Treatment of ophthalmologic diseases

Abstract

To provide methods, uses, bispecific antibodies (for use), medicaments or pharmaceutical formulations for the treatment of patients suffering from an ocular vascular disease.SOLUTION: The invention provides a bispecific antibody which binds to human vascular endothelial growth factor (VEGF) and to human angiopoietin-2 (ANG-2). The bispecific antibody is administered (is to be administered) intravitreally every 8 weeks or less frequently.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 various disorders, including solid tumors, intraocular neovascular syndromes such as proliferative retinopathy 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, G. K. (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 parental mouse antibody as bevacizumab (Avastin®). However, it has been affinity matured to provide a more potent binding to VEGF-A (WO 98 / 45331). Since systemic blockade of VEGF-A is known to be associated with an increased risk of certain adverse events, ranibizumab therefore lacks the Fc portion in order to reduce the risk of systemic exposure and systemic toxicity. It is an anti-angiogenic agent approved for the treatment of "exudative" age-related macular degeneration (neovascular age-related macular degeneration), a common form of age-related vision loss.

[0004] The corneal angiogenesis assay demonstrated that both ANG-1 and ANG-2 have similar effects and act synergistically with VEGF to promote the growth of new blood vessels (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 angiogenic at high concentrations in vitro (Kim, I., et al., Oncogene 19 (2000) 4549-52). ANG-2 acts as an anti-apoptotic survival factor for endothelial cells through activation of Tie2 via the PI-3 kinase and Akt pathways during apoptosis due to serum starvation at high concentrations (Kim, I., et al., Oncogene 19 (2000) 4549-52).

[0005] Ocular vascular diseases such as "exudative" age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR) are caused by abnormal choroidal or retinal angiogenesis, respectively. Hemorrhage and leakage from these blood 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), are caused by abnormal retinal leakage, leading to retinal swelling and impairment of visual function. These conditions are the leading causes of vision loss in industrialized countries. The retina consists of well-defined layers of neuronal, glial, and vascular components, and relatively small disturbances such as vascular proliferation or edema can cause significant loss of visual function. Hereditary retinal degenerations, such as retinitis pigmentosa (RP), are also associated with vascular abnormalities such as arteriolar narrowing and vascular atrophy. These affect many individuals, one in 3500, and are characterized by progressive night blindness, loss of visual fields, optic nerve atrophy, attenuation of arterioles, and central visual field defects that often progress to total blindness.

[0006] Ischemic retinopathy is characterized by a reduction or dysfunction of the retinal vascular structure, 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. The growth of these abnormal new blood vessels is what creates most of the threat to vision, because they can leak, bleed, or cause scarring, which can ultimately lead to retinal detachment. Current treatments for ischemic retinopathy seek to stop the growth of diseased blood vessels, but do not address the underlying ischemia that drives the growth of diseased blood vessels. Furthermore, the standard treatment for diabetic retinopathy, an ischemic retinopathy that affects millions of people, involves the destruction of a portion of the retina using a laser in an attempt to stop the growth of new blood vessels and preserve the central visual field. Strategies are used to block the function of vascular endothelial growth factor (VEGF), a major promoter of abnormal blood vessel growth and leakage. In the short term, anti-VEGF antibody therapy can improve vision, but does not address the underlying ischemia and, in fact, can worsen the condition because it inhibits the growth of all blood vessels, including beneficial collateral vessels. There are also serious concerns about systemic exposure to these drugs in the elderly and / or diabetic patients, in whom new blood vessel growth may be required in ischemic brain, heart, or limbs.

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

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

[0009] In one embodiment of the invention, the increase in letters of 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 after the start of treatment, respectively.

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

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

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

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

[0014] These time points are extremely early, and typically, in neovascular age-related macular degeneration, the maximum increase is not reached until about 6 to 9 months, and in diabetic macular edema, until 9 to 12 months.

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

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

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

[0018] In one embodiment of the present invention, the bispecific antibody that binds to human VEGF and human ANG2 is a bispecific and 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 the heavy-chain variable domain, the CDR3H region of SEQ ID NO: 1, the CDR2H region of SEQ ID NO: 2, and the CDR1H region of SEQ ID NO: 3, and, in the light-chain variable domain, the CDR3L region of SEQ ID NO: 4, the CDR2L region of SEQ ID NO: 5, and the CDR1L region of SEQ ID NO: 6; ii) the second antigen-binding site that specifically binds to ANG-2 comprises, in the heavy-chain variable domain, the CDR3H region of SEQ ID NO: 9, the CDR2H region of SEQ ID NO: 10, and the CDR1H region of SEQ ID NO: 11, and, in the light-chain variable domain, the CDR3L region of SEQ ID NO: 12, the CDR2L region of SEQ ID NO: 13, and the CDR1L region of SEQ ID NO: 14; iii) 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 the Kabat EU index).

[0019] In one embodiment of the invention, a patient suffering from an ocular vascular disease has not previously received treatment (e.g., monotherapy) with an anti-VEGF antibody (is untreated).

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

[0021] In one embodiment of the invention, the ocular vascular disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a dosing schedule of every 8 weeks (Q8W) at regular intervals after the start of treatment.

[0022] In one embodiment of the invention, the ocular vascular disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a dosing schedule of every 12 weeks (Q12W) at regular intervals after the start of treatment. In one embodiment of the invention, there is a first 1-dose Q8W cycle before the regular Q12W dosing schedule after the start of treatment.

[0023] In one embodiment of the invention, the ocular vascular disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a dosing schedule that extends the dosing interval if the disease is stably absent or shortens the interval if disease activity is present after the start of treatment. In one embodiment of the invention, such a dosing schedule includes the patient receiving dosing at Q4W or Q8W or Q12W or Q16W depending on the patient's disease state. In one embodiment of the invention, the stable absence of the disease is - an increase in the central subfield thickness (CST) of less than 50 μm; and / or - a decrease in the best corrected visual acuity (BCVA / ETDRS) of less than 5 letters determined as, and the disease activity is - An increased central foveal thickness (CST) of more than 50 μm; and / or - A decreased best corrected visual acuity (BCVA / ETDRS) of more than 5 letters is determined as such.

[0024] In one embodiment of the present invention, the ocular vascular disease is age-related macular degeneration, and the treatment of a patient suffering from age-related macular degeneration includes a dosing schedule that, after the start of treatment, extends the dosing interval if the disease is stably absent or shortens the interval if disease activity is present. In one embodiment of the present invention, such a dosing schedule includes the patient receiving dosing at Q4W or Q8W or Q12W or Q16W, depending on the patient's disease state. In one embodiment of the present invention, the stable absence of disease is - An increased central foveal thickness (CST) of less than 50 μm; and / or - A decreased best corrected visual acuity (BCVA / ETDRS) of less than 5 letters is determined as such, and disease activity is - An increased central foveal thickness (CST) of more than 50 μm; and / or - A decreased best corrected visual acuity (BCVA / ETDRS) of more than 5 letters is determined as such.

Brief Description of the Drawings

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[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 to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), wherein the bispecific antibody is administered intravitreally at 8-week intervals or less frequently (scheduled to be administered) (in one embodiment, at 9-week intervals or less frequently; in one embodiment, at 10-week intervals or less frequently; in one embodiment, at 11-week intervals or less frequently; in one embodiment, at 12-week intervals or less frequently; in one embodiment, at 13-week intervals or less frequently; in one embodiment, at 14-week intervals or less frequently; in one embodiment, at 15-week intervals or less frequently).

[0027] One embodiment of the present invention is a method 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 to 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, in one embodiment 15 letters or more) in the best corrected visual acuity measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the best corrected visual acuity (BCVA) letter score of the patient before dosing with the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally every 8 weeks or less frequently (scheduled to be administered) (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 present invention is 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 to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), wherein the patient has an improvement in visual acuity after administration of the bispecific VEGF / ANG2 antibody as measured by an increase of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, 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 pre-dose BCVA letter score of the patient for the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally every 8 weeks or less frequently (scheduled to be administered) (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 present invention, the increase in letters of 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 after the start of treatment, respectively.

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

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

[0032] One embodiment of the present invention is a bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in the treatment of ocular vascular diseases, and the bispecific antibody is administered intravitreally (scheduled to be administered) at intervals of 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 present invention is a bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in the treatment of patients with ocular vascular diseases, where the patient has an increase of 12 letters or more (in one embodiment, 13 letters or more, in one embodiment, 14 letters or more, in one embodiment, 15 letters or more) in the best corrected visual acuity measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the best corrected visual acuity letter score of the patient before dosing with the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally at intervals of every 8 weeks or less frequently (scheduled to be administered) (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 present invention is a bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in the treatment of patients suffering from ocular vascular diseases, wherein the patient shows an improvement in visual acuity after (intravitreal) administration of the bispecific VEGF / ANG2 antibody as measured by an increase of 12 letters or more in best corrected visual acuity (BCVA) (13 letters or more in one embodiment, 14 letters or more in one embodiment, 15 letters or more in one embodiment) of best corrected visual acuity measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart as compared to the BCVA letter score of the patient before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally at 8-week intervals or less frequently (scheduled to be administered) (in one embodiment, at 9-week intervals or less frequently; in one embodiment, at 10-week intervals or less frequently; in one embodiment, at 11-week intervals or less frequently; in one embodiment, at 12-week intervals or less frequently; in one embodiment, at 13-week intervals or less frequently; in one embodiment, at 14-week intervals or less frequently; in one embodiment, at 15-week intervals or less frequently).

[0035] In one embodiment of the present invention, the increase in letters of 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 after the start of treatment, respectively.

[0036] In one embodiment of the present invention, the increase in letters of the 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 after the start of treatment, respectively.

[0037] In one embodiment of the present invention, such bispecific antibodies (for use) are used to extend the time to re-treatment and / or to extend the time to visual acuity decline, where re-treatment with the bispecific antibody is (in one embodiment using a spectral domain optical coherence tomography (SD-OCT)) an increase in the central foveal thickness (CST) of 50 μm or more; and / or a decrease in the best corrected visual acuity (BCVA / ETDRS) of 5 letters or more is administered in the case of disease activity determined as such.

[0038] One embodiment of the present invention is a pharmaceutical or pharmaceutical formulation comprising a bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in the treatment of ocular vascular diseases, where the bispecific antibody is administered intravitreally at 8-week intervals or less frequently (is scheduled to be administered) (in one embodiment, at 9-week intervals or less frequently; in one embodiment, at 10-week intervals or less frequently; in one embodiment, at 11-week intervals or less frequently; in one embodiment, at 12-week intervals or less frequently; in one embodiment, at 13-week intervals or less frequently; in one embodiment, at 14-week intervals or less frequently; in one embodiment, at 15-week intervals or less frequently).

[0039] One embodiment of the present invention is a pharmaceutical or pharmaceutical formulation comprising a bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in the treatment of patients suffering from ocular vascular diseases, where the patient has an increase of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, in one embodiment 15 letters or more) in the best corrected visual acuity measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the best corrected visual acuity (BCVA) letter score of the patient before dosing with the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally every 8 weeks or less frequently (scheduled to be administered) (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 present invention is a pharmaceutical or pharmaceutical formulation comprising a bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in the treatment of patients suffering from ocular vascular diseases, where the patient shows an improvement in 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, in one embodiment 15 letters or more) measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the pre-dose BCVA letter score of the patient. In one embodiment, the bispecific antibody is administered intravitreally at 8-week intervals or less frequently (is scheduled to be administered) (in one embodiment, at 9-week intervals or less frequently; in one embodiment, at 10-week intervals or less frequently; in one embodiment, at 11-week intervals or less frequently; in one embodiment, at 12-week intervals or less frequently; in one embodiment, at 13-week intervals or less frequently; in one embodiment, at 14-week intervals or less frequently; in one embodiment, at 15-week intervals or less frequently).

[0041] In one embodiment of the present invention, the increase in letters of 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 the start of treatment.

[0042] In one embodiment of the present invention, the increase in letters of the 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 the start of treatment.

[0043] In one embodiment of the present invention, such a pharmaceutical or pharmaceutical formulation is used to extend the time to re-treatment and / or to extend the time to vision loss, where re-treatment with a bispecific antibody is (using a spectral domain optical coherence tomography (SD-OCT) in one embodiment) an increase in the central foveal thickness (CST) of the retina of 50 μm or more; and / or a decrease in the best corrected visual acuity (BCVA / ETDRS) of 5 letters or more is administered in the case of disease activity determined as such.

[0044] One embodiment of the present invention is the use of a bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for the manufacture of a pharmaceutical for use in the treatment of ocular vascular diseases, where the bispecific antibody is administered intravitreally at 8-week intervals or less frequently (scheduled to be administered) (in one embodiment, at 9-week intervals or less frequently; in one embodiment, at 10-week intervals or less frequently; in one embodiment, at 11-week intervals or less frequently; in one embodiment, at 12-week intervals or less frequently; in one embodiment, at 13-week intervals or less frequently; in one embodiment, at 14-week intervals or less frequently; in one embodiment, at 15-week intervals or less frequently).

[0045] One embodiment of the present invention is the use of a bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for the manufacture of a medicament for use in the treatment of ocular vascular diseases, where the patient has an increase of 12 letters or more (13 letters or more in one embodiment, 14 letters or more in one embodiment, 15 letters or more in one embodiment) in the best corrected visual acuity measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the best corrected visual acuity (BCVA) letter score of the patient before dosing with the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally at 8-week intervals or less frequently (scheduled to be administered) (in one embodiment, at 9-week intervals or less frequently; in one embodiment, at 10-week intervals or less frequently; in one embodiment, at 11-week intervals or less frequently; in one embodiment, at 12-week intervals or less frequently; in one embodiment, at 13-week intervals or less frequently; in one embodiment, at 14-week intervals or less frequently; in one embodiment, at 15-week intervals or less frequently).

[0046] One embodiment of the present invention is the use of a bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for the manufacture of a medicament for use in the treatment of ocular vascular diseases, where the patient shows an improvement in visual acuity after (intravitreal) administration of the bispecific VEGF / ANG2 antibody as measured by an increase of 12 letters or more (in one embodiment 13 letters or more, in one embodiment 14 letters or more, in one embodiment 15 letters or more) in the best corrected visual acuity (BCVA) compared to the BCVA letter score of the patient before dosing with the bispecific VEGF / ANG2 antibody, using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart. In one embodiment, the bispecific antibody is administered intravitreally at 8-week intervals or less frequently (is scheduled to be administered) (in one embodiment at 9-week intervals or less frequently; in one embodiment at 10-week intervals or less frequently; in one embodiment at 11-week intervals or less frequently; in one embodiment at 12-week intervals or less frequently; in one embodiment at 13-week intervals or less frequently; in one embodiment at 14-week intervals or less frequently; in one embodiment at 15-week intervals or less frequently).

[0047] In one embodiment of the present invention, the increase in letters of 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 after the start of treatment, respectively.

[0048] In one embodiment of the present invention, the increase in letters of the 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 after the start of treatment, respectively.

[0049] In one embodiment of the present invention, the pharmaceutical is used to extend the time to re-treatment and / or to extend the time to vision loss, where re-treatment with the bispecific antibody is (using a spectral domain optical coherence tomography (SD-OCT) in one embodiment) an increase in the central foveal thickness (CST) of the retina of 50 μm or more; and / or a decrease in the best corrected visual acuity (BCVA / ETDRS) of 5 letters or more administered in the case of disease activity determined as such.

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

[0051] Such a method, use, bispecific antibody (for use), pharmaceutical, or pharmaceutical formulation may include administering a first dose (“start of treatment”), followed by sequential administration of a second dose or more of a therapeutically effective amount of the bispecific antibody, pharmaceutical, or pharmaceutical formulation (e.g., administration every 3 to 7 months; in one embodiment, the start of treatment includes administration every 3 to 4 months, in one embodiment, the start of treatment includes administration every 4 to 5 months; in one embodiment, the start of treatment includes administration every 4 to 6 months; in one embodiment, the start of treatment includes administration at least every 4 months; in one embodiment, the start of treatment includes administration every 5 to 7 months, in one embodiment, the start of treatment includes administration every 6 months).

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

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

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

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

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

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

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

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

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

[0061] The terms "ocular vascular diseases" and "vascular eye diseases" are used interchangeably herein and include, but are not limited to, intraocular angiogenesis syndromes such as diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, neovascular glaucoma, retinal vein (branch) 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, G.K., (eds.), 2nd edition, Marcel Dekker, New York (1994), pp. 1625-1710). Ocular vascular disorders as used herein refer to any condition characterized by the altered or uncontrolled growth of new blood vessels and invasion into ocular tissue structures such as the retina or cornea. In one embodiment, the ocular vascular disease is selected from the group consisting of exudative age-related macular degeneration (exudative 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, or branch retinal vein occlusion or hemiretinal vein occlusion, retinitis, conjunctivitis, uveitis, choroiditis, choroidal neovascularization (CNV) secondary to intraocular inflammation (including but not limited to secondary histoplasmosis or presumed histoplasmosis or choroiditis), myopic choroidal neovascularization (mCNV), and choroidal neovascularization secondary to trauma, retinopathy of prematurity, and iris rubeosis / neovascular glaucoma, and other ophthalmic diseases (where the eye disease or disorder is accompanied by ocular neovascularization, vascular leakage, and / or retinal edema).Therefore, the anti-VEGF / ANG2 bispecific antibodies for the uses and methods described herein are useful for the prevention and treatment of exudative age-related macular degeneration, neovascular age-related macular degeneration, cystoid macular edema, diabetic macular edema, non-proliferative diabetic retinopathy, proliferative diabetic retinopathy, and uveitis, and preferably exudative age-related macular degeneration, neovascular age-related macular degeneration, and preferably diabetic macular edema, cystoid macular edema, non-proliferative diabetic retinopathy, and proliferative diabetic retinopathy, and particularly for the prevention and treatment of exudative age-related macular degeneration. In some embodiments, the ocular vascular disease is selected from the group consisting of exudative age-related macular degeneration (exudative 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 causative of) corneal angiogenesis include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, metabolic disorders due to contact lenses, atopic keratitis, superior limbic keratitis, pterygium, dry keratitis, Sjogren's syndrome, rosacea, phlyctenosis, syphilis, mycobacterial infections, steatosis, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex virus infections, herpes zoster infections, protozoal infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal corneal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's granulomatosis, scleritis, Stevens-Johnson disease, pemphigoid, radial keratotomy, and corneal transplant rejection.

[0063] Diseases / conditions associated with (or which may cause) angiogenesis of the retina / choroid 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 (papilla) pits, Sturge-Weber disease, pars planitis, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, trauma, and laser complications. Other diseases include those associated with rubeosis (neovascularization of the angle) and diseases caused by abnormal proliferation of vascular connective or fibrous tissue, including all forms of proliferative vitreoretinopathy.

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

[0065] Macular degeneration is a condition mainly seen in the elderly, where detachment, atrophy, and sometimes bleeding occur at the center of the inner limiting membrane of the eye, known as the macular region of the retina. This can lead to a central visual field defect, which involves the 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 defect (blindness) in people over 50 years old in the United States today. Some macular dystrophies that occur in younger individuals are sometimes referred to as macular degeneration, and the term is generally used to refer to age-related macular degeneration (AMD or ARMD).

[0066] As used herein, "age-related macular degeneration (AMD)" refers to a serious eye condition when the small central part of the retina known as the macula deteriorates. AMD includes exudative AMD and neovascular AMD. Exudative AMD (also called wet AMD or neovascular AMD) is characterized by abnormal blood vessel growth from the choroid under the macula. This is called choroidal neovascularization. These blood vessels leak blood and fluid (downward and) into the retina, causing visual distortion such as the appearance of wavy lines (due to retinal elevation) and blind spots and central visual field defects. These abnormal blood vessels eventually form scars, resulting in a permanent central visual field defect. Symptoms of age-related macular degeneration include a dark, blurred area in the center of the visual field; and lost or changed color vision. Age-related macular degeneration can be detected by a regular eye examination. One of the most common early signs of macular degeneration is the presence of drusen, small yellow deposits under the retina, and pigment clumps.

[0067] Progressive age-related macular degeneration, which is associated with significant vision loss, has two disease forms: dry and exudative. Central geographic atrophy, which is the dry form of progressive age-related macular degeneration, results from atrophy of the subretinal retinal pigment epithelium layer, which causes vision loss through a reduction in photoreceptors (rods and cones) in the central part of the eye. Since there is no treatment available for this condition, vitamin supplements, along 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 vision in some patients.

[0068] Retinitis pigmentosa (RP) is a group of hereditary eye conditions. In the progression of the symptoms of retinitis pigmentosa, night blindness generally occurs several years or even decades before visual field constriction. Many people with retinitis pigmentosa do not become legally blind until their 40s or 50s and retain some vision throughout their lives. Others become completely blind from retinitis pigmentosa, sometimes as early as in childhood. The progression of retinitis pigmentosa varies in each case. Retinitis pigmentosa is a type of hereditary retinal dystrophy, a group of hereditary disorders that lead to progressive vision loss due to abnormalities in the photoreceptors (rods and cones) or retinal pigment epithelium (RPE) of the retina. The affected individual first experiences abnormal dark adaptation or night blindness (nyctalopia), followed by a reduction in the peripheral visual field (also known as constriction of the visual field) and sometimes a central visual field defect in the later stages of the disease.

[0069] Macular edema occurs when fluid and protein deposits collect above or below the macula of the eye, which is the central area of the retina involved in good vision, causing thickening and swelling of the retina. 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 eye. This area contains densely packed cones that enable a person to see the shape, color, and details directly in the line of sight by providing a sharp and clear central vision. 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 develops 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 of the eye, leading to thickening and swelling (edema) of the retina. 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 eye. The main symptoms of diabetic macular edema include, but are not limited to, blurred vision, floaters, decreased contrast sensitivity, double vision, and ultimately vision loss. The pathophysiology of diabetic macular edema is typically characterized by the breakdown of the inner blood-retinal barrier that normally prevents the movement of fluid into the retina, allowing fluid to accumulate within the retinal tissue, and the presence of retinal thickening. Diabetic macular edema is currently diagnosed during an eye examination consisting of a visual acuity test that measures the smallest letters a person can read on a standard eye chart, a dilated eye examination to look for signs of the disease, a contrast examination such as optical coherence tomography (OCT) or fluorescein angiography (FA), and tonometry, which 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 major categories: focal and diffuse. Focal diabetic macular edema is characterized by discrete and distinct areas of leakage within the macula that have sufficient blood flow within the macula. Diffuse diabetic macular edema results from leakage of the entire capillary bed surrounding the macula, which is caused by the breakdown of the inner blood-retinal barrier of the eye. In addition to focal and diffuse, diabetic macular edema can also be classified clinically based on examination findings into clinically significant macular edema (CSME), diabetic macular edema that is not clinically significant, and diabetic macular edema associated with the center-involved (CI) fovea (CSME-CI). The present invention includes methods for treating diabetic macular edema of the above categories.

[0071] The best corrected visual acuity (BCVA) is determined by taking each letter score using a method adapted from the Early Treatment Diabetic Retinopathy Study [ETDRS] protocol at a distance of 4 meters (using an ETDRS-like chart for diabetic retinopathy).

[0072] Disease activity is determined, for example, through a decrease in BCVA / ETDR letter score and / or through macular thickening by spectral domain optical coherence tomography (SD-OCT) including the center of the macula as the central subfoveal thickness (CST). In one preferred embodiment, the central subfoveal thickness (CST) is determined using spectral domain optical coherence tomography (SD-OCT). In one preferred embodiment, the central subfoveal thickness is measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ device; in one preferred embodiment, the central subfoveal thickness is measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ device; in one embodiment, the central subfoveal thickness is measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ device; in one embodiment, the central subfoveal thickness is measured by spectral domain optical coherence tomography (SD-OCT) using an Optvue™ device. As used herein, the term "patient having" refers to a human who exhibits one or more symptoms or signs of an ocular vascular disease as described herein and / or who has been diagnosed with an ocular vascular disease as described herein. The term "patient having" also includes, for example, a subject who, prior to treatment, exhibits (or has exhibited) one or more signs of an ocular vascular disease, such as retinal neovascularization, angiogenesis, vascular leakage, retinal thickening of the fovea, hard yellow subfoveal 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, double vision, and ultimate vision loss.

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

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

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

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

[0077] One embodiment of the present invention is a treatment method, use, bispecific antibody (for use), pharmaceutical, or pharmaceutical formulation as described herein, where the ocular vascular disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a dosing schedule at regular 8-week intervals (Q8W) after the start of treatment (in one embodiment, the start of treatment includes administration every 5 to 7 months; in one embodiment, the start of treatment includes administration every 6 months).

[0078] One embodiment of the present invention is a treatment method, use, bispecific antibody (for use), pharmaceutical, or pharmaceutical formulation as described herein, where the ocular vascular disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a dosing schedule at regular 12-week intervals (Q12W) after the start of treatment (in one embodiment, the start of treatment includes administration every 5 to 7 months; in one embodiment, the start of treatment includes administration every 6 months). In one embodiment, there is an initial 1-dose Q8W cycle before the regular Q12W dosing schedule after the start of treatment.

[0079] One embodiment of the present invention is a treatment method, use, bispecific antibody (for use), pharmaceutical, or pharmaceutical formulation as described herein, wherein the ocular vascular disease is diabetic macular edema, and the treatment of a patient with diabetic macular edema includes a dosing schedule that, after initiation of treatment, extends the dosing interval if the disease is stably absent or shortens the interval if disease activity is present (in one embodiment, initiation of treatment includes dosing every 3 to 7 months; in one embodiment, initiation of treatment includes dosing every 3 to 5 months; in one embodiment, initiation of treatment includes dosing at least every 4 months; in one embodiment, initiation of treatment includes dosing every 4 to 6 months). In one embodiment, such a dosing schedule includes the patient receiving dosing of Q4W or Q8W or Q12W or Q16W depending on the patient's disease state. In one embodiment, stable absence of the disease is - an increase in central subfield thickness (CST) of less than 50 μm, - a decrease in best corrected visual acuity (BCVA / ETDRS) of less than 5 letters as determined, and disease activity is - an increase in central subfield thickness (CST) of 50 μm or more, - a decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more as determined.

[0080] In one embodiment, stable absence of the disease is - the central subfield thickness (CST) is less than about 300 μm (in one embodiment, less than 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ instrument; in one embodiment, less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ instrument; in one embodiment, less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ instrument; in one embodiment, less than 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ instrument) as determined, and disease activity is - The central foveal thickness (CST) of the retina exceeds about 300 μm (in one embodiment, it exceeds 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ device; in one embodiment, it exceeds 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ device; in one embodiment, it exceeds 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ device; in one embodiment, it exceeds 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ device). is determined as.

[0081] One embodiment of the present invention is a treatment method, use, bispecific antibody (for use), pharmaceutical, or pharmaceutical formulation as described herein, wherein the ocular vascular disease is age-related macular degeneration (in one embodiment, exudative age-related macular degeneration), and the treatment of a patient suffering from age-related macular degeneration (in one embodiment, exudative age-related macular degeneration) includes a dosing schedule that, after the start of treatment, extends the dosing interval if the disease is stably absent or shortens the interval if disease activity is present (in one embodiment, the start of treatment includes dosing every 3 to 7 months; in one embodiment, the start of treatment includes dosing every 3 to 5 months; in one embodiment, the start of treatment includes dosing at least every 4 months; in one embodiment, the start of treatment includes dosing every 4 to 6 months). In one embodiment, such a dosing schedule includes the patient receiving dosing of Q4W or Q8W or Q12W or Q16W depending on the patient's disease state. In one embodiment, the stable absence of the disease is - an increase in the central foveal thickness (CST) of less than 50 μm; and / or - a decrease in the best corrected visual acuity (BCVA / ETDRS) of less than 5 letters is determined as, and the disease activity is - an increase in the central foveal thickness (CST) of 50 μm or more; and / or - a decrease in the best corrected visual acuity (BCVA / ETDRS) of 5 letters or more is determined as

[0082] In one embodiment, the stable absence of disease is - the central subfield thickness (CST) is less than about 300 μm (less than 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ instrument in one embodiment; less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ instrument in one embodiment; less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ instrument in one embodiment; less than 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ instrument in one embodiment) determined as, and the disease activity is - the central subfield thickness (CST) is greater than about 300 μm (greater than 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ instrument in one embodiment; greater than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ instrument in one embodiment; greater than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ instrument in one embodiment; greater than 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ instrument in one embodiment) determined as

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

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

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

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

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

[0088] As used within this application, the term "valence" indicates the presence of a specific number of binding sites within an antibody molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" indicate the presence of two, four, and six binding sites, respectively, within an antibody molecule. The bispecific antibodies according to the present invention are preferably "bivalent".

[0089] As used herein, "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 term "antibody" is synonymous and refers to an antibody having 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 International Publication No. WO 2010 / 040508, International Publication No. WO 2011 / 117329, International Publication No. WO 2012 / 131078, International Publication No. WO 2015 / 083978, International Publication No. WO 2017 / 197199, and International Publication No. WO 2014 / 009465. International Publication No. WO 2014 / 009465 describes bispecific anti-VEGF / ANG2 antibodies that are specially designed for the treatment of ocular vascular diseases. The bispecific anti-VEGF / ANG2 antibodies of International Publication No. WO 2014 / 009465 (which is incorporated herein by reference in its entirety) are particularly useful for the treatment and treatment planning of ocular vascular diseases as described herein.

[0091] In one embodiment, a bispecific antibody that binds to 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 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 the heavy-chain variable domain, the CDR3H region of SEQ ID NO: 1, the CDR2H region of SEQ ID NO: 2, and the CDR1H region of SEQ ID NO: 3, and, in the light-chain variable domain, the CDR3L region of SEQ ID NO: 4, the CDR2L region of SEQ ID NO: 5, and the CDR1L region of SEQ ID NO: 6; ii) the second antigen-binding site that specifically binds to ANG-2 comprises, in the heavy-chain variable domain, the CDR3H region of SEQ ID NO: 9, the CDR2H region of SEQ ID NO: 10, and the CDR1H region of SEQ ID NO: 11, and, in the light-chain variable domain, the CDR3L region of SEQ ID NO: 12, the CDR2L region of SEQ ID NO: 13, and the 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 Kabat EU index).

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

[0093] In one embodiment, such a bispecific anti-VEGF / ANG2 antibody i) The first antigen-binding site that specifically binds to VEGF comprises the amino acid sequence of SEQ ID NO: 7 as the heavy chain variable domain VH and the amino acid sequence of SEQ ID NO: 8 as the light chain variable domain VL. 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 aspect of the invention, such a bispecific and bivalent antibody according to the invention a) the heavy and light chains of a first full-length antibody that specifically binds to VEGF; b) the modified heavy and modified light chains of a second full-length antibody that specifically binds to ANG-2 (where the constant domains CL and CH1 are exchanged with each other) characterized by comprising.

[0095] This bispecific and bivalent antibody format for a bispecific antibody that specifically binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) is described in International Publication No. WO 2009 / 080253 (including a knob-into-hole type modified CH3 domain). Antibodies based on this bispecific and bivalent antibody format have been named CrossMAb.

[0096] In one embodiment, such a bispecific and bivalent anti-VEGF / ANG2 antibody is 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 characterized by comprising.

[0097] In one embodiment, such a bispecific and bivalent anti-VEGF / ANG2 antibody is characterized by comprising 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 and bivalent anti-VEGF / ANG2 antibody is faricimab.

[0098] Accordingly, one embodiment of the present invention is a bispecific and bivalent 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, characterized by comprising 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 and bivalent anti-VEGF / ANG2 antibody is faricimab.

[0099] In one embodiment, the CH3 domains of the bispecific and bivalent antibodies according to the present invention are modified by the "knob-into-hole" technology, which is detailed with several examples in, for example, WO 96 / 027011, Ridgway J.B., et al., Protein Eng 9 (1996) 617-621; and Merchant, A.M., et al., Nat Biotechnol 16 (1998) 677-681. In this method, the interaction surfaces of the two CH3 domains are modified to increase the heterodimerization of both heavy chains containing these two CH3 domains. The two CH3 domains (of the two heavy chains) can each be a "knob", while the other is a "hole". The introduction of disulfide bridges stabilizes the heterodimer (Merchant, A.M, 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 present invention, the bispecific anti-VEGF / ANG2 antibody according to the present invention the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain contact at an interface that includes the original interface between the CH3 domains of the antibody; wherein the interface is modified to facilitate the formation of the bispecific antibody, and the modification herein a) the CH3 domain of one heavy chain is modified, such that within the original interface of the CH3 domain of the other heavy chain that contacts the original interface of the CH3 domain of one heavy chain within the bispecific antibody, an amino acid residue is substituted with an amino acid residue having a larger side chain volume, thereby creating a protrusion within the interface of the CH3 domain of one heavy chain, which can be located within a cavity within the interface of the CH3 domain of the other heavy chain, and b) the CH3 domain of the other heavy chain is modified, Thereby, within the original interface of the second CH3 domain that contacts the original interface of the first CH3 domain in the bispecific antibody, the amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the interface of the second CH3 domain, into which a protrusion within the interface of the first CH3 domain can be positioned characterized in that.

[0101] Therefore, the bispecific anti-VEGF / ANG2 antibody for use described herein preferably 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 the interface, including modifications within the original interface between the CH3 domains of the antibody; where i) in the CH3 domain of one heavy chain, the amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion within the interface of one heavy chain's CH3 domain, which can be positioned within the cavity within the interface of the other heavy chain's CH3 domain, and ii) in the CH3 domain of the other heavy chain, the amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the interface of the second CH3 domain, into which a protrusion within the interface of the first CH3 domain can be positioned characterized in that.

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

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

[0104] In one aspect of the present invention, both CH3 domains are further modified by introducing cysteine (C) as an amino acid at the corresponding positions of each CH3 domain, whereby a disulfide bridge can be formed 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 inter-chain disulfide bridge may be used between the CH3 domains by introducing an S354C mutation in one CH3 domain and a Y349C mutation in the other CH3 domain (Merchant, A.M, et al., Nature Biotech 16 (1998) 677-681).

[0106] In another preferred embodiment, the bispecific antibody comprises S354C and T366W mutations in one of the two CH3 domains, and Y349C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. In another preferred embodiment, the bispecific antibody comprises Y349C, T366W mutations in one of the two CH3 domains, and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains (additional Y349C or S354C mutations within one CH3 domain and additional S354C or Y349C mutations within the other CH3 domain form an inter-chain disulfide bridge) (numbering is always according to the Kabat EU index) (Kabat, E.A., 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 modification to enhance heterodimerization are contemplated as alternative methods of the present invention and are described, for example, in International Publication No. 96 / 27011, International Publication No. 98 / 050431, European Patent No. 1870459, International Publication No. 2007 / 110205, International Publication No. 2007 / 147901, International Publication No. 2009 / 089004, International Publication No. 2010 / 129304, International Publication No. 2011 / 90754, International Publication No. 2011 / 143545, International Publication No. 2012 / 058768, International Publication No. 2013 / 157954, and International Publication No. 2013 / 096291.

[0108] In one embodiment, the heterodimerization approach described in European Patent No. 1870459A1 is used instead. This approach is based on the introduction of substitutions / mutations of charged amino acids with opposite charges at specific amino acid positions at the interface within the CH3 / CH3 domain between both heavy chains. One preferred embodiment for said multispecific antibody is the mutation of amino acids R409D and K370E within the CH3 domain of one heavy chain of the multispecific antibody, and the mutation of amino acids D399K and E357K within the CH3 domain of the other heavy chain (numbering according to the Kabat EU index).

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

[0110] In one embodiment, the heterodimerization approach described in International Publication No. WO 2013 / 157953 is used instead. In one embodiment, the CH3 domain of one heavy chain contains the mutation of amino acid T366K, and the CH3 domain of the other heavy chain contains the mutation of amino acid L351D. In a further embodiment, the CH3 domain of one heavy chain further contains the mutation of amino acid L351K.

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

[0112] In one embodiment, the heterodimerization approach described in International Publication No. WO 2012 / 058768 is used instead. In one embodiment, the CH3 domain of one heavy chain contains the mutations of amino acids L351Y and Y407A, and the CH3 domain of the other heavy chain contains the mutations of amino acids T366A and K409F. In a further embodiment, the CH3 domain of the other heavy chain further contains the mutation of an amino acid at position T411, D399, S400, F405, N390, or K392. In one embodiment, the mutation of the amino acid is 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 selected from the group consisting of.

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

[0114] In one embodiment, the heterodimerization approach described in International Publication No. WO 2011 / 143545 is used instead. In one embodiment, the amino acid modifications according to International Publication No. WO 2011 / 143545 are introduced at positions selected from the group consisting of 368 and 409 within the CH3 domain of the heavy chain.

[0115] In one embodiment, the heterodimerization approach described in International Publication No. WO 2011 / 090762, which also uses the above-mentioned knob-into-hole, is used instead. In one embodiment, the CH3 domain of one heavy chain comprises the mutation of amino acid T366W, and the CH3 domain of the other heavy chain comprises the mutation of amino acid Y407A. In one embodiment, the CH3 domain of one heavy chain comprises the mutation of amino acid T366Y, and the CH3 domain of the other heavy chain comprises the mutation of amino acid Y407T.

[0116] In one embodiment, the multispecific antibody is an antibody of the IgG2 isotype, and the heterodimerization approach described in International Publication No. WO 2010 / 129304 is used instead.

[0117] In one embodiment, the heterodimerization approach described in International Publication No. WO 2009 / 089004 is used instead. In one embodiment, the CH3 domain of one heavy chain comprises an amino acid substitution of K392 or N392 with a negatively charged amino acid (in one embodiment, glutamic acid (E) or aspartic acid (D); in a further embodiment, the mutation K392D or N392D), and the CH3 domain of the other heavy chain comprises an amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (in one embodiment, lysine (K) or arginine (R); in a further embodiment, the substitution D399K, E356K, D356K, or E357K; in yet a further embodiment, the mutation D399K or E356K). In a further embodiment, the CH3 domain of one heavy chain further comprises an amino acid substitution of K409 or R409 with a negatively charged amino acid (in one embodiment, glutamic acid (E) or aspartic acid (D); in a further embodiment, the mutation K409D or R409D). In a further embodiment, the CH3 domain of one heavy chain further or alternatively comprises an amino acid substitution of 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 International Publication No. WO 2007 / 147901 is used instead. In one embodiment, the CH3 domain of one heavy chain comprises the mutations of amino acids K253E, D282K, and K322D, and the CH3 domain of the other heavy chain comprises the mutations of amino acids D239K, E240K, and K292D.

[0119] In one embodiment, the heterodimerization approach described in International Publication No. WO 2007 / 110205 is used instead.

[0120] In one embodiment, a bispecific antibody that binds to 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 to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, where i) the first antigen-binding site that specifically binds to VEGF comprises, in the heavy chain variable domain, the CDR3H region of SEQ ID NO: 1, the CDR2H region of SEQ ID NO: 2, and the CDR1H region of SEQ ID NO: 3, and, in the light chain variable domain, the CDR3L region of SEQ ID NO: 4, the CDR2L region of SEQ ID NO: 5, and the CDR1L region of SEQ ID NO: 6; ii) the second antigen-binding site that specifically binds to ANG-2 comprises, in the heavy chain variable domain, the CDR3H region of SEQ ID NO: 9, the CDR2H region of SEQ ID NO: 10, and the CDR1H region of SEQ ID NO: 11, and, in the light chain variable domain, the CDR3L region of SEQ ID NO: 12, the CDR2L region of SEQ ID NO: 13, and the CDR1L region of SEQ ID NO: 14; iii) 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 the Kabat EU index); iv) the heavy chain constant region comprises the T366W mutation in one CH3 domain and the mutations T366S, L368A, and Y407V in the other CH3 domain (numbering according to the Kabat EU index).

[0121] In one embodiment, a bispecific antibody that binds to 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 to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, where i) The first antigen-binding site that specifically binds to VEGF comprises, in the heavy-chain variable domain, the CDR3H region of SEQ ID NO: 1, the CDR2H region of SEQ ID NO: 2, and the CDR1H region of SEQ ID NO: 3, and, in the light-chain variable domain, the CDR3L region of SEQ ID NO: 4, the CDR2L region of SEQ ID NO: 5, and the CDR1L region of SEQ ID NO: 6; ii) The second antigen-binding site that specifically binds to ANG-2 comprises, in the heavy-chain variable domain, the CDR3H region of SEQ ID NO: 9, the CDR2H region of SEQ ID NO: 10, and the CDR1H region of SEQ ID NO: 11, and, in the light-chain variable domain, the CDR3L region of SEQ ID NO: 12, the CDR2L region of SEQ ID NO: 13, and the CDR1L region of SEQ ID NO: 14; iii) 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 the Kabat EU index); iv) The heavy-chain constant region comprises the S354C and T366W mutations in one CH3 domain and the Y349C, T366S, L368A, and Y407V mutations in the other CH3 domain (numbering according to the Kabat EU index).

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

[0123] In one embodiment, such a bispecific anti-VEGF / ANG2 antibody i) The first antigen-binding site that specifically binds to VEGF comprises the amino acid sequence of SEQ ID NO: 7 as the heavy-chain variable domain VH and the amino acid sequence of SEQ ID NO: 8 as the light-chain variable domain VL, 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 characterized in that.

[0124] In one aspect of the present invention, such a bispecific and bivalent antibody according to the present invention is a) the heavy and light chains of a first full-length antibody that specifically binds to VEGF; b) the 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 exchanged with each other) characterized by comprising.

[0125] As used herein, the term "VEGF" refers to 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 the signal peptide), which is a 165-amino acid human vascular endothelial growth factor (VEGF / VEGF-A), and related vascular endothelial growth factor isoforms 121, 189, and 206, as described in Leung, D.W., et al., Science 246 (1989) 1306-9; Houck et al., Mol. Endocrin. 5 (1991) 1806 -1814; Keck, P.J., et al., Science 246 (1989) 1309-12 and Connolly, D.T., et al., J. Biol. Chem. 264 (1989) 20017-24, and also refers to the natural alleles and processing forms of such growth factors. VEGF is involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with tumors and ocular disorders (Ferrara, N., et al., Endocr. Rev. 18 (1997) 4-25; Berkman, R.A.,et al., J. Clin. Invest. 91 (1993) 153-159; Brown, L.F., et al., Human Pathol. 26 (1995) 86-91; Brown, L.F., et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J., et al., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, H.F., et al., Am. J. Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoprotein isolated from several sources and contains several isoforms. VEGF exhibits highly specific mitogenic activity for endothelial cells. VEGF antagonists / inhibitors inhibit the binding of VEGF to its receptor VEGFR. Known VEGF antagonists / inhibitors include bispecific anti-VEGF / ANG2 antibodies as described in International Publication No. WO 2014 / 009465.

[0126] As used herein, the term "ANG-2" refers to human angiopoietin-2 (ANG-2) (also abbreviated as ANGPT2 or ANG2) (SEQ ID NO: 25), as described, for example, in Maisonpierre, P.C., et al, Science 277 (1997) 55-60 and Cheung, A.H., 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 that is selectively expressed within vascular endothelium (Yancopoulos, G.D., et al., Nature 407 (2000) 242-48). There are currently four established members of the angiopoietin family. Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent widely diverged counterparts of the same locus in mouse and human (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 an agonist and an antagonist, respectively, in tissue culture experiments (see Davis, S., et al., Cell 87 (1996) 1161-69 for ANG-1; Maisonpierre, P.C., et al., Science 277 (1997) 55-60 for ANG-2). All known angiopoietins bind primarily 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, P.C., et al., Science 277 (1997) 55-60). An ANG2 antagonist / inhibitor inhibits the binding of ANG2 to its receptor, TIE2. Known ANG2 antagonists / inhibitors include bispecific anti-VEGF / ANG2 antibodies such as those described in International Publication No. WO 2014 / 009465.

[0127] The antigen-binding site of the bispecific antibody of the present invention contains six complementarity-determining regions (CDRs) that contribute to changing the degree 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 ranges of the CDRs and the framework regions (FRs) are determined by comparison with an edited amino acid sequence database, and such regions are defined by the degree of variability between sequences.

[0128] The antibody of the present invention includes an immunoglobulin constant region derived from one or more immunoglobulin classes of human origin, and such immunoglobulin classes include the IgG, IgM, IgA, IgD, and IgE classes, and, in the case of IgG and IgA, their subclasses IgG1 and IgG4.

[0129] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single amino acid composition.

[0130] The term "chimeric antibody" refers to an antibody that typically comprises a variable region, i.e., a binding region, derived from one source or species, prepared by recombinant DNA technology, and at least a part of a constant region derived from a different source or species. A chimeric antibody comprising a murine variable region and a human constant region is preferred. Other preferred forms of "chimeric antibody" encompassed by the present invention are those in which the constant region has been modified or changed from the constant region of the original antibody to give rise to the properties according to the present invention, particularly properties relating to binding to C1q and / or binding to an Fc receptor (FcR). Such chimeric antibodies are also referred to as "class-switch antibodies". A chimeric antibody is an immunoglobulin gene expression product that includes a DNA segment encoding an immunoglobulin variable region and a DNA segment encoding an immunoglobulin constant region. Methods for producing chimeric antibodies include conventional recombinant DNA techniques and gene transfection techniques well known in the art. See, for example, Morrison, S.L., 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 "complementary determining regions" (CDRs) have been modified to include the CDRs of an immunoglobulin having a different specificity compared to the parental 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, M.S., et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to the CDRs showing sequences that recognize the antigens described above for chimeric antibodies. Other forms of "humanized antibody" encompassed by the present invention are those in which the constant region has been modified or changed from the constant region of the original antibody, thereby giving rise to the properties according to the present invention, particularly properties relating to binding to C1q and / or binding to an Fc receptor (FcR).

[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 art of the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable of generating a complete repertoire of human antibodies or selected human antibodies without the production of endogenous immunoglobulins during immunization. Introduction of a human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies upon challenge with an antigen (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 be made 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 the preparation of human monoclonal antibodies (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, A.L., p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95).As already described for the chimeric and humanized antibodies of the present invention, the term "human antibody" as used herein also includes, for example, antibodies whose constant regions have been modified by "class switching", i.e., changes or mutations in the Fc portion (e.g., mutations from IgG1 to IgG4 and / or IgG1 / IgG4), to give rise to the properties described for the present invention, particularly properties related to binding to C1q and / or binding to FcR.

[0133] The term "recombinant antibody" as used herein is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, e.g., antibodies isolated from host cells such as NS0 cells or CHO cells or from animals transgenic for human immunoglobulin genes (e.g., mice), or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant antibodies include variable and constant regions in rearranged form. The recombinant antibodies according to the present invention have been subjected to somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that are derived from and related to human germline VH and VL sequences but may not naturally occur in the human antibody germline repertoire in vivo.

[0134] As used herein, the "variable domain" (the variable domain of the light chain (VL), the variable domain of the heavy chain (VH)) refers to each pair of the light chain and the heavy chain that is directly involved in the binding of the antibody to the antigen. The variable domains of the human light chain and heavy chain have the same general structure, and each domain includes four framework (FR) regions whose sequences are widely conserved and are 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 structures. The CDRs within each chain maintain 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 the antibody play a particularly important role in the binding specificity / affinity of the antibodies described in the present invention, and thus, further objects of the present invention are provided.

[0135] As used herein, the terms "hypervariable region" or "antigen-binding portion of an antibody" refer to the amino acid residues of the antibody that are involved in binding to the antigen. The hypervariable regions include amino acid residues derived 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 the antibody include the domains of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The CDRs on each chain are separated by such framework amino acids. In particular, the CDR3 of the heavy chain is the region that most contributes to the binding to the antigen. The CDR regions and FR regions are determined according to the standard definitions of Kabat, E.A., 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, in the direction from the N-terminus to the C-terminus of the antibody, a polypeptide consisting of a variable heavy chain domain (VH), a constant heavy chain domain 1 (C1), an antibody hinge region (HR), a constant heavy chain domain 2 (CH2), and a constant heavy chain domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and optionally, in the case of a subclass IgE antibody, a constant heavy chain domain 4 (CH4). Preferably, a "full-length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2, and CH3 in the direction from the N-terminus to the C-terminus. A "full-length antibody light chain" is a polypeptide consisting of a variable light chain domain (VL) and a constant light chain domain (CL), abbreviated as VL-CL, in the direction from the N-terminus to the C-terminus of the antibody. The constant light chain domain (CL) can be κ (kappa) or λ (lambda). The two full-length antibody chains are linked to each other via inter-polypeptide disulfide bonds between the CL domain and the CH1 domain and between the hinge regions of the full-length antibody heavy chains. Examples of typical full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE. The full-length antibodies described in the present invention can be of a single species, can be of human origin, for example, or they can be chimeric antibodies or humanized antibodies. The full-length antibodies described in the present invention contain two antigen-binding sites each formed by a pair of VH and VL, both of which specifically bind to the same antigen. The C-terminus of the heavy or light chain of the above-mentioned full-length antibody refers to the last amino acid at the C-terminus of the above-mentioned heavy or light chain. The N-terminus of the heavy or light chain of the above-mentioned full-length antibody refers to the last amino acid at the N-terminus of the above-mentioned heavy or light chain.

[0137] As used herein, the term "constant region" refers to the sum of the domains of an antibody other than the variable regions. The constant regions are not directly involved in binding to an antigen, but exhibit various effector functions. Depending on the amino acid sequences of their heavy-chain constant regions, antibodies are classified into the classes IgA, IgD, IgE, IgG, and IgM, and some of these 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 found in all five antibody classes are called κ (kappa) and λ (lambda).

[0138] As used herein, the terms "human-derived constant region" or "human constant region" refer to the heavy chain constant region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG4, and / or the light chain constant regions κ or λ. Such constant regions are well known in 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 the location within the application and the numbering of mutations, the EU numbering system (EU index) described in Kabat, E.A., 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 "numbering according to Kabat's EU index".

[0139] In one embodiment, the bispecific antibody described in the present invention has 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 C-terminal lysine (Lys447) of the Fc region may or may not be present.

[0140] In one embodiment, the 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 constant portion of the heavy chain amino acid sequence of SEQ ID NO: 17 and the heavy chain amino acid sequence of SEQ ID NO: 18. In a further embodiment, a C-terminal glycine (Gly446) is present. In a further embodiment, a C-terminal glycine (Gly446) and a C-terminal lysine (Lys447) are present.

[0141] Unless otherwise specified herein, the numbering of amino acid residues in the constant region is according to the EU numbering system, also called the Kabat EU index, as described in Kabat, E.A. 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 antibody according to the invention is of the human IgG1 subclass having the mutations L234A (Leu235Ala), L235A (Leu234Ala) and P329G (Pro329Gly). Such antibodies exhibit reduced binding to FcR (in particular they no longer show 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] The previously described Pro329Ala mutation only removes two-thirds of the FcγRIII sandwich interaction, while Pro329Gly in the antibodies described in the present invention completely abrogates the binding of the Fc portion to FcγRIII. This is particularly useful because the binding to FcγRIII is involved in ADCC (antibody-dependent cell cytotoxicity) which can lead to cell death and be beneficial in the treatment of cancer diseases, but can also cause serious side effects in the treatment based on antibodies for other vascular or immunological diseases. Thus, the antibodies described in the present invention of the IgG1 subclass having the mutations L234A, L235A, and P329G, and of the IgG4 subclass having the mutations S228P, L235E, and P329G are particularly useful because neither of them shows any binding to FcRγI, FcRγII, and FcRγIII anymore.

[0144] An “effective amount” of an agent, such as a pharmaceutical formulation or a bispecific anti-VEGF / ANG2 antibody, refers to an amount effective in achieving a desired therapeutic or prophylactic result for the dosage and duration required to achieve such result.

[0145] In one embodiment of the present invention, a bispecific antibody, pharmaceutical, or pharmaceutical formulation as described herein is administered via intravitreal application, for example via intravitreal injection (“intravitreally” administered). This can be carried out according to standard procedures known in the art. See, for example, 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, the therapeutic kit of the invention may contain one or more doses of the described bispecific antibody present in a pharmaceutical or a pharmaceutical formulation, an instrument suitable for intravitreal injection of the pharmaceutical or the pharmaceutical formulation, and an instruction manual detailing the appropriate subject and protocol for performing the injection. In these embodiments, the pharmaceutical or the pharmaceutical formulation is typically administered to a subject in need of treatment via intravitreal injection. This can be carried out according to standard procedures known in the art. See, for example, 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 administration route selected, the bispecific antibodies as described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0148] [Table 1] TIFF2025084976000003.tif118161

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

[0150] 2A. A bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in the treatment of a patient with an 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 the best corrected visual acuity measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the best corrected visual acuity (BCVA) letter score of the patient before dosing with the bispecific VEGF / ANG2 antibody.

[0151] 2B. A bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in the treatment of a patient with an ocular vascular disease, wherein the patient shows improvement in visual acuity after administration of the bispecific VEGF / ANG2 antibody as measured by 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 the best corrected visual acuity measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the best corrected visual acuity (BCVA) letter score of the patient before dosing with the bispecific VEGF / ANG2 antibody.

[0152] 3. The bispecific antibody is administered (scheduled to 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), the bispecific antibody according to any one of Embodiments 2A - 2B (for use).

[0153] 4. The increase in the letters of BCVA BCVA / ETDRS 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 after the start of treatment, the bispecific antibody according to any one of Embodiments 1 - 3 (for use).

[0154] 5. The increase in the letters of BCVA BCVA / ETDRS 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 after the start of treatment, the bispecific antibody according to any one of Embodiments 1 - 3 (for use).

[0155] 6. The bispecific antibody is used to extend the time until re-treatment and / or to extend the time until a decrease in vision (e.g., best corrected visual acuity (BCVA) BCVA / ETDRS), and re-treatment is determined by a central foveal thickness (CST) increased by 50 μm or more (using a spectral domain optical coherence tomography (SD-OCT) in one embodiment); and / or a best corrected visual acuity (BCVA / ETDRS) decreased by 5 letters or more, and is considered necessary in the case of disease activity, the bispecific antibody according to any one of Embodiments 1 to 5 (for use).

[0156] 7. The bispecific antibody is administered at each administration every 3 to 7 months after the start of treatment (in one embodiment, the start of treatment includes administration every 3 to 5 months; in one embodiment, the start of treatment includes administration every 4 months; in one embodiment, the start of treatment includes administration every 5 to 7 months; in one embodiment, the start of treatment includes administration every 6 months), the bispecific antibody according to any one of Embodiments 1 to 6 (for use).

[0157] 8. The ocular vascular disease is selected from the group consisting of exudative age-related macular degeneration (exudative 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, or secondary to branch retinal vein occlusion or hemiretinal vein occlusion, retinitis, conjunctivitis, uveitis, choroiditis, choroidal neovascularization (CNV) secondary to intraocular inflammation (including ocular histoplasmosis or presumed histoplasmosis or choroiditis); myopic choroidal neovascularization (mCNV), and choroidal neovascularization secondary to trauma, retinopathy of prematurity, and iris neovascularization / neovascular glaucoma, the bispecific antibody according to any one of Embodiments 1 to 7 (for use).

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

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

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

[0161] 12. The bispecific antibody according to any one of embodiments 1 to 11, wherein the bispecific antibody is administered every 10 to 12 weeks.

[0162] 13. The bispecific antibody according to any one of embodiments 1 to 11, wherein the bispecific antibody is administered every 11 to 13 weeks.

[0163] 14. The bispecific antibody according to any one of embodiments 1 to 11, wherein the bispecific antibody is administered every 12 to 14 weeks.

[0164] 15. The bispecific antibody according to any one of embodiments 1 to 11, wherein the bispecific antibody is administered every 13 to 15 weeks.

[0165] 16. The bispecific antibody according to any one of embodiments 1 to 11, wherein the bispecific antibody is administered every 14 to 16 weeks.

[0166] 17. The bispecific antibody that binds to human VEGF and human ANG2 is a bispecific and 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, where i) The first antigen-binding site that specifically binds to VEGF includes, in the heavy-chain variable domain, the CDR3H region of SEQ ID NO: 1, the CDR2H region of SEQ ID NO: 2, and the CDR1H region of SEQ ID NO: 3, and, in the light-chain variable domain, the CDR3L region of SEQ ID NO: 4, the CDR2L region of SEQ ID NO: 5, and the CDR1L region of SEQ ID NO: 6; ii) The second antigen-binding site that specifically binds to ANG-2 includes, in the heavy-chain variable domain, the CDR3H region of SEQ ID NO: 9, the CDR2H region of SEQ ID NO: 10, and the CDR1H region of SEQ ID NO: 11, and, in the light-chain variable domain, the CDR3L region of SEQ ID NO: 12, the CDR2L region of SEQ ID NO: 13, and the CDR1L region of SEQ ID NO: 14; iii) The bispecific antibody is the bispecific antibody according to any one of embodiments 1 to 16 (for use) comprising 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 the Kabat EU index).

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

[0168] 19. The bispecific antibody that binds to human VEGF and human ANG2 is the bispecific antibody according to embodiment 18 (for use) comprising 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 is the bispecific antibody according to any one of embodiments 17 to 19 (for use) administered at a dose of about 5 to 7 mg (in each treatment).

[0170] 21. The bispecific antibody is administered at a dose of about 6 mg (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, and is the bispecific antibody for use as described in any one of Embodiments 17 to 19.

[0171] 22. The bispecific antibody is administered at a concentration of about 30 mg / ml, and is the bispecific antibody for use as described in any one of Embodiments 20 to 21.

[0172] 23. The bispecific antibody is administered at a concentration of about 120 mg / ml, and is the bispecific antibody for use as described in any one of Embodiments 20 to 21.

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

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

[0175] 26. The ocular vascular disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a dosing schedule of every 8 weeks (Q8W) at regular intervals after the start of treatment (in one embodiment, the start of treatment includes administration every 5 to 7 months; in one embodiment, the start of treatment includes administration every 6 months), and is the bispecific antibody for use as described in Embodiments 1 to 25.

[0176] 27. The ocular vascular disease is diabetic macular edema, and the treatment of patients suffering from diabetic macular edema includes a dosing schedule of every fixed 12 weeks (Q12W) after the start of treatment (in one embodiment, the start of treatment includes administration every 5 to 7 months; in one embodiment, the start of treatment includes administration every 6 months), the bispecific antibody described in Embodiments 1 to 26 (for use).

[0177] 28. Before the fixed Q12W dosing schedule after the start of treatment, there is a first 1-dose Q8W cycle, the bispecific antibody described in Embodiment 27 (for use).

[0178] 29. The ocular vascular disease is diabetic macular edema, and the treatment of patients suffering from diabetic macular edema includes a dosing schedule that extends the dosing interval if the disease is stably absent or shortens the interval if there is disease activity after the start of treatment (in one embodiment, the start of treatment includes administration every 3 to 7 months; in one embodiment, the start of treatment includes administration every 4 to 6 months), the bispecific antibody described in Embodiments 1 to 28 (for use).

[0179] 30. Such a dosing schedule includes the patient receiving dosing of Q8W or Q12W or Q16W according to the patient's disease state (in one embodiment, dosing of Q4W or Q8W or Q12W or Q16W according to the patient's disease state), the bispecific antibody described in Embodiment 29 (for use).

[0180] 31. The stable absence of the disease is - an increase in the central subfield thickness (CST) of less than 50 μm; and / or - a decrease in the best corrected visual acuity (BCVA / ETDRS) of less than 5 letters determined as, and the disease activity is - an increase in the central subfield thickness (CST) of 50 μm or more; and / or - a decrease in the best corrected visual acuity (BCVA / ETDRS) of 5 letters or more determined as, the bispecific antibody described in Embodiment 29 or 30 (for use).

[0181] 32. The stable absence of disease is - when the central foveal thickness (CST) is less than about 300 μm (in one embodiment, less than 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ instrument; in one embodiment, less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ instrument; in one embodiment, less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ instrument; in one embodiment, less than 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ instrument) determined as, and the disease activity is - when the central foveal thickness (CST) exceeds about 300 μm (in one embodiment, exceeds 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ instrument; in one embodiment, exceeds 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ instrument; in one embodiment, exceeds 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ instrument; in one embodiment, exceeds 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ instrument) determined as, the method according to embodiment 29 or 30.

[0182] 33. The ocular vascular disease is age-related macular degeneration (in one embodiment, exudative age-related macular degeneration), and the treatment of a patient suffering from age-related macular degeneration (in one embodiment, exudative age-related macular degeneration) is, after the start of treatment (in one embodiment, the start of treatment includes administration every 3 to 7 months; in one embodiment, the start of treatment includes administration every 4 to 6 months), to extend the dosing interval if the disease is stably absent or to shorten the interval if disease activity is present, the bispecific antibody according to embodiments 1 to 32 (for use) comprising a dosing schedule.

[0183] 34. Such a dosing schedule includes the patient receiving dosing at Q8W or Q12W or Q16W (in one embodiment, dosing at Q4W or Q8W or Q12W or Q16W) depending on the patient's disease state, for the bispecific antibody described in embodiment 33 (for use).

[0184] 35. The stable absence of disease is - an increase in the central subfield thickness (CST) of less than 50 μm; and / or - a decrease in the best corrected visual acuity (BCVA / ETDRS) of less than 5 letters as determined, and the disease activity is - an increase in the central subfield thickness (CST) of 50 μm or more; and / or - a decrease in the best corrected visual acuity (BCVA / ETDRS) of 5 letters or more as determined, for the bispecific antibody described in embodiment 33 or 34 (for use).

[0185] 36. The stable absence of disease is - the central subfield thickness (CST) being less than about 300 μm (in one embodiment, less than 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ instrument; in one embodiment, less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ instrument; in one embodiment, less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ instrument; in one embodiment, less than 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ instrument) as determined, and the disease activity is -The central foveal retinal thickness (CST) exceeds about 300 μm (in one embodiment, it exceeds 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ device; in one embodiment, it exceeds 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ device; in one embodiment, it exceeds 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ device; in one embodiment, it exceeds 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ device). The bispecific antibody according to embodiment 33 or 34, as determined for use.

[0186] The embodiments of the present invention are listed below: 1. A method 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 to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), wherein the bispecific antibody is administered intravitreally at intervals of 8 weeks or less frequently (in one embodiment, at intervals of 9 weeks or less frequently; in one embodiment, at intervals of 10 weeks or less frequently; in one embodiment, at intervals of 11 weeks or less frequently; in one embodiment, at intervals of 12 weeks or less frequently; in one embodiment, at intervals of 13 weeks or less frequently; in one embodiment, at intervals of 14 weeks or less frequently; in one embodiment, at intervals of 15 weeks or less frequently).

[0187] 2A. A method for treating a patient suffering from an ocular vascular disorder, the method comprising administering to the patient an effective amount of a bispecific antibody that binds to 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, in one embodiment, 15 letters or more) in the best corrected visual acuity as measured using an Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart compared to the best corrected visual acuity (BCVA) letter score of the patient before dosing with the bispecific VEGF / ANG2 antibody.

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

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

[0190] 4. An increase in the letter of the BCVA / ETDRS letter score is measured at the 4th week, and / or the 8th week, and / or the 12th week, and / or the 16th week, and / or the 20th week, and / or the 24th week after the start of treatment, according to the method described in any one of Embodiments 1 to 3.

[0191] 5. An increase in the letter of the BCVA / ETDRS letter score is measured at the 45th week, and / or the 46th week, and / or the 47th week, and / or the 48th week, and / or the 49th week, and / or the 50th week, and / or the 51st week, and / or the 52nd week, and / or the 53rd week, and / or the 54th week, and / or the 55th week, and / or the 56th week, and / or the 57th week, and / or the 58th week, and / or the 59th week, and / or the 60th week after the start of treatment, according to the method described in any one of Embodiments 1 to 3.

[0192] 6. The bispecific antibody is used to extend the time to re-treatment and / or to extend the time to vision loss. Re-treatment with the bispecific antibody is administered when the disease activity is determined as an increase in the central foveal thickness (CST) of 50 μm or more (using a spectral domain optical coherence tomography (SD-OCT) in one embodiment); and / or a decrease in the best corrected visual acuity (BCVA / ETDRS) of 5 letters or more, according to the method described in any one of Embodiments 1 to 5.

[0193] 7. The bispecific antibody is administered at a dose every 3 to 7 months after the start of treatment (in one embodiment, the start of treatment includes a dose every 3 to 5 months; in one embodiment, the start of treatment includes a dose every 4 months; in one embodiment, the start of treatment includes a dose every 5 to 7 months; in one embodiment, the start of treatment includes a dose every 6 months), according to the method described in any one of Embodiments 1 to 6.

[0194] 8. The ocular vascular disease is selected from the group consisting of exudative age-related macular degeneration (exudative 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, or secondary to branch retinal vein occlusion, or secondary to hemiretinal vein occlusion, retinitis, conjunctivitis, uveitis, choroiditis, choroidal neovascularization (CNV) secondary to intraocular inflammation (including ocular histoplasmosis or presumed histoplasmosis or secondary to choroiditis); myopic choroidal neovascularization (mCNV), and choroidal neovascularization secondary to trauma, retinopathy of prematurity, and iris rubeosis / neovascular glaucoma, and is the method according to any one of Embodiments 1 to 7.

[0195] 9. The ocular vascular disease is diabetic macular edema (DME), and is the method according to any one of Embodiments 1 to 7.

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

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

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

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

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

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

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

[0203] 17. A bispecific antibody that binds to human VEGF and human ANG2 is a bispecific and 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, where i) The first antigen-binding site that specifically binds to VEGF comprises, in the heavy-chain variable domain, the CDR3H region of SEQ ID NO: 1, the CDR2H region of SEQ ID NO: 2, and the CDR1H region of SEQ ID NO: 3, and, in the light-chain variable domain, the CDR3L region of SEQ ID NO: 4, the CDR2L region of SEQ ID NO: 5, and the CDR1L region of SEQ ID NO: 6; ii) The second antigen-binding site that specifically binds to ANG-2 comprises, in the heavy-chain variable domain, the CDR3H region of SEQ ID NO: 9, the CDR2H region of SEQ ID NO: 10, and the CDR1H region of SEQ ID NO: 11, and, in the light-chain variable domain, the CDR3L region of SEQ ID NO: 12, the CDR2L region of SEQ ID NO: 13, and the CDR1L region of SEQ ID NO: 14; iii) 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 the Kabat EU index), and the method according to any one of Embodiments 1 to 16.

[0204] 18. i) The first antigen-binding site that specifically binds to VEGF comprises the amino acid sequence of SEQ ID NO: 7 as the heavy-chain variable domain VH and the amino acid sequence of SEQ ID NO: 8 as the light-chain variable domain VL, ii) The second antigen-binding site that specifically binds to ANG-2 is the method according to embodiment 17, which 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 bispecific antibody that binds to human VEGF and human ANG2 is the method according to embodiment 18, which 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 bispecific antibody is administered at a dose of about 5-7 mg (in each treatment), according to any one of embodiments 17-19.

[0207] 21. The bispecific antibody is administered at a dose of about 6 mg (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), according to any one of embodiments 17-19.

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

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

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

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

[0212] 26. The ocular vascular disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a dosing schedule that is regular every 8 weeks (Q8W) after the start of treatment (in one embodiment, the start of treatment includes administration every 5 to 7 months; in one embodiment, the start of treatment includes administration every 6 months), the method described in Embodiments 1 to 25.

[0213] 27. The ocular vascular disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a dosing schedule that is regular every 12 weeks (Q12W) after the start of treatment (in one embodiment, the start of treatment includes administration every 5 to 7 months; in one embodiment, the start of treatment includes administration every 6 months), the method described in Embodiments 1 to 26.

[0214] 28. The method described in Embodiment 27, wherein there is a first 1-dose Q8W cycle before the regular Q12W dosing schedule after the start of treatment.

[0215] 29. The ocular vascular disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a dosing schedule that, after the start of treatment, extends the dosing interval if the disease is stably absent or shortens the interval if disease activity is present (in one embodiment, the start of treatment includes administration every 3 to 7 months; in one embodiment, the start of treatment includes administration every 4 to 6 months), the method described in Embodiments 1 to 28.

[0216] 30. Such a dosing schedule includes the patient receiving dosing of Q8W or Q12W or Q16W according to the patient's disease state (in one embodiment, dosing of Q4W or Q8W or Q12W or Q16W according to the patient's disease state), the method described in Embodiment 29.

[0217] 31. The stable absence of the disease is - an increase in the central subfield thickness (CST) of less than 50 μm; and / or - a decrease in the best corrected visual acuity (BCVA / ETDRS) of less than 5 letters determined as, and the disease activity is - an increase in the central subfield thickness (CST) of 50 μm or more; and / or The best corrected visual acuity (BCVA / ETDRS) decreased by 5 letters or more as determined by the method according to embodiment 28 or 29

[0218] 32. The stable absence of the disease is - the central subfield thickness (CST) is less than about 300 μm (in one embodiment, less than 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ instrument; in one embodiment, less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ instrument; in one embodiment, less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ instrument; in one embodiment, less than 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ instrument) determined as, and the disease activity is - the central subfield thickness (CST) is greater than about 300 μm (in one embodiment, greater than 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ instrument; in one embodiment, greater than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ instrument; in one embodiment, greater than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ instrument; in one embodiment, greater than 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ instrument) as determined by the method according to embodiment 28 or 29

[0219] 33. Treatment of patients suffering from age-related macular degeneration (exudative age-related macular degeneration in one embodiment) includes a dosing schedule that, after initiation of treatment (in one embodiment, initiation of treatment includes administration every 3 to 7 months; in one embodiment, initiation of treatment includes administration every 4 to 6 months), extends the dosing interval if the disease is stably absent or shortens the interval if disease activity is present, as described in Methods of Embodiments 1-32.

[0220] 34. Such a dosing schedule includes the patient receiving dosing at Q8W or Q12W or Q16W (in one embodiment, Q4W or Q8W or Q12W or Q16W dosing) depending on the patient's disease state, as described in the method of Embodiment 33.

[0221] 35. Stable absence of the disease is determined as - an increase in central subfield thickness (CST) of less than 50 μm; and / or - a decrease in best corrected visual acuity (BCVA / ETDRS) of less than 5 letters and disease activity is determined as - an increase in central subfield thickness (CST) of 50 μm or more; and / or - a decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more as described in the method of Embodiment 33 or 34.

[0222] 36. Stable absence of the disease is - the central subfield thickness (CST) being less than about 300 μm (in one embodiment, less than 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ instrument; in one embodiment, less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ instrument; in one embodiment, less than 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ instrument; in one embodiment, less than 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ instrument) determined as, and disease activity is - the central subfield thickness (CST) of the retina exceeds about 300 μm (in one embodiment, exceeds 325 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis™ instrument; in one embodiment, exceeds 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus™ instrument; in one embodiment, exceeds 315 μm as measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon™ instrument; in one embodiment, exceeds 295 μm as measured by spectral domain optical coherence tomography (SD-OCT) using an Optovue™ instrument) determined as, the method according to embodiment 33 or 34.

[0223] Examples Treatment of patients suffering from ocular vascular diseases using a bispecific antibody that binds to human VEGF and human ANG2 Example 1A: Efficacy and duration of treatment of patients suffering from diabetic macular edema (DME) Objective The first objective The first objective of this study was as follows: To evaluate the efficacy of 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, compared to a placebo, in untreated patients with central-involved diabetic macular edema (CI-DME). The sterile, colorless to slightly brownish preservative-free solution of RO6867461 for intravitreal administration at a dose of either 1.5 mg or 6 mg every 4 weeks was used. The concentration of the bispecific antibody was approximately 120 mg / ml. This bispecific anti-VEGF / ANG2 antibody in this specification is named RO6867461 or RG7716 or VEGFang2-0016, or faricimab. The generation of this antibody VEGFang2-0016 is also detailed in International Publication No. WO 2014 / 009465, which is incorporated herein by reference.

[0224] The second objective The second objective of this clinical trial was as follows: To examine the pharmacokinetics and anatomical outcomes to provide information on the mechanism of action of RO6867461. To examine the formation of anti-RO6867461 antibodies in plasma. To explore the duration of the effect of RO6867461.

[0225] Exploratory objectives The exploratory objectives of this clinical trial were as follows: To explore the predicted effect of previous intravitreal anti-VEGF antibody treatment on the efficacy of RO6867461. To evaluate the efficacy and safety of RO6867461 compared to the active drug in patients with diabetic macular edema involving the fovea who had previously received intravitreal anti-VEGF antibody treatment. To evaluate the effect of RO6867461 on plasma marker levels of angiogenesis and inflammation. To examine the concentration of RO6867461 in aqueous humor samples (optional) and vitreous humor (optional), and to examine biomarkers of angiogenesis and inflammation if sample volume permits. To evaluate the improvement of the diabetic retinopathy (DR) severity score.

[0226] Trial design This was a multi-center, randomized, active drug-controlled, double-blind, parallel-group, 36-week trial using multiple doses in patients with diabetic macular edema involving the fovea. The three groups in this trial were as follows: 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 principal investigator of the trial.

[0228] Number of patients Patients up to a maximum of 210 were randomized.

[0229] Approximately 150 untreated patients and approximately 60 patients who had been previously treated intravitreally with an anti-VEGF antibody participated in the clinical trial.

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

[0231] Target population Male and female patients 18 years of age or older with diabetic macular edema having a fovea.

[0232] Selection criteria / Exclusion criteria Selection criteria Patients must meet the following criteria to participate in the clinical trial:

[0233] Eye criteria for the study eye: Macular edema associated with diabetic retinopathy, defined as macular thickening by spectral domain optical coherence tomography (SD-OCT) including the central part of the macula: Central subfield thickness (CST) of 325 μm or more in the foveal region using Spectralis (trademark) (Heidelberg Engineering) at screening (if Spectralis (trademark) is not available, the following devices and foveal region retinal thickness thresholds were acceptable: 315 μm or more in the foveal region for Cirrus (trademark), 315 μm or more in the foveal region for Topcon, 295 μm or more in the foveal region for Optovue (trademark)).

[0234] On day 1, having a best-corrected visual acuity (BCVA) letter score of 73 - 24 letters (including the boundary value) in the Early Treatment Diabetic Retinopathy Study (ETDRS)-like chart (equivalent to 20 / 40 - 20 / 320 in Snellen notation), with vision decreased mainly due to diabetic macular edema.

[0235] A transparent light-transmitting body and appropriate pupil dilation that enable acquisition of high-quality retinal images for a definitive diagnosis.

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

[0237] Ability and willingness to prepare written informed consent and comply with the clinical trial protocol according to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and local regulations. Alternatively, a legally authorized representative must consent to the patient in accordance with the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use and local regulations.

[0238] 18 years of age or older.

[0239] Women who are not postmenopausal (i.e., amenorrhea not induced by treatment, confirmed by follicle-stimulating hormone, for more than 12 months if not receiving hormone replacement therapy), or who have consented 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 at least 4 weeks after the last dose.

[0240] Abstinence is only acceptable if it is in line with the patient's preferred and normal lifestyle. Periodic abstinence (e.g., calendar method, ovulation method, basal body temperature method, or post-ovulation method) and withdrawal method were not acceptable contraceptive methods.

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

[0242] For males, consent to use barrier contraception during the treatment period of at least 4 weeks after the last dose of the investigational drug.

[0243] Patients must refrain from attempting to participate in any other clinical trials involving the investigational medicinal product (IMP) or device until the completion of the current trial.

[0244] Exclusion criteria Patients meeting any of the following criteria were excluded from participation in the trial:

[0245] Eye criteria for the study eye: Any signs of high-risk proliferative diabetic retinopathy as defined below: Any vitreous hemorrhage or preretinal hemorrhage. Neovascularization of more than half of the disc area within a region equivalent to the standard 7-field ETDRS using mydriatics in clinical examination. Neovascularization of more than one-third of the disc area in clinical examination. Any treatment with anti-VEGF antibodies into the vitreous within 3 months prior to Day 1. Any extensive retinal photocoagulation (PRP) treatment prior to Day 1. Any macular laser photocoagulation within 3 months prior to Day 1. History of retinal vitreous surgery. Any treatment with intravitreal or periocular corticosteroids within 3 months prior to Day 1. Any history of use of Iluvien® or Ozurdex® implants prior to Day 1 would not be tolerated. Any cataract surgery or treatment of complications of cataract surgery using steroids within 3 months prior to Day 1. History of incisional glaucoma surgery. Uncontrolled glaucoma (e.g., progressive visual field loss, or intraocular pressure [IOP] of more than 25 mmHg despite treatment with glaucoma medications).

[0246] Concurrent eye conditions in the study eye: History of rubeosis Any current eye disease or eye disease history other than diabetic macular edema that can disrupt the evaluation of the macula or affect central vision (e.g., age-related macular degeneration, retinal vein occlusion, uveitis, retinitis pigmentosa, histoplasmosis, active or inactive cytomegalovirus, pathologic myopia, retinal detachment, macular traction, macular hole, severe cataract). Any current eye condition (e.g., foveal atrophy, pigment abnormalities, dense central foveal hard exudates, extraretinal conditions) in which, in the opinion of the principal investigator of the clinical trial, visual acuity decline will not be improved by the resolution of macular edema. Any active eye infection on Day 1. Any active intraocular inflammation on Day 1 (grade is minimal or more).

[0247] Characteristics of the other eye: Any treatment with an anti-VEGF antibody within 7 days prior to Day 1. Any retinal condition in which, in the opinion of the principal investigator of the clinical trial, treatment with an anti-VEGF antibody may be required within 7 days from Day 1.

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

[0249] Untreated diabetes, or initiation of oral antidiabetic drugs or insulin within 4 months prior to Day 1, or anticipated change of antidiabetic drugs during the trial period. Renal insufficiency that requires renal transplantation, hemodialysis, or peritoneal dialysis within 6 months prior to Day 1, or is expected to require hemodialysis or peritoneal dialysis at any point during the trial. Medical history of other diseases, metabolic dysfunction, conditions where the use of the investigational drug is contraindicated, or physical examination findings or clinical laboratory findings that may reasonably be suspected to affect the interpretation of the trial results, or may place the patient at high risk in the treatment of complications according to the opinion of the responsible investigator of the trial. Women of childbearing potential, positive pregnancy blood test. Women who are breastfeeding. Systemic use of corticosteroids within 1 month prior to Day 1. Any known allergies to the investigational drug, fluorescein, any component of the formulation used, mydriatic eye drops, or any anesthetic and antibacterial eye drops used. Any other restrictions due to the use of the investigational drug. Any treatment with the investigational drug within 3 months prior to Day 1.

[0250] Duration of the trial The total duration of the trial was up to 40 weeks for each patient who participated as follows (from screening to end of the trial): Screening: up to 4 weeks. Baseline: Day 1. Treatment administration period of the trial: from Day 1 to Week 20. Observation period: from Week 20 to up to Week 36. Requirements for follow-up observation regarding safety: during the observation period and for 7 days after the administration of ranibizumab.

[0251] End of the trial The end of the trial was defined as the day on which the last patient's last observation (LPLO) was conducted. LPLO was expected to be conducted 36 weeks after the last patient participated.

[0252] Evaluation Items for Efficacy and Pharmacokinetics The population for the primary analysis was treatment-naive patients. Additional analyses may be conducted in the overall population and in patients previously treated with intravitreal anti-VEGF antibodies.

[0253] The primary efficacy evaluation item for this clinical trial was the mean change in BCVA (ETDRS letters) from baseline at week 24 in treatment-naive patients.

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

[0255] Exploratory evaluation items The exploratory evaluation items in this clinical trial included, but were not limited to, the following: BCVA: Difference in the mean change in best-corrected visual acuity (BCVA) from baseline between treatment-naive patients and patients previously administered intravitreal anti-VEGF antibodies (difference in the effect of RO6867461).

[0256] Exploratory evaluation items related to persistence: Time to an increase in central foveal area 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 values at week 20. Time to retreatment with 0.3 mg of ranibizumab after week 20.

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

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

[0259] Best corrected visual acuity The best corrected visual acuity at a test distance of 4 meters at the start was measured by trained and certified vision examiners who were blinded to the assignment of the investigational drug group before mydriasis.

[0260] Best corrected visual acuity was measured by using a set of three Precision Vision (trademark) or the Light House distance visual acuity charts (revised ETDRS charts 1, 2, and R). A vision manual was provided to the principal investigator of the trial. Certifications for the vision examiners and the vision examination room were obtained before any vision examinations were conducted.

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

[0262]

Table 2

[0263] The main evaluation items of efficacy are shown in Figure 1. Figure 1 shows the change in best-corrected visual acuity over time from baseline to week 24 for patients who have not been treated previously. VA2 refers to the bispecific anti-VEGF / ANG2 antibody RO6867461 containing the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 (doses of 6.0 mg or 1.5 mg are administered intravitreally), and RBZ refers to ranibizumab (Lucentis®) (a dose of 0.3 mg is administered intravitreally).

[0264] Change in central subfield thickness (CST) from baseline (study eye) An important secondary evaluation item was the change in central subfield thickness (CST) from baseline. The results are shown in Figure 2. The bispecific anti-VEGF / ANG2 antibody RO6867461 containing the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 (doses of 6.0 mg or 1.5 mg are administered intravitreally) was compared with ranibizumab (Lucentis®) (a dose of 0.3 mg is administered intravitreally). This second anatomical evaluation item directly supports the primary evaluation item of best-corrected visual acuity.

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

[0266] The numerical values of best-corrected visual acuity and central subfield thickness obtained at week 24 were compared with the numerical values obtained at the visit at week 20. The numerical values of best-corrected visual acuity and central subfield thickness obtained at weeks 28, 32, and 36 were compared with the numerical values obtained at week 24.

[0267] If a patient met both of the following criteria, the patient received a single dose of 0.3 mg of ranibizumab and the study was terminated: · The central subfield thickness increased by 50 μm or more. ·The best corrected visual acuity decreased by 5 letters or more due to diabetic macular edema.

[0268] The results are shown in Figure 3: Figure 3 shows the time to re-treatment after dosing was discontinued (20 weeks later or after dosing every 6 months = time point after the last intravitreal (IVT) administration) based on disease activity evaluated by both a best corrected visual acuity that decreased by 5 letters or more and a central foveal area retinal thickness that increased by 50 μm or more. The bispecific anti-VEGF / ANG2 antibody RO6867461 (doses of 6.0 mg or 1.5 mg were administered intravitreally), which contains 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®) (a dose of 0.3 mg was administered intravitreally).

[0269] For overview, Figure 4 shows a graphical comparison of 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 of Patients with Diabetic Macular Edema (DME) In a further trial similar to the trial described above under Example 1A, patients suffering from diabetic macular edema (e.g., diabetic macular edema including the fovea (CI-DME)) are treated with a bispecific antibody that binds to human VEGF and human ANG2 and comprises the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. For treatment, a placebo such as aflibercept and / or ranibizumab and / or brolucizumab will be used. Patients include those who have not received treatment with an anti-VEGF antibody (who have not been previously treated with anti-VEGF antibody monotherapy, e.g., using aflibercept and / or ranibizumab and / or brolucizumab), and also a group of patients who have been previously treated with anti-VEGF antibody monotherapy. The name of each bispecific antibody that binds to human VEGF and human ANG2 is RO6867461 or RG7716. Use vials of a sterile, colorless to slightly brown preservative-free solution of RO6867461 for intravitreal administration at either a dose of 1.5 mg or 6 mg.

[0271] Use one or more of the following dosing regimens: a) Patients suffering from diabetic macular edema will be treated with a fixed dosing regimen every 8 weeks after the start of treatment (e.g., initially injections every 6 months). b) Patients suffering from diabetic macular edema will be treated with a fixed dosing regimen every 12 weeks after the start of treatment (e.g., initially injections every 6 months) (one regimen includes first a cycle of dosing every 8 weeks). c) Patients suffering from diabetic macular edema will be treated with a dosing prescription regimen that extends the injection interval when the disease is stably absent or shortens the interval when disease activity is present after the start of treatment (e.g., initially injections every 3 to 7 months). Such a prescription regimen includes, for example, the patient receiving dosing of Q4W / Q8W / Q12W / Q16W depending on the patient's disease state.

[0272] The evaluation of disease stability will be based on the best corrected visual acuity (BCVA), the central subfield thickness (CST), and the retinal thickness based on optical coherence tomography (OCT). The evaluation items and results will be evaluated as described in Example 1A, for example. The primary evaluation items will be from week 45 to week 60.

[0273] In one embodiment, a patient suffering from diabetic macular edema is untreated (has not been previously treated with monotherapy with an anti-VEGF antibody, such as aflibercept and / or ranibizumab and / or brolucizumab).

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

[0275] In one embodiment, a patient suffering from diabetic macular edema will be treated with a regular dosing schedule every 8 weeks after the start of treatment (for example, initially with injections every 6 months).

[0276] In one embodiment, a patient suffering from diabetic macular edema will be treated with a regular dosing schedule every 12 weeks after the start of treatment (in one embodiment, including a first cycle of dosing every 8 weeks).

[0277] In one embodiment, a patient suffering from diabetic macular edema will be treated with a dosing prescription plan that extends the injection interval if the disease is stably absent or shortens the interval if disease activity is present after the start of treatment (for example, initially with injections every 3 - 7 months). In one embodiment, such a prescription plan includes the patient receiving dosing of Q4W / Q8W / Q12W / Q16W depending on the patient's disease state.

[0278] In one embodiment, a patient suffering from age-related macular degeneration will be treated with a dosing regimen that extends the injection interval if the disease is stable after initiation of treatment (e.g., initially every 3 - 4 months) or shortens the interval if disease activity is present. In one embodiment, such a dosing regimen will include the patient receiving Q4W / Q8W / Q12W / Q16W dosing, depending on the patient's disease state.

[0279] Example 2A: Efficacy and Duration of Treatment of Patients with Age-Related Macular Degeneration (AMD) Objectives and Evaluation Items This study evaluated the efficacy, safety, and pharmacokinetics of RO6867461 administered at 12-week and 16-week intervals 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 (the generation of this antibody, VEGFang2-0016, is also detailed in International Publication No. WO 2014 / 009465, which is incorporated by reference). The name of this bispecific anti-VEGF / ANG2 antibody in this specification is RO6867461 or RG7716 or VEGFang2-0016 or faricimab.

[0280] A summary of the specific objectives and corresponding evaluation items for the study is shown below.

[0281] Objectives and Corresponding Evaluation Items Primary Efficacy Objectives · To evaluate the efficacy of RO6867461 on visual acuity when administered at 12-week and 16-week intervals.

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

[0283] Second Efficacy Objective: 1) To evaluate the efficacy of RO6867461 against additional visual acuity evaluation items. Corresponding evaluation items · Average value of the change from the baseline of the best corrected visual acuity over time using an ETDRS-like chart. · Ratio of patients with an increase of 15 letters or more, 10 letters or more, 5 letters or more, or 0 letters or more from the baseline of the best corrected visual acuity over time. · Ratio of patients avoiding a decrease of 15 letters or more, 10 letters or more, 5 letters or more, or 0 letters or more from the baseline of the best corrected visual acuity over time. · Ratio of patients with a best corrected visual acuity of 20 / 40 or better over time. · Ratio of patients with a best corrected visual acuity of 20 / 200 or worse over time.

[0284] 2) To evaluate the efficacy of RO6867461 against anatomical evaluation items using SD-OCT. Corresponding evaluation items · Average value of the change from the baseline of the retinal thickness in the foveal region over time. · Average value of the change from the baseline of the average value of the retinal thickness in the foveal region (1 mm in diameter) over time. · Ratio of patients with intraretinal fluid, subretinal fluid, cysts, or retinal pigment epithelial detachment over time.

[0285] 3) To evaluate the efficacy of RO6867461 against anatomical evaluation items using fundus fluorescein angiography (FFA). Corresponding evaluation items · Average value of the change from the baseline in the entire area of choroidal neovascularization (CNV) at week 40 and week 52. · Average value of the change from the baseline in the entire area of choroidal neovascularization components at week 40 and week 52. · Average value of the change from the baseline in the entire area of leakage at week 40 and week 52.

[0286] Purpose of exploring efficacy · Investigate the incidence of disease activity at week 24. Corresponding evaluation items · The proportion of patients with disease activity at week 24.

[0287] Purpose of safety · Evaluate the safety of multiple doses of RO6867461 intravitreally at 12-week intervals and 16-week intervals. Corresponding evaluation items · Incidence and severity of ocular adverse events. · Incidence and severity of adverse events other than the eye. · Other safety data, including but not limited to reasons for withdrawal from the clinical trial, clinical data, concomitant medications, vital signs, and physical examination results, will be listed and summarized by description.

[0288] Purpose of exploring pharmacokinetics / pharmacodynamics 1) Evaluate the systemic pharmacokinetic profile of RO6867461. Corresponding evaluation items · Plasma concentration of RO6867461 at specific time points. 2) Evaluate the profiles of RO6867461, ranibizumab, free VEGF-A, and Ang-2 in the aqueous humor. · Relationship between the concentration or pharmacokinetic parameters of RO6867461 in the aqueous humor and the concentrations of free VEGF-A and Ang-2. Corresponding evaluation items · Relationship between the ranibizumab concentration or pharmacokinetic parameters in the aqueous humor and the concentrations of free VEGF-A and Ang-2. · Time course of the concentrations of free VEGF-A and Ang-2 in the aqueous humor.

[0289] Purpose of immunogenicity · Examine the formation of anti-RO6867461 antibodies in plasma. Corresponding evaluation items · Incidence of anti-drug antibodies during the clinical trial.

[0290] Purpose of exploring biomarkers To evaluate the response to RO6867461, explore the levels of angiogenesis and inflammation biomarker candidates in aqueous humor at baseline and additional time points. Corresponding evaluation items · The relationship between the primary and secondary evaluation items and the aqueous humor concentration of biomarker candidates. Abbreviations used above: ADA = anti-drug antibody; Ang-2 = angiopoietin-2; BCVA = best corrected visual acuity; CFT = central foveal thickness; CNV = choroidal neovascularization; CST = central foveal retinal 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] Trial design (Figure 5 presents an overview of the trial design) Description of the trial This was a Phase II, multi-center, randomized, placebo-controlled, double-masked, parallel-group, 52-week trial to investigate the efficacy, safety, and pharmacokinetics of RO6867461 administered to untreated patients with neovascular age-related macular degeneration (nAMD) at 12-week intervals and 16-week intervals.

[0292] Approximately 75 patients participated and were 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 (injections at weeks 24, 36, and 48; 3 injections) until week 48. · Group B (Q16W): 6 mg of RO6867461 is injected intravitreally every 4 weeks (4 injections) until week 12, and then 6 mg of RO6867461 is injected intravitreally every 16 weeks (injections at weeks 28 and 44; 2 injections) until week 48. The evaluation defined by the protocol for disease activity at week 24 requires that Group B patients with active disease (refer to the following criteria) switch to a dosing schedule of 6 mg of RO6867461 every 12 weeks for the remainder of the trial. Injections are initiated at week 24 and repeated at weeks 36 and 48. · Group C (control group): 0.5 mg of ranibizumab (intravitreally) every 4 weeks for 48 weeks (13 injections). Only one eye will be selected as the study eye. The total duration of the trial for each patient will be up to 56 weeks and will be classified as follows: · Screening: Up to 4 weeks before randomization or on the same day as randomization. · Randomization: Day 1. · Administration of study treatment: From day 1 to week 48. · Final visit: Week 52.

[0293] Patients underwent screening tests within 4 weeks prior to administration of the study treatment. Visits for screening tests and the visit at week 1 / day 1 (randomization) may be combined if all evaluations (other than informed consent) are completed within 48 hours of the visit date. During screening (or the combined visit date for the screening test visit / day 1 visit), including central reading of fundus photography (FP), spectral domain optical coherence tomography (SD-OCT), and fundus fluorescein angiography (FFA), the eligibility of patients was evaluated to ensure that choroidal neovascularization secondary to age-related macular degeneration met the predefined eye criteria in the trial. Patients considered ineligible based on screening results for any of the following reasons were eligible for rescreening: · Uncontrolled blood pressure · Administrative reasons (e.g., unable to schedule day 1 within 28 days of the screening test visit) ·Does not meet the eligibility criteria for the study eye (in which case, the patient may be eligible to participate in the second eye after the initial screening period).

[0294] At the time of re-screening, an assessment of the patient's visit at all screening examinations was performed (excluding the acquisition of images for fundus fluorescein angiography). However, eligible fundus fluorescein angiography (FFA) images from the central reading center were taken within 4 weeks prior to the new Day 1 visit (randomization).

[0295] On Day 1, eligible patients received an initial intravitreal injection of either RO6867461 or ranibizumab according to the above randomization plan and established standard dosing procedures. Patients returned to the eye clinic 7 days after the initial intravitreal injection and then every 4 weeks for study treatment administration and evaluation as outlined in the activity plan in the protocol. To maintain blinding throughout the study period, sham intravitreal injections were delivered to the patients randomized to Groups A and B.

[0296] All patients were evaluated for disease activity at Week 24. Patients randomized to Group B who had active disease (see criteria below) at Week 24 were switched to a Q12W dosing schedule of 6 mg of RO6867461 for the remainder of the study period, 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 foveal retinal thickness (central foveal retinal thickness greater than 50 μm on Spectralis® OCT) compared to the mean value of central foveal retinal thickness over the last two visits (Weeks 16 and 20), or ·An increase in central foveal retinal thickness of 75 μm or more compared to the lowest central foveal retinal thickness recorded at either Week 16 or Week 20, or · Compared with the mean value of the best corrected visual acuity over the most recent two visits (at 16 weeks and 20 weeks) due to the disease activity of neovascular age-related macular degeneration, a decrease in the best corrected visual acuity (BCVA) of at least 5 letters, or · Compared with the highest best corrected visual acuity recorded at either 16 weeks or 20 weeks due to the disease activity of neovascular age-related macular degeneration, a decrease in the best corrected visual acuity of 10 letters or more, or · The presence of new macular hemorrhage due to the activity of neovascular age-related macular degeneration.

[0298] The patient will return for the last visit at week 52. After the last visit, adverse events should be followed up as outlined in the protocol. Assessments conducted in the case of unscheduled visits are determined by the principal investigator of the clinical trial.

[0299] Number of patients: Approximately 75 untreated patients with neovascular age-related macular degeneration were expected to participate in this trial in the United States and be randomized.

[0300] Target population Eligibility criteria Patients met the following criteria for participation in the trial: Eye criteria for the trial eye. · Untreated choroidal neovascularization secondary to age-related macular degeneration (neovascular age-related macular degeneration). · Subfoveal or juxtafoveal choroidal neovascularization with a foveal component (such as demonstrated by subretinal fluid, subretinal hyperreflective material, evidence of leakage, or hemorrhage) related to choroidal neovascular activity by fundus fluorescein angiography or spectral domain optical coherence tomography. ·All types of choroidal neovascularization lesions (predominantly classic, minimally classic, or occult) associated with: The total lesion size (including blood, atrophy, fibrosis, and neovascularization) is 6 disc areas or less as determined by fundus fluorescein angiography, and the area of the choroidal neovascularization component is 50% or more of the total lesion size as determined by fundus fluorescein angiography, and active choroidal neovascularization (evidence of leakage) as confirmed by fundus fluorescein angiography, and exudates of choroidal neovascularization (presence of fluid) as confirmed by spectral domain optical coherence tomography. ·A clear light-transmitting body and appropriate pupil dilation that enable the acquisition of high-quality retinal images for a definitive diagnosis. General Criteria ·Signature on the informed consent form. ·Being 50 years of age or older on Day 1. ·In the judgment of the principal investigator of the clinical trial, being able to comply with the clinical trial protocol. ·For women of childbearing potential: Agreeing to maintain abstinence (refraining from sexual intercourse with the opposite sex) or using 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 the investigational treatment. ·The patient must refrain from entering and participating in any other clinical trial, including investigational medicinal products (IMP) or devices, until the completion of the current clinical trial.

[0301] Exclusion Criteria Patients who met any of the following criteria were excluded from participating in the clinical trial: Eye Criteria for the Study Eye ·Choroidal neovascularization caused by causes other than age-related macular degeneration, such as ocular histoplasmosis, trauma, pathologic myopia, retinitis pigmentosa, choroidal rupture, or uveitis. ·Central serous chorioretinopathy at the time of screening. ·Retinal pigment epithelial tear involving the macula. ·In fundus fluorescein angiography, subretinal hemorrhage that exceeds 50% of the total lesion area and / or includes foveal fibrosis or atrophy that exceeds 50% of the total lesion area and / or involves the fovea. · Any pretreatment or combination treatment for choroidal neovascularization, including (but not limited to) intravitreal treatment (steroids, anti-vascular endothelial growth factor [VEGF] antibodies, tissue plasminogen activator, ocriplasmin, perfluoropropane gas, air), pharmacological intervention around the eyeball, photocoagulation with an argon laser, verteporfin photodynamic therapy, diode laser, transpupillary thermotherapy, or surgical intervention. · Cataract surgery within 3 months from baseline evaluation (day 1). · Any other intraocular surgery (transscleral pars plana vitrectomy, glaucoma surgery, corneal transplantation, radiation therapy). · Previous intravitreal treatment (including anti-VEGF antibody drugs), excluding the management of cataract complications using intravitreal treatment with steroids. · Previous pharmacological intervention around the eyeball for other retinal diseases. Current ocular condition · Any co-existing intraocular condition in the study eye (e.g., amblyopia, aphakia, retinal detachment, cataract, diabetic retinopathy, or macular disease, or a preretinal membrane with traction) that, in the opinion of the principal investigator of the clinical trial, either reduces the likelihood of visual improvement or may require medical or surgical intervention during the course of the clinical trial. · Active intraocular inflammation (grade or more) in the study eye on day 1 (before randomization). · The highest corrected visual acuity letter score of 73 to 24 letters (including the boundary value) on the Early Treatment Diabetic Retinopathy Study (ETDRS) - like chart in the study eye on day 1 (equivalent to 20 / 40 to 20 / 320 in Snellen notation). · Current vitreous hemorrhage in the study eye. · Uncontrolled glaucoma in the study eye (e.g., progressive visual field loss, or an intraocular pressure [IOP] of 25 mmHg or more as defined, despite treatment with glaucoma medications). · The equivalent spherical refractive error showing myopia exceeding 8 diopters in the study eye. · A 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 1 month before screening. · Uncontrolled blood pressure (defined as systolic blood pressure > 180 mmHg and / or diastolic blood pressure > 100 mmHg at patient rest [blood pressure]). If the patient's initial 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 by hypertensive medication, the patient should have been taking the same medication continuously for at least 30 days prior to Day 1. · Stroke or myocardial infarction within 3 months before Day 1. · History of other diseases, metabolic dysfunction, use of investigational drug is contraindicated, or may affect the interpretation of the trial results, or the patient is at high risk for treatment complications according to the opinion of the principal investigator, with a condition that is reasonably suspected, physical examination findings, or clinical laboratory findings. · Pregnant, breastfeeding, or planning to become pregnant during the trial. Women of childbearing potential must have a negative pregnancy urine test within 28 days before the start of the trial procedure. If the pregnancy urine test is positive, it must be confirmed by a pregnancy serum test. · Known allergy to ranibizumab, fluorescein, any component of the formulation used, mydriatic eye drops, or any anesthetic and antibacterial eye drops used. · Treatment with investigational therapy within 3 months before the start of the trial procedure.

[0302] End of the trial The end of the trial was defined as the day on which the last patient's last visit (LPLV) was conducted. The LPLV was expected to be conducted 52 weeks after the last patient participated.

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

[0304] Test article of investigational drug RO6867461 drug (120 mg / mL) is provided as a sterile, colorless to slightly brownish liquid and contains no preservatives. Vials of RO6867461 solution without preservatives for intravitreal administration at a dose of 6 mg per vial were used. The concentration of the bispecific antibody was approximately 120 mg / ml.

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

[0306] Patients were given a 50 μL intravitreal injection of RO6867461 or ranibizumab or placebo administration to the study eye according to a randomization plan as described below. · Group A (Q12W): Intravitreal injection of 6 mg of RO6867461 every 4 weeks until week 12 (4 injections), then intravitreal injection of 6 mg of RO6867461 every 12 weeks until week 48 (injections at weeks 24, 36, and 48; 3 injections). · Group B (Q16W): Intravitreal injection of 6 mg of RO6867461 every 4 weeks until week 12 (4 injections), then intravitreal injection of 6 mg of RO6867461 every 16 weeks until week 48 (injections at weeks 28 and 44; 2 injections). · 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] Evaluation of the study When several evaluations are performed simultaneously, the following order was suggested at the discretion of the principal investigator of the study. The order may be adjusted to optimize the time management of the site personnel and patients, except when it is stated explicitly as obligatory (i.e., in the italicized text). · Vital signs · Blood sample collection: In the hospital visits where fundus fluorescein angiography is performed, blood sample collection and angiography can be performed from the same venous cannula. The blood sample must be collected before angiography. · Eye evaluation and imaging. Best corrected visual acuity: The best corrected visual acuity must be performed before pupil dilation. At the time of the screening visit and the visit on Day 1, in patients who may fail the screening as a result of the letter score of the best corrected visual acuity, in order to avoid unnecessary examinations, it may be performed before vital signs and blood sample collection. Slit-lamp microscopy. Pupil dilation. SD-OCT. Fundus photography (+ infrared reflectance). Fundus fluorescein angiography. Binocular indirect ophthalmoscopy with high magnification under pupil dilation. Intraocular pressure: There is an obligation to perform it after all imaging evaluations, and the same method should be used throughout the trial period.

[0309] · Collection of aqueous humor sample (optional) Disease-specific evaluation Unless otherwise specified in the activity plan (Appendix 1), all eye evaluations were performed on both eyes.

[0310] Best corrected visual acuity The best corrected visual acuity at a test distance starting from 4 meters was measured by a trained and certified visual acuity (VA) examiner blinded to the allocation of the study eye treatment before pupil dilation.

[0311] The best corrected visual acuity was measured using a set of distance visual acuity charts (Revised ETDRS Charts 1, 2, and R) from Precision Vision (trademark) or the Light House. A visual acuity procedure manual was provided to the principal investigator of the study. Approval of the visual acuity examiner and the visual acuity examination room was obtained before any visual acuity examination was performed.

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

[0313] Additional eye evaluations The following are examples of additional eye evaluations performed during the trial: · Slit lamp microscopy (a scale for grading hyperemia per cell and vitreous hemorrhage density is detailed in Addendum 2). · Binocular indirect ophthalmoscopy with high magnification after pupil dilation. · Intraocular pressure

[0314] The method of measuring intraocular pressure used for the patients was consistent throughout the trial. The intraocular pressure of both eyes was measured after all imaging.

[0315] At the time of the trial treatment visit, the intraocular pressure was measured before the administration of the trial treatment and 30 (±15) minutes after the administration of the treatment to the study eye. If the intraocular pressure is 30 mmHg or higher, the intraocular pressure should be re-evaluated 30 (±15) minutes later. If the intraocular pressure continues to rise, treatment was performed at the discretion of the trial responsible physician. · Evaluation of counting fingers vision For each patient, the perfusion fluid of the optic nerve head after treatment in the study eye was evaluated by testing counting fingers vision, hand movement, or light perception as appropriate immediately after the administration of the trial treatment (within a maximum of 15 minutes after the administration of the treatment).

[0316] Eye imaging The Central Reading Center provided the Central Reading Center Manual and teaching materials for the ocular imaging required in the clinical trial to the site. Before the trial images were obtained, the site personnel and the imaging system (where applicable) were approved by the Reading Center as specified in the Central Reading Center Manual. Ocular images of all trial subjects were obtained only by the equipment approved / registered at the trial site and by the personnel trained and certified by the Central Reading Center. Copies of the ocular images of all trial subjects were transferred to the Central Reading Center for storage and for independent analysis (including for confirmation of the eligibility of the specified image-related criteria).

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

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

[0319] Results Best corrected visual acuity (BCVA) and duration of increase in BCVA (time to re-treatment to maintain increase in BCVA) The primary efficacy endpoints are shown in Figure 6. Figure 6 shows the change in BCVA from baseline over time up to week 40. RO6867461 refers to the bispecific anti-VEGF / ANG2 antibody RO6867461 (a dose of 6.0 mg administered intravitreally either Q12W or Q16W) that includes 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 at a dose of 0.3 mg Q4W. The initial increase in best corrected visual acuity was fully maintained in the Q12W or Q16W groups of RO6867461 and was maintained in a similar range in the Q4W group of ranibizumab (Lucentis®).

[0320] Change in central subfield thickness (CST) from baseline (study eye) An important secondary endpoint was the change in central subfield thickness (CST) from baseline. The results are shown in Figure 7. The bispecific anti-VEGF / ANG2 antibody RO6867461 (a dose of 6.0 mg administered intravitreally either Q12W or Q16W) that includes 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®) (administered intravitreally at a dose of 0.3 mg Q4W). This anatomical secondary endpoint directly supported the primary endpoint of best corrected visual acuity. The central subfield thickness decreased more with RO6867461, the bispecific anti-VEGF / ANG2 antibody, than with ranibizumab during the treatment period.

[0321] Example 2B: Efficacy and duration of treatment in patients with age-related macular degeneration (AMD) In a further trial similar to the trial described above under Example 2A, patients suffering from age-related macular degeneration (e.g., exudative 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 and that comprises the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. As the active agent in the treatment, for example, aflibercept and / or ranibizumab and / or brolucizumab will be used. Patients include those who have not been treated with an anti-VEGF antibody (who have not been previously treated with monotherapy with an anti-VEGF antibody, e.g., aflibercept and / or ranibizumab and / or brolucizumab) and also a group of patients who have been previously treated with monotherapy with an anti-VEGF antibody, e.g., aflibercept and / or ranibizumab and / or brolucizumab. The name of each bispecific antibody that binds to human VEGF and human ANG2 is RO6867461 or RG7716. Use a vial of a sterile, colorless to brownish tint preservative-free solution of RO6867461 for intravitreal administration at either a dose of 1.5 mg or 6 mg.

[0322] For example, use the following dosing schedule: Patients suffering from age-related macular degeneration will be treated using a dosing regimen where, after the start of treatment (e.g., initially using injections every 3 to 7 months), the injection interval is extended if the disease is stable or shortened if there is disease activity. Such a dosing regimen will include, for example, the patient receiving a Q4W / Q8W / Q12W / Q16W dosing, depending on the patient's disease state.

[0323] The evaluation of disease stability will be based on best corrected visual acuity (BCVA) and central foveal retinal thickness, and also retinal thickness based on optical coherence tomography (OCT). The evaluation items and results will be evaluated, for example, as described in Example 1A. The primary evaluation items will be from week 45 to week 60.

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

[0325] Affinity of Ang2 solution, including assessment of species cross-reactivity Solution affinity measures the affinity of an interaction by determining the concentration of free interacting pairs in an equilibrium mixture. The solution affinity assay was maintained at a constant concentration of <vegf-ang-2>It includes mixing the bispecific antibody with ligands (= Ang2) at various concentrations. An antibody with the maximum possible resonance units (e.g., 17,000 resonance units (RU)) was 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 were HBS-P (pH 7.4). The flow cell was set at 25 °C, the sample block was set at 12 °C, and priming was performed twice using the running buffer. To create a calibration curve, increasing concentrations of Ang2 were added to the immobilized <vegf-ang2>It was injected into a Biacore (trademark) flow cell containing a bispecific antibody. The amount of bound Ang2 was determined as resonance units (RU) and plotted against the concentration. Each ligand ( <vegf-ang-2>Solutions of the bispecific antibody at 11 concentrations from 0 to 200 nM were incubated with 10 nM Ang2 and allowed to reach equilibrium at room temperature. The free Ang2 concentration was determined from a calibration curve generated before and after measuring the response of a solution containing a known amount of Ang2. A four-parameter fit was set up using XLfit4 (IDBS software) with Model 201, using the 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 for 30 seconds using a 0.85% H 3 PO 4 solution at a flow rate of 30 μl / min. The difference in bulk refractive index was corrected by subtracting the response obtained from the blank-coupled surface. The results are shown in Table 6.

[0326] Affinity at the steady state of FcRn For the measurement of FcRn, bispecific antibodies against each other were compared using the steady-state affinity. Human FcRn was diluted in coupling buffer (10 μg / ml, sodium acetate pH 5.0) and immobilized on a C1 chip (GE Healthcare BR-1005-35) by a targeting immobilization procedure using a Biacore™ Wizard until a final response of 200 RU was achieved. The flow cell was set at 25 °C, the sample block was set at 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 at 30 μl / min and various samples were continuously injected onto the chip surface while selecting an association time of 180 seconds. The surface was regenerated with PBS-T (pH 8) injected at a flow rate of 30 μl / min for 60 seconds. The difference in bulk refractive index was corrected by subtracting the response obtained from the blank surface. Buffer injections were also subtracted (= double control). For the calculation of the steady-state affinity, the method of Bia-Evaluation software was used. Briefly, the numerical value of resonance units (RU maximum value) was plotted against the analyzed concentration to obtain a dose-response curve. The upper asymptote was calculated based on a two-parameter fit, and the maximum half-value RU value and thus the affinity could be determined. The results are shown in Figure 5 and Table 7. Similarly, the affinity for cynomolgus monkey, mouse, and rabbit FcRn can also be determined.

[0327] Measurement of FcγRIIIa For the measurement of FcγRIIIa, a direct binding assay was used. A capture system of approximately 3000 resonance units (RU) (1 μg / ml penta-His; Qiagen) was coupled onto a CM5 chip (GE Healthcare BR-1005-30) at pH 5.0 by using an amine coupling kit supplied by GE Healthcare. The sample and sample buffer were HBS-P+ pH 7.4. The flow cell was set at 25 °C (the sample block was set at 12 °C) and primed twice with the running buffer. The FcγRIIIa-His receptor was captured by injecting a 100 nM solution for 60 seconds at a flow rate of 5 μl / min. Binding was measured by injecting a 100 nM bispecific antibody or a single specific control antibody (anti-Dig antibody for antibodies of IgG1 and IgG4 subclasses) for 180 seconds at a flow rate of 30 μl / min. The surface was regenerated by washing with a glycine (pH 2.5) solution for 120 seconds at a flow rate of 30 μl / min. Since the binding of FcγRIIIa deviates from the Langmuir 1:1 model, binding only / no binding was determined using this assay. Similarly, the 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 abolishes further detectable binding to FcγRIIIa.

[0328] <vegf-ang-2>Evaluation of the Binding of VEGF and Ang-2 Independently to Bispecific Antibodies A capture system of approximately 3500 resonance units (RU) (10 μg / ml goat anti-human IgG; GE Healthcare Biosciences, Sweden) was coupled onto a CM4 chip (GE Healthcare BR-1005-34) at pH 5.0 by 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 temperature of the flow cell was set at 25 °C and the temperature of the sample block was set at 12 °C. Prior to capture, the flow cell was primed twice with running buffer.

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

[0330] 1. Inject human VEGF at a concentration of 1.200 nM for 180 seconds (to identify the single binding of the antigen) 2. Inject human Ang2 at a concentration of 100 nM for 180 seconds (to identify the single binding of the antigen) 3. Inject human VEGF at a concentration of 200 nM for 180 seconds, and then further inject human Ang2 at a concentration of 100 nM for 180 seconds (to identify the binding of Ang2 in the presence of VEGF) 4. Inject human Ang2 at a concentration of 100 nM for 180 seconds, and then further inject human VEGF at a concentration of 200 nM (to identify the binding of VEGF in the presence of Ang2) 5. Simultaneously inject human VEGF at a concentration of 200 nM and human Ang2 at a concentration of 100 nM for 180 seconds (to identify the binding of VEGF and Ang2 simultaneously)

[0331] The surface was 3 mM MgCl 2 It was regenerated by washing the solution at a flow rate of 30 μl / min for 60 seconds. The difference in bulk refractive index was corrected by subtracting the response obtained from the goat anti-human IgG surface.

[0332] If the final signals obtained as a result of Approaches 3, 4, and 5 are equal to or similar to the sum of the individual final signals of Approaches 1 and 2, the bispecific antibody can bind to both antigens independently of each other. The results are shown in the following table, where VEGFang2-0016 (= RO6867461) is shown to be able to bind to VEGF and ANG2 independently of each other.

[0333] <vegf-ang-2>Evaluation of Simultaneous Binding to VEGF and Ang2 for Bispecific Antibody First, VEGF (20 μg / ml) of approximately 1600 resonance units (RU) was coupled onto a CM4 chip (GE Healthcare BR-1005-34) at pH 5.0 by 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) at pH 7.4. The flow cell was set at 25°C, the sample block was set at 12°C, and primed twice with running buffer. Second, a solution of 50 nM bispecific antibody was injected at a flow rate of 30 μl / min for 180 seconds. Third, hANG-2 was injected at a flow rate of 30 μl / min for 180 seconds. The binding response of hAng-2 depends on the amount of bispecific antibody bound to VEGF and indicates simultaneous binding. The surface was regenerated by washing with 0.85% H 3 PO 4 solution at a flow rate of 30 μl / min for 60 seconds. Simultaneous binding was pre-bound to VEGF <vegf-ang-2>Shown by additional specific binding signals of hAng2 to bispecific antibodies.

[0334] [Table 3]

[0335] [Table 4]

[0336] [Table 5]

[0337] [Table 6]

[0338] [Table 7] < / vegf>

Claims

[Claim 1] A pharmaceutical composition for use in the treatment of diabetic macular edema (DME), comprising faricimab, a bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), A loading dose of 6 mg of faricimab will be administered intravitreally (IVT) every 12 weeks for 3 to 7 doses, followed by 6 mg of faricimab administered IVT every 12 weeks. Pharmaceutical compositions.

Images