9 to 15 months (in one embodiment, 6 to 9 months; in one embodiment, 6 to 12 months) after initiation of treatment.

[0017] In one embodiment of the invention, the ocular vascular disease is wet age-related macular degeneration (wet AMD) or neovascular age-related macular degeneration (nAMD), and the letter increase in the BCVA / ETDRS letter score is at 24 weeks, and / or 25 weeks, and / or 26 weeks, and / or 27 weeks, and / or 28 weeks, and / or 29 weeks, and / or 30 weeks, and / or 31 weeks, and / or 32 weeks, and / or 33 weeks, and / or 34 weeks, respectively, after initiation of treatment. and / or 35th week, and / or 36th week, and / or 37th week, and / or 38th week, and / or 39th week, and / or 40th week, and / or 41st week, and / or 42nd week, and / or 43rd week, and / or 44th week, and / or 45th week, and / or 46th week, and / or 47th week, and / or 48th week, and / or 49th week, and / or 50th week, and / or 51st week, and / or 52nd week, and / or 53rd week.

[0018] In one embodiment of the invention, the bispecific antibody that binds to human VEGF and human ANG2 is a bispecific, bivalent anti-VEGF / ANG2 antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, wherein i) the first antigen-binding site that specifically binds to VEGF comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO: 3, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO: 5, and a CDR1L region of SEQ ID NO: 6; ii) the second antigen-binding site that specifically binds to ANG-2 comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 9, a CDR2H region of SEQ ID NO: 10, and a CDR1H region of SEQ ID NO: 11, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ ID NO: 14; iii) The bispecific antibody comprises a heavy chain constant region of the human IgG1 subclass containing the mutations I253A, H310A, and H435A and the mutations L234A, L235A, and P329G (numbering according to the EU index of Kabat).

[0019] In one embodiment of the invention, the patient suffering from ocular vascular disease has not previously been treated (is treatment naive) with an anti-VEGF antibody (eg, as monotherapy).

[0020] In one embodiment of the invention, the patient suffering from ocular vascular disease has previously undergone treatment (eg, monotherapy) with an anti-VEGF antibody.

[0021] In one embodiment of the invention, the ocular vascular disease is diabetic macular edema, and treatment of patients with diabetic macular edema involves a consistent every eight weeks (Q8W) dosing regimen after treatment initiation.

[0022] In one embodiment of the invention, the ocular vascular disease is diabetic macular edema, and treatment of patients with diabetic macular edema comprises a consistent every 12 weeks (Q12W) dosing regimen after initiation of treatment. In one embodiment of the invention, after initiation of treatment, the consistent Q12W dosing regimen is preceded by an initial cycle of Q8W with one dose.

[0023] In one embodiment of the invention, the ocular vascular disease is diabetic macular edema, and treating patients with diabetic macular edema involves a dosing regimen in which, after initiation of treatment, the dosing interval is extended if stable disease-free or shortened if disease activity is present. In one embodiment of the invention, such a dosing regimen involves the patient receiving Q4W or Q8W or Q12W or Q16W dosing, depending on the patient's disease status. In one embodiment of the invention, stable disease-free status is defined as: -Central retinal thickness (CST) increased by less than 50 μm; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by less than 5 letters and disease activity is determined as -Central retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by 5 letters or more is determined as follows.

[0024] In one embodiment of the present invention, the ocular vascular disease is age-related macular degeneration, and the treatment of patients with age-related macular degeneration comprises a dosing regimen in which, after initiation of treatment, the administration interval is extended if stable disease-free or shortened if disease activity is present. In one embodiment of the present invention, such a dosing regimen comprises the patient receiving Q4W or Q8W or Q12W or Q16W dosing, depending on the patient's disease status. In one embodiment of the present invention, stable disease-free status is defined as: -Central retinal thickness (CST) increased by less than 50 μm; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by less than 5 letters and disease activity is determined as -Central retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by 5 letters or more is determined as follows. [Brief explanation of the drawings]

[0025] [Figure 1] Change in best corrected visual acuity (BCVA) in treated diabetic macular edema patients (untreated patients) over time from baseline to week 24. VA2 refers to the bispecific anti-VEGF / ANG2 antibody RO6867461 (6.0 mg or 1.5 mg dose administered intravitreally) comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, and RBZ refers to ranibizumab (Lucentis®) (0.3 mg dose administered intravitreally). [Figure 2]Central retinal thickness (CST) measured by spectral-domain optical coherence tomography (SD OCT). Change in central retinal thickness from baseline to week 24 in treated diabetic macular edema patients (untreated patients) over time. The bispecific anti-VEGF / ANG2 antibody RO6867461 (6.0 mg or 1.5 mg dose administered intravitreally), comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, was compared to ranibizumab (Lucentis®) (0.3 mg dose administered intravitreally). [Figure 3] Based on disease activity assessed by both a loss of best-corrected visual acuity of ≥ 5 letters and an increase in central foveal retinal thickness of ≥ 50 μm, time to required retreatment (after dosing was stopped (20 weeks or every 6 months = time after last intravitreal (IVT) administration)). The bispecific anti-VEGF / ANG2 antibody RO6867461 (6.0 mg or 1.5 mg dose administered intravitreally) comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 was compared to ranibizumab (Lucentis®) (0.3 mg dose administered intravitreally). [Figure 4] Graphical comparison with other treatment options for diabetic macular edema based on published results (comparison agents Lucentis® (ranibizumab), Eylea® (aflibercept), brolucizumab, and VA2 (RO6867461 / RG7716)). [Figure 5] Overview of the clinical trial design for the evaluation of the bispecific antibody RO6867461, administered every 12 and 16 weeks, in patients with neovascular age-related macular degeneration (nAMD). [Figure 6] Increase in best-corrected visual acuity from baseline in patients with neovascular age-related macular degeneration (nAMD) comparing bispecific antibody RO6867461 (comprising the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20) (6.0 mg administered intravitreally every 12 and 16 weeks) with ranibizumab (Lucentis®) (0.3 mg dose administered intravitreally every 4 weeks). [Figure 7] Change in baseline central retinal thickness (measured via OCT) in patients with neovascular age-related macular degeneration (nAMD) comparing bispecific antibody RO6867461 (comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20) (6.0 mg administered intravitreally every 12 and 16 weeks) with ranibizumab (Lucentis®) (0.3 mg dose administered intravitreally every 4 weeks).

[0026] Detailed Description of the Invention According to one aspect of the present invention, there is provided a method of treating a patient suffering from an ocular vascular disease, the method comprising administering to the patient an effective amount of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2); The bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0027] One embodiment of the invention is a method of treating a patient suffering from an ocular vascular disease, the method comprising the step of administering to the patient an effective amount of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), wherein the patient experiences an increase of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, and in one embodiment 15 letters or more) in best corrected visual acuity (BCVA) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's BCVA letter score prior to administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0028] One embodiment of the invention is a method of treating a patient suffering from an ocular vascular disease, the method comprising the step of administering to the patient an effective amount of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), wherein the patient exhibits improved visual acuity after administration of the bispecific VEGF / ANG2 antibody as measured by an increase in best corrected visual acuity (BCVA) of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, and in one embodiment 15 letters or more) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0029] In one embodiment of the invention, the letter increase in the BCVA / ETDRS letter score is measured at 4 weeks, and / or 8 weeks, and / or 12 weeks, and / or 16 weeks, and / or 20 weeks, and / or 24 weeks, respectively, after initiation of treatment.

[0030] In one embodiment of the invention, the letter increase in the BCVA / ETDRS letter score is at 24 weeks, and / or 25 weeks, and / or 26 weeks, and / or 27 weeks, and / or 28 weeks, and / or 29 weeks, and / or 30 weeks, and / or 31 weeks, and / or 32 weeks, and / or 33 weeks, and / or 34 weeks, and / or 35 weeks, and / or 36 weeks, and / or 37 weeks, and / or 38 weeks, and / or 39 weeks, and / or 40 weeks after the start of treatment, respectively. and / or at 41st week, and / or 42nd week, and / or 43rd week, and / or 44th week, and / or 45th week, and / or 46th week, and / or 47th week, and / or 48th week, and / or 49th week, and / or 50th week, and / or 51st week, and / or 52nd week, and / or 53rd week, and / or 54th week, and / or 55th week, and / or 56th week, and / or 57th week, and / or 58th week, and / or 59th week, and / or 60th week. In one embodiment of the invention, the letter increase in the BCVA / ETDRS letter scores is measured at 45 weeks, and / or 46 weeks, and / or 47 weeks, and / or 48 weeks, and / or 49 weeks, and / or 50 weeks, and / or 51 weeks, and / or 52 weeks, and / or 53 weeks, and / or 54 weeks, and / or 55 weeks, and / or 56 weeks, and / or 57 weeks, and / or 58 weeks, and / or 59 weeks, and / or 60 weeks, respectively, after initiation of treatment.

[0031] In one embodiment of the invention, the method is used to extend the time to retreatment and / or to extend the time to vision loss, and retreatment with the bispecific antibody comprises: an increase in foveal retinal thickness of 50 μm or more (in one embodiment using spectral-domain optical coherence tomography (SD-OCT)); and / or A decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more It is administered in the event of disease activity determined as

[0032] One embodiment of the invention is a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in treating ocular vascular disease, wherein the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0033] One embodiment of the invention is a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in treating a patient suffering from ocular vascular disease, wherein the patient has an increase of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, and in one embodiment 15 letters or more) in best corrected visual acuity (BCVA) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score prior to administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0034] One embodiment of the invention is a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in treating a patient suffering from ocular vascular disease, wherein the patient exhibits improved visual acuity after (intravitreal) administration of the bispecific VEGF / ANG2 antibody as measured by an increase in best corrected visual acuity (BCVA) of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, and in one embodiment 15 letters or more) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0035] In one embodiment of the invention, the letter increase in the BCVA / ETDRS letter score is measured at 4 weeks, and / or 8 weeks, and / or 12 weeks, and / or 16 weeks, and / or 20 weeks, and / or 24 weeks, respectively, after initiation of treatment.

[0036] In one embodiment of the invention, the letter increase in the BCVA / ETDRS letter scores is measured at 45 weeks, and / or 46 weeks, and / or 47 weeks, and / or 48 weeks, and / or 49 weeks, and / or 50 weeks, and / or 51 weeks, and / or 52 weeks, and / or 53 weeks, and / or 54 weeks, and / or 55 weeks, and / or 56 weeks, and / or 57 weeks, and / or 58 weeks, and / or 59 weeks, and / or 60 weeks, respectively, after initiation of treatment.

[0037] In one embodiment of the invention, such a bispecific antibody (for use) is used to extend the time to retreatment and / or to extend the time to vision loss, wherein retreatment with the bispecific antibody comprises: an increase in central retinal thickness (CST) of 50 μm or more (in one embodiment using spectral-domain optical coherence tomography (SD-OCT)); and / or A decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more It is administered in the event of disease activity determined as

[0038] One embodiment of the invention is a medicament or pharmaceutical formulation comprising a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in treating ocular vascular disease, wherein the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0039] One embodiment of the invention is a medicament or pharmaceutical formulation comprising a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in treating a patient suffering from ocular vascular disease, wherein the patient has an increase of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, and in one embodiment 15 letters or more) in best corrected visual acuity (BCVA) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score prior to administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0040] One embodiment of the invention is a medicament or pharmaceutical formulation comprising a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in treating a patient suffering from ocular vascular disease, wherein the patient exhibits improved visual acuity after (intravitreal) administration of the bispecific anti-VEGF / ANG2 antibody as measured by an increase in best corrected visual acuity (BCVA) of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, and in one embodiment 15 letters or more) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0041] In one embodiment of the invention, the letter increase in the BCVA / ETDRS letter score is measured at 4 weeks, and / or 8 weeks, and / or 12 weeks, and / or 16 weeks, and / or 20 weeks, and / or 24 weeks, respectively, after initiation of treatment.

[0042] In one embodiment of the invention, the letter increase in the BCVA / ETDRS letter scores is measured at 45 weeks, and / or 46 weeks, and / or 47 weeks, and / or 48 weeks, and / or 49 weeks, and / or 50 weeks, and / or 51 weeks, and / or 52 weeks, and / or 53 weeks, and / or 54 weeks, and / or 55 weeks, and / or 56 weeks, and / or 57 weeks, and / or 58 weeks, and / or 59 weeks, and / or 60 weeks, respectively, after initiation of treatment.

[0043] In one embodiment of the invention, such a medicament or pharmaceutical formulation is used to extend the time to retreatment and / or to extend the time to vision loss, wherein retreatment with the bispecific antibody comprises: an increase in central retinal thickness (CST) of 50 μm or more (in one embodiment using spectral-domain optical coherence tomography (SD-OCT)); and / or A decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more It is administered in the event of disease activity determined as

[0044] One embodiment of the invention is the use of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for the manufacture of a medicament for use in treating ocular vascular disease, wherein the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0045] One embodiment of the invention is the use of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for the manufacture of a medicament for use in treating ocular vascular disease, wherein the patient has an increase of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, and in one embodiment 15 letters or more) in best corrected visual acuity (BCVA) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score prior to administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0046] One embodiment of the invention is the use of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for the manufacture of a medicament for use in treating ocular vascular disease, wherein the patient exhibits improved visual acuity after (intravitreal) administration of the bispecific VEGF / ANG2 antibody as measured by an increase in best corrected visual acuity (BCVA) of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, and in one embodiment 15 letters or more) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0047] In one embodiment of the invention, the letter increase in the BCVA / ETDRS letter score is measured at 4 weeks, and / or 8 weeks, and / or 12 weeks, and / or 16 weeks, and / or 20 weeks, and / or 24 weeks, respectively, after initiation of treatment.

[0048] In one embodiment of the invention, the letter increase in the BCVA / ETDRS letter scores is measured at 45 weeks, and / or 46 weeks, and / or 47 weeks, and / or 48 weeks, and / or 49 weeks, and / or 50 weeks, and / or 51 weeks, and / or 52 weeks, and / or 53 weeks, and / or 54 weeks, and / or 55 weeks, and / or 56 weeks, and / or 57 weeks, and / or 58 weeks, and / or 59 weeks, and / or 60 weeks, respectively, after initiation of treatment.

[0049] In one embodiment of the invention, the medicament is used to extend the time to retreatment and / or to extend the time to vision loss, wherein retreatment with the bispecific antibody comprises: an increase in central retinal thickness (CST) of 50 μm or more (in one embodiment using spectral-domain optical coherence tomography (SD-OCT)); and / or A decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more It is administered in the event of disease activity determined as

[0050] In one embodiment, the determination of best corrected visual acuity in such a method, use, bispecific antibody (for use), medicament or pharmaceutical preparation is based on an eye chart adapted from the Early Treatment Diabetic Retinopathy Study (ETDRS) protocol and assessed starting at a distance of 4 meters.

[0051] Such methods, uses, bispecific antibodies (for use), medicaments, or pharmaceutical formulations may comprise administering an initial dose ("initiation of treatment"), followed by sequential administration of one or more second therapeutically effective doses of the bispecific antibody, medicament, or pharmaceutical formulation (e.g., administration every 3 to 7 months; in one embodiment initiation of treatment comprises administration every 3 to 4 months, in one embodiment initiation of treatment comprises administration every 4 to 5 months; in one embodiment initiation of treatment comprises administration every 4 to 6 months; in one embodiment initiation of treatment comprises administration at least every 4 months; in one embodiment initiation of treatment comprises administration every 5 to 7 months, in one embodiment initiation of treatment comprises administration every 6 months).

[0052] In one embodiment of the invention, the bispecific antibody, medicament, or pharmaceutical formulation is administered every 10 to 12 weeks (after treatment has begun).

[0053] In one embodiment of the invention, the bispecific antibody, medicament, or pharmaceutical formulation is administered every 11 to 13 weeks (after treatment initiation).

[0054] In one embodiment of the invention, the bispecific antibody, medicament, or pharmaceutical formulation is administered every 12 to 14 weeks (after treatment has begun).

[0055] In one embodiment of the invention, the bispecific antibody, medicament, or pharmaceutical formulation is administered every 13 to 15 weeks (after treatment initiation).

[0056] In one embodiment of the invention, the bispecific antibody, medicament, or pharmaceutical formulation is administered every 14 to 16 weeks (after treatment initiation).

[0057] In one embodiment of the invention, the bispecific antibody, medicament, or pharmaceutical formulation is administered every 10-11 weeks, or every 11-12 weeks, or every 12-13 weeks, or every 13-14 weeks, or every 14-15 weeks, or every 15-16 weeks (each after initiation of treatment).

[0058] In one embodiment of the invention, the bispecific antibody, medicament, or pharmaceutical formulation is administered every 10 weeks, or every 11 weeks, or every 12 weeks, or every 13 weeks, or every 14 weeks, or every 16 weeks (respectively after initiation of treatment).

[0059] In one embodiment of the invention, the bispecific antibody, medicament, or pharmaceutical formulation is administered at a dose of about 5-7 mg (per treatment). In one embodiment, the bispecific antibody is administered at a dose of 6 mg + / - 10% (per treatment). In one embodiment, the bispecific antibody is administered at a dose of about 6 mg (per treatment) (in one embodiment, at a dose of 6 mg (per treatment)).

[0060] In one embodiment of the invention, the bispecific antibody, medicament, or pharmaceutical formulation is administered at a concentration of about 30 mg / ml of bispecific antibody. In one embodiment of the invention, the bispecific antibody, medicament, or pharmaceutical formulation is administered at a concentration of about 120 mg / ml of bispecific antibody.

[0061] The terms "ocular vascular disease" and "vascular eye disease" are used synonymously herein and include, but are not limited to, intraocular neovascular syndromes such as diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, neovascular glaucoma, branch retinal vein occlusion, central retinal vein occlusion, macular degeneration, age-related macular degeneration, retinitis pigmentosa, retinal angiomatous proliferation, macular telangiectasia, ischemic retinopathy, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, and retinal degeneration (Garner, A., Vascular diseases, In: Pathobiology of ocular disease, A dynamic approach, Garner, A., and Klintworth, GK, (eds.), 2nd edition, Marcel Dekker, New York (1994), pp. 1625-1710). As used herein, ocular vascular disease refers to any condition characterized by the altered or uncontrolled growth of new blood vessels and their invasion into ocular tissue structures such as the retina or cornea. In one embodiment, the ocular vascular disease includes wet age-related macular degeneration (wet AMD), neovascular age-related macular degeneration (nAMD), diabetic macular edema (DME), cystoid macular edema (CME), non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), macular edema secondary to central retinal vein occlusion, secondary to hemiretinal vein occlusion, or secondary to branch retinal vein occlusion, retinitis, conjunctivitis, uveitis, choroiditis, ocular vascular disease, and the like. choroidal neovascularization (CNV) secondary to ocular inflammation (including secondary to ocular histoplasmosis or presumed histoplasmosis or choroiditis); myopic choroidal neovascularization (mCNV), and choroidal neovascularization secondary to trauma, retinopathy of prematurity, and rubeosis iridis / neovascular glaucoma, and other ophthalmic diseases (ocular diseases or disorders associated with ocular neovascularization, vascular leakage, and / or retinal edema).Thus, the anti-VEGF / ANG2 bispecific antibodies for the uses and methods described herein are useful for the prevention and treatment of wet age-related macular degeneration, neovascular age-related macular degeneration, cystoid macular edema, diabetic macular edema, nonproliferative diabetic retinopathy, proliferative diabetic retinopathy, and uveitis, and preferably wet age-related macular degeneration, neovascular age-related macular degeneration, and preferably diabetic macular edema, cystoid macular edema, nonproliferative diabetic retinopathy, and proliferative diabetic retinopathy, and also particularly wet age-related macular degeneration. In some embodiments, the ocular vascular disease is selected from the group consisting of wet age-related macular degeneration (wet AMD), neovascular age-related macular degeneration (nAMD), (diabetic) macular edema, retinal vein occlusion, retinopathy of prematurity, and diabetic retinopathy.

[0062] Other diseases / conditions associated with (or that may cause) corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, metabolic disorders due to contact lenses, atopic keratitis, superior limbal keratitis, pterygium, keratitis sicca, Sjogren's syndrome, acne rosacea, fuchtelinosis, syphilis, mycobacterial infection, fatty degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex virus infection, herpes zoster infection, protozoal infection, Kaposi's sarcoma, Mooren's ulcer, Therrien's peripheral corneal degeneration, peripheral keratolysis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis nodosa, trauma, Wegener's granulomatosis, scleritis, Steven-Johnson disease, pemphigoid, radial keratotomy, and corneal transplant rejection.

[0063] Diseases / conditions associated with (or that may cause) retinal / choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid artery occlusion, chronic uveitis / vitreitis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinitis pigmentosa, retinal edema (including macular edema), Eales' disease, Behcet's disease, infections causing retinitis / choroiditis, presumed ocular histoplasmosis, Best's disease, myopia, optic nerve (disk) pits, Stuttgart disease, pars planitis, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications. Other diseases include, but are not limited to, diseases associated with rubeosis (angular neovascularization) and diseases caused by abnormal proliferation of fibrous or vascular tissue, including all forms of proliferative vitreoretinopathy.

[0064] Retinopathy of prematurity (ROP) is an eye disease that affects premature infants. It is thought to be caused by uncontrolled growth of retinal blood vessels, which can lead to scarring and retinal detachment. Retinopathy of prematurity can be mild and resolve spontaneously, but in severe cases, it can lead to blindness. Therefore, all premature babies are at risk for ROP, and very low birth weight is an additional risk factor. Both oxygen toxicity and relative hypoxia can contribute to the development of ROP.

[0065] Macular degeneration is a condition that primarily affects elderly people, in which the center of the eye's internal limiting membrane, known as the macular region of the retina, experiences detachment, atrophy, and sometimes bleeding. This can lead to loss of central vision, which can include an inability to see fine details, read, or recognize faces. According to the American Academy of Ophthalmology, it is the leading cause of central visual field loss (blindness) in people over the age of 50 in the United States today. Some macular dystrophies that occur in younger individuals are sometimes referred to as macular degeneration, a term commonly referred to as age-related macular degeneration (AMD or ARMD).

[0066] As used herein, "age-related macular degeneration (AMD)" refers to a serious eye condition that occurs when the small central portion of the retina, known as the macula, deteriorates. AMD includes wet AMD and neovascular AMD. Wet AMD (also known as wet AMD, or wAMD, or neovascular AMD, or nAMD) is characterized by the proliferation of abnormal blood vessels from the choroid under the macula. This is called choroidal neovascularization. These blood vessels leak blood and fluid down into the retina, causing visual distortion (elevated retina and) straight lines appearing wavy, as well as blind spots and central visual field defects. These abnormal blood vessels eventually form scars, resulting in permanent loss of central vision. Symptoms of age-related macular degeneration include a dark, blurred area in the center of vision and lost or altered color vision. Age-related macular degeneration can be detected by a routine eye examination. One of the most common early signs of macular degeneration is the presence of drusen, tiny yellow deposits and pigmented masses under the retina.

[0067] Progressive age-related macular degeneration, which is responsible for significant visual loss, has two types: dry and wet.Dry progressive age-related macular degeneration, central geographic atrophy, is caused by the atrophy of the retinal pigment epithelium layer under the retina, which causes a decrease in visual acuity through the loss of photoreceptors (rods and cones) in the central part of the eye.Since no treatment is available for this condition, vitamin supplements with high doses of antioxidants, lutein, and zeaxanthin have been demonstrated by the National Eye Institute and others to slow the progression of dry macular degeneration and improve visual acuity in some patients.

[0068] Retinitis pigmentosa (RP) is a group of inherited eye conditions. In the progression of RP symptoms, night blindness typically precedes visual field constriction by years or even decades. Many people with RP do not become legally blind until their 40s or 50s and retain some vision throughout their lives. Others become completely blind from RP, sometimes as early as childhood. The progression of RP varies from case to case. Retinitis pigmentosa is a type of inherited retinal dystrophy, a group of inherited disorders that result in progressive vision loss due to abnormalities in the retinal photoreceptors (rods and cones) or the retinal pigment epithelium (RPE). Affected individuals first experience impaired dark adaptation or night blindness (night blindness), followed by reduced peripheral vision (also known as visual field constriction) and sometimes central visual field loss late in the course of the disease.

[0069] Macular edema occurs when fluid and protein deposits collect above or below the macula, the central region of the retina responsible for good vision, causing the retina to thicken and swell. The swelling can distort a person's central vision because the macula is located near the center of the retina at the back of the eyeball. This area contains densely packed cones that provide sharp, clear central vision, allowing a person to see shape, color, and detail directly in the line of sight. Cystoid macular edema is a type of macular edema that involves the formation of cysts.

[0070] As used herein, "diabetic macular edema" (DME) refers to a serious eye condition that occurs in people with diabetes (type 1 or type 2). Macular edema occurs when blood vessels in the retina leak into the macula, causing fluid and protein deposits to collect above or below the macula, resulting in retinal thickening and swelling (edema). The swelling can distort a person's central vision because the macula is located near the center of the retina at the back of the eyeball. Primary symptoms of diabetic macular edema include, but are not limited to, blurred vision, floaters, decreased contrast vision, double vision, and ultimately decreased visual acuity. The pathology of diabetic macular edema is characterized by a breakdown of the inner blood-retinal barrier, which normally prevents fluid from moving into the retina, allowing fluid to accumulate in the retinal tissue, and the presence of retinal thickening. Diabetic macular edema is currently diagnosed during an eye examination, which consists of a visual acuity test to determine the smallest letter a person can read on a standard eye chart, a dilated pupil to check for signs of disease, an angiogram such as optical coherence tomography (OCT) or fluorescein angiography (FA), and tonometry, a device that measures intraocular pressure. The following tests are also performed to determine treatment: optical coherence tomography (OCT), fluorescein angiography, and color stereo fundus photography. Diabetic macular edema can generally be characterized into two broad categories: focal and diffuse. Focal diabetic macular edema is characterized by separate, distinct, specific areas of leakage within the macula with sufficient blood flow within the macula. Diffuse diabetic macular edema results from leakage of the entire capillary bed surrounding the macula, resulting from breakdown of the inner blood-retinal barrier of the eye. In addition to focal and diffuse diabetic macular edema, diabetic macular edema is also classified based on clinical examination findings into clinically significant macular edema (CSME), clinically insignificant diabetic macular edema, and diabetic macular edema involving the fovea (CSME-CI). The present invention includes methods for treating the above categories of diabetic macular edema.

[0071] Best-corrected visual acuity (BCVA) was determined using a method adapted from the Early Treatment Diabetic Retinopathy Study [ETDRS] protocol at a distance of 4 meters (using an ETDRS-like chart) to generate each letter score.

[0072] Disease activity is determined, for example, via a decrease in BCVA / ETDR letter scores and / or via macular thickening by spectral-domain optical coherence tomography (SD-OCT), including the center of the macula as central foveal thickness (CST) (also known as central subfoveal thickness). In one preferred embodiment, central foveal thickness (CST) is determined using spectral-domain optical coherence tomography (SD-OCT). In one preferred embodiment, foveal region retinal thickness is measured by spectral-domain optical coherence tomography (SD-OCT) using a Spectralis™ instrument; in one preferred embodiment, foveal region retinal thickness is measured by spectral-domain optical coherence tomography (SD-OCT) using a Cirrus™ instrument; in one embodiment, foveal region retinal thickness is measured by spectral-domain optical coherence tomography (SD-OCT) using a Topcon™ instrument; in one embodiment, foveal region retinal thickness is measured by spectral-domain optical coherence tomography (SD-OCT) using an Optvue™ instrument. As used herein, the term "patient suffering from" refers to a human who exhibits one or more symptoms or signs of an ocular vascular disease as described herein and / or has been diagnosed with an ocular vascular disease as described herein. The term "patient suffering from" may also include, for example, a subject who, prior to treatment, exhibits (or has exhibited) one or more symptoms of ocular vascular disease, such as retinal vascularization, neovascularization, vascular leakage, foveal retinal thickening, hard, yellow foveal exudate with adjacent retinal thickening, and retinal thickening of at least one disc area (any part of which is within one disc diameter of the fovea), blurred vision, floaters, decreased contrast vision, diplopia, and eventual decreased visual acuity.

[0073] As used herein, the term "patient suffering from" may also include a subset of a population that may be more susceptible to diabetic macular edema or age-related macular degeneration, or that may exhibit elevated levels of biomarkers associated with diabetic macular edema or age-related macular degeneration. For example, a "subject in need thereof" may include a subject who has had diabetes for 10 years or more, or who frequently exhibits elevated blood glucose levels or elevated fasting blood glucose levels. In certain embodiments, the term "patient suffering from" includes subjects who have or have been diagnosed with diabetes before or at the time of administration of a bispecific anti-VEGF / ANG2 antibody. In certain embodiments, the term "patient suffering from" includes subjects who are over 50 years of age before or at the time of administration of an anti-VEGF / ANG2 antibody. In some embodiments, the term "patient suffering from" includes subjects who are smokers or who have high blood pressure or high cholesterol.

[0074] The present invention includes methods or bispecific antibodies (for use), medicaments or pharmaceutical formulations for treating, preventing, or reducing the severity of ocular vascular disease comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific anti-VEGF / ANG2 antibody (or a medicament or pharmaceutical formulation comprising a bispecific anti-VEGF / ANG2 antibody), wherein the bispecific antibody, medicament, or pharmaceutical formulation comprising such a bispecific anti-VEGF / ANG2 antibody is administered (intravitreally) to the subject in multiple doses, for example, as part of a particular therapeutic dosing regimen.

[0075] One embodiment of the invention is a method of treatment, use, bispecific antibody (for use), medicament, or pharmaceutical formulation as described herein, wherein the patient suffering from ocular vascular disease has not previously been treated (e.g., is treatment naive) with an anti-VEGF antibody (e.g., as monotherapy).

[0076] One embodiment of the invention is a method of treatment, use, bispecific antibody (for use), medicament, or pharmaceutical formulation as described herein, wherein the patient suffering from ocular vascular disease has previously been treated with an anti-VEGF antibody (e.g., as monotherapy).

[0077] One embodiment of the invention is a method of treatment, use, bispecific antibody (for use), medicament, or pharmaceutical formulation as described herein, wherein the ocular vascular disease is diabetic macular edema, and wherein treating patients with diabetic macular edema comprises a consistent every 8 weeks (Q8W) dosing regimen following initiation of treatment (in one embodiment, initiation of treatment comprises dosing every 5-7 months; in one embodiment, initiation of treatment comprises dosing every 6 months).

[0078] One embodiment of the invention is a method of treatment, use, bispecific antibody (for use), medicament, or pharmaceutical formulation as described herein, wherein the ocular vascular disease is diabetic macular edema, and wherein treating patients with diabetic macular edema comprises a consistent every 12 weeks (Q12W) dosing regimen after initiation of treatment (in one embodiment, initiation of treatment comprises dosing every 5-7 months; in one embodiment, initiation of treatment comprises dosing every 6 months). In one embodiment, after initiation of treatment, the consistent Q12W dosing regimen is preceded by an initial cycle of Q8W with one dose.

[0079] One embodiment of the invention is a method of treatment, use, bispecific antibody (for use), medicament, or pharmaceutical formulation as described herein, wherein the ocular vascular disease is diabetic macular edema, and wherein treating a patient with diabetic macular edema comprises a dosing regimen in which, after initiation of treatment, the dosing interval is extended in the event of stable disease absence, or the interval is shortened in the event of disease activity (in one embodiment, initiation of treatment comprises dosing every 3-7 months; in one embodiment, initiation of treatment comprises dosing every 3-5 months; in one embodiment, initiation of treatment comprises dosing at least every 4 months; in one embodiment, initiation of treatment comprises dosing every 4-6 months). In one embodiment, such a dosing regimen comprises the patient receiving Q4W or Q8W or Q12W or Q16W dosing, depending on the patient's disease status. In one embodiment, stable disease absence is -Central retinal thickness (CST) increased by less than 50 μm, -Best corrected visual acuity (BCVA / ETDRS) reduced by less than 5 letters and disease activity is determined as -central retinal thickness (CST) increased by 50 μm or more, -Best corrected visual acuity (BCVA / ETDRS) reduced by 5 letters or more is determined as follows.

[0080] In one embodiment, stable disease freedom is defined as: - Central retinal thickness (CST) of less than about 300 μm (in one embodiment, less than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, less than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). and disease activity is determined as - Central retinal thickness (CST) of greater than about 300 μm (in one embodiment, greater than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, greater than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). is determined as follows.

[0081] One embodiment of the invention is a method of treatment, use, bispecific antibody (for use), medicament, or pharmaceutical formulation as described herein, wherein the ocular vascular disease is age-related macular degeneration (in one embodiment, wet age-related macular degeneration), and wherein treating a patient with age-related macular degeneration (in one embodiment, wet age-related macular degeneration) comprises a dosing regimen in which, after initiation of treatment, the dosing interval is extended in the event of stable disease absence, or the interval is shortened in the event of disease activity (in one embodiment, initiation of treatment comprises dosing every 3-7 months; in one embodiment, initiation of treatment comprises dosing every 3-5 months; in one embodiment, initiation of treatment comprises dosing at least every 4 months; in one embodiment, initiation of treatment comprises dosing every 4-6 months). In one embodiment, such a dosing regimen comprises the patient receiving Q4W or Q8W or Q12W or Q16W dosing, depending on the patient's disease status. In one embodiment, stable disease absence is -Central retinal thickness (CST) increased by less than 50 μm; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by less than 5 letters and disease activity is determined as -Central retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by 5 letters or more is determined as follows.

[0082] In one embodiment, stable disease freedom is defined as: - Central retinal thickness (CST) of less than about 300 μm (in one embodiment, less than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, less than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). and disease activity is determined as - Central retinal thickness (CST) of greater than about 300 μm (in one embodiment, greater than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, greater than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). is determined as follows.

[0083] In one embodiment, the ocular vascular disease in such method, use, bispecific antibody (for use), medicament or pharmaceutical preparation is wet age-related macular degeneration (neovascular age-related macular degeneration).

[0084] As used herein, "antibody" refers to a binding protein that comprises an antigen-binding site. As used herein, the term "binding site" or "antigen-binding site" refers to the region(s) of an antibody molecule to which a ligand actually binds. The term "antigen-binding site" comprises an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) (a VH / VL pair).

[0085] Antibody specificity refers to the selective recognition of an antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific.

[0086] A "bispecific antibody" according to the present invention is an antibody with two different antigen-binding specificities. The antibody of the present invention is specific for two different antigens: VEGF as the first antigen and ANG-2 as the second antigen.

[0087] As used herein, the term "monospecific" antibody refers to an antibody that has one or more binding sites, each of which binds to the same epitope of the same antigen.

[0088] The term "valent" as used within this application refers to the presence of a specific number of binding sites within an antibody molecule. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, within an antibody molecule. Bispecific antibodies according to the invention are preferably "bivalent."

[0089] As used herein, the terms "bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2)," "bispecific anti-VEGF / ANG2 antibody," and "bispecific <vegf ang2>The terms "antibody" and "antibody" are synonymous and refer to antibodies that have at least two different antigen binding sites: a first antigen binding site that binds to VEGF and a second antigen binding site that binds to ANG2.

[0090] Bispecific anti-VEGF / ANG2 antibodies are described, for example, in WO 2010040508, WO 2011 / 117329, WO 2012 / 131078, WO 2015 / 083978, WO 2017 / 197199, and WO 2014 / 009465. WO 2014 / 009465 describes bispecific anti-VEGF / ANG2 antibodies specifically designed for the treatment of ocular vascular disease. The bispecific anti-VEGF / ANG2 antibodies of WO 2014 / 009465 (which is incorporated herein in its entirety) are particularly useful for the treatment and treatment regimens of ocular vascular disease as described herein.

[0091] In one embodiment, the bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) is a bispecific anti-VEGF / ANG2 antibody comprising a first antigen-binding site that specifically binds human VEGF and a second antigen-binding site that specifically binds human ANG-2, wherein i) the first antigen-binding site that specifically binds to VEGF comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO: 3, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO: 5, and a CDR1L region of SEQ ID NO: 6; ii) the second antigen-binding site that specifically binds to ANG-2 comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 9, a CDR2H region of SEQ ID NO: 10, and a CDR1H region of SEQ ID NO: 11, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ ID NO: 14; iii) The bispecific antibody comprises a heavy chain constant region of the human IgG1 subclass containing the mutations I253A, H310A, and H435A and the mutations L234A, L235A, and P329G (numbering according to the EU index of Kabat).

[0092] In one embodiment, such a bispecific anti-VEGF / ANG2 antibody is bivalent.

[0093] In one embodiment, such a bispecific anti-VEGF / ANG2 antibody comprises: i) the first antigen-binding site that specifically binds to VEGF comprises a heavy chain variable domain VH having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain VL having the amino acid sequence of SEQ ID NO: 8; ii) The second antigen-binding site that specifically binds to ANG-2 is characterized by comprising the amino acid sequence of SEQ ID NO: 15 as the heavy chain variable domain VH and the amino acid sequence of SEQ ID NO: 16 as the light chain variable domain VL.

[0094] In one embodiment of the invention, such a bispecific and bivalent antibody according to the invention comprises: a) the heavy and light chains of a first full-length antibody that specifically binds to VEGF; b) Modified heavy and light chains of a second full-length antibody that specifically binds to ANG-2 (where the constant domains CL and CH1 are swapped with each other). The present invention is characterized by comprising:

[0095] This bispecific, bivalent antibody format for a bispecific antibody that specifically binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) (containing a knob-into-hole modified CH3 domain) is described in WO 2009 / 080253. Antibodies based on this bispecific, bivalent antibody format are termed cross-linked monoclonal antibodies (CrossMAbs).

[0096] In one embodiment, such a bispecific, bivalent anti-VEGF / ANG2 antibody comprises: a) the amino acid sequence of SEQ ID NO: 17 as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO: 18 as the light chain of the first full-length antibody; and b) the amino acid sequence of SEQ ID NO: 19 as the modified heavy chain of the second full-length antibody, and the amino acid sequence of SEQ ID NO: 20 as the modified light chain of the second full-length antibody. The present invention is characterized by comprising:

[0097] In one embodiment, such a bispecific, bivalent anti-VEGF / ANG2 antibody is characterized in that it comprises the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In one preferred embodiment, the bispecific, bivalent anti-VEGF / ANG2 antibody is faricimab.

[0098] Thus, one embodiment of the present invention is a bispecific, bivalent antibody comprising a first antigen-binding site that specifically binds human VEGF and a second antigen-binding site that specifically binds human ANG-2, characterized in that the first antigen-binding site comprises the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In one preferred embodiment, the bispecific, bivalent anti-VEGF / ANG2 antibody is faricimab.

[0099] In one embodiment, the CH3 domains of the bispecific, bivalent antibodies of the invention are modified by the "knob-into-hole" technique, as described, for example, in WO 96 / 027011; Ridgway JB, et al., Protein Eng 9 (1996) 617-621; and Merchant, AM, et al., Nat Biotechnol 16 (1998) 677-681. In this method, the interaction surfaces of the two CH3 domains are modified to increase heterodimerization of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be a "knob," while the other is a "hole." The introduction of disulfide bridges stabilizes the heterodimer (Merchant, AM, et al., Nature Biotech 16 (1998) 677-681; Atwell, S., et al. J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

[0100] In a preferred embodiment of the invention, the bispecific anti-VEGF / ANG2 antibody according to the invention comprises: the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain each contact at an interface that comprises the natural interface between antibody CH3 domains; wherein the interface is modified to promote the formation of a bispecific antibody, wherein the modification comprises: a) the CH3 domain of one heavy chain is modified; This allows the CH3 domain of one heavy chain in the bispecific antibody to contact the CH3 domain of the other heavy chain in its native interface. an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protrusion in the interface of the CH3 domain of one heavy chain, which can be positioned in a cavity in the interface of the CH3 domain of the other heavy chain; and b) the CH3 domain of the other heavy chain is modified; This results in the formation of a second CH3 domain in the native interface that contacts the native interface of the first CH3 domain in the bispecific antibody. The amino acid residues are replaced with amino acid residues having smaller side chain volumes, thereby creating a cavity in the interface of the second CH3 domain into which the protrusion in the interface of the first CH3 domain can be positioned. It is characterized by:

[0101] Thus, bispecific anti-VEGF / ANG2 antibodies for use as described herein preferably comprise: the CH3 domain of the heavy chain of the full-length antibody of a) and the CH3 domain of the heavy chain of the full-length antibody of b) each contact at an interface that contains an alteration within the original interface between the CH3 domains of the antibody; where: i) in the CH3 domain of one heavy chain, The amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protrusion in the interface of the CH3 domain of one heavy chain, which can locate in a cavity in the interface of the CH3 domain of the other heavy chain, and ii) in the CH3 domain of the other heavy chain, The amino acid residues are replaced with amino acid residues having smaller side chain volumes, which creates a cavity in the interface of the second CH3 domain into which the protrusion in the interface of the first CH3 domain can be positioned. It is characterized by:

[0102] Preferably, said amino acid residues with larger side chain volumes are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).

[0103] Preferably, said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).

[0104] In one embodiment of the present invention, both CH3 domains are further modified by the introduction of cysteine ​​(C) as an amino acid at the corresponding position in each CH3 domain, thereby allowing the formation of a disulfide bridge between both CH3 domains.

[0105] In one embodiment, the bispecific antibody comprises a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, Y407V mutations in the CH3 domain of the "hole chain." For example, an additional interchain disulfide bridge between the CH3 domains may be used by introducing a S354C mutation in one CH3 domain and a Y349C mutation in the other CH3 domain (Merchant, AM, et al., Nature Biotech 16 (1998) 677-681).

[0106] In another preferred embodiment, the bispecific antibody comprises the mutations S354C and T366W in one of the two CH3 domains and the mutations Y349C, T366S, L368A, Y407V in the other of the two CH3 domains. In another preferred embodiment, the bispecific antibody comprises the mutations Y349C, T366W in one of the two CH3 domains and the mutations S354C, T366S, L368A, Y407V in the other of the two CH3 domains (the additional Y349C or S354C mutation in one CH3 domain and the additional S354C or Y349C mutation in the other CH3 domain form an interchain disulfide bridge) (numbering always according to the EU index of Kabat) (Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0107] Other techniques for CH3 modifications that enhance heterodimerization are contemplated as alternatives of the present invention and are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, and WO 2013 / 096291.

[0108] In one embodiment, the heterodimerization approach described in EP 1 870 459 A1 is used instead. This approach is based on the introduction of oppositely charged charged amino acid substitutions / mutations at specific amino acid positions in the interface between both heavy chains in the CH3 / CH3 domains. One preferred embodiment for such multispecific antibodies is the mutation of amino acids R409D and K370E in the CH3 domain of one heavy chain of the multispecific antibody and the mutation of amino acids D399K and E357K in the CH3 domain of the other heavy chain (numbering according to Kabat EU index).

[0109] In another embodiment, the multispecific antibody comprises a mutation of the amino acid T366W in the CH3 domain of the "knob chain" and a mutation of the amino acids T366S, L368A and Y407V in the CH3 domain of the "hole chain", and further a mutation of the amino acids R409D and K370E in the CH3 domain of the "knob chain" and a mutation of the amino acids D399K and E357K in the CH3 domain of the "hole chain".

[0110] In one embodiment, the heterodimerization approach described in WO 2013 / 157953 is used instead. In one embodiment, the CH3 domain of one heavy chain comprises a mutation of amino acid T366K, and the CH3 domain of the other heavy chain comprises a mutation of amino acid L351D. In a further embodiment, the CH3 domain of one heavy chain further comprises a mutation of amino acid L351K.

[0111] In a further embodiment, the CH3 domain of the other heavy chain further comprises an amino acid mutation selected from Y349E, Y349D, and L368E (in one embodiment L368E).

[0112] In one embodiment, the heterodimerization approach described in WO 2012 / 058768 is used instead. In one embodiment, the CH3 domain of one heavy chain comprises amino acid mutations L351Y and Y407A, and the CH3 domain of the other heavy chain comprises amino acid mutations T366A and K409F. In a further embodiment, the CH3 domain of the other heavy chain further comprises amino acid mutations at positions T411, D399, S400, F405, N390, or K392. In one embodiment, the amino acid mutations are: a) T411N, T411R, T411Q, T411K, T411D, T411E and 411W, b) D399R, D399W, D399Y and D399K, c) S400E, S400D, S400R and S400K, d) F405I, F405M, F405T, F405S, F405V and F405W, e) N390R, N390K and N390D, f) K392V, K392M, K392R, K392L, K392F and K392E is selected from the group consisting of:

[0113] In a further embodiment, the CH3 domain of one heavy chain comprises mutations of amino acids L351Y and Y407A, and the CH3 domain of the other heavy chain comprises mutations of amino acids T366V and K409F. In a further embodiment, the CH3 domain of one heavy chain comprises mutations of amino acids Y407A, and the CH3 domain of the other heavy chain comprises mutations of amino acids T366A and K409F. In a further embodiment, the CH3 domain of the other heavy chain further comprises mutations of amino acids K392E, T411E, D399R, and S400R.

[0114] In one embodiment, the heterodimerization approach described in WO 2011 / 143545 is used instead, hi one embodiment, the amino acid modifications according to WO 2011 / 143545 are introduced at positions selected from the group consisting of 368 and 409 in the CH3 domain of the heavy chain.

[0115] In one embodiment, the heterodimerization approach described in WO 2011 / 090762, which also uses the knob-into-hole approach described above, is used instead. In one embodiment, the CH3 domain of one heavy chain contains a mutation of amino acid T366W, and the CH3 domain of the other heavy chain contains a mutation of amino acid Y407A. In one embodiment, the CH3 domain of one heavy chain contains a mutation of amino acid T366Y, and the CH3 domain of the other heavy chain contains a mutation of amino acid Y407T.

[0116] In one embodiment, the multispecific antibody is of the IgG2 isotype and the heterodimerization approach described in WO 2010 / 129304 is used instead.

[0117] In one embodiment, the heterodimerization approach described in WO 2009 / 089004 is used instead. In one embodiment, the CH3 domain of one heavy chain comprises a substitution of the amino acid K392 or N392 with a negatively charged amino acid (in one embodiment, glutamic acid (E) or aspartic acid (D); in a further embodiment, a K392D or N392D mutation), and the CH3 domain of the other heavy chain comprises a substitution of the amino acid D399, E356, D356, or E357 with a positively charged amino acid (in one embodiment, lysine (K) or arginine (R); in a further embodiment, a D399K, E356K, D356K, or E357K substitution; in an even further embodiment, a D399K or E356K mutation). In a further embodiment, the CH3 domain of one heavy chain further comprises a substitution of the amino acid at K409 or R409 with a negatively charged amino acid (in one embodiment, glutamic acid (E) or aspartic acid (E); in a further embodiment, a K409D or R409D mutation). In a further embodiment, the CH3 domain of one heavy chain also or alternatively comprises a substitution of the amino acid at K439 and / or K370 with a negatively charged amino acid (in one embodiment, glutamic acid (E) or aspartic acid (D)).

[0118] In one embodiment, the heterodimerization approach described in WO 2007 / 147901 is used instead. In one embodiment, the CH3 domain of one heavy chain contains the following mutations: amino acids K253E, D282K, and K322D, and the CH3 domain of the other heavy chain contains the following mutations: amino acids D239K, E240K, and K292D.

[0119] In one embodiment, the heterodimerization approach described in WO 2007 / 110205 is used instead.

[0120] In one embodiment, the bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) is a bispecific anti-VEGF / ANG2 antibody comprising a first antigen-binding site that specifically binds human VEGF and a second antigen-binding site that specifically binds human ANG-2, wherein: i) the first antigen-binding site that specifically binds to VEGF comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO: 3, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO: 5, and a CDR1L region of SEQ ID NO: 6; ii) the second antigen-binding site that specifically binds to ANG-2 comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 9, a CDR2H region of SEQ ID NO: 10, and a CDR1H region of SEQ ID NO: 11, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ ID NO: 14; iii) the bispecific antibody comprises a heavy chain constant region of the human IgG1 subclass containing the mutations I253A, H310A, and H435A and the mutations L234A, L235A, and P329G (numbering according to the EU index of Kabat); iv) The heavy chain constant region contains a T366W mutation in one CH3 domain and T366S, L368A, Y407V mutations in the other CH3 domain (numbering according to EU index of Kabat).

[0121] In one embodiment, the bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) is a bispecific anti-VEGF / ANG2 antibody comprising a first antigen-binding site that specifically binds human VEGF and a second antigen-binding site that specifically binds human ANG-2, wherein i) the first antigen-binding site that specifically binds to VEGF comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO: 3, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO: 5, and a CDR1L region of SEQ ID NO: 6; ii) the second antigen-binding site that specifically binds to ANG-2 comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 9, a CDR2H region of SEQ ID NO: 10, and a CDR1H region of SEQ ID NO: 11, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ ID NO: 14; iii) the bispecific antibody comprises a heavy chain constant region of the human IgG1 subclass containing the mutations I253A, H310A, and H435A and the mutations L234A, L235A, and P329G (numbering according to the EU index of Kabat); iv) The heavy chain constant region contains S354C and T366W mutations in one CH3 domain and Y349C, T366S, L368A, and Y407V mutations in the other CH3 domain (numbering according to EU index of Kabat).

[0122] In one embodiment, such a bispecific anti-VEGF / ANG2 antibody is bivalent.

[0123] In one embodiment, such a bispecific anti-VEGF / ANG2 antibody comprises: i) the first antigen-binding site that specifically binds to VEGF comprises a heavy chain variable domain VH having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain VL having the amino acid sequence of SEQ ID NO: 8; ii) the second antigen-binding site that specifically binds to ANG-2 comprises the amino acid sequence of SEQ ID NO: 15 as the heavy chain variable domain VH and the amino acid sequence of SEQ ID NO: 16 as the light chain variable domain VL; It is characterized by:

[0124] In one embodiment of the invention, such a bispecific and bivalent antibody according to the invention comprises: a) the heavy and light chains of a first full-length antibody that specifically binds to VEGF; b) Modified heavy and light chains of a second full-length antibody that specifically binds to ANG-2 (where the constant domains CL and CH1 are swapped with each other). The present invention is characterized by comprising:

[0125] As used herein, the term "VEGF" refers to human vascular endothelial growth factor (VEGF / VEGF-A), which is a 165-amino acid human vascular endothelial growth factor (amino acids 27-191 of the precursor sequence of human VEGF165: SEQ ID NO: 24; amino acids 1-26 represent a signal peptide), and to the VEGF-A protein described in Leung, DW, et al., Science 246 (1989) 1306-9; Houck et al., Mol. Endocrin. 5 (1991) 1806-1814; Keck, PJ, et al., Science 246 (1989) 1309-12 and Connolly, DT, et al., J. Biol. Chem. 264 (1989) It refers to the naturally occurring allelic and processed forms of vascular endothelial growth factor, along with the related 121, 189, and 206 isoforms of such growth factors, as described in US Pat. No. 6,200,777, ... VEGF is involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with tumors and intraocular disorders (Ferrara, N., et al., Endocr. Rev. 18 (1997) 4-25; Berkman, RA, et al., J. Clin. Invest. 91 (1993) 153-159; Brown, LF, et al., Human Pathol. 26 (1995) 86-91; Brown, LF, et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J., et al., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, HF, et al., Am. J. Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoprotein isolated from several sources and includes several isoforms. VEGF exhibits highly specific mitogenic activity against endothelial cells. VEGF antagonists / inhibitors inhibit the binding of VEGF to its receptor, VEGFR. Known VEGF antagonists / inhibitors include bispecific anti-VEGF / ANG2 antibodies, such as those described in WO 2014 / 009465.

[0126] The term "ANG-2" as used herein refers to human angiopoietin-2 (ANG-2) (also abbreviated as ANGPT2 or ANG2) (SEQ ID NO: 25), as described, for example, in Maisonpierre, PC, et al., Science 277 (1997) 55-60 and Cheung, AH, et al., Genomics 48 (1998) 389-91. Angiopoietin-1 (SEQ ID NO: 26) and angiopoietin-2 were discovered as ligands for Tie, a tyrosine kinase family selectively expressed in vascular endothelium (Yancopoulos, GD, et al., Nature 407 (2000) 242-48). There are currently four defined members of the angiopoietin family. Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent widely diverged counterparts of the same gene locus in mice and humans (Kim, I., et al., FEBS Let, 443 (1999) 353-56; Kim, I., et al., J Biol Chem 274 (1999) 26523-28). ANG-1 and ANG-2 were initially identified as agonists and antagonists, respectively, in tissue culture experiments (for ANG-1, see Davis, S., et al., Cell 87 (1996) 1161-69; for ANG-2, see Maisonpierre, PC, et al., Science 277 (1997) 55-60). All known angiopoietins primarily bind to their receptor, TIE2 (SEQ ID NO: 27), and both Ang-1 and Ang-2 bind to TIE2 with an affinity (Kd) of 3 nM (Maisonpierre, PC, et al., Science 277 (1997) 55-60). ANG2 antagonists / inhibitors inhibit the binding of ANG2 to its receptor, TIE2. Known ANG2 antagonists / inhibitors include bispecific anti-VEGF / ANG2 antibodies, such as those described in WO 2014 / 009465.

[0127] The antigen-binding site of the bispecific antibodies of the present invention contains six complementarity-determining regions (CDRs), which contribute to varying degrees of affinity of the binding site for the antigen. There are three heavy chain variable domain CDRs (CDRH1, CDRH2, and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2, and CDRL3). The extent of the CDRs and framework regions (FRs) was determined by comparison with a compiled amino acid sequence database, and such regions are defined by the degree of variability between sequences.

[0128] The antibodies of the present invention comprise immunoglobulin constant regions derived from one or more immunoglobulin classes of human origin, including the IgG, IgM, IgA, IgD, and IgE classes, and in the case of IgG and IgA, their subclasses IgG1 and IgG4.

[0129] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.

[0130] The term "chimeric antibody" refers to an antibody, usually prepared by recombinant DNA technology, comprising a variable region, i.e., a binding region, derived from one source or species and at least a portion of a constant region derived from a different source or species. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Another preferred form of "chimeric antibody" encompassed by the present invention is one in which the constant region has been modified or altered from that of the original antibody to confer the properties according to the invention, particularly with respect to binding to C1q and / or Fc receptors (FcRs). Such chimeric antibodies are also referred to as "class-switched antibodies." Chimeric antibodies are the product of immunoglobulin gene expression, comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques, which are well known in the art. See, for example, Morrison, SL, et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.

[0131] The term "humanized antibody" refers to an antibody in which the framework or "complementarity-determining regions" (CDRs) have been modified to contain CDRs from an immunoglobulin with different specificity compared to the parent immunoglobulin. In a preferred embodiment, murine CDRs are grafted into the framework regions of a human antibody to prepare a "humanized antibody." See, for example, Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, MS, et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those exhibiting antigen-recognition sequences as described above for chimeric antibodies. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant regions have been modified or changed from those of the original antibody to thereby confer the properties according to the invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0132] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the state of the art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies or selected human antibodies in the absence of endogenous immunoglobulin production. Introduction of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Brueggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodies may also be generated in phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole, A., et al. and Boerner, P., et al. are also available for preparing human monoclonal antibodies (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, AL, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95).As already described for the chimeric and humanized antibodies according to the invention, the term "human antibody" as used herein also includes such antibodies whose constant region has been modified, for example by "class switching", i.e. by changing or mutating the Fc part (e.g. from IgG1 to IgG4 and / or IgG1 / IgG4 mutations), in order to generate the properties according to the invention, in particular with regard to binding to C1q and / or to FcR.

[0133] As used herein, the term "recombinant antibody" is intended to include all human antibodies that have been prepared, expressed, created, or isolated by recombinant means, e.g., antibodies isolated from host cells such as NSO or CHO cells, or from animals (e.g., mice) transgenic for human immunoglobulin genes, or expressed using a recombinant expression vector transfected into a host cell. Such recombinant antibodies comprise variable and constant regions in rearranged forms. Recombinant antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist in the human antibody germline repertoire in vivo.

[0134] As used herein, the term "variable domain" (light chain variable domain (VL) and heavy chain variable domain (VH)) refers to each pair of light and heavy chains that are directly involved in binding the antibody to the antigen. Human light and heavy chain variable domains have the same general structure, and each domain contains four framework (FR) regions whose sequences are extensively conserved and connected by three "hypervariable regions" (i.e., complementarity-determining regions (CDRs)). The framework regions adopt a β-sheet structure, and the CDRs may form loops connecting the β-sheet structure. The CDRs within each chain are held in their three-dimensional structure by the framework regions and, together with the CDRs of the other chain, form the antigen-binding site. The CDR3 regions of the heavy and light chains of an antibody play a particularly important role in determining the binding specificity / affinity of the antibodies described herein and therefore provide a further object of the present invention.

[0135] As used herein, the term "hypervariable region" or "antigen-binding portion of an antibody" refers to the amino acid residues of an antibody that are involved in antigen binding. The hypervariable region contains amino acid residues from the "complementarity-determining regions" or "CDRs." The "framework" or "FR" regions are the variable domain regions other than the hypervariable region residues as defined herein. Thus, the light and heavy chains of an antibody contain, from N- to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs on each chain are separated by such framework amino acids. In particular, CDR3 of the heavy chain is the region most responsible for antigen binding. The CDR and FR regions are determined according to the standard definitions in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0136] The term "full-length antibody" refers to an antibody consisting of two "full-length antibody heavy chains" and two "full-length antibody light chains." A "full-length antibody heavy chain" is a polypeptide consisting of, from N- to C-terminus of the antibody, a heavy chain variable domain (VH), antibody heavy chain constant domain 1 (C1), antibody hinge region (HR), antibody heavy chain constant domain 2 (CH2), and antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and optionally, in the case of subclass IgE antibodies, antibody heavy chain constant domain 4 (CH4). Preferably, a "full-length antibody heavy chain" is a polypeptide consisting of, from N- to C-terminus of the antibody, VH, CH1, HR, CH2, and CH3. A "full-length antibody light chain" is a polypeptide consisting of, from N- to C-terminus of the antibody, a light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated as VL-CL. The antibody light chain constant domain (CL) can be κ (kappa) or λ (lambda). Two full-length antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL and CH1 domains and between the hinge regions of the full-length antibody heavy chains. Typical full-length antibody examples are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE. Full-length antibodies according to the present invention may be derived from a single species, e.g., human, or they may be chimerized or humanized. Full-length antibodies according to the present invention comprise two antigen-binding sites, each formed by a VH / VL pair, both of which specifically bind to the same antigen. The C-terminus of the heavy or light chain of the full-length antibody refers to the last amino acid at the C-terminus of the heavy or light chain. The N-terminus of the heavy or light chain of the full-length antibody refers to the last amino acid at the N-terminus of the heavy or light chain.

[0137] The term "constant region" as used within this application refers to the sum of the domains of an antibody other than the variable region. The constant region is not directly involved in binding to an antigen but exhibits various effector functions. Depending on the amino acid sequence of the constant region of their heavy chain, antibodies are classified into classes IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses, such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The light chain constant regions that can be found in all five antibody classes are called κ (kappa) and λ (lambda).

[0138] The term "constant region derived from human origin" or "human constant region" as used in the present application refers to the heavy chain constant region and / or the light chain constant region kappa or lambda of a human antibody of the subclass IgG1, IgG2, IgG3, or IgG4. Such constant regions are well known in the state of the art and are described, for example, by Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson, G., and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). For numbering of positions and mutations within the application, the EU numbering system (EU index) as set forth in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used and is referred to as "Kabat EU index numbering."

[0139] In one embodiment, the bispecific antibodies according to the invention have a constant region of the human IgG1 subclass (derived from the human IgG1 subclass), however, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and the C-terminal lysine (Lys447) of the Fc region may or may not be present.

[0140] In one embodiment, a bispecific antibody as described herein is of the IgG1 isotype / subclass and comprises the heavy chain constant domain of SEQ ID NO: 23, or the heavy chain amino acid sequence of SEQ ID NO: 17 and the constant portion of the heavy chain amino acid sequence of SEQ ID NO: 18. In one embodiment, a C-terminal glycine (Gly446) is further present. In one embodiment, a C-terminal glycine (Gly446) and a C-terminal lysine (Lys447) are further present.

[0141] Unless otherwise specified herein, the numbering of amino acid residues in the constant region is according to the EU numbering system, also known as the EU index of Kabat, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0142] In one embodiment, the bispecific antibodies according to the invention are of the human IgG1 subclass with the mutations L234A (Leu235Ala), L235A (Leu234Ala) and P329G (Pro329Gly). Such antibodies exhibit reduced FcR binding (in particular, they no longer exhibit any binding to FcRγI, FcRγII and FcRγIII). This is particularly useful for reducing possible side effects, such as thrombosis (Meyer, T., et al., J. Thromb. Haemost. 7 (2009) 171-81).

[0143] While the previously described Pro329Ala mutation eliminates only two-thirds of the FcγRIIIa sandwich interaction, the Pro329Gly mutation in the antibodies described herein completely confers binding of the Fc portion to FcγRIII. This is particularly useful because binding to FcγRIII leads to cell death and is involved in ADCC (antibody-dependent cellular cytotoxicity), which can be beneficial in the treatment of cancer diseases, but can also cause serious side effects in antibody-based treatments of other vascular or immunological diseases. Thus, antibodies described herein of the IgG1 subclass with the mutations L234A, L235A, and P329G and the IgG4 subclass with the mutations S228P, L235E, and P329G are particularly useful because they no longer exhibit any binding to FcRγI, FcRγII, and FcRγIII.

[0144] An "effective amount" of an agent, eg, a pharmaceutical formulation or a bispecific anti-VEGF / ANG2 antibody, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0145] In one embodiment of the invention, a bispecific antibody, medicament, or pharmaceutical formulation as described herein is administered via intravitreal application, e.g., via intravitreal injection (administered "intravitreally"). This can be performed according to standard procedures known in the art. See, e.g., Ritter et al., J. Clin. Invest. 116 (2006) 3266-76; Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25 (1999) 196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-5.

[0146] In some embodiments, therapeutic kits of the invention may contain one or more doses of the described bispecific antibody present in a medicament or pharmaceutical formulation, equipment suitable for intravitreal injection of the medicament or pharmaceutical formulation, and instructions detailing the appropriate target and protocol for performing the injection. In these embodiments, the medicament or pharmaceutical formulation is typically administered to a subject in need of treatment via intravitreal injection. This can be performed according to standard procedures known in the art. See, e.g., Ritter et al., J. Clin. Invest. 116 (2006) 3266-76; Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25 (1999) 196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-5.

[0147] Regardless of the route of administration selected, the bispecific antibodies as described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0148] [Table 1] TIFF2023099094000003.tif118161

[0149] The following are embodiments of the present invention: 1. A bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in treating ocular vascular disease, wherein the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0150] 2A. A bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in treating a patient suffering from ocular vascular disease, wherein the patient has an increase of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, 15 letters or more) in best corrected visual acuity (BCVA) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score prior to administration of the bispecific VEGF / ANG2 antibody.

[0151] 2B. A bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in treating a patient suffering from ocular vascular disease, wherein the patient exhibits improved visual acuity after administration of the bispecific VEGF / ANG2 antibody as measured by an increase in best corrected visual acuity (BCVA) of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, 15 letters or more) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody.

[0152] 3. The bispecific antibody (for use) according to any one of embodiments 2A-2B, wherein the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0153] 4. The bispecific antibody (for use) according to any one of embodiments 1 to 3, wherein the increase in BCVA / ETDRS letters is measured at 4 weeks, and / or 8 weeks, and / or 12 weeks, and / or 16 weeks, and / or 20 weeks, and / or 24 weeks, respectively, after the start of treatment.

[0154] 5. BCVA. The bispecific antibody (for use) according to any one of embodiments 1 to 3, wherein the increase in BCVA / ETDRS letters is measured at 45 weeks, and / or 46 weeks, and / or 47 weeks, and / or 48 weeks, and / or 49 weeks, and / or 50 weeks, and / or 51 weeks, and / or 52 weeks, and / or 53 weeks, and / or 54 weeks, and / or 55 weeks, and / or 56 weeks, and / or 57 weeks, and / or 58 weeks, and / or 59 weeks, and / or 60 weeks, respectively, after the start of treatment.

[0155] 6. The bispecific antibody (for use) of any one of embodiments 1 to 5, wherein the bispecific antibody is used to extend the time to retreatment and / or to extend the time to decline in visual acuity (e.g. best corrected visual acuity (BCVA) BCVA / ETDRS), where retreatment would be necessary in the event of disease activity determined (in one embodiment using Spectral Domain Optical Coherence Tomography (SD-OCT)) as follows: a central foveal thickness (CST) increase of 50 μm or more; and / or a decline in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more.

[0156] 7. The bispecific antibody (for use) according to any one of embodiments 1 to 6, wherein said bispecific antibody is administered in doses every 3 to 7 months after initiation of treatment (in one embodiment, initiation of treatment comprises administration every 3 to 5 months; in one embodiment, initiation of treatment comprises administration every 4 months; in one embodiment, initiation of treatment comprises administration every 5 to 7 months, and in one embodiment, initiation of treatment comprises administration every 6 months).

[0157] 8. The bispecific antibody (for use) of any one of embodiments 1 to 7, wherein the ocular vascular disease is selected from the group of wet age-related macular degeneration (wet AMD), neovascular age-related macular degeneration, diabetic macular edema (DME), cystoid macular edema (CME), non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), macular edema secondary to central retinal vein occlusion, secondary to hemiretinal vein occlusion, or secondary to branch retinal vein occlusion, retinitis, conjunctivitis, uveitis, choroiditis, choroidal neovascularization (CNV) secondary to intraocular inflammation (including secondary to ocular histoplasmosis or presumed histoplasmosis or choroiditis); myopic choroidal neovascularization (mCNV), and choroidal neovascularization secondary to trauma, retinopathy of prematurity, and rubeosis iridis / neovascular glaucoma.

[0158] 9. The bispecific antibody (for use) according to any one of embodiments 1 to 7, wherein the ocular vascular disease is diabetic macular edema (DME).

[0159] 10. The bispecific antibody (for use) according to any one of embodiments 1 to 7, wherein the ocular vascular disease is wet age-related macular degeneration (wet AMD) or neovascular age-related macular degeneration (nAMD).

[0160] 11. The bispecific antibody (for use) according to any one of embodiments 1 to 10, wherein the bispecific antibody binding to VEGF and human ANG-2 is a VEGF antagonist / inhibitor and an ANG2 antagonist / inhibitor, or inhibits the binding of VEGF to its receptor VEGFR and inhibits the binding of ANG2 to its receptor TIE2.

[0161] 12. The bispecific antibody (for use) according to any one of embodiments 1 to 11, wherein said bispecific antibody is administered every 10 to 12 weeks.

[0162] 13. The bispecific antibody (for use) according to any one of embodiments 1 to 11, wherein said bispecific antibody is administered every 11 to 13 weeks.

[0163] 14. The bispecific antibody (for use) according to any one of embodiments 1 to 11, wherein said bispecific antibody is administered every 12 to 14 weeks.

[0164] 15. The bispecific antibody (for use) according to any one of embodiments 1 to 11, wherein said bispecific antibody is administered every 13 to 15 weeks.

[0165] 16. The bispecific antibody (for use) according to any one of embodiments 1 to 11, wherein said bispecific antibody is administered every 14 to 16 weeks.

[0166] 17. A bispecific antibody that binds to human VEGF and human ANG2 is a bispecific, bivalent anti-VEGF / ANG2 antibody that comprises a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, wherein i) the first antigen-binding site that specifically binds to VEGF comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO: 3, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO: 5, and a CDR1L region of SEQ ID NO: 6; ii) the second antigen-binding site that specifically binds to ANG-2 comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 9, a CDR2H region of SEQ ID NO: 10, and a CDR1H region of SEQ ID NO: 11, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ ID NO: 14; iii) The bispecific antibody (for use) according to any one of embodiments 1 to 16, wherein the bispecific antibody comprises a heavy chain constant region of the human IgG1 subclass comprising the mutations I253A, H310A, and H435A and the mutations L234A, L235A, and P329G (numbering according to EU index of Kabat).

[0167] 18.i) the first antigen-binding site that specifically binds to VEGF comprises a heavy chain variable domain VH having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain VL having the amino acid sequence of SEQ ID NO: 8; ii) The bispecific antibody (for use) of embodiment 17, wherein the second antigen-binding site that specifically binds to ANG-2 comprises the amino acid sequence of SEQ ID NO: 15 for the heavy chain variable domain VH and the amino acid sequence of SEQ ID NO: 16 for the light chain variable domain VL.

[0168] 19. The bispecific antibody (for use) according to embodiment 18, wherein the bispecific antibody that binds to human VEGF and human ANG2 comprises the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0169] 20. The bispecific antibody (for use) according to any one of embodiments 17 to 19, wherein the bispecific antibody is administered in a dose of about 5 to 7 mg (in each treatment).

[0170] 21. The bispecific antibody (for use) according to any one of embodiments 17 to 19, wherein the bispecific antibody is administered at a dose of about 6 mg (in each treatment) (in one embodiment, at a dose of 6 mg + / - 10% (in each treatment)); (in one embodiment, at a dose of about 6 mg (in each treatment)).

[0171] 22. The bispecific antibody (for use) according to any one of embodiments 20 to 21, wherein the bispecific antibody is administered at a concentration of about 30 mg / ml.

[0172] 23. The bispecific antibody (for use) according to any one of embodiments 20 to 21, wherein the bispecific antibody is administered at a concentration of about 120 mg / ml.

[0173] 24. The bispecific antibody (for use) according to any one of embodiments 1 to 23, wherein the patient suffering from ocular vascular disease has not previously received treatment (e.g. monotherapy) with an anti-VEGF antibody (is treatment-naive).

[0174] 25. The bispecific antibody (for use) according to any one of embodiments 1 to 24, wherein the patient suffering from ocular vascular disease has previously received treatment (e.g. monotherapy) with an anti-VEGF antibody.

[0175] 26. The bispecific antibody (for use) according to embodiments 1 to 25, wherein the ocular vascular disease is diabetic macular edema, and wherein treatment of patients with diabetic macular edema comprises a consistent every 8 weeks (Q8W) dosing regimen after initiation of treatment (in one embodiment, initiation of treatment comprises administration every 5 to 7 months; in one embodiment, initiation of treatment comprises administration every 6 months).

[0176] 27. The bispecific antibody (for use) according to embodiments 1 to 26, wherein the ocular vascular disease is diabetic macular edema, and wherein treatment of patients with diabetic macular edema comprises a consistent every 12 weeks (Q12W) dosing regimen after initiation of treatment (in one embodiment, initiation of treatment comprises administration every 5 to 7 months; in one embodiment, initiation of treatment comprises administration every 6 months).

[0177] 28. The bispecific antibody (for use) according to embodiment 27, wherein after treatment initiation, a steady Q12W dosing regimen is preceded by an initial cycle of one dose Q8W.

[0178] 29. The bispecific antibody (for use) according to embodiments 1 to 28, wherein the ocular vascular disease is diabetic macular edema, and wherein treatment of patients with diabetic macular edema comprises a dosing regimen in which, after initiation of treatment, the interval between doses is extended in the absence of stable disease, or the interval is shortened in the presence of disease activity (in one embodiment, initiation of treatment comprises dosing every 3 to 7 months; in one embodiment, initiation of treatment comprises dosing every 4 to 6 months).

[0179] 30. The bispecific antibody (for use) according to embodiment 29, wherein such dosing regimen comprises the patient receiving Q8W or Q12W or Q16W dosing, depending on the patient's disease state (in one embodiment Q4W or Q8W or Q12W or Q16W dosing, depending on the patient's disease state).

[0180] 31. Stable absence of disease is -Central retinal thickness (CST) increased by less than 50 μm; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by less than 5 letters and disease activity is determined as -Central retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by 5 letters or more 31. The bispecific antibody (for use) according to embodiment 29 or 30, wherein the bispecific antibody (for use) is determined as follows:

[0181] 32. Stable absence of disease is - Central retinal thickness (CST) of less than about 300 μm (in one embodiment, less than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, less than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). and disease activity is determined as - Central retinal thickness (CST) of greater than about 300 μm (in one embodiment, greater than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, greater than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). 31. The method of embodiment 29 or 30, wherein the

[0182] 33. The bispecific antibody (for use) according to embodiments 1 to 32, wherein the ocular vascular disease is age-related macular degeneration (in one embodiment wet age-related macular degeneration), and wherein treatment of patients suffering from age-related macular degeneration (in one embodiment wet age-related macular degeneration) comprises a dosing regimen, where, after initiation of treatment (in one embodiment, initiation of treatment comprises administration every 3 to 7 months; in one embodiment, initiation of treatment comprises administration every 4 to 6 months), the interval between doses is extended in the stable absence of disease, or the interval is shortened in the presence of disease activity.

[0183] 34. The bispecific antibody (for use) according to embodiment 33, wherein such dosing regimen comprises the patient receiving Q8W or Q12W or Q16W dosing, depending on the patient's disease state (in one embodiment Q4W or Q8W or Q12W or Q16W dosing, depending on the patient's disease state).

[0184] 35. Stable absence of disease is -Central retinal thickness (CST) increased by less than 50 μm; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by less than 5 letters and disease activity is determined as -Central retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by 5 letters or more 35. The bispecific antibody (for use) of embodiment 33 or 34, wherein the bispecific antibody (for use) is determined as follows:

[0185] 36. Stable absence of disease is - Central retinal thickness (CST) of less than about 300 μm (in one embodiment, less than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, less than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). and disease activity is determined as - Central retinal thickness (CST) of greater than about 300 μm (in one embodiment, greater than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, greater than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). 35. The bispecific antibody (for use) of embodiment 33 or 34, wherein the bispecific antibody (for use) is determined as follows:

[0186] The following are embodiments of the present invention: 1. A method of treating a patient suffering from an ocular vascular disease, the method comprising administering to the patient an effective amount of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), wherein the bispecific antibody is administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0187] 2A. A method of treating a patient suffering from ocular vascular disease, the method comprising administering to the patient an effective amount of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), wherein the patient has an increase of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, and in one embodiment 15 letters or more) in best corrected visual acuity (BCVA) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score prior to administration of the bispecific VEGF / ANG2 antibody.

[0188] 2B. A method of treating a patient suffering from ocular vascular disease, the method comprising administering to the patient an effective amount of a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), wherein the patient exhibits improved visual acuity after administration of the bispecific VEGF / ANG2 antibody as measured by an increase in best corrected visual acuity (BCVA) of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, and in one embodiment 15 letters or more) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody.

[0189] 3. The method of any one of embodiments 2A-2B, wherein the bispecific antibody is (will be) administered intravitreally every 8 weeks or less frequently (in one embodiment, every 9 weeks or less frequently; in one embodiment, every 10 weeks or less frequently; in one embodiment, every 11 weeks or less frequently; in one embodiment, every 12 weeks or less frequently; in one embodiment, every 13 weeks or less frequently; in one embodiment, every 14 weeks or less frequently; in one embodiment, every 15 weeks or less frequently).

[0190] 4. The method of any one of embodiments 1-3, wherein the letter increase in the BCVA / ETDRS letter score is measured at 4 weeks, and / or 8 weeks, and / or 12 weeks, and / or 16 weeks, and / or 20 weeks, and / or 24 weeks, respectively, after initiation of treatment.

[0191] 5. The method of any one of embodiments 1-3, wherein the letter increase in BCVA / ETDRS letter score is measured at 45 weeks, and / or 46 weeks, and / or 47 weeks, and / or 48 weeks, and / or 49 weeks, and / or 50 weeks, and / or 51 weeks, and / or 52 weeks, and / or 53 weeks, and / or 54 weeks, and / or 55 weeks, and / or 56 weeks, and / or 57 weeks, and / or 58 weeks, and / or 59 weeks, and / or 60 weeks, respectively, after initiation of treatment.

[0192] 6. The method of any one of embodiments 1-5, wherein the bispecific antibody is used to extend the time to retreatment and / or to extend the time to visual acuity decline, and retreatment with the bispecific antibody is administered upon disease activity as determined (in one embodiment using Spectral Domain Optical Coherence Tomography (SD-OCT)) as an increase in central retinal thickness (CST) of 50 μm or more; and / or a decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more.

[0193] 7. The method of any one of embodiments 1-6, wherein said bispecific antibody is administered in doses every 3 to 7 months after initiation of treatment (in one embodiment, initiation of treatment comprises administration every 3 to 5 months; in one embodiment, initiation of treatment comprises administration every 4 months; in one embodiment, initiation of treatment comprises administration every 5 to 7 months, and in one embodiment, initiation of treatment comprises administration every 6 months).

[0194] 8. The method of any one of embodiments 1-7, wherein the ocular vascular disease is selected from the group consisting of wet age-related macular degeneration (wet AMD), neovascular age-related macular degeneration, diabetic macular edema (DME), cystoid macular edema (CME), non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), macular edema secondary to central retinal vein occlusion, secondary to hemiretinal vein occlusion, or secondary to branch retinal vein occlusion, retinitis, conjunctivitis, uveitis, choroiditis, choroidal neovascularization (CNV) secondary to intraocular inflammation (including secondary to ocular histoplasmosis or presumed histoplasmosis or choroiditis); myopic choroidal neovascularization (mCNV), and choroidal neovascularization secondary to trauma, retinopathy of prematurity, and rubeosis iridis / neovascular glaucoma.

[0195] 9. The method of any one of embodiments 1-7, wherein the ocular vascular disease is diabetic macular edema (DME).

[0196] 10. The method of any one of embodiments 1 to 7, wherein the ocular vascular disease is wet age-related macular degeneration (wet AMD) or neovascular age-related macular degeneration (nAMD).

[0197] 11. The method of any one of embodiments 1 to 10, wherein the bispecific antibody that binds VEGF and human ANG-2 is a VEGF antagonist / inhibitor and an ANG2 antagonist / inhibitor, or inhibits the binding of VEGF to its receptor, VEGFR, and inhibits the binding of ANG2 to its receptor, TIE2.

[0198] 12. The method of any one of embodiments 1 to 11, wherein the bispecific antibody is administered every 10 to 12 weeks.

[0199] 13. The method of any one of embodiments 1 to 11, wherein the bispecific antibody is administered every 11 to 13 weeks.

[0200] 14. The method of any one of embodiments 1 to 11, wherein the bispecific antibody is administered every 12 to 14 weeks.

[0201] 15. The method of any one of embodiments 1 to 11, wherein the bispecific antibody is administered every 13 to 15 weeks.

[0202] 16. The method of any one of embodiments 1 to 11, wherein the bispecific antibody is administered every 14 to 16 weeks.

[0203] 17. A bispecific antibody that binds to human VEGF and human ANG2 is a bispecific, bivalent anti-VEGF / ANG2 antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, wherein i) the first antigen-binding site that specifically binds to VEGF comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of SEQ ID NO: 3, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO: 5, and a CDR1L region of SEQ ID NO: 6; ii) the second antigen-binding site that specifically binds to ANG-2 comprises, in a heavy chain variable domain, a CDR3H region of SEQ ID NO: 9, a CDR2H region of SEQ ID NO: 10, and a CDR1H region of SEQ ID NO: 11, and, in a light chain variable domain, a CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ ID NO: 14; iii) The method according to any one of embodiments 1 to 16, wherein the bispecific antibody comprises a heavy chain constant region of the human IgG1 subclass comprising the mutations I253A, H310A, and H435A, and the mutations L234A, L235A, and P329G (numbering according to EU index of Kabat).

[0204] 18.i) the first antigen-binding site that specifically binds to VEGF comprises a heavy chain variable domain VH having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain VL having the amino acid sequence of SEQ ID NO: 8; ii) the second antigen-binding site that specifically binds to ANG-2 comprises the amino acid sequence of SEQ ID NO: 15 as the heavy chain variable domain VH and the amino acid sequence of SEQ ID NO: 16 as the light chain variable domain VL.

[0205] 19. The method of embodiment 18, wherein the bispecific antibody that binds to human VEGF and human ANG2 comprises the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0206] 20. The method of any one of embodiments 17-19, wherein the bispecific antibody is administered at a dose of about 5-7 mg (in each treatment).

[0207] 21. The method of any one of embodiments 17 to 19, wherein the bispecific antibody is administered at a dose of about 6 mg (in each treatment) (in one embodiment, at a dose of 6 mg + / - 10% (in each treatment)); (in one embodiment, at a dose of about 6 mg (in each treatment)).

[0208] 22. The method of any one of embodiments 20-21, wherein the bispecific antibody is administered at a concentration of about 30 mg / ml.

[0209] 23. The method of any one of embodiments 20-21, wherein the bispecific antibody is administered at a concentration of about 120 mg / ml.

[0210] 24. The method of any one of embodiments 1 to 23, wherein the patient suffering from ocular vascular disease has not previously received treatment (e.g., monotherapy) with an anti-VEGF antibody (is treatment-naive).

[0211] 25. The method of any one of embodiments 1 to 24, wherein the patient suffering from ocular vascular disease has previously received treatment (e.g., monotherapy) with an anti-VEGF antibody.

[0212] 26. The method of any one of embodiments 1 to 25, wherein the ocular vascular disease is diabetic macular edema, and treating patients with diabetic macular edema comprises a consistent every 8 weeks (Q8W) dosing regimen after initiation of treatment (in one embodiment, initiation of treatment comprises administration every 5-7 months; in one embodiment, initiation of treatment comprises administration every 6 months).

[0213] 27. The method of embodiments 1-26, wherein the ocular vascular disease is diabetic macular edema, and treating patients with diabetic macular edema comprises a consistent every 12 weeks (Q12W) dosing regimen after initiation of treatment (in one embodiment, initiation of treatment comprises administration every 5-7 months; in one embodiment, initiation of treatment comprises administration every 6 months).

[0214] 28. The method of embodiment 27, wherein after treatment initiation, a steady Q12W dosing regimen is preceded by an initial cycle of one dose of Q8W.

[0215] 29. The method of embodiments 1-28, wherein the ocular vascular disease is diabetic macular edema, and treating patients with diabetic macular edema comprises a dosing regimen in which, after initiation of treatment, the dosing interval is extended in the absence of stable disease or the interval is shortened in the presence of disease activity (in one embodiment, initiation of treatment comprises dosing every 3-7 months; in one embodiment, initiation of treatment comprises dosing every 4-6 months).

[0216] 30. The method of embodiment 29, wherein such dosing regimen comprises the patient receiving Q8W or Q12W or Q16W dosing, depending on the patient's disease state (in one embodiment, Q4W or Q8W or Q12W or Q16W dosing, depending on the patient's disease state).

[0217] 31. Stable absence of disease is -Central retinal thickness (CST) increased by less than 50 μm; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by less than 5 letters and disease activity is determined as -Central retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by 5 letters or more 30. The method of embodiment 28 or 29, wherein the .alpha.-to-.alpha.

[0218] 32. Stable absence of disease is - Central retinal thickness (CST) of less than about 300 μm (in one embodiment, less than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, less than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). and disease activity is determined as - Central retinal thickness (CST) of greater than about 300 μm (in one embodiment, greater than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, greater than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). 30. The method of embodiment 28 or 29, wherein the .alpha.-to-.alpha.

[0219] 33. The method of embodiments 1-32, wherein the treatment of a patient suffering from age-related macular degeneration (in one embodiment, wet age-related macular degeneration) comprises a dosing regimen in which, after initiation of treatment (in one embodiment, initiation of treatment comprises dosing every 3-7 months; in one embodiment, initiation of treatment comprises dosing every 4-6 months), the dosing interval is extended in the absence of stable disease, or the interval is shortened in the presence of disease activity.

[0220] 34. The method of embodiment 33, wherein such dosing regimen comprises the patient receiving Q8W or Q12W or Q16W dosing, depending on the patient's disease state (in one embodiment, Q4W or Q8W or Q12W or Q16W dosing, depending on the patient's disease state).

[0221] 35. Stable absence of disease is -Central retinal thickness (CST) increased by less than 50 μm; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by less than 5 letters and disease activity is determined as -Central retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) reduced by 5 letters or more 35. The method of embodiment 33 or 34, wherein the

[0222] 36. Stable absence of disease is - Central retinal thickness (CST) of less than about 300 μm (in one embodiment, less than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, less than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, less than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). and disease activity is determined as - Central retinal thickness (CST) of greater than about 300 μm (in one embodiment, greater than 325 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Spectralis™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Cirrus™ instrument; in one embodiment, greater than 315 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with a Topcon™ instrument; in one embodiment, greater than 295 μm as measured by Spectral Domain Optical Coherence Tomography (SD-OCT) with an OptoView™ instrument). 35. The method of embodiment 33 or 34, wherein the

[0223] Example Treatment of patients with ocular vascular disease using a bispecific antibody that binds human VEGF and human ANG2 Example 1A: Efficacy and Duration of Treatment in Patients with Diabetic Macular Edema (DME) the purpose First Objective The primary objectives of this trial were to: To evaluate the efficacy of a bispecific antibody that binds human VEGF and human ANG2 (this antibody, VEGFang2-0016, and its production are also described in detail in International Publication No. WO 2014 / 009465, which is incorporated by reference), comprising the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, compared with an active agent in treatment-naive patients with foveal-involving diabetic macular edema (CI-DME). This bispecific anti-VEGF / ANG2 antibody is referred to herein as RO6867461, RG7716, VEGFang2-0016, or faricimab. Vials of a sterile, colorless to brownish, preservative-free solution of RO6867461 for intravitreal administration at doses of either 1.5 mg or 6 mg every four weeks were used. The concentration of the bispecific antibody was approximately 120 mg / ml.

[0224] Second Objective The secondary objectives of this trial were: To investigate the pharmacokinetics and anatomical outcomes that provide information on the mechanism of action of RO6867461. To investigate the formation of anti-RO6867461 antibodies in plasma. To explore the duration of effect of RO6867461.

[0225] exploratory purpose The exploratory objectives of this study were to: To explore the prospective effect of prior treatment with an intravitreal anti-VEGF antibody on the efficacy of RO6867461. To evaluate the efficacy and safety of RO6867461 compared to active control in patients with diabetic macular edema involving the fovea who have previously been treated with an intravitreal anti-VEGF antibody. To evaluate the effect of RO6867461 on plasma levels of markers of angiogenesis and inflammation. To examine the concentration of RO6867461 in aqueous humor samples (optional) and vitreous humor (optional), as well as biomarkers of angiogenesis and inflammation, if sample volume permits. To evaluate improvement in diabetic retinopathy (DR) severity scores.

[0226] Clinical trial design This was a multi-dose, multicenter, randomized, active-controlled, double-blind, three-arm, parallel-group, 36-week trial in patients with diabetic macular edema with a foveal fovea. The three arms of the trial were: Group A: 0.3 mg of ranibizumab (intravitreal) Group B: 1.5 mg of RO6867461 (intravitreal) Group C: 6 mg of RO6867461 (intravitreal)

[0227] Only one eye was selected as the study eye. If both eyes met all eligibility criteria, the eye with the worse best-corrected visual acuity was designated as the study eye. If both eyes met all eligibility criteria and had the same best-corrected visual acuity letter score on Day 1, the selection of the study eye was determined by the investigator.

[0228] Number of patients A maximum of 210 patients were randomized.

[0229] Approximately 150 treatment-naive patients and approximately 60 patients who had previously been treated intravitreally with anti-VEGF antibodies participated in the trial.

[0230] Approximately 50 treatment-naive patients were randomized to each group (1:1:1 randomization scheme), and approximately 30 patients who had previously been treated intravitreally with anti-VEGF antibodies were randomized to groups A and C.

[0231] Target Population Male and female patients aged 18 years or older with diabetic macular edema involving the fovea.

[0232] Inclusion / Exclusion Criteria Selection Criteria Patients must meet the following criteria for participation in the trial:

[0233] Ocular criteria for the study eye: Macular edema associated with diabetic retinopathy, defined as macular thickening by spectral-domain optical coherence tomography (SD-OCT) involving the central macula: central retinal thickness (CST) of 325 μm or greater using Spectralis™ (Heidelberg) at screening (if Spectralis™ was unavailable, the following device and central retinal thickness thresholds were acceptable: central retinal thickness of 315 μm or greater for Cirrus™, central retinal thickness of 315 μm or greater for Topcon, and central retinal thickness of 295 μm or greater for Optoview™).

[0234] A best-corrected visual acuity (BCVA) letter score of 73 to 24 letters (including borderline) on an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart on day 1 (equivalent to a Snellen index of 20 / 40-20 / 320) was primarily due to diabetic macular edema.

[0235] Clear optics and adequate pupil dilation to allow for the acquisition of high-quality retinal images for definitive diagnosis.

[0236] General criteria: Diagnosis of diabetes mellitus (DM: type 1 or type 2) as defined by the World Health Organization and / or the American Diabetes Association.

[0237] Be able and willing to provide written informed consent and comply with the clinical trial protocol in accordance with the International Council for Harmonisation (ICH) and local regulations. Alternatively, a legally authorized representative must consent the patient in accordance with ICH and local regulations.

[0238] 18 years of age or older.

[0239] Women who are not postmenopausal (i.e., have had non-treatment-induced amenorrhea confirmed by follicle-stimulating hormone for 12 months or more if not receiving hormone replacement therapy), or who have agreed to sterilization (absence of ovaries and / or uterus) to maintain abstinence, or who use a combined contraceptive method with a failure rate of less than 1% per year throughout the treatment period and for at least 4 weeks after the last dose.

[0240] Abstinence is acceptable only if it is in line with the patient's preferred and usual lifestyle. Periodic abstinence (e.g., calendar, ovulation, thermogenic, or postovulatory methods) and pull-out methods were not acceptable methods of contraception.

[0241] Examples of contraceptive methods with expected failure rates of less than 1% per year include vasectomy, hormonal implants, the proper use of combined oral or injectable hormonal contraceptives, and certain intrauterine devices. Alternatively, two methods (e.g., two barrier methods such as a condom and a cervical cap) may be combined to achieve a failure rate of less than 1% per year; barrier methods should always be supplemented with the use of spermicide.

[0242] For men, agree to use barrier contraception during treatment for at least 4 weeks after the last dose of study drug.

[0243] Patients must refrain from participating in any other clinical trials involving investigational medicinal products (IMPs) or devices until completion of the current trial.

[0244] Exclusion criteria Patients who met any of the following criteria were excluded from participating in the trial:

[0245] Ocular criteria for the study eye: Any signs of high-risk proliferative diabetic retinopathy, defined as: Any intravitreal or preretinal hemorrhage. Clinical examination revealed neovascularization of at least half of the papillary area within an area equivalent to a standard 7-field ETDRS using mydriatic medication. Clinical examination reveals neovascularization of more than one-third of the nipple area. Any treatment with intravitreal anti-VEGF antibodies within 3 months prior to Day 1. Any extensive retinal photocoagulation (PRP) procedure prior to day 1. Any macular laser photocoagulation within the 3 months prior to Day 1. History of vitreoretinal surgery. Treatment with any intravitreal or periocular corticosteroids within 3 months prior to Day 1. History of use of any Iluvien® or Ozardex® implants prior to Day 1 will not be allowed. Treatment of any cataract surgery or complication of cataract surgery with steroids within the 3 months prior to Day 1. History of incisional glaucoma surgery. Uncontrolled glaucoma (e.g., progressive visual field loss or intraocular pressure [IOP] of 25 mmHg or greater despite treatment with glaucoma medications).

[0246] Concomitant ocular conditions in the study eye: History of rubeosis Any current or history of eye disease other than diabetic macular edema that may confound macular evaluation or affect central vision (e.g., age-related macular degeneration, retinal vein occlusion, uveitis, angioid streaks, histoplasmosis, active or inactive cytomegalovirus, pathological myopia, retinal detachment, macular traction, macular hole, severe cataract). Any current ocular condition (e.g., foveal atrophy, pigmentary abnormalities, dense foveal hard exudates, extraretinal conditions) in which, in the investigator's opinion, resolution of macular edema would not improve visual acuity loss. Any active eye infection on day 1. Any active intraocular inflammation (grade slight or greater) on day 1.

[0247] Other eye characteristics: Treatment with any anti-VEGF antibody within 7 days prior to Day 1. Any retinal condition that, in the opinion of the investigator, may require treatment with an anti-VEGF antibody within Day 1 to 7 days.

[0248] General criteria: Any systemic anti-VEGF antibodies within the 6 months prior to Day 1. Any major illness or major surgery within the month prior to Day 1. Any febrile illness within the week prior to Day 1. Any stroke or myocardial infarction within the 12 months prior to Day 1. Uncontrolled blood pressure (blood pressure; defined as a systolic blood pressure greater than 180 mmHg and / or a diastolic blood pressure greater than 100 mmHg in a patient at rest). If the patient's first reading exceeds these values, a second reading may be taken 30 minutes or more later on the same day or on another day during the screening period. If the patient's blood pressure needs to be controlled with hypertensive medication, the patient should have been taking the same medication continuously for at least 1 month prior to Day 1. Patients with glycosylated hemoglobin HbA1c greater than 12% at screening.

[0249] Untreated diabetes, or initiation of oral antidiabetic medication or insulin within 4 months prior to Day 1, or anticipated change in antidiabetic medication during the study period. Renal failure requiring kidney transplantation, hemodialysis, or peritoneal dialysis within the 6 months prior to Day 1, or anticipated to require hemodialysis or peritoneal dialysis at any time during the study. History of other diseases, metabolic dysfunction, conditions that contraindicate the use of the investigational drug, or physical or clinical examination findings that may affect the interpretation of the trial results or that, in the opinion of the investigator, place the patient at high risk for treatment complications. Women of childbearing potential, positive pregnancy blood test. Breastfeeding women. Use of systemic corticosteroids within the month prior to Day 1. Any known hypersensitivity to the active drug, fluorescein, any component of the formulation used, mydriatic eye drops, or any anesthetic and antibacterial eye drops used. Any other limitations due to the use of active drugs. Any treatment with an investigational drug within the 3 months prior to Day 1.

[0250] Clinical trial period The total duration of the trial was up to 40 weeks for each patient enrolled as follows (from screening to study completion): Screening: Up to 4 weeks. Baseline: Day 1. Study treatment administration period: Day 1 through Week 20. Observation period: from week 20 to a maximum of week 36. Safety follow-up required: During the observation period and for 7 days after administration of ranibizumab.

[0251] End of clinical trial The end of the trial was defined as the date of last patient last observation (LPLO), which was expected to occur 36 weeks after the last patient enrolled.

[0252] Efficacy and pharmacokinetic endpoints The primary analysis population was treatment-naive patients. Additional analyses may be performed in the total population and in patients previously treated with intravitreal anti-VEGF antibodies.

[0253] The primary efficacy endpoint for this trial was the mean change in BCVA (ETDRS letter) from baseline to week 24 in treatment-naive patients.

[0254] Anatomical endpoints assessed by SD-OCT: Mean change from baseline in foveal thickness at 24 weeks. Mean change from baseline in mean central foveal retinal thickness (1 mm diameter) at 24 weeks. Proportion of patients with resolution of subretinal and intraretinal fluid at 24 weeks. Anatomical endpoints assessed by fundus fluorescein angiography (FFA). Proportion of patients with resolution of macular leakage at 24 weeks. Change from baseline in size of the foveal avascular zone at 24 weeks.

[0255] Exploratory endpoints Exploratory endpoints in this trial included, but were not limited to, the following: BCVA: Difference in mean change in best-corrected visual acuity (BCVA) from baseline between untreated patients and patients previously treated with intravitreal anti-VEGF antibodies (difference in effect of RO6867461).

[0256] Exploratory endpoints related to durability: Time to an increase in central retinal thickness of 50 μm or more and / or a decrease in best-corrected visual acuity of 5 letters or more due to diabetic macular edema compared to the value at week 20. Time to retreatment with 0.3 mg ranibizumab after week 20.

[0257] result The primary efficacy analysis included all randomized patients, who were categorized according to the treatment assigned at randomization.

[0258] The primary efficacy variable was the change in best-corrected visual acuity from baseline to week 24. The primary efficacy analysis was performed using a mixed model for repeated measures (MMRM) model.

[0259] best corrected visual acuity Best-corrected visual acuity at a baseline test distance of 4 meters was measured before pupil dilation by a trained, certified ophthalmologist blinded to study drug group assignment.

[0260] Best-corrected visual acuity was measured using a set of three Precision Vision™ or Lighthouse distance charts (revised ETDRS charts 1, 2, and R). A visual acuity manual was provided to the investigator. Certification of the vision examiner and vision testing laboratory was obtained before any vision tests were administered.

[0261] The best-corrected visual acuity examiner was blinded to the study eye and treatment assignment and would only perform refraction and best-corrected visual acuity assessments (e.g., visual acuity specification manual). The best-corrected visual acuity examiner was also blinded to the best-corrected visual acuity letter scores from the patient's previous visit and knew only the patient's refraction data from the previous visit. The best-corrected visual acuity examiner was not permitted to perform any other duties, including direct patient care.

[0262] [Table 2]

[0263] The primary efficacy endpoint is shown in Figure 1, which depicts the primary efficacy endpoint: change in best-corrected visual acuity from baseline to week 24 over time for previously treatment-naive patients. VA2 refers to the bispecific anti-VEGF / ANG2 antibody RO6867461 (6.0 mg or 1.5 mg dose administered intravitreally) comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, and RBZ refers to ranibizumab (Lucentis®) (0.3 mg dose administered intravitreally).

[0264] Change in central retinal thickness (CST) from baseline (test eye) The key secondary endpoint was the change from baseline in central subretinal thickness (CST). Results are shown in Figure 2. The bispecific anti-VEGF / ANG2 antibody RO6867461 (6.0 mg or 1.5 mg administered intravitreally), comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, was compared to ranibizumab (Lucentis®) (0.3 mg administered intravitreally). This secondary anatomical endpoint directly supports the primary endpoint of best-corrected visual acuity.

[0265] Duration / time to retreatment Criteria for ranibizumab treatment during the observation period At each visit after the last dose of study treatment (week 20 visit), best corrected visual acuity was assessed and SD-OCT angiography was performed (except at week 26 visit).

[0266] Best corrected visual acuity and foveal retinal thickness values ​​obtained at week 24 were compared with those obtained at the week 20 visit. Best corrected visual acuity and foveal retinal thickness values ​​obtained at weeks 28, 32, and 36 were compared with those obtained at week 24.

[0267] Patients received a single dose of 0.3 mg ranibizumab and completed the study if they met both of the following criteria: -Increase of 50 μm or more in foveal region retinal thickness. A loss of best corrected visual acuity of 5 letters or more due to diabetic macular edema.

[0268] The results are shown in Figure 3, which shows the time to retreatment after dosing was discontinued (after 20 weeks or every 6 months = time point after the last intravitreal (IVT) administration) based on disease activity assessed by both a loss of best-corrected visual acuity of ≥ 5 letters and an increase in central retinal thickness of ≥ 50 μm. The bispecific anti-VEGF / ANG2 antibody RO6867461 (6.0 mg or 1.5 mg dose administered intravitreally) comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 was compared to ranibizumab (Lucentis®) (0.3 mg dose administered intravitreally).

[0269] For overview, Figure 4 shows a graphical comparison to other treatment options for diabetic macular edema based on published results (comparing the following drugs: Lucentis® (ranibizumab), Eylea® (aflibercept), brolucizumab, and VA2 (RO6867461 / RG7716)).

[0270] Example 1B: Efficacy and Duration of Treatment in Patients with Diabetic Macular Edema (DME) In a further clinical trial similar to that described above under Example 1A, patients suffering from diabetic macular edema (e.g., diabetic macular edema involving the fovea (CI-DME)) are treated with a bispecific antibody that binds to human VEGF and human ANG2, comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. Active agents in the treatment may include, for example, aflibercept and / or ranibizumab and / or brolucizumab. Patients include those who have not been previously treated with an anti-VEGF antibody monotherapy (e.g., have not been previously treated with an anti-VEGF antibody monotherapy, e.g., aflibercept and / or ranibizumab and / or brolucizumab), as well as patients who have previously been treated with an anti-VEGF antibody monotherapy. The names of the respective bispecific antibodies that bind to human VEGF and human ANG2 are RO6867461 and RG7716. Vials of sterile, colorless to brownish, preservative-free solution of RO6867461 for intravitreal administration at either a 1.5 mg or 6 mg dose are used.

[0271] Use one or more of the following regimens: a) Patients with diabetic macular edema will be treated with a consistent every 8 weeks dosing regimen after initiation of treatment (e.g., injections every 6 months initially), b) Patients with diabetic macular edema will be treated with consistent 12-week dosing (one regimen initially includes one cycle of 8-week dosing) after initiation of treatment (e.g., injections every 6 months initially), c) Patients with diabetic macular edema will be treated with a dosing regimen in which, after initiation of treatment (e.g., injections every 3-7 months initially), the injection intervals will be increased in the absence of stable disease or decreased in the presence of disease activity. Such regimens include, for example, patients receiving Q4W / Q8W / Q12W / Q16W dosing, depending on the patient's disease status.

[0272] Assessment of disease stability will be based on best-corrected visual acuity (BCVA) and central foveal retinal thickness (CST), as well as retinal thickness based on optical coherence tomography (OCT). Endpoints and outcomes will be assessed, for example, as described in Example 1A. The primary endpoint will be from week 45 to week 60.

[0273] In one embodiment, the patient with diabetic macular edema is treatment naive (has not been previously treated with anti-VEGF antibody monotherapy, e.g., aflibercept and / or ranibizumab and / or brolucizumab).

[0274] In one embodiment, the patient with diabetic macular edema has been previously treated with anti-VEGF antibody monotherapy, such as aflibercept and / or ranibizumab and / or brolucizumab.

[0275] In one embodiment, patients with diabetic macular edema will be treated with a regular every 8 weeks dosing regimen after initiating treatment (eg, injections every 6 months initially).

[0276] In one embodiment, patients with diabetic macular edema will be treated with a fixed 12-week dosing regimen (including, in one embodiment, one cycle of 8-week dosing initially) after initiating treatment (e.g., injections every 6 months initially).

[0277] In one embodiment, patients with diabetic macular edema will be treated with a dosing regimen in which, after initiation of treatment (e.g., injections every 3-7 months initially), the interval between injections will be increased in the absence of stable disease, or decreased in the presence of disease activity. In one embodiment, such a regimen involves the patient receiving Q4W / Q8W / Q12W / Q16W dosing, depending on the patient's disease status.

[0278] In one embodiment, a patient with age-related macular degeneration will be treated with a dosing regimen in which, after initiation of treatment (e.g., injections every 3-4 months initially), the interval between injections will be increased in the absence of stable disease, or decreased in the presence of disease activity. In one embodiment, such a regimen involves the patient receiving Q4W / Q8W / Q12W / Q16W dosing, depending on the patient's disease status.

[0279] Example 2A: Efficacy and Duration of Treatment in Patients with Age-Related Macular Degeneration (AMD) Objectives and evaluation items This clinical trial evaluated the efficacy, safety, and pharmacokinetics of RO6867461 administered every 12 and 16 weeks to patients with neovascular age-related macular degeneration (nAMD). RO6867461 is a bispecific antibody that binds to human VEGF and human ANG2, and includes the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. (This antibody, VEGFang2-0016, and its production are also described in detail in WO 2014 / 009465, which is incorporated by reference.) This bispecific anti-VEGF / ANG2 antibody is referred to herein as RO6867461, RG7716, VEGFang2-0016, or faricimab.

[0280] The specific objectives and corresponding endpoints for the trial are outlined below.

[0281] Objectives and corresponding evaluation items Primary efficacy objective To evaluate the effect of RO6867461 on visual acuity when administered every 12 weeks and every 16 weeks.

[0282] Corresponding evaluation items Mean change from baseline in best corrected visual acuity at week 40 using an ETDRS-like chart.

[0283] Secondary validity objectives: 1) To evaluate the efficacy of RO6867461 on additional visual acuity endpoints. Corresponding evaluation items Mean change from baseline in best corrected visual acuity over time using an ETDRS-like chart. The proportion of patients with an increase from baseline in best-corrected visual acuity of 15 or more letters, 10 or more letters, 5 or more letters, or 0 or more letters over time. The proportion of patients who avoid a loss of 15 or more letters, 10 or more letters, 5 or more letters, or 0 or more letters from baseline in best-corrected visual acuity over time. Proportion of patients with best-corrected visual acuity of 20 / 40 or better over time. Proportion of patients with best-corrected visual acuity of 20 / 200 or worse over time.

[0284] 2) To evaluate the efficacy of RO6867461 on anatomical endpoints using SD-OCT. Corresponding evaluation items Mean change from baseline in central foveal retinal thickness over time. The mean change from baseline in mean central foveal retinal thickness (1 mm diameter) over time. The proportion of patients with intraretinal fluid, subretinal fluid, cysts, or retinal pigment epithelial detachment over time.

[0285] 3) To evaluate the efficacy of RO6867461 on anatomical endpoints using fundus fluorescein angiography (FFA). Corresponding evaluation items Mean change from baseline in total area of ​​choroidal neovascularization (CNV) at weeks 40 and 52. Mean change from baseline in total area of ​​choroidal neovascularization at weeks 40 and 52. Mean change from baseline in total area of ​​leakage at weeks 40 and 52.

[0286] Purpose of exploring effectiveness To investigate the incidence of disease activity at 24 weeks. Corresponding evaluation items Proportion of patients with active disease at 24 weeks.

[0287] Safety Objectives To evaluate the safety of multiple intravitreal doses of RO6867461 administered at 12-week and 16-week intervals. Corresponding evaluation items Incidence and severity of ocular adverse events. Incidence and severity of non-ocular adverse events. Other safety data will also be listed and summarized narratively, including, but not limited to, reasons for withdrawal from the study, clinical data, concomitant medications, vital signs, and physical examination results.

[0288] Purpose: to explore pharmacokinetics / pharmacodynamics 1) To evaluate the systemic pharmacokinetic profile of RO6867461. Corresponding evaluation items - Plasma concentrations of RO6867461 at a specific time point. 2) To evaluate the RO6867461, ranibizumab, free VEGF-A, and Ang-2 profiles in aqueous humor. The relationship between the concentration or pharmacokinetic parameters of RO6867461 in aqueous humor and the concentrations of free VEGF-A and Ang-2. Corresponding evaluation items · The relationship between ranibizumab concentrations or pharmacokinetic parameters in aqueous humor and the concentrations of free VEGF-A and Ang-2. Time course of free VEGF-A and Ang-2 concentrations in aqueous humor.

[0289] Immunogenicity objectives To investigate the formation of anti-RO6867461 antibodies in plasma. Corresponding evaluation items -The incidence of anti-drug antibodies during the clinical trial.

[0290] Purpose of biomarker discovery To explore the levels of candidate biomarkers of angiogenesis and inflammation in aqueous humor at baseline and additional time points to assess response to RO6867461. Corresponding evaluation items The relationship between the primary and secondary endpoints and aqueous humor concentrations of candidate biomarkers. Abbreviations used above: ADA = anti-drug antibody; Ang-2 = angiopoietin-2; BCVA = best corrected visual acuity; CFT = foveal thickness; CNV = choroidal neovascularization; CST = central subretinal thickness; ETDRS = Early Treatment Diabetic Retinopathy Study; FFA = fundus fluorescein angiography; IVT = intravitreal; PK = pharmacokinetics; SD-OCT = spectral domain optical coherence tomography; VEGF-A = vascular endothelial growth factor A.

[0291] Clinical trial design (Figure 5 presents an overview of the clinical trial design) Explanation of the clinical trial This was a Phase II, multicenter, randomized, active-controlled, subject- and endpoint assessor-blinded, parallel-group, 52-week trial to investigate the efficacy, safety, and pharmacokinetics of RO6867461 administered every 12 and 16 weeks to treatment-naïve patients with neovascular age-related macular degeneration (nAMD).

[0292] Approximately 75 patients were enrolled and randomized in a 2:2:1 ratio to one of three treatment groups: Group A (Q12W): 6 mg of RO6867461 intravitreally (IVT) every 4 weeks (4 injections) until week 12, then 6 mg of RO6867461 intravitreally every 12 weeks until week 48 (injections at weeks 24, 36, and 48; 3 injections). Arm B (Q16W): 6 mg intravitreal RO6867461 every 4 weeks (4 injections) until Week 12, then 6 mg intravitreal RO6867461 every 16 weeks (injections at Weeks 28 and 44; 2 injections) until Week 48. A protocol-defined assessment of disease activity at Week 24 required Arm B patients with active disease (see criteria below) to switch to a dosing regimen of 6 mg RO6867461 every 12 weeks for the remainder of the study, with injections starting at Week 24 and repeated at Weeks 36 and 48. Group C (comparison group): Ranibizumab 0.5 mg (intravitreous) every 4 weeks for 48 weeks (13 injections). Only one eye will be selected as the study eye. The total duration of the study in each patient will be up to 56 weeks and will be categorized as follows: Screening: up to 4 weeks prior to or on the same day as randomization. Randomization: Day 1. Study treatment administration: Day 1 through Week 48. Last visit: 52 weeks.

[0293] Patients underwent screening within 4 weeks prior to administration of study treatment. The screening visit and the Week 1 / Day 1 (randomization) visit could be combined, provided all assessments (other than informed consent) were completed within 48 hours. Patients were assessed for eligibility during screening (or the combined Screening / Day 1 visit), including central review of fundus photography (FP), spectral-domain optical coherence tomography (SD-OCT), and fundus fluorescein angiography (FFA) to ensure choroidal neovascularization secondary to age-related macular degeneration met the pre-specified ocular criteria for the study. Patients deemed ineligible based on screening results for any of the following reasons could be rescreened: Uncontrolled blood pressure Administrative reasons (e.g., inability to schedule Day 1 within 28 days of the screening visit) -Do not meet the eligibility criteria for the study eye (in which case, patients may be eligible to participate in the second eye after the initial screening period).

[0294] At the time of rescreening, evaluation of all screening visits was performed (except for fundus fluorescein angiography image collection), except for eligible central reading center fundus fluorescein angiography (FFA) images taken within 4 weeks before the new Day 1 visit (randomization).

[0295] On Day 1, eligible patients received their first intravitreal dose of either RO6867461 or ranibizumab according to the randomization plan described above and established standard dosing procedures. Patients returned to the eye clinic 7 days after their first intravitreal dose and every 4 weeks thereafter for study treatment administration and evaluations as outlined in the protocol schedule. To maintain blinding throughout the study period, intravitreal placebo was delivered to patients randomized to Groups A and B.

[0296] All patients were assessed for disease activity at week 24. Patients randomized to Arm B who had active disease (see criteria below) at week 24 were switched to a dosing regimen of 6 mg RO6867461 Q12W for the remainder of the study, with injections starting at week 24 and repeated at weeks 36 and 48.

[0297] A determination of active disease was made if any of the following criteria were met: An increase in central retinal thickness (central retinal thickness of more than 50 μm on Spectralis® OCT) compared to the mean central retinal thickness over the last two visits (weeks 16 and 20), or An increase in central retinal thickness of 75 μm or more compared to the lowest central retinal thickness recorded at either week 16 or week 20, or A decrease in best-corrected visual acuity (BCVA) of at least 5 letters compared to the mean of best-corrected visual acuity over the last two visits (weeks 16 and 20) due to neovascular age-related macular degeneration disease activity, or A decrease in best-corrected visual acuity of 10 letters or more compared to the highest best-corrected visual acuity recorded at either Week 16 or Week 20 due to neovascular age-related macular degeneration disease activity, or Presence of new macular hemorrhage due to active neovascular age-related macular degeneration.

[0298] Patients will return for a final visit at Week 52. After the final visit, adverse events should be followed as outlined in the protocol. Assessments performed in the event of unscheduled visits will be adjudicated by the investigator.

[0299] Patient population: Approximately 75 treatment-naive patients with neovascular age-related macular degeneration were expected to participate and be randomized in this US trial.

[0300] Target Population Eligibility Criteria Patients met the following criteria for inclusion in the trial: ocular criteria for the study eye; · Untreated choroidal neovascularization secondary to age-related macular degeneration (neovascular age-related macular degeneration). Subfoveal or parafoveal choroidal neovascularization with a foveal component (as evidenced by subretinal fluid, subretinal hyperreflective material, evidence of leakage, or hemorrhage) associated with choroidal neovascularization by fundus fluorescein angiography or spectral-domain optical coherence tomography. Any type of choroidal neovascular lesion (predominantly classic, minimally classic, or occult), with: total lesion size (including blood, atrophy, fibrosis, and neovascularization) ≤ 6 disc areas by fundus fluorescein angiography, and choroidal neovascular area ≥ 50% of the total lesion size by fundus fluorescein angiography, and active choroidal neovascularization (evidence of leakage) confirmed by fundus fluorescein angiography, and choroidal neovascular exudate (presence of fluid) confirmed by spectral-domain optical coherence tomography. · Clear optics and adequate pupil dilation to allow for good quality retinal images to be obtained and a definitive diagnosis to be made. general criteria ·Signing an informed consent form. - Be 50 years of age or older on Day 1. In the investigator's judgment, compliance with the clinical trial protocol is possible. For women of childbearing potential: Agree to maintain abstinence (abstain from heterosexual intercourse) or use a contraceptive method with a failure rate of less than 1% per year throughout the treatment period and for at least 28 days after the last dose of study treatment. Patients must not be willing to participate in any other clinical trials involving investigational new medicines (IMPs) or devices until the completion of the current trial.

[0301] Exclusion criteria Patients who met any of the following criteria were excluded from participating in the trial: Ocular criteria for the study eye Choroidal neovascularization due to causes other than age-related macular degeneration, such as ocular histoplasmosis, trauma, pathologic myopia, angioid streaks, choroidal rupture, or uveitis. Central serous chorioretinopathy at screening. ·Retinal pigment epithelial tears, including the macula. Subretinal hemorrhage on fundus fluorescein angiography, exceeding 50% of the total lesion area and / or involving foveal fibrosis or atrophy exceeding 50% of the total lesion area and / or involving the fovea. Any pre- or concomitant treatment for choroidal neovascularization, including (but not limited to) intravitreal treatments (steroids, anti-vascular endothelial growth factor [VEGF] antibodies, tissue plasminogen activator, ocriplasmin, octafluoropropane gas, air), periocular pharmacological interventions, argon laser photocoagulation, verteporfin photodynamic therapy, diode laser, transpupillary thermotherapy, or surgical intervention. Cataract surgery within 3 months of the baseline assessment (Day 1). Any other intraocular surgery (pars plana vitrectomy, glaucoma surgery, corneal transplant, radiation therapy). Previous intravitreal treatment (including anti-VEGF antibody medications), except for management of cataract complications using intravitreal steroid treatment. Previous periocular pharmacological intervention for other retinal diseases. Current eye condition Any concurrent ocular condition in the study eye that, in the opinion of the investigator, either reduces the likelihood of visual improvement or may require medical or surgical intervention during the course of the study (e.g., amblyopia, aphakia, retinal detachment, cataract, diabetic retinopathy, or maculopathy, or preretinal membrane with traction). Active intraocular inflammation (minimal grade or worse) in the study eye on Day 1 (before randomization). Best corrected visual acuity letter score of 73 to 24 letters (including borderline) on an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart on Day 1 (equivalent to 20 / 40 to 20 / 320 on the Snellen scale). Current intravitreous hemorrhage in the study eye. Uncontrolled glaucoma in the study eye (defined as, for example, progressive visual field loss or intraocular pressure [IOP] of 25 mmHg or greater despite treatment with glaucoma medications). The spherical equivalent of a refractive error indicating more than 8 diopters of myopia in the study eye. · History of idiopathic or autoimmune-related uveitis in either eye. Active infectious conjunctivitis, keratitis, scleritis, or endophthalmitis in either eye on Day 1 (before randomization). general criteria Any major illness or major surgery within one month prior to screening. Uncontrolled blood pressure (defined as a systolic blood pressure greater than 180 mmHg and / or a diastolic blood pressure greater than 100 mmHg when the patient is at rest [blood pressure]). If the patient's first reading exceeds these values, a second reading may be taken later on the same day or on another day during the screening period. If the patient's blood pressure is controlled with hypertensive medication, the patient should have been taking the same medication for at least 30 consecutive days prior to Day 1. - Stroke or myocardial infarction within 3 months prior to Day 1. History of other diseases, metabolic dysfunction, physical examination findings, or clinical laboratory findings that contraindicate the use of the investigational drug or that reasonably suspect a condition that may affect the interpretation of the trial results or that, in the opinion of the investigator, places the patient at high risk for complications of the procedure. Pregnant, nursing, or planning to become pregnant during the study. Women of childbearing potential must have a negative urine pregnancy test result within 28 days before the start of study treatment. A positive urine pregnancy test must be confirmed by a serum pregnancy test. Known hypersensitivity to ranibizumab, fluorescein, any component of the formulation used, mydriatic eye drops, or any anesthetic or antibacterial eye drops used. Treatment with an investigational therapy within 3 months prior to the start of study treatment.

[0302] End of clinical trial The end of the trial was defined as the date of the last patient's last visit (LPLV), which was expected to occur 52 weeks after the last patient enrolled.

[0303] Clinical trial period The total duration of the trial, from screening of the first patient to the end of the trial, was expected to be approximately 18-19 months.

[0304] Investigational Drug Test Products The RO6867461 drug product (120 mg / mL) is supplied as a sterile, colorless to brownish liquid and contains no preservatives. Vials of sterile, colorless to brownish, preservative-free RO6867461 solution for intravitreal administration in 6 mg doses were used. The concentration of the bispecific antibody was approximately 120 mg / mL.

[0305] Dosage and administration, RO6867461, ranibizumab, and placebo.

[0306] Patients received a 50 μL intravitreal injection of RO6867461 or ranibizumab or placebo in the study eye according to the randomization plan, as described below. Group A (Q12W): 6 mg of RO6867461 administered intravitreously every 4 weeks until week 12 (4 injections), then 6 mg of RO6867461 administered intravitreously every 12 weeks until week 48 (injections at weeks 24, 36, and 48; 3 injections). Group B (Q16W): 6 mg of RO6867461 administered intravitreously every 4 weeks until week 12 (four injections), then 6 mg of RO6867461 administered intravitreously every 16 weeks until week 48 (two injections at weeks 28 and 44). Group C (comparison group): intravitreal injection of 0.5 mg of ranibizumab every 4 weeks for 48 weeks (13 injections).

[0307] Only one eye was selected as the study eye.

[0308] Clinical trial evaluation When several assessments are performed simultaneously, the following order is suggested at the investigator's discretion. The order may be adjusted to optimize time management of site personnel and patients, except where explicitly stated as mandatory (i.e., in italicized text). Vital signs Blood Sampling: At visits where fundus fluorescein angiography is performed, blood sampling and angiography can be performed through the same venous cannula. The blood sample must be collected before the angiography. ·Ocular evaluation and imaging. Best Corrected Visual Acuity: Best corrected visual acuity must be performed before pupil dilation. At the screening visit and at the Day 1 visit, best corrected visual acuity may be performed before vital signs and blood sampling to avoid unnecessary investigations in patients who may have screen failures as a result of a best corrected visual acuity letter score. Slit-lamp examination. Pupils dilated. SD-OCT. Fundus photography (+infrared reflectance). Fundus fluorescein angiography. Binocular indirect high-magnification fundus examination with pupil dilation. Intraocular pressure: Mandatory after all imaging evaluations and should be used throughout the trial.

[0309] Aqueous humor sampling (optional) Disease-specific evaluation Unless otherwise specified in the activity schedule (Addendum 1), all ocular evaluations were performed on both eyes.

[0310] best corrected visual acuity Best-corrected visual acuity at test distances starting at 4 meters was measured before pupil dilation by a trained and certified visual acuity (VA) examiner blinded to study ocular treatment assignment.

[0311] Best corrected visual acuity was measured using a set of PrecisionVision™ or Lighthouse distance visual acuity charts (Revised ETDRS Charts 1, 2, and R). A visual acuity procedure manual was provided to the investigator. Vision examiner and vision laboratory approval was obtained before any vision tests were performed.

[0312] The best-corrected visual acuity examiner will be blinded to the study eye and treatment assignment and will perform the refraction and best-corrected visual acuity assessment (e.g., visual acuity specification manual). The best-corrected visual acuity examiner will also be blinded to the best-corrected visual acuity letter scores from the patient's previous visit and may only know the patient's refraction data that is known from the previous visit.

[0313] Additional ocular evaluation Additional ocular evaluations performed during the trial include: Slit-lamp examination (scales for grading redness and intravitreal hemorrhage density per cell are detailed in Appendix 2) Binocular indirect ophthalmoscopy with high magnification with pupil dilation. Intraocular pressure

[0314] The intraocular pressure measurement method used in patients was consistent throughout the trial. Intraocular pressure measurements in both eyes were performed after all imaging.

[0315] At the study treatment visit, intraocular pressure was performed before study treatment administration and 30 (± 15) minutes after treatment administration to the study eye, and if intraocular pressure was 30 mmHg or greater, intraocular pressure should be reassessed 30 (± 15) minutes later. If intraocular pressure continued to rise, treatment was administered at the investigator's discretion. Finger counting visual acuity assessment Post-treatment optic nerve head irrigation fluid in the study eye was assessed for each patient immediately after administration of the study treatment (within a maximum of 15 minutes after treatment administration) by testing finger counting visual acuity, hand movements, or light perception, as appropriate.

[0316] Ocular Imaging The central reading center provided the site with the central reading center manual and educational materials for ocular imaging required for the trial. Before study images were acquired, site personnel and imaging systems (if applicable) were authorized by the reading center as specified in the central reading center manual. Ocular images of all study subjects were acquired only by trained and central reading center-certified personnel with authorized / registered equipment at the study site. Copies of all study subject ocular images were forwarded to the central reading center for archival and independent analysis (including confirmation of eligibility for specified image-related criteria).

[0317] Assessment of disease activity at 24 weeks All patients were evaluated for disease activity at week 24. Patients randomized to Arm B who had active disease (see criteria below) at week 24 were switched to a 6 mg RO6867461 Q12W dosing regimen for the remainder of the study, with injections starting at week 24 and repeated at weeks 36 and 48.

[0318] Active disease was determined if any of the following criteria were met: An increase in central retinal thickness of more than 50 μm on Spectralis OCT compared to the mean central retinal thickness over the last two visits (weeks 16 and 20), or An increase in central retinal thickness of 75 μm or more compared to the lowest central retinal thickness recorded at either week 16 or week 20, or A decrease in best-corrected visual acuity of at least 5 letters compared to the average of best-corrected visual acuity over the last two visits (weeks 16 and 20) due to neovascular age-related macular degeneration disease activity, or A decrease in best-corrected visual acuity of 10 letters or more compared to the highest best-corrected visual acuity recorded at either Week 16 or Week 20 due to neovascular age-related macular degeneration disease activity, or Presence of new macular hemorrhage due to active neovascular age-related macular degeneration.

[0319] result Best corrected visual acuity (BCVA) and duration of BCVA gain (time to retreatment to maintain BCVA gain) The primary efficacy endpoint is shown in Figure 6. Figure 6 shows the primary efficacy endpoint: change in BCVA from baseline to week 40 over time. RO6867461 refers to the bispecific anti-VEGF / ANG2 antibody RO6867461 (6.0 mg dose administered intravitreally either Q12W or Q16W) comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, and ranibizumab (Lucentis®) was administered intravitreally Q4W at a dose of 0.3 mg. The initial gain in best-corrected visual acuity was fully maintained in the RO6867461 Q12W or Q16W groups and to a similar extent in the ranibizumab (Lucentis®) Q4W group.

[0320] Change in central retinal thickness (CST) from baseline (test eye) The key secondary endpoint was the change from baseline in central subretinal thickness (CST). Results are shown in Figure 7. The bispecific anti-VEGF / ANG2 antibody RO6867461, comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 (6.0 mg administered intravitreally either Q12W or Q16W), was compared with ranibizumab (Lucentis®) (0.3 mg administered intravitreally Q4W). This anatomical secondary endpoint directly supports the primary endpoint of best-corrected visual acuity. Central subretinal thickness decreased more with the bispecific anti-VEGF / ANG2 antibody RO6867461 than with ranibizumab during treatment initiation.

[0321] Example 2B: Efficacy and Duration of Treatment in Patients with Age-Related Macular Degeneration (AMD) In a further clinical trial similar to that described above under Example 2A, patients suffering from age-related macular degeneration (e.g., wet age-related macular degeneration (wAMD), particularly neovascular age-related macular degeneration) are treated with a bispecific antibody that binds to human VEGF and human ANG2, comprising the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. Active agents in the treatment may include, for example, aflibercept and / or ranibizumab and / or brolucizumab. Patients include patients who have not been treated with an anti-VEGF antibody (i.e., have not previously been treated with anti-VEGF antibody monotherapy, e.g., aflibercept and / or ranibizumab and / or brolucizumab), as well as patients who have previously been treated with anti-VEGF antibody monotherapy, e.g., aflibercept and / or ranibizumab and / or brolucizumab. The bispecific antibodies that bind to human VEGF and human ANG2 are named RO6867461 and RG7716, respectively. Vials of sterile, colorless to brownish, preservative-free solution of RO6867461 for intravitreal administration are available at either a 1.5 mg or 6 mg dose.

[0322] For example, the following dosing regimen is used: Patients with age-related macular degeneration will be treated with a dosing regimen in which, after initiation of treatment (e.g., with injections every 3-7 months initially), the interval between injections will be extended if there is stable disease absence, or the interval will be shortened if there is disease activity. Such regimens include, for example, having the patient receive Q4W / Q8W / Q12W / Q16W dosing, depending on the patient's disease status.

[0323] Assessment of disease stability will be based on best-corrected visual acuity (BCVA) and foveal retinal thickness, as well as optical coherence tomography (OCT)-based retinal thickness. Endpoints and outcomes will be assessed, for example, as described in Example 1A. The primary endpoint will be from week 45 to week 60.

[0324] Example 3 Binding of anti-VEGF / ANG2 antibodies to VEGF, Ang2, FcγR, and FcRn Kinetic affinity of VEGF isoforms, including assessment of species cross-reactivity Approximately 12,000 resonance units (RU) of the capture system (10 μg / ml goat anti-human F(ab)'2; order number: 28958325; GE Healthcare Biosciences, Sweden) were coupled onto a CM5 chip (GE Healthcare BR-1005-30) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. The sample and system buffer was PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween® 20) pH 7.4. The flow cell was set to 25°C (the sample block was set to 12°C) and primed twice with running buffer. Bispecific antibodies were captured by injecting a 50 nM solution at a flow rate of 5 μl / min for 30 s. Association was measured by injecting various concentrations of human hVEGF121, mouse mVEGF120, or rat rVEGF164 in solution for 300 seconds at a flow rate of 30 μl / min, starting at 300 nM at a 1:3 dilution. The dissociation phase was monitored for up to 1200 seconds and triggered by switching from the sample solution to running buffer. The surface was regenerated by washing with glycine pH 2.1 solution for 60 seconds at a flow rate of 30 μl / min. Bulk refractive index differences were corrected by subtracting the response obtained from the goat anti-human F(ab)'2 surface. A blank injection was also subtracted (= double referencing). The apparent K D For the calculation of the kinetic parameters, the Langmuir 1:1 model was used. The results are shown in Table 5.

[0325] Affinity of Ang2 solutions, including assessment of species cross-reactivity Solution affinity measures the affinity of an interaction by determining the concentration of the free interacting pair in the equilibrium mixture. Solution affinity assays are performed using a constant concentration of <vegf-ang-2>The bispecific antibody was mixed with various concentrations of ligand (=Ang2). The maximum possible resonance units (e.g., 17,000 resonance units (RU)) of antibody were immobilized on the surface of a CM5 chip (GE Healthcare BR-1005-30) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. The sample and system buffer was HBS-P (pH 7.4). The flow cell was set at 25°C, the sample block at 12°C, and primed twice with running buffer. To generate a calibration curve, increasing concentrations of Ang2 were added to the immobilized <vegf-ang2>The bispecific antibody was injected into a Biacore™ flow cell. The amount of bound Ang2 was determined as resonance units (RU) and plotted against the concentration. <vegf-ang-2>Solutions of the bispecific antibody (11 concentrations ranging from 0 to 200 nM) were incubated with 10 nM Ang2 and allowed to equilibrate at room temperature. Free Ang2 concentrations were determined from a calibration curve constructed before and after measuring the response of solutions containing known amounts of Ang2. A four-parameter fit was constructed using XLfit4 (IDBS software) using model 201, with free Ang2 concentration as the y-axis and the concentration of the antibody used for inhibition as the x-axis. Affinity was calculated by determining the inflection point of this curve. The surface was regenerated by washing once with 0.85% H3PO4 solution at a flow rate of 30 μl / min for 30 seconds. Differences in bulk refractive index were corrected by subtracting the response obtained from a blank bound surface. The results are shown in Table 6.

[0326] Steady-state affinity of FcRn Steady-state affinity was used to compare the bispecific antibodies against each other for FcRn measurements. Human FcRn was diluted in coupling buffer (10 μg / ml, sodium acetate pH 5.0) and immobilized onto a C1 chip (GE Healthcare BR-1005-35) using a targeted immobilization procedure using the Biacore™ Wizard to a final response of 200 RU. The flow cell was set to 25°C, the sample block was set to 12°C, and primed twice with running buffer. The sample and system buffer was PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween® 20), pH 6.0. To evaluate various IgG concentrations for each antibody, concentrations of 62.5 nM, 125 nM, 250 nM, and 500 nM were prepared. The flow rate was set to 30 μl / min, and the various samples were injected sequentially over the chip surface, selecting an association time of 180 seconds. The surface was regenerated with PBS-T (pH 8) injected for 60 seconds at a flow rate of 30 μl / min. Bulk refractive index differences were corrected by subtracting the response obtained from a blank surface. Buffer injections were also subtracted (=duplicate control). The steady-state affinity was calculated using the method of the Bia-Evaluation software. Briefly, the number of resonance units (RU max) was plotted against the analyzed concentration to obtain a dose-response curve. Based on a two-parameter fit, an upper asymptote was calculated, allowing the half-maximal RU value, and thus the affinity, to be determined. The results are shown in Figure 5 and Table 7. Similarly, affinity for cynomolgus monkey, mouse, and rabbit FcRn could also be determined.

[0327] Measurement of FcγRIIIa A direct binding assay was used to measure FcγRIIIa. Approximately 3,000 resonance units (RU) of capture system (1 μg / ml penta-His; Qiagen) were coupled onto a CM5 chip (GE Healthcare BR-1005-30) at pH 5.0 using an amine coupling kit provided by GE Healthcare. The sample and sample buffer were HBS-P + pH 7.4. The flow cell was set to 25°C (sample block set to 12°C) and primed twice with running buffer. The FcγRIIIa-His receptor was captured by injecting a 100 nM solution for 60 s at a flow rate of 5 μl / min. Binding was measured by injecting 100 nM of bispecific antibodies or monospecific control antibodies (anti-Dig antibody for IgG1 and IgG4 subclass antibodies) for 180 s at a flow rate of 30 μl / min. The surface was regenerated by washing with glycine (pH 2.5) solution at a flow rate of 30 μl / min for 120 seconds. Because FcγRIIIa binding differs from the Langmuir 1:1 model, binding only / no binding was determined using this assay. Similarly, binding of FcγRIa and FcγRIIa can be determined. The results are shown in Figure 6, where it can be seen that introduction of the mutation P329G LALA resulted in no further detectable binding to FcγRIIIa.

[0328] <vegf-ang-2>Evaluation of independent VEGF and Ang-2 binding to bispecific antibodies Approximately 3500 resonance units (RU) of the capture system (10 μg / ml goat anti-human IgG; GE Healthcare Biosciences, Sweden) were coupled onto a CM4 chip (GE Healthcare BR-1005-34) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. The sample and system buffer was PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween® 20) pH 7.4. The flow cell temperature was set to 25°C, and the sample block temperature was set to 12°C. Prior to capture, the flow cell was primed twice with running buffer.

[0329] The bispecific antibodies were captured by injecting a 10 nM solution for 60 seconds at a flow rate of 5 μl / min. Independent binding of each ligand to the bispecific antibodies was analyzed by determining the effective binding capacity for each ligand added either sequentially or simultaneously (flow rate of 30 μl / min).

[0330] Human VEGF was injected at a concentration of 1.200 nM for 180 seconds (to identify the sole binding of the antigen). 2. Human Ang2 was injected at a concentration of 100 nM for 180 seconds (to identify the sole binding of the antigen). 3. Human VEGF at a concentration of 200 nM was injected for 180 seconds, followed by a further injection of human Ang2 at a concentration of 100 nM for 180 seconds (to identify Ang2 binding in the presence of VEGF). 4. Human Ang2 was injected at a concentration of 100 nM for 180 seconds, followed by a further injection of human VEGF at a concentration of 200 nM (to identify VEGF binding in the presence of Ang2). 5. Human VEGF at a concentration of 200 nM and human Ang2 at a concentration of 100 nM were co-injected for 180 seconds (to simultaneously identify the binding of VEGF and Ang2).

[0331] The surface was regenerated by washing for 60 seconds with 3 mM MgCl2 solution at a flow rate of 30 μl / min. Bulk refractive index differences were corrected by subtracting the response obtained from the goat anti-human IgG surface.

[0332] A bispecific antibody is capable of binding to both antigens independently of each other if the final signal obtained as a result of approaches 3, 4, and 5 is equal to or similar to the sum of the individual final signals of approaches 1 and 2. The results are shown in the table below, which demonstrates that VEGFang2-0016 (=RO6867461) is capable of binding to VEGF and ANG2 independently of each other.

[0333] <vegf-ang-2>Evaluation of simultaneous binding to VEGF and Ang2 for bispecific antibodies First, approximately 1600 resonance units (RU) of VEGF (20 μg / ml) was coupled onto a CM4 chip (GE Healthcare BR-1005-34) at pH 5.0 using an amine coupling kit provided by GE Healthcare. The sample and system buffer was PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween® 20) pH 7.4. The flow cell was set at 25°C, and the sample block was set at 12°C and primed twice with running buffer. Second, a 50 nM solution of the bispecific antibody was injected for 180 seconds at a flow rate of 30 μl / min. Third, hANG-2 was injected for 180 seconds at a flow rate of 30 μl / min. The binding response of hAng-2 depended on the amount of bispecific antibody bound to VEGF, indicating simultaneous binding. The surface was regenerated by washing with 0.85% H3PO4 solution for 60 seconds at a flow rate of 30 μl / min. Simultaneous binding was observed for pre-bound VEGF. <vegf-ang-2>As shown by the additional specific binding signal of hAng2 to the bispecific antibody.

[0334] [Table 3]

[0335] [Table 4]

[0336] [Table 5]

[0337] [Table 6]

[0338] [Table 7] < / vegf>

Claims

[Claim 1] 1. A pharmaceutical composition for use in the treatment of diabetic macular edema (DME) comprising faricimab, a bispecific antibody that binds human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), A pharmaceutical composition comprising an initial dose of 6 mg of faricimab administered intravitreally (IVT) 3 to 7 times monthly, followed by 6 mg of faricimab IVT every 16 weeks.

Images