Treatment of ophthalmic 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 murine antibody as bevacizumab (Avastin®). However, it has been affinity matured to provide a stronger binding to VEGF-A (WO 98 / 45331). Since the 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 visual impairment.

[0004] Corneal angiogenesis assays showed that both ANG-1 and ANG-2 had similar effects and promoted the proliferation of new blood vessels by acting synergistically with VEGF (Asahara, T., et al., Circ. Res. 83 (1998) 233-40). The possibility of a dose-dependent endothelial response arose from the observation that ANG-2 may also be angiogenic at high concentrations in vitro (Kim, I., et al., Oncogene 19 (2000) 4549-52). At high concentrations, ANG-2 acts as an apoptotic survival factor for endothelial cells during apoptosis induced by serum depletion, through the activation of Tie2 via PI-3 kinase and the Akt pathway (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 neovascularization of the choroid or retina, respectively. Bleeding and leakage from these blood vessels can lead to retinal dysfunction and decreased vision. 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 impaired visual function. These conditions are the main causes of vision loss in industrialized countries. The retina consists of well-defined layers of neuronal, glial, and vascular components, and even relatively minor damage, such as that seen in vascular proliferation or edema, can lead to significant vision loss. Hereditary retinal degenerations, such as retinitis pigmentosa (RP), also involve vascular abnormalities, including arteriole stenosis and vascular atrophy. These conditions affect a large number of individuals, about 1 in 3,500, and are characterized by progressive night blindness, visual field defects, optic nerve atrophy, weakening of arterioles, and central visual field defects, which often progress to total blindness.

[0006] Ischemic retinopathy is characterized by a reduction or dysfunction of the retinal vascular structure, which results in decreased blood flow and hypoxia. The retina responds to hypoxia by generating signals to proliferate new blood vessels, but these new vessels are usually fragile and disorderly. It is the proliferation of these abnormal new blood vessels that creates the majority of the threat to vision because they can leak, bleed, or scar, which can ultimately lead to retinal detachment. Current treatments for ischemic retinopathy seek to stop the proliferation of diseased blood vessels, but do not address the underlying ischemia that drives the proliferation. Furthermore, standard treatment for diabetic retinopathy, an ischemic retinopathy affecting millions of people, involves laser-assisted partial destruction of the retina in an attempt to destroy ischemic tissue in order to stop the proliferation of new blood vessels and preserve central vision. Strategies are being used to block the function of vascular endothelial growth factor (VEGF), a major promoter of abnormal blood vessel proliferation and leakage. In the short term, anti-VEGF antibody therapy can improve vision, but it does not address the underlying ischemia and, in fact, inhibits the growth of all blood vessels, including beneficial collateral vessels, potentially worsening the condition. Furthermore, systemic exposure to these drugs is a serious concern in elderly and / or diabetic patients where new vascular growth may be necessary 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), drug, or pharmaceutical formulation is provided for the treatment of a patient suffering from an ophthalmic vascular disease, the method comprising the step of 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 intravitreously every 8 weeks or less (in one embodiment, every 9 weeks or less; in one embodiment, every 10 weeks or less; in one embodiment, every 11 weeks or less; in one embodiment, every 12 weeks or less; in one embodiment, every 13 weeks or less; in one embodiment, every 14 weeks or less; in one embodiment, every 15 weeks or less; in one embodiment, every 16 weeks or less).

[0008] One aspect of the present invention is a method, use, bispecific antibody (for use), pharmaceutical, or pharmaceutical formulation (for use) for the treatment of / for the treatment of a patient suffering from ophthalmic vascular disease, the method, use, bispecific antibody (for use), pharmaceutical, or pharmaceutical formulation (for use) comprising the step of administering to a 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 (13 letters or more in one embodiment, 14 letters or more in one embodiment, and 15 letters or more in one embodiment) in best corrected visual acuity (BCVA) as measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally every 8 weeks or less. One embodiment of the present invention is a method for treating a patient with ophthalmic vascular disease, comprising the step of 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 improvement in visual acuity after administration of the bispecific VEGF / ANG2 antibody, as measured by an increase of 12 or more letters (13 or more letters in one embodiment, 14 or more letters in another embodiment, and 15 or more letters in another embodiment) in best-corrected visual acuity (BCVA) letter score measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best-corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally every 8 weeks or less.

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

[0010] In one embodiment of the present invention, the increase in letters of the BCVA / ETDRS letter score occurs at 24 weeks and / or 25 weeks and / or 26 weeks and / or 27 weeks and / or 28 weeks and / or 29 weeks and / or 30 weeks and / or 31 weeks and / or 32 weeks and / or 33 weeks and / or 34 weeks and / or 35 weeks and / or 36 weeks and / or 37 weeks and / or 38 weeks and / or 39 weeks and / or 40 weeks after the start of treatment, respectively. Measurements are taken intermittently and / or at weeks 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and / or 60. In one embodiment of the present invention, ocular vascular diseases are 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), nonproliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), macular edema secondary to central retinal vein occlusion, hemiretinal vein occlusion, or branch retinal vein occlusion, retinitis, conjunctivitis, uveitis, choroiditis, choroidal neovascularization (CNV) secondary to intraocular inflammation (including secondary to ocular histoplasmosis or presumptive 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 ophthalmic vascular disease is diabetic macular edema (DME), and the increase in letters of the BCVA / ETDRS letter score is measured approximately 9–15 months after the start of treatment (9–14 months in one embodiment, and 9–12 months in another embodiment).

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

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

[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 letters of the BCVA / ETDRS letter score is measured approximately 9–15 months after the start of treatment (6–9 months in one embodiment, and 6–12 months in another embodiment).

[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 the increase in letters of the BCVA / ETDRS letter score occurs at 24 weeks, and / or 25 weeks, and / or 26 weeks, and / or 27 weeks, and / or 28 weeks, and / or 29 weeks, and / or 30 weeks, and / or 31 weeks, and / or 32 weeks, and / or 33 weeks, and / or 34 weeks, respectively, after the start of treatment. Measurements are taken at / or weeks 35, and / or 36, and / or 37, and / or 38, and / or 39, and / or 40, and / or 41, and / or 42, and / or 43, and / or 44, and / or 45, and / or 46, and / or 47, and / or 48, and / or 49, and / or 50, and / or 51, and / or 52, and / or 53.

[0018] In one embodiment of the present invention, a bispecific antibody that binds to human VEGF and human ANG2 is a bispecific, bivalent anti-VEGF / ANG2 antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, 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 antibodies contain the heavy chain constant regions of human IgG1 subclasses including mutants I253A, H310A, and H435A, and mutants L234A, L235A, and P329G (numbered according to Kabat's EU index).

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

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

[0021] In one embodiment of the present invention, the ophthalmic vascular disease is diabetic macular edema, and the treatment of a patient with diabetic macular edema includes a consistent 8-week (Q8W) medication schedule after the start of treatment.

[0022] In one embodiment of the present invention, the ophthalmic vascular disease is diabetic macular edema, and the treatment of a patient with diabetic macular edema includes a fixed 12-week (Q12W) medication schedule after the initiation of treatment. In one embodiment of the present invention, after the initiation of treatment, there is an initial 1-dose Q8W cycle prior to the fixed Q12W medication schedule.

[0023] In one embodiment of the present invention, the ocular vascular disease is diabetic macular edema, and the treatment of a patient with diabetic macular edema includes a medication plan in which, after the start of treatment, the dosing interval is extended if the disease is stable and absent, or shortened if disease activity is present. In one embodiment of the present invention, such a medication plan includes the patient receiving medication at Q4W, Q8W, Q12W, or Q16W, depending on the patient's disease state. In one embodiment of the present invention, stable disease absence is defined as - Foveal retinal thickness (CST) increased by less than -50 μm; and / or Best corrected visual acuity (BCVA / ETDRS) with a decrease of less than -5 letters It was determined that the disease activity was, - An increased central foveal thickness (CST) of more than 50 μm; and / or - A decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more 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 the disease is - An increased central foveal thickness (CST) of less than 50 μm; and / or - A decrease in best corrected visual acuity (BCVA / ETDRS) of less than 5 letters is determined as such, and the disease activity is - An increased central foveal thickness (CST) of more than 50 μm; and / or - A decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more is determined as such.

Brief Description of the Drawings

[0025] [Figure 1] Change in best corrected visual acuity (BCVA) over time from baseline to week 24 in patients with diabetic macular edema (untreated patients) treated. VA2 refers to the bispecific anti-VEGF / ANG2 antibody RO6867461 (a dose of 6.0 mg or 1.5 mg is administered intravitreally) containing the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, and RBZ refers to ranibizumab (Lucentis®) (a dose of 0.3 mg is administered intravitreally). [Figure 2]Foveal retinal thickness (CST) as measured by spectral domain optical coherence tomography (SD OCT). Changes in foveal retinal thickness from baseline to 24 weeks in treated (untreated) patients with diabetic macular edema. The bispecific anti-VEGF / ANG2 antibody RO6867461 (administered intravitreally at doses of 6.0 mg or 1.5 mg), containing the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, was compared with ranibizumab (Lucentis®) (administered intravitreally at a dose of 0.3 mg). [Figure 3] Based on disease activity assessed by both a maximum corrected visual acuity decrease of 5 letters or more and a foveal retinal thickness increase of 50 μm or more, the time to required retreatment (after discontinuation of medication (20 weeks or after 6 monthly doses = time after the last intravitreal (IVT) administration)) was used. The bispecific anti-VEGF / ANG2 antibody RO6867461 (administered intravitreally at doses of 6.0 mg or 1.5 mg), containing the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, was compared with ranibizumab (Lucentis®) (administered intravitreally at doses of 0.3 mg). [Figure 4] A graphical comparison of diabetic macular edema with other treatment options based on published results (comparing drugs: Lucentis® (ranibizumab), Eylea® (aflibercept), brolucizumab, and VA2 (RO6867461 / RG7716)). [Figure 5] An overview of the trial design for evaluating the bispecific antibody RO6867461 administered at 12-week and 16-week intervals in patients with neovascular age-related macular degeneration (nAMD). [Figure 6] This study compared the bispecific antibody RO6867461 (containing the amino acid sequences of SEQ ID NOs. 17, 18, 19, and 20) (6.0 mg administered intravitreally at 12-week and 16-week intervals) with ranibizumab (Lucentis®) (0.3 mg administered intravitreally at 4-week intervals) in patients with neovascular age-related macular degeneration (nAMD) in terms of the increase in best corrected visual acuity from baseline. [Figure 7] Changes in baseline foveal retinal thickness (measured via OCT) in patients with neovascular age-related macular degeneration (nAMD) compared between the bispecific antibody RO6867461 (containing amino acid sequences of SEQ ID NOs. 17, 18, 19, and 20) (6.0 mg administered intravitreally at 12-week and 16-week intervals) and ranibizumab (Lucentis®) (0.3 mg administered intravitreally at 4-week intervals).

[0026] Detailed description of the invention According to one aspect of the present invention, a method for treating a patient suffering from an ophthalmic vascular disease is provided, 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 is administered intravitreously every 8 weeks or less (or is planned to be administered every 9 weeks or less in one embodiment; every 10 weeks or less in one embodiment; every 11 weeks or less in one embodiment; every 12 weeks or less in one embodiment; every 13 weeks or less in one embodiment; every 14 weeks or less in one embodiment; every 15 weeks or less in one embodiment).

[0027] One embodiment of the present invention is a method for treating a patient suffering from an ophthalmic 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 (13 letters or more in one embodiment, 14 letters or more in another embodiment, and 15 letters or more in another embodiment) in best corrected visual acuity (BCVA) as measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally every 8 weeks or less (or is planned to be administered every 9 weeks or less; every 10 weeks or less; every 11 weeks or less; every 12 weeks or less; every 13 weeks or less; every 14 weeks or less; every 15 weeks or less).

[0028] One embodiment of the present invention is a method for treating a patient suffering from an ophthalmic 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 exhibits an improvement in visual acuity after administration of the bispecific VEGF / ANG2 antibody, as measured by an increase of 12 or more letters (13 or more letters in one embodiment, 14 or more letters in another embodiment, and 15 or more letters in another embodiment) in best-corrected visual acuity as measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best-corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreously every 8 weeks or less (or is planned to be administered every 9 weeks or less; every 10 weeks or less; every 11 weeks or less; every 12 weeks or less; every 13 weeks or less; every 14 weeks or less; every 15 weeks or less).

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

[0030] In one embodiment of the present invention, the increase in letters of the BCVA / ETDRS letter score occurs at 24 weeks and / or 25 weeks, and / or 26 weeks, and / or 27 weeks, and / or 28 weeks, and / or 29 weeks, and / or 30 weeks, and / or 31 weeks, and / or 32 weeks, and / or 33 weeks, and / or 34 weeks, and / or 35 weeks, and / or 36 weeks, and / or 37 weeks, and / or 38 weeks, and / or 39 weeks, and / or 40 weeks after the start of treatment. Measurements are taken intermittently and / or at weeks 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and / or 60. In one embodiment of the present invention, the increase in letters in 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.

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

[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 disease, the bispecific antibody being administered intravitreously every 8 weeks or less (or is planned to be administered every 9 weeks or less in one embodiment; every 10 weeks or less in one embodiment; every 11 weeks or less in one embodiment; every 12 weeks or less in one embodiment; every 13 weeks or less in one embodiment; every 14 weeks or less in one embodiment; every 15 weeks or less in one embodiment).

[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 ophthalmic vascular disease, wherein the patient has an increase of 12 or more letters (13 or more letters in one embodiment, 14 or more letters in another embodiment, and 15 or more letters in another embodiment) in best corrected visual acuity (BCVA) as measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreally every 8 weeks or less (or is planned to be administered every 9 weeks or less; every 10 weeks or less; every 11 weeks or less; every 12 weeks or less; every 13 weeks or less; every 14 weeks or less; every 15 weeks or less).

[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 with ophthalmic vascular disease, wherein the patients show improvement in visual acuity after (intravitreal) administration of the bispecific VEGF / ANG2 antibody, as measured by an increase of 12 or more letters (13 or more letters in one embodiment, 14 or more letters in another embodiment, and 15 or more letters in another embodiment) in best-corrected visual acuity (BCVA) letter score measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best-corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreously every 8 weeks or less (or is planned to be administered every 9 weeks or less; every 10 weeks or less; every 11 weeks or less; every 12 weeks or less; every 13 weeks or less; every 14 weeks or less; every 15 weeks or less).

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

[0036] In one embodiment of the present invention, the increase in letters in 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.

[0037] In one embodiment of the present invention, such a bispecific antibody (for use) is used to extend the time to retreatment and / or to extend the time to vision deterioration, wherein retreatment with the bispecific antibody is An increase in foveal retinal thickness (CST) of 50 μm or more (using spectral domain optical coherence tomography (SD-OCT) in one embodiment); and / or Decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more It is administered when the disease activity is determined to be as follows.

[0038] One embodiment of the present invention is a pharmaceutical or pharmaceutical preparation comprising a bispecific antibody bound to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for use in the treatment of ocular vascular disease, wherein the bispecific antibody is administered intravitreally every 8 weeks or less (in one embodiment, every 9 weeks or less; in one embodiment, every 10 weeks or less; in one embodiment, every 11 weeks or less; in one embodiment, every 12 weeks or less; in one embodiment, every 13 weeks or less; in one embodiment, every 14 weeks or less; in one embodiment, every 15 weeks or less).

[0039] One embodiment of the present invention is a pharmaceutical or pharmaceutical preparation 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 a patient suffering from ophthalmic vascular disease, wherein the patient has an increase of 12 letters or more (13 letters or more in one embodiment, 14 letters or more in another embodiment, and 15 letters or more in another embodiment) in best corrected visual acuity (BCVA) as measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreously every 8 weeks or less (or is planned to be administered every 9 weeks or less; every 10 weeks or less; every 11 weeks or less; every 12 weeks or less; every 13 weeks or less; every 14 weeks or less; every 15 weeks or less).

[0040] One embodiment of the present invention is a pharmaceutical or pharmaceutical preparation 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 a patient suffering from ophthalmic vascular disease, wherein the patient exhibits improvement in visual acuity after (intravitreal) administration of the bispecific anti-VEGF / ANG2 antibody, as measured by an increase of 12 or more letters (13 or more letters in one embodiment, 14 or more letters in another embodiment, and 15 or more letters in another embodiment) in best-corrected visual acuity (BCVA) letter score measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best-corrected visual acuity (BCVA) letter score before administration of the bispecific anti-VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreously every 8 weeks or less (or is planned to be administered every 9 weeks or less; every 10 weeks or less; every 11 weeks or less; every 12 weeks or less; every 13 weeks or less; every 14 weeks or less; every 15 weeks or less).

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

[0042] In one embodiment of the present invention, the increase in letters in 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 retreatment and / or the time to vision deterioration, wherein retreatment using a bispecific antibody is An increase in foveal retinal thickness (CST) of 50 μm or more (using spectral domain optical coherence tomography (SD-OCT) in one embodiment); and / or Decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more It is administered when the disease activity is determined to be as follows.

[0044] One embodiment of the present invention is the use of a bispecific antibody conjugating to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2) for the manufacture of a pharmaceutical product for use in the treatment of ocular vascular disease, wherein the bispecific antibody is administered intravitreally every 8 weeks or less (or is intended to be administered every 9 weeks or less in one embodiment; every 10 weeks or less in one embodiment; every 11 weeks or less in one embodiment; every 12 weeks or less in one embodiment; every 13 weeks or less in one embodiment; every 14 weeks or less in one embodiment; or every 15 weeks or less in one embodiment).

[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 pharmaceutical for use in the treatment of ophthalmic vascular disease, wherein the patient has an increase of 12 or more letters (13 or more letters in one embodiment, 14 or more letters in another embodiment, and 15 or more letters in another embodiment) in best corrected visual acuity (BCVA) as measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreously every 8 weeks or less (or is planned to be administered every 9 weeks or less; every 10 weeks or less; every 11 weeks or less; every 12 weeks or less; every 13 weeks or less; every 14 weeks or less; every 15 weeks or less).

[0046] One embodiment of the present invention is the use of a bispecific antibody conjugating 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 ophthalmic vascular disease, wherein the patient shows improvement in visual acuity after (intravitreal) administration of the bispecific VEGF / ANG2 antibody, as measured by an increase of 12 or more letters (13 or more letters in one embodiment, 14 or more letters in another embodiment, and 15 or more letters in another embodiment) in best-corrected visual acuity as measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best-corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody. In one embodiment, the bispecific antibody is administered intravitreously every 8 weeks or less (or is planned to be administered every 9 weeks or less; every 10 weeks or less; every 11 weeks or less; every 12 weeks or less; every 13 weeks or less; every 14 weeks or less; every 15 weeks or less).

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

[0048] In one embodiment of the present invention, the increase in letters in 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.

[0049] In one embodiment of the present invention, the pharmaceutical is used to extend the time to retreatment and / or to extend the time to vision deterioration, wherein retreatment using the bispecific antibody is An increase in foveal retinal thickness (CST) of 50 μm or more (using spectral domain optical coherence tomography (SD-OCT) in one embodiment); and / or Decrease in best corrected visual acuity (BCVA / ETDRS) of 5 letters or more It is administered when the disease activity is determined to be as follows.

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

[0051] Such methods, uses, bispecific antibodies (for use), pharmaceuticals, or pharmaceutical preparations may include administering an initial dose ("initial dose") followed by sequential administration of a second or more therapeutically effective doses of the bispecific antibody, pharmaceutical, or pharmaceutical preparation (e.g., 3-7 doses). Each month Administration; in one embodiment, the start of treatment is 3-4 Each month In one embodiment, the administration is performed, and the treatment is initiated at 4-5 Each month The administration includes; in one embodiment, the initiation of treatment is 4-6 Each month The administration includes; in one embodiment, the initiation of treatment is at least 4 Each month The administration includes; in one embodiment, the initiation of treatment is 5-7 Each month In one embodiment, the administration is included, and the initiation of treatment is 6 Each month (Including administration).

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

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

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

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

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

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

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

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

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

[0061] The terms “ocular vascular disease” and “vascular eye disease” are used herein as synonyms and include, but are not limited to, intraocular neovascular 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 angiomatosis, macular telangiectasia, ischemic retinopathy, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, and retinal degeneration (Garner, A., Vascular diseases, In: Pathobiology of ocular disease, A dynamic approach, Garner, A., and Klintworth, GK, (eds.), 2nd edition, Marcel Dekker, New York (1994), pp. 1625-1710). As used herein, ocular vascular disorders refer to any medical condition characterized by the altered or uncontrolled proliferation of new blood vessels and their invasion into ocular tissue structures such as the retina or cornea. In one embodiment, ocular vascular disorders include exudative age-related macular degeneration (exudative AMD), neovascular age-related macular degeneration (nAMD), diabetic macular edema (DME), cystic macular edema (CME), nonproliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), macular edema secondary to central retinal vein occlusion, hemiretinal vein occlusion, or branch retinal vein occlusion, retinitis, conjunctivitis, uveitis, choroiditis, and ocular conditions. The following are selected from the group consisting of choroidal neovascularization (CNV) secondary to internal inflammation (including secondary to ocular histoplasmosis or presumptive histoplasmosis or choroiditis); myopic choroidal neovascularization (mCNV); choroidal neovascularization secondary to trauma, retinopathy of prematurity, and iris rubeosis / neovascular glaucoma; and other ophthalmic diseases (in which the eye disease or disorder is accompanied by ocular neovascularization, vascular leakage, and / or retinal edema).Therefore, the anti-VEGF / ANG2 bispecific antibodies for use 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, nonproliferative 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, nonproliferative diabetic retinopathy, and proliferative diabetic retinopathy, and especially exudative age-related macular degeneration. In some embodiments, the ophthalmic 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 potentially causing) corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens-induced metabolic disorders, atopic keratitis, superior limbal keratitis, pterygium, keratitis sicca, Sjögren's syndrome, rosacea, fuchterinosis, syphilis, mycobacterial infection, fatty degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex virus infection, herpes zoster infection, protozoal infection, Kaposi's sarcoma, Mohren's ulcer, peri-Terien's corneal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's granulomatosis, scleritis, Stevens-Johnson disease, bullous pemphigoid, radial keratotomy, and corneal transplant rejection.

[0063] Diseases / conditions associated with (or potentially causing) retinal / choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoma, syphilis, pseudoxanthoma elastica, Paget's disease, venous occlusion, arterial occlusion, carotid occlusion, chronic uveitis / vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinitis pigmentosa, retinal edema (including macular edema), Eels' disease, Behçet's disease, infections causing retinitis / choroiditis, presumed ocular histoplasmosis, Best's disease, myopia, optic fovea, Stuttgart disease, ciliary body squamous cellulitis, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications. Other diseases include, but are not limited to, diseases associated with rubeosis (angiovascular neovascularization) and diseases caused by abnormal proliferation of vascular connective tissue or fibrous tissue, including all types of proliferative vitreoretinopathy.

[0064] Retinopathy of prematurity (ROP) is an eye disease that develops in premature infants. It is thought to be caused by the uncontrolled proliferation 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 is an additional risk factor. Both oxygen toxicity and relative hypoxia can contribute to the development of ROP.

[0065] Macular degeneration is a condition primarily seen in older adults, characterized by detachment, atrophy, and sometimes hemorrhage in the central part of the eye's internal limiting membrane, known as the macula of the retina. This can lead to central visual field defects, such as difficulty seeing fine details, reading, or recognizing faces. According to the American Academy of Ophthalmology, it is the leading cause of central visual field defects (blindness) in people over 50 in the United States today. Some macular dystrophys that develop in younger individuals are sometimes referred to as macular degeneration, and the term is commonly known as age-related macular degeneration (AMD or ARMD).

[0066] As used herein, “age-related macular degeneration (AMD)” refers to a serious eye condition resulting from the deterioration of the small central part of the retina known as the macula. AMD includes exudative AMD and neovascular AMD. Exudative AMD (also called wAMD or nAMD) is characterized by the abnormal proliferation of blood vessels from the submacula choroid. This is called choroidal neovascularization. These vessels leak blood and fluid (downward and into) the retina, causing visual distortion (such as retinal elevation and wavy lines), as well as blind spots and central visual field defects. These abnormal vessels eventually form scars, resulting in permanent loss of central visual field. Symptoms of age-related macular degeneration include a dark, hazy area in the center of the visual field; and lost or altered color vision. Age-related macular degeneration can be detected by a routine eye examination. One of the most common early signs of macular degeneration is the presence of drusen, which are small yellow deposits under the retina, and pigment clumps.

[0067] Progressive age-related macular degeneration (AMD), which is associated with significant vision loss, has two subtypes: dry and exudative. Dry AMD, or central geographic atrophy, arises from atrophy of the retinal pigment epithelium layer beneath the retina, leading to vision loss through a decrease 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 shown by the National Eye Institute and others to slow the progression of dry AMD and improve vision in some patients.

[0068] Retinitis pigmentosa (RP) is a group of hereditary eye conditions. In the progression of retinitis pigmentosa, night blindness typically 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 childhood. The progression of retinitis pigmentosa varies from case to 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. In affected individuals, the first symptoms are abnormalities in dark adaptation or night blindness, followed by a decrease in peripheral vision (also known as visual field constriction) and sometimes central vision loss later in the course of the disease.

[0069] Macular edema occurs when fluid and protein deposits accumulate above or below the macula of the eye, the central region of the retina involved in good vision, causing thickening and swelling of the retina. This swelling can distort a person's central vision because the macula is located near the center of the retina at the back of the eyeball. This region contains densely packed cones that allow a person to see shapes, colors, and details directly in their line of sight, resulting in 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 mellitus (type 1 or type 2). Macular edema occurs when blood vessels in the retina leak into the macula, causing fluid and protein deposits to accumulate above or below the macula of the eye, leading to retinal thickening and swelling (edema). This swelling can distort a person's central vision because the macula is located near the center of the retina at the back of the eyeball. The main symptoms of diabetic macular edema include, but are not limited to, blurred vision, floaters, decreased contrast vision, diplopia, and ultimately decreased visual acuity. The pathophysiology of diabetic macular edema is characterized by the disruption of the medial blood-retinal barrier, which normally prevents fluid from moving into the retina, allowing fluid to accumulate in the retinal tissue, and the presence of retinal thickening. Diabetic macular edema is currently diagnosed during an eye examination, which consists of a visual acuity test measuring the smallest letters a person can read on a standard visual acuity chart, a pupillary dilation test to check for signs of the disease, angiography such as optical coherence tomography (OCT) or fluorescein angiography (FA), and intraocular pressure measurement using an instrument that measures intraocular pressure. The following tests are also performed to determine treatment: optical coherence tomography (OCT), fluorescein angiography, and color stereotactic fundus photography. Diabetic macular edema can be broadly characterized into two major categories: focal and diffuse. Focal diabetic macular edema is characterized by specific, distinct, and separate areas of leakage within the macula, where there is sufficient blood flow within the macula. Diffuse diabetic macular edema results from leakage of the entire capillary bed surrounding the macula, resulting from a disruption of the medial blood-retinal barrier of the eye. In addition to focal and diffuse types, diabetic macular edema is also classified, based on clinical laboratory findings, into clinically significant macular edema (CSME), clinically insignificant diabetic macular edema, and diabetic macular edema associated with the fovea, including the fovea (CSME-CI). The present invention includes methods for treating diabetic macular edema of the above categories.

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

[0072] Disease activity is determined, for example, through a decrease in the BCVA / ETDR letter score and / or through macular thickening including the center of the macula as foveal region retinal thickness (CST) (also known as subfoveal central thickness) by spectral domain optical coherence tomography (SD-OCT). In one preferred embodiment, foveal region retinal thickness (CST) is determined using spectral domain optical coherence tomography (SD-OCT). In one preferred embodiment, the foveal retinal thickness is measured by spectral-domain optical coherence tomography (SD-OCT) using a Spectralis® instrument; in one preferred embodiment, the foveal retinal thickness is measured by spectral-domain optical coherence tomography (SD-OCT) using a Cirrus® instrument; in one embodiment, the foveal retinal thickness is measured by spectral-domain optical coherence tomography (SD-OCT) using a Topcon® instrument; and in one embodiment, the foveal retinal thickness is measured by spectral-domain optical coherence tomography (SD-OCT) using an Optvue® instrument. The term “patient suffering from” as used herein means a person who exhibits one or more symptoms or signs of an ophthalmic vascular disease as described herein and / or has been diagnosed with an ophthalmic vascular disease as described herein. The term “patients suffering from” may also include, for example, subjects who, prior to treatment, exhibit (or have exhibited) one or more signs of ophthalmic vascular disease, such as retinal angiogenesis, neovascularization, vascular leakage, foveal retinal thickening, hard, yellowish foveal exudate with thickening of adjacent retinas, and retinal thickening of at least one foveal area (any portion thereof being within one foveal diameter), blurred vision, floaters, decreased contrast vision, diplopia, and eventual decreased visual acuity.

[0073] As used herein, the term “patients with” may also include a subset of individuals who are susceptible to diabetic macular edema or age-related macular degeneration, or who may exhibit elevated levels of biomarkers associated with diabetic macular edema or age-related macular degeneration. For example, “subjects requiring it” may include subjects who have had diabetes for more than 10 years or who frequently exhibit high blood glucose or high fasting blood glucose levels. In certain embodiments, the term “patients with” may include subjects who have diabetes or have been diagnosed with diabetes prior to or at the time of administration of the bispecific anti-VEGF / ANG2 antibody. In certain embodiments, the term “patients with” may include subjects who are over 50 years of age prior to or at the time of administration of the anti-VEGF / ANG2 antibody. In some embodiments, the term “patients with” may include subjects who are smokers or who have hypertension or high cholesterol.

[0074] The present invention includes a method or a bispecific antibody (for use), a pharmaceutical or pharmaceutical preparation for treating, preventing, or reducing the severity of an ophthalmic vascular disease, comprising the step of administering a therapeutically effective dose of a bispecific anti-VEGF / ANG2 antibody (or a pharmaceutical or pharmaceutical preparation containing a bispecific anti-VEGF / ANG2 antibody) to a subject in need thereof, wherein such a bispecific antibody, pharmaceutical, or pharmaceutical preparation containing a bispecific anti-VEGF / ANG2 antibody is administered (intravitreal) to the subject in multiple doses, for example, as part of a specific therapeutic dosing plan.

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

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

[0077] One embodiment of the present invention is a treatment method, use, bispecific antibody (for use), drug, or pharmaceutical preparation as described herein, wherein the ophthalmic disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a consistent 8-week (Q8W) dosing schedule after the initiation of treatment (in one embodiment, the initiation of treatment is 5-7 Each month The administration includes; in one embodiment, the initiation of the treatment is 6 Each month (Including administration).

[0078] One embodiment of the present invention is a treatment method, use, bispecific antibody (for use), drug, or pharmaceutical preparation as described herein, wherein the ocular vascular disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a consistent 12-week (Q12W) medication schedule after the initiation of treatment (in one embodiment, the initiation of treatment is 5-7 Each month The administration includes; in one embodiment, the initiation of the treatment is 6 Each month (Including administration). In one embodiment, after the initiation of treatment, there is a Q8W cycle of the first dose before a consistent Q12W dosing schedule.

[0079] One embodiment of the present invention is a treatment method, use, bispecific antibody (for use), drug, or pharmaceutical preparation as described herein, wherein the ophthalmic disease is diabetic macular edema, and the treatment of a patient suffering from diabetic macular edema includes a drug regimen in which, after the initiation of treatment, the dosing interval is extended if the disease is stable and absent, or shortened if disease activity is present (in one embodiment, the initiation of treatment is 3-7 Each month The administration includes; in one embodiment, the initiation of treatment is 3-5 Each month The administration includes; in one embodiment, the initiation of the treatment is at least 4 Each month The administration includes; in one embodiment, the initiation of treatment is 4-6 Each month(including administration). In one embodiment, such a medication plan includes the patient receiving medication at Q4W, Q8W, Q12W, or Q16W, depending on the patient's disease state. In one embodiment, the absence of stable disease is -Foveal retinal thickness (CST) increased by less than 50 μm. Best corrected visual acuity (BCVA / ETDRS) with a decrease of less than -5 letters It was determined that the disease activity was, Foveal retinal thickness (CST) increased by more than -50 μm. -Best corrected visual acuity (BCVA / ETDRS) with a decrease of 5 letters or more It is decided as such.

[0080] In one embodiment, the absence of disease in a stable state is - Foveal region retinal thickness (CST) is less than approximately 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) It was determined that the disease activity was, - Foveal region retinal thickness (CST) exceeds approximately 300 μm (In one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis® instrument, it exceeds 325 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using an OptoVue® instrument, it exceeds 295 μm) It is decided as such.

[0081] One embodiment of the present invention is a treatment method, use, bispecific antibody (for use), drug, or pharmaceutical preparation as described herein, wherein the ocular disease is age-related macular degeneration (exudative age-related macular degeneration in one embodiment), and the treatment of a patient suffering from age-related macular degeneration (exudative age-related macular degeneration in one embodiment) includes a drug regimen in which, after the initiation of treatment, the dosing interval is extended if the disease is stable and absent, or shortened if disease activity is present (in one embodiment, the initiation of treatment is 3-7 Each month The administration includes; in one embodiment, the initiation of treatment is 3-5 Each month The administration includes; in one embodiment, the initiation of the treatment is at least 4 Each month The administration includes; in one embodiment, the initiation of treatment is 4-6 Each month (including administration). In one embodiment, such a medication plan includes the patient receiving medication at Q4W, Q8W, Q12W, or Q16W, depending on the patient's disease state. In one embodiment, the absence of stable disease is - Foveal retinal thickness (CST) increased by less than -50 μm; and / or Best corrected visual acuity (BCVA / ETDRS) with a decrease of less than -5 letters It was determined that the disease activity was, -Fovear retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) with a decrease of 5 letters or more It is decided as such.

[0082] In one embodiment, the absence of disease in a stable state is - Foveal region retinal thickness (CST) is less than approximately 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) It was determined that the disease activity was, - Foveal region retinal thickness (CST) exceeds approximately 300 μm (In one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis® instrument, it exceeds 325 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using an OptoVue® instrument, it exceeds 295 μm) It is decided as such.

[0083] In one embodiment, the ocular vascular disease in such a method, use, bispecific antibody (for use), drug, 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 (or group) of the antibody molecule to which the ligand actually binds. The term "antigen-binding site" includes the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL) (VH / VL pair).

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

[0086] The "bispecific antibody" according to the present invention is an antibody having two different antigen-binding specificities. The antibody of the present invention is specific to two different antigens: VEGF as the primary antigen and ANG-2 as the secondary antigen.

[0087] As used herein, the term “single-specific” antibody refers to an antibody that has one or more binding sites that bind to the same epitope of the same antigen.

[0088] The term "valency" as used in this application refers to the presence of a specific number of binding sites within an antibody molecule. Therefore, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, within the antibody molecule. The bispecific antibody according to the present invention is preferably "bivalent."

[0089] The terms used herein refer to "bispecific antibodies that bind to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2)," "bispecific anti-VEGF / ANG2 antibodies," and "bispecific antibodies." <vegf ang2>The term "antibody" is synonymous and refers to an antibody that has 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 Nos. 2010040508, 2011 / 117329, 2012 / 131078, 2015 / 083978, 2017 / 197199, and 2014 / 009465. International Publication No. 2014 / 009465 describes a bispecific anti-VEGF / ANG2 antibody specifically designed for the treatment of ophthalmic vascular diseases. The bispecific anti-VEGF / ANG2 antibody of International Publication No. 2014 / 009465 (which is incorporated in its entirety herein) is particularly useful for the treatment and treatment planning of ophthalmic 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, 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 antibodies contain the heavy chain constant regions of human IgG1 subclasses including mutants I253A, H310A, and H435A, and mutants L234A, L235A, and P329G (numbered according to Kabat's 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 is 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 present invention, such a bispecific and bivalent antibody according to the present invention is a) Heavy and light chains of a first full-length antibody that specifically binds to VEGF; b) Modified heavy and light chains of a second full-length antibody that specifically binds to ANG-2 (where the constant domains CL and CH1 are swapped with each other). It is characterized by including.

[0095] This bispecific, 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. 2009 / 080253 (including a modified CH3 domain of the knob-into-hole type). Antibodies based on this bispecific, bivalent antibody format are named Cross-MAb monoclonal antibodies.

[0096] In one embodiment, such a bispecific, 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. It is characterized by including.

[0097] In one embodiment, such a bispecific, 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, bivalent anti-VEGF / ANG2 antibody is falisimab.

[0098] Accordingly, one embodiment of the present invention is a bispecific, 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, bivalent anti-VEGF / ANG2 antibody is falisimab.

[0099] In one embodiment, the CH3 domain of the bispecific, bivalent antibody according to the present invention is modified by the “knob-into-hole” technique, as detailed with several examples in, for example, International Publication No. 96 / 027011, Ridgway JB, et al., Protein Eng 9 (1996) 617-621; and Merchant, AM, et al., Nat Biotechnol 16 (1998) 677-681. In this method, the interaction surface of the two CH3 domains is modified to increase heterodimerization of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be a “knob” while the other is a “hole”. The introduction of disulfide bridges stabilizes heterodimers (Merchant, AM, et al., Nature Biotech 16 (1998) 677-681; Atwell, S., et al. J. Mol. Biol. 270 (1997) 26-35) and increases yield.

[0100] In a preferred embodiment of the present invention, the bispecific anti-VEGF / ANG2 antibody according to the present invention is The CH3 domain of one heavy chain and the CH3 domain of the other heavy chain are in contact at the interface, including the original interface between the CH3 domains of the antibody; Here, the interface is modified to promote the formation of bispecific antibodies, and this modification is: a) The CH3 domain of one of the heavy chains has been modified, This allows the CH3 domain of one heavy chain in the bispecific antibody to come into contact with the original interface of the CH3 domain of the other heavy chain, An amino acid residue is substituted with an amino acid residue having a larger side chain volume, which creates a protrusion within the interface of the CH3 domain of one heavy chain, which can then be located within the cavity at the interface of the CH3 domain of the other heavy chain, and b) The CH3 domain of the other heavy chain is modified, This allows the original interface of the second CH3 domain to come into contact with the original interface of the first CH3 domain within the bispecific antibody. The amino acid residue is substituted with an amino acid residue having a smaller side chain volume, which creates a cavity within the interface of the second CH3 domain, allowing the protrusion within the interface of the first CH3 domain to be located within this cavity. It is characterized by the following:

[0101] Therefore, the bispecific anti-VEGF / ANG2 antibody for use described herein is preferably, The CH3 domains of the heavy chain of the full-length antibody in a) and the CH3 domains of the heavy chain of the full-length antibody in b) are in contact at an interface, each containing a modification within the original interface between the CH3 domains of the antibody; Here, i) In the CH3 domain of one of the heavy chains, The amino acid residue is substituted with an amino acid residue having a larger side chain volume, which creates a protrusion within the interface of the CH3 domain of one heavy chain, which can then be located within the cavity at the interface of the CH3 domain of the other heavy chain, and ii) In the CH3 domain of the other heavy chain, The amino acid residues are substituted with amino acid residues having smaller side chain volumes, which creates a cavity within the interface of the second CH3 domain, allowing the protrusions within the interface of the first CH3 domain to be located within it. It is characterized by the following:

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

[0103] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and 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 in each CH3 domain, thereby enabling the formation of a disulfide bridge between both CH3 domains.

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

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

[0107] Other techniques for CH3 modification that enhance heterodimerization are considered alternatives to the present invention and are described, for example, in International Publication Nos. 96 / 27011, 98 / 050431, European Patent No. 1870459, International Publication Nos. 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012 / 058768, 2013 / 157954, and 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 a substitution / mutation of a charged amino acid having opposite charges at a specific amino acid position in the interface within the CH3 / CH3 domains between both heavy chains. One preferred embodiment for the multispecific antibody is the mutation of amino acids R409D and K370E in the CH3 domain of one heavy chain of the multispecific antibody, and the mutation of amino acids D399K and E357K in the CH3 domain of the other heavy chain (numbered according to the Kabat EU index).

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

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

[0111] In further embodiments, the CH3 domain of the other heavy chain further includes amino acid mutations selected from Y349E, Y349D, and L368E (L368E in one embodiment).

[0112] In one embodiment, the heterodimerization approach described in International Publication No. 2012 / 058768 is used instead. In one embodiment, the CH3 domain of one heavy chain contains mutations in amino acids L351Y and Y407A, and the CH3 domain of the other heavy chain contains mutations in amino acids T366A and K409F. In a further embodiment, the CH3 domain of the other heavy chain further contains amino acid mutations at positions T411, D399, S400, F405, N390, or K392. In one embodiment, the aforementioned amino acid mutations are a) T411N, T411R, T411Q, T411K, T411D, T411E, and 411W, b) D399R, D399W, D399Y, and D399K, c) S400E, S400D, S400R and S400K, d) F405I, F405M, F405T, F405S, F405V and F405W, e) N390R, N390K and N390D, f) K392V, K392M, K392R, K392L, K392F and K392E Selected from the group consisting of .

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

[0114] In one embodiment, the heterodimerization approach described in International Publication No. 2011 / 143545 is used instead. In one embodiment, the amino acid modification according to International Publication No. 2011 / 143545 is introduced at a position 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. 2011 / 090762, which also uses the knob-into-hole method described above, is used instead. In one embodiment, the CH3 domain of one heavy chain contains a mutation for amino acid T366W, and the CH3 domain of the other heavy chain contains a mutation for amino acid Y407A. In another embodiment, the CH3 domain of one heavy chain contains a mutation for amino acid T366Y, and the CH3 domain of the other heavy chain contains a mutation for amino acid Y407T.

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

[0117] In one embodiment, the heterodimerization approach described in International Publication No. 2009 / 089004 is used instead. In one embodiment, the CH3 domain of one heavy chain includes the substitution of the amino acid K392 or N392 using a negatively charged amino acid (in one embodiment, glutamic acid (E) or aspartic acid (D); in a further embodiment, a mutation of K392D or N392D), and the CH3 domain of the other heavy chain includes the substitution of the amino acid D399, E356, D356, or E357 using a positively charged amino acid (in one embodiment, lysine (K) or arginine (R); in a further embodiment, a substitution of D399K, E356K, D356K, or E357K; in yet another embodiment, a mutation of D399K or E356K). In further embodiments, the CH3 domain of one heavy chain further includes the substitution of the K409 or R409 amino acid using a negatively charged amino acid (in one embodiment, glutamic acid (E) or aspartic acid (E); in further embodiments, a mutation of K409D or R409D). In further embodiments, the CH3 domain of one heavy chain further or alternatively includes the substitution of the K439 and / or K370 amino acid using 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. 2007 / 147901 is used instead. In one embodiment, the CH3 domain of one heavy chain contains mutations in amino acids K253E, D282K, and K322D, and the CH3 domain of the other heavy chain contains mutations in amino acids D239K, E240K, and K292D.

[0119] In one embodiment, the heterodimerization approach described in International Publication No. 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 contains the heavy chain constant region of the human IgG1 subclass, including mutants I253A, H310A, and H435A, and mutants L234A, L235A, and P329G (numbered according to Kabat's EU index); iv) In the heavy chain constant region, the T366W mutation is contained in one CH3 domain, and the T366S, L368A, and Y407V mutations are contained in the other CH3 domain (numbering is based on Kabat's 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 contains the heavy chain constant region of the human IgG1 subclass, including mutants I253A, H310A, and H435A, and mutants L234A, L235A, and P329G (numbered according to Kabat's EU index); iv) In the heavy chain constant region, the S354C and T366W mutations are contained in one CH3 domain, and the Y349C, T366S, L368A, and Y407V mutations are contained in the other CH3 domain (numbering is 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 is 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 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. It is characterized by the following:

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

[0125] As used herein, the term "VEGF" refers to human vascular endothelial growth factor (VEGF / VEGF-A), which is a 165-amino acid human vascular endothelial growth factor (amino acids 27-191 of the precursor sequence of human VEGF165: SEQ ID NO: 24; amino acids 1-26 represent a signal peptide), and also to human vascular endothelial growth factor (VEGF / VEGF-A), as described in Leung, DW, et al., Science 246 (1989) 1306-9; Houck et al., Mol. Endocrin. 5 (1991) 1806-1814; Keck, PJ, et al., Science 246 (1989) 1309-12 and Connolly, DT, et al., J. Biol. Chem. 264 (1989). This refers to the native alleles and processing forms of vascular endothelial growth factor, along with related isoforms 121, 189, and 206, as described in 20017-24. VEGF is involved in regulating normal and abnormal angiogenesis and neovascularization associated with tumors and intraocular disorders (Ferrara, N., et al., Endocr. Rev. 18 (1997) 4-25; Berkman, RA, et al., J. Clin. Invest. 91 (1993) 153-159; Brown, LF, et al., Human Pathol. 26 (1995) 86-91; Brown, LF, et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J., et al., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, HF, et al., Am. J. Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoprotein isolated from several sources and includes several isoforms. VEGF exhibits highly specific mitotic activity against endothelial cells. VEGF antagonists / inhibitors inhibit the binding of VEGF to its receptor, VEGFR. Known VEGF antagonists / inhibitors include bispecific anti-VEGF / ANG2 antibodies, such as those described in International Publication No. 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, PC, et al., Science 277 (1997) 55-60 and Cheung, AH, et al., Genomics 48 (1998) 389-91. Angiopoietin-1 (SEQ ID NO: 26) and angiopoietin-2 were discovered as ligands for Tie, a tyrosine kinase family selectively expressed in vascular endothelium (Yancopoulos, GD, et al., Nature 407 (2000) 242-48). There are currently four confirmed members in the angiopoietin family. Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent broadly branched counterparts at 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 agonist and antagonist, respectively, in tissue culture experiments (see Davis, S., et al., Cell 87 (1996) 1161-69 for ANG-1; and Maisonpierre, PC, et al., Science 277 (1997) 55-60 for ANG-2). All known angiopoietins primarily bind to their receptor, TIE2 (SEQ ID NO: 27), and both Ang-1 and Ang-2 bind to TIE2 with an affinity (Kd) of 3 nM (Maisonpierre, PC, et al., Science 277 (1997) 55-60). ANG2 antagonists / inhibitors inhibit the binding of ANG2 to its receptor, TIE2. Known ANG2 antagonists / inhibitors include bispecific anti-VEGF / ANG2 antibodies, such as those described in International Publication No. 2014 / 009465.

[0127] The antigen-binding site of the bispecific antibody of the present invention contains six complementarity-determining regions (CDRs) that contribute to altering the degree of affinity of the binding site to the antigen. There are three heavy-chain variable domain CDRs (CDRH1, CDRH2, and CDRH3) and three light-chain variable domain CDRs (CDRL1, CDRL2, and CDRL3). The extent of the CDRs and framework regions (FRs) is determined by comparison with an edited amino acid sequence database, and these regions are defined by the degree of variability between sequences.

[0128] The antibody of the present invention comprises an immunoglobulin constant region derived from one or more immunoglobulin classes of human origin, 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 terms "monoclonal antibody" or "monoclonal antibody composition" refer to preparations of antibody molecules having a single amino acid composition.

[0130] The term "chimeric antibody" typically refers to an antibody prepared by recombinant DNA technology that includes a variable region, i.e., a binding region, derived from one source or species, and at least a portion of a constant region derived from a different source or species. Chimeric antibodies containing a mouse variable region and a human constant region are preferred. Other preferred forms of "chimeric antibodies" encompassed by the present invention are those in which the constant region has been modified or altered from the constant region of the original antibody to produce properties according to the present invention, particularly properties relating to binding to C1q and / or Fc receptor (FcR). Such chimeric antibodies are also called "class-switched antibodies." A chimeric antibody is an immunoglobulin gene expression product containing 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 technology and gene transfection technology, which are well known in the art. For example, see Morrison, SL, et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244.

[0131] The term “humanized antibody” refers to an antibody in which the framework or “complementarity-determining region” (CDR) has been modified to include a CDR of an immunoglobulin having different specificity compared to the parent immunoglobulin. In a preferred embodiment, a mouse CDR is transplanted into the framework region of a human antibody to prepare a “humanized antibody.” See, for example, Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, MS, et al., Nature 314 (1985) 268-270. Particularly preferred CDRs are those that show a sequence recognizing the antigen described above for chimeric antibodies. Other forms of “humanized antibodies” encompassed by the present invention are those in which the constant region has been modified or altered from the constant region of the original antibody, thereby resulting in the properties according to the present invention, particularly those relating to binding to C1q and / or 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 cutting edge of the art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that can generate a complete repertoire of human antibodies or selected human antibodies during immunization without the production of endogenous immunoglobulins. Introducing a human germline immunoglobulin gene array into such germline mutant mice would likely induce the production of human antibodies upon antigen challenge (see, for example, Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Brueggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodies may also be produced in a phage display library (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole, A., et al. and Boerner, P., et al. can also be used for the preparation of human monoclonal antibodies (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, AL, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95).As already described with respect to the chimeric antibodies and humanized antibodies described in the present invention, the term “human antibody” as used herein also includes antibodies in which the constant region has been modified, for example, by “class switching,” i.e., a change or mutation of the Fc portion (e.g., from IgG1 to IgG4 and / or a mutation of IgG1 / IgG4), to produce the properties described in the present invention, particularly the properties relating to binding to C1q and / or binding to FcR.

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

[0134] In this specification, the “variable domains” (variable domains of the light chain (VL) and variable domains of the heavy chain (VH)) refer to the light and heavy chain pairs directly involved in antibody binding to the antigen. The variable domains of the human light and heavy chains have the same general structure, and each domain contains four framework (FR) regions whose sequences are widely conserved, connected by three “hypervariable regions” (i.e., complementarity-determining regions (CDRs)). The framework regions take the form of 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 through the framework regions and, together with the CDRs of other chains, 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 antibody described in this invention, and therefore provide further objectives for the present invention.

[0135] As used herein, the terms “hypervariable region” or “antigen-binding region of an antibody” refer to the amino acid residues of the antibody involved in binding to the antigen. The hypervariable region includes amino acid residues derived from the “complementarity-determining region,” or “CDR.” The “framework,” or “FR,” region is a variable domain region other than the hypervariable region residues as defined herein. Therefore, the light and heavy chains of an antibody contain the domains 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, CDR3 of the heavy chain is the region that contributes most to binding to the antigen. The CDR and FR regions are determined according to the standard definitions in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

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

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

[0138] As used in this application, the terms “human-derived constant region” or “human constant region” refer to the heavy chain constant region and / or light chain constant region κ or λ of a human antibody of subclass IgG1, IgG2, IgG3, or IgG4. Such constant regions are well known in the most advanced field and have been described, for example, by Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, e.g., Johnson, G., and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218; Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). For the numbering of positions and mutations within the application, the EU numbering system (EU Index) described in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used, and is referred to as "Kabat's EU Index numbering."

[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) of the Fc region, or the C-terminal glycine (Gly446) and C-terminal lysine (Lys447), may or may not be present.

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

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

[0142] In one embodiment, the bispecific antibody described in the present invention is a human IgG1 subclass having the mutants L234A(Leu235Ala), L235A(Leu234Ala), and P329G(Pro329Gly). Such antibodies exhibit reduced binding to FcR (in particular, they no longer bind at all to FcRγI, FcRγII, and FcRγIII). This is particularly useful in reducing potential side effects, such as thrombosis (Meyer, T., et al., J. Thromb. Haemost. 7 (2009) 171-81).

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

[0144] The “effective dose” of a drug, such as a pharmaceutical preparation or a bispecific anti-VEGF / ANG2 antibody, refers to the effective amount in the dose and duration required to achieve the desired therapeutic or preventive outcome.

[0145] In one embodiment of the present invention, a bispecific antibody, drug, or pharmaceutical formulation as described herein is administered via intravitreous application, for example, by intravitreous injection ("intravitreous" administration). 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 present invention may include one or more described doses of bispecific antibodies present in the pharmaceutical or pharmaceutical formulation, an instrument suitable for intravitreal injection of the pharmaceutical or pharmaceutical formulation, and instructions detailing appropriate subjects and protocols for administering the injection. In these embodiments, the pharmaceutical or pharmaceutical formulation is typically administered to subjects 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 route of administration selected, the bispecific antibodies described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0148] [Table 1] TIFF0007852107000002.tif118161

[0149] Embodiments of the present invention are listed below: 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 disease, the bispecific antibody is administered intravitreally every 8 weeks or less (or is planned to be administered every 9 weeks or less in one embodiment; every 10 weeks or less in one embodiment; every 11 weeks or less in one embodiment; every 12 weeks or less in one embodiment; every 13 weeks or less in one embodiment; every 14 weeks or less in one embodiment; every 15 weeks or less in one embodiment).

[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 patients with ocular vascular disease, wherein the patient has an increase of 12 or more letters (13 or more letters in one embodiment, 14 or more letters in another embodiment, and 15 or more letters) in best corrected visual acuity (BCVA) letter score, as measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best corrected visual acuity (BCVA) letter score before administration of 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 patients with ophthalmic 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 or more letters (13 or more letters in one embodiment, 14 or more letters in another embodiment, and 15 or more letters) in best-corrected visual acuity as measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best-corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody.

[0152] 3. The bispecific antibody described in any one of Embodiments 2A to 2B is administered (or is planned to be administered) intravitreously every 8 weeks or less (in one embodiment, every 9 weeks or less; in one embodiment, every 10 weeks or less; in one embodiment, every 11 weeks or less; in one embodiment, every 12 weeks or less; in one embodiment, every 13 weeks or less; in one embodiment, every 14 weeks or less; in one embodiment, every 15 weeks or less).

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

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

[0155] 6. The bispecific antibody described in any one of Embodiments 1 to 5 (for use) is used to extend the time to retreatment and / or to extend the time to a decline in visual acuity (e.g., best corrected visual acuity (BCVA) BCVA / ETDRS), and retreatment is deemed necessary in cases of disease activity determined as a foveal retinal thickness (CST) increase of 50 μm or more (using spectral domain optical coherence tomography (SD-OCT) in one embodiment); and / or a best corrected visual acuity (BCVA / ETDRS) decrease of 5 letters or more.

[0156] 7. The bispecific antibody is administered 3 to 7 days after the start of treatment. Each month It is administered by administration (in one embodiment, the start of treatment is 3-5 Each month The administration includes; in one embodiment, the initiation of the procedure is 4 Each month The administration includes; in one embodiment, the initiation of treatment is 5-7 Each month In one embodiment, including administration, the initiation of treatment is 6 Each month A bispecific antibody (for use) as described in any one of Embodiments 1 to 6, including administration.

[0157] 8. Ocular vascular diseases are 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), nonproliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), macular edema secondary to central retinal vein occlusion, hemiretinal vein occlusion, or branch retinal vein occlusion, retinitis, conjunctivitis, uveitis, choroiditis, choroidal neovascularization (CNV) secondary to intraocular inflammation (including secondary to ocular histoplasmosis or presumptive histoplasmosis or choroiditis); myopic choroidal neovascularization (mCNV); and choroidal neovascularization secondary to trauma, retinopathy of prematurity, and iris rubeosis / neovascular glaucoma, and a bispecific antibody (for use) as described in any one of Embodiments 1 to 7.

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

[0159] 10. A bispecific antibody (for use) 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. A bispecific antibody that binds to VEGF and human ANG-2 is a VEGF antagonist / inhibitor and an ANG2 antagonist / inhibitor, or a bispecific antibody according to any one of Embodiments 1 to 10 that inhibits the binding of VEGF to its receptor VEGFR and inhibits the binding of ANG2 to its receptor TIE2.

[0161] 12. The bispecific antibody described above is a bispecific antibody (for use) according to any one of Embodiments 1 to 11, administered every 10 to 12 weeks.

[0162] 13. The bispecific antibody described above is a bispecific antibody (for use) according to any one of Embodiments 1 to 11, administered every 11 to 13 weeks.

[0163] 14. The bispecific antibody described in any one of Embodiments 1 to 11 is administered every 12 to 14 weeks.

[0164] 15. The bispecific antibody described above is a bispecific antibody (for use) according to any one of Embodiments 1 to 11, administered every 13 to 15 weeks.

[0165] 16. The bispecific antibody described above is a bispecific antibody (for use) according to any one of Embodiments 1 to 11, administered every 14 to 16 weeks.

[0166] 17. A bispecific antibody that binds to human VEGF and human ANG2 is a bispecific, bivalent anti-VEGF / ANG2 antibody that includes a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2. 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) A bispecific antibody (for use) according to any one of Embodiments 1 to 16, comprising the heavy chain constant region of a human IgG1 subclass including mutants I253A, H310A, and H435A and mutants L234A, L235A, and P329G (numbered according to Kabat's EU index).

[0167] 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) A bispecific antibody (for use) according to Embodiment 17, wherein 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.

[0168] 19. 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, as described in Embodiment 18 (for use).

[0169] 20. The bispecific antibody described above is administered in a dose of approximately 5-7 mg (in each treatment), as described in any one of Embodiments 17-19 (for use).

[0170] 21. The bispecific antibody described in any one of Embodiments 17 to 19 (for use), administered in a dose of approximately 6 mg (in each treatment) (in one embodiment, in a dose of 6 mg + / - 10% (in each treatment); (in one embodiment, in a dose of approximately 6 mg (in each treatment)).

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

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

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

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

[0175] 26. The ocular vascular disease is diabetic macular edema, and the treatment of patients with diabetic macular edema includes a consistent 8-week (Q8W) medication schedule after the start of treatment (in one embodiment, the start of treatment is 5-7 weeks). Each month The administration includes; in one embodiment, the initiation of the treatment is 6 Each month A bispecific antibody (for use) according to Embodiments 1 to 25, including administration.

[0176] 27. The ocular vascular disease is diabetic macular edema, and the treatment of patients with diabetic macular edema includes a consistent 12-week (Q12W) medication schedule after the start of treatment (in one embodiment, the start of treatment is 5-7 Each month The administration includes; in one embodiment, the initiation of the treatment is 6 Each month A bispecific antibody (for use) according to Embodiments 1 to 26, including administration.

[0177] 28. A bispecific antibody (for use) as described in Embodiment 27, wherein, after the initiation of treatment, there is an initial one-dose Q8W cycle before a consistent Q12W dosing schedule.

[0178] 29. The ocular vascular disease is diabetic macular edema, and the treatment of patients with diabetic macular edema includes a medication plan in which, after the start of treatment, the dosing interval is extended if the disease is stable and not present, or shortened if disease activity is present (in one embodiment, the start of treatment is 3-7 Each month The administration includes; in one embodiment, the initiation of treatment is 4-6 Each month A bispecific antibody (for use) according to Embodiments 1 to 28, including administration.

[0179] 30. Such a medication plan includes the patient receiving medication Q8W, Q12W, or Q16W depending on the patient's disease state (in one embodiment, Q4W, Q8W, Q12W, or Q16W depending on the patient's disease state), the bispecific antibody (for use) as described in Embodiment 29.

[0180] 31. The absence of disease in a stable state is - Foveal retinal thickness (CST) increased by less than -50 μm; and / or Best corrected visual acuity (BCVA / ETDRS) with a decrease of less than -5 letters It was determined that the disease activity was, -Fovear retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) with a decrease of 5 letters or more A bispecific antibody (for use) according to Embodiment 29 or 30, determined to be as follows.

[0181] 32. The absence of disease in a stable state is - Foveal region retinal thickness (CST) is less than approximately 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) It was determined that the disease activity was, - Foveal region retinal thickness (CST) exceeds approximately 300 μm (In one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis® instrument, it exceeds 325 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using an OptoVue® instrument, it exceeds 295 μm) The method according to embodiment 29 or 30, which is determined as follows.

[0182] 33. The ocular vascular disease is age-related macular degeneration (exudative age-related macular degeneration in one embodiment), and treatment for patients suffering from age-related macular degeneration (exudative age-related macular degeneration in one embodiment) is performed after the start of treatment (in one embodiment, treatment is performed from 3 to 7 Each month The administration includes; in one embodiment, the initiation of treatment is 4-6 Each month A bispecific antibody (for use) according to Embodiments 1 to 32, including a drug administration plan that extends the administration interval if the disease is stable and absent, or shortens the interval if disease activity is present.

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

[0184] 35. The absence of disease in a stable state is - Foveal retinal thickness (CST) increased by less than -50 μm; and / or Best corrected visual acuity (BCVA / ETDRS) with a decrease of less than -5 letters It was determined that the disease activity was, -Fovear retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) with a decrease of 5 letters or more A bispecific antibody (for use) according to Embodiment 33 or 34, determined to be as follows.

[0185] 36. The absence of disease in a stable state is - Foveal region retinal thickness (CST) is less than approximately 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) It was determined that the disease activity was, - Foveal region retinal thickness (CST) exceeds approximately 300 μm (In one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis® instrument, it exceeds 325 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using an OptoVue® instrument, it exceeds 295 μm) A bispecific antibody (for use) according to Embodiment 33 or 34, determined to be as follows.

[0186] Embodiments of the present invention are listed below: 1. A method for treating a patient suffering from ocular vascular disease, comprising the step of 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 intravitreously every 8 weeks or less (in one embodiment, every 9 weeks or less; in one embodiment, every 10 weeks or less; in one embodiment, every 11 weeks or less; in one embodiment, every 12 weeks or less; in one embodiment, every 13 weeks or less; in one embodiment, every 14 weeks or less; in one embodiment, every 15 weeks or less).

[0187] 2A. A method for treating a patient with ocular vascular disease, comprising the step of 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 (13 letters or more in one embodiment, 14 letters or more in one embodiment, and 15 letters or more in one embodiment) in best corrected visual acuity (BCVA) as measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody.

[0188] 2B. A method for treating a patient with ophthalmic vascular disease, comprising the step of 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 or more letters (13 or more letters in one embodiment, 14 or more letters in one embodiment, and 15 or more letters in one embodiment) in best-corrected visual acuity as measured using an Early Treatment Study for Diabetic Retinopathy (ETDRS)-like chart, compared to the patient's best-corrected visual acuity (BCVA) letter score before administration of the bispecific VEGF / ANG2 antibody.

[0189] 3. The method according to any one of Embodiments 2A to 2B, wherein the bispecific antibody is administered intravitreously every 8 weeks or less (in one embodiment, every 9 weeks or less; in one embodiment, every 10 weeks or less; in one embodiment, every 11 weeks or less; in one embodiment, every 12 weeks or less; in one embodiment, every 13 weeks or less; in one embodiment, every 14 weeks or less; in one embodiment, every 15 weeks or less).

[0190] 4. The method according to any one of Embodiments 1 to 3, wherein 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.

[0191] 5. The method according to any one of Embodiments 1 to 3, wherein the increase in 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.

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

[0193] 7. The bispecific antibody is administered 3 to 7 days after the start of treatment. Each month It is administered by administration (in one embodiment, the start of treatment is 3-5 Each month The administration includes; in one embodiment, the initiation of the procedure is 4 Each month The administration includes; in one embodiment, the initiation of treatment is 5-7 Each month In one embodiment, including administration, the initiation of treatment is 6 Each month The method according to any one of Embodiments 1 to 6 (including administration).

[0194] 8. The method according to any one of Embodiments 1 to 7, wherein 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), nonproliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), macular edema secondary to central retinal vein occlusion, hemiretinal vein occlusion, or branch retinal vein occlusion, retinitis, conjunctivitis, uveitis, choroiditis, choroidal neovascularization (CNV) secondary to intraocular inflammation (including secondary to ocular histoplasmosis or presumptive histoplasmosis or choroiditis); myopic choroidal neovascularization (mCNV); and choroidal neovascularization secondary to trauma, retinopathy of prematurity, and iris rubeosis / neovascular glaucoma.

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

[0196] 10. The method 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).

[0197] 11. The method according to any one of Embodiments 1 to 10, wherein 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 its receptor VEGFR and inhibits the binding of ANG2 to its receptor TIE2.

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

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

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

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

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

[0203] 17. A bispecific antibody that binds to human VEGF and human ANG2 is a bispecific, bivalent anti-VEGF / ANG2 antibody that includes a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2. 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 method according to any one of Embodiments 1 to 16, wherein the bispecific antibody comprises the heavy chain constant region of a human IgG1 subclass including mutants I253A, H310A, and H435A and mutants L234A, L235A, and P329G (numbered according to Kabat's EU index).

[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 method according to Embodiment 17, wherein 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.

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

[0206] 20. The method according to any one of Embodiments 17 to 19, wherein the bispecific antibody is administered in a dose of approximately 5 to 7 mg (in each treatment).

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

[0208] 22. The method according to any one of Embodiments 20 to 21, wherein the bispecific antibody is administered at a concentration of approximately 30 mg / ml.

[0209] 23. The method according to any one of Embodiments 20 to 21, wherein the bispecific antibody is administered at a concentration of approximately 120 mg / ml.

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

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

[0212] 26. The ocular vascular disease is diabetic macular edema, and the treatment of patients with diabetic macular edema includes a consistent 8-week (Q8W) medication schedule after the start of treatment (in one embodiment, the start of treatment is 5-7 weeks). Each month The administration includes; in one embodiment, the initiation of the treatment is 6 Each month The method according to Embodiments 1 to 25, including administration.

[0213] 27. The ocular vascular disease is diabetic macular edema, and the treatment of patients with diabetic macular edema includes a consistent 12-week (Q12W) medication schedule after the start of treatment (in one embodiment, the start of treatment is 5-7 Each month The administration includes; in one embodiment, the initiation of the treatment is 6 Each month The method according to Embodiments 1 to 26, including administration.

[0214] 28. The method according to Embodiment 27, wherein, after the initiation of treatment, there is an initial dose-based Q8W cycle before a consistent Q12W medication schedule.

[0215] 29. The ocular vascular disease is diabetic macular edema, and the treatment of patients with diabetic macular edema includes a medication plan in which, after the start of treatment, the dosing interval is extended if the disease is stable and not present, or shortened if disease activity is present (in one embodiment, the start of treatment is 3-7 Each month The administration includes; in one embodiment, the initiation of treatment is 4-6 Each month The method according to Embodiments 1 to 28, including administration.

[0216] 30. The method according to Embodiment 29, wherein such a medication plan includes the patient receiving medication Q8W, Q12W, or Q16W depending on the patient's disease state (in one embodiment, medication Q4W, Q8W, Q12W, or Q16W depending on the patient's disease state).

[0217] 31. The absence of disease in a stable state is - Foveal retinal thickness (CST) increased by less than -50 μm; and / or Best corrected visual acuity (BCVA / ETDRS) with a decrease of less than -5 letters It was determined that the disease activity was, -Fovear retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) with a decrease of 5 letters or more The method according to embodiment 28 or 29, which is determined as follows.

[0218] 32. The absence of disease in a stable state is - Foveal region retinal thickness (CST) is less than approximately 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) It was determined that the disease activity was, - Foveal region retinal thickness (CST) exceeds approximately 300 μm (In one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis® instrument, it exceeds 325 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using an OptoVue® instrument, it exceeds 295 μm) The method according to embodiment 28 or 29, which is determined as follows.

[0219] 33. Treatment of patients with age-related macular degeneration (exudative age-related macular degeneration in one embodiment) is performed after the start of treatment (in one embodiment, the start of treatment is 3-7 Each month The administration includes; in one embodiment, the initiation of treatment is 4-6 Each month The method according to Embodiments 1 to 32, including a drug administration plan that extends the administration interval if the disease is stable and not present, or shortens the interval if disease activity is present.

[0220] 34. The method according to Embodiment 33, wherein such a medication plan includes the patient receiving medication Q8W, Q12W, or Q16W depending on the patient's disease state (in one embodiment, medication Q4W, Q8W, Q12W, or Q16W depending on the patient's disease state).

[0221] 35. The absence of disease in a stable state is - Foveal retinal thickness (CST) increased by less than -50 μm; and / or Best corrected visual acuity (BCVA / ETDRS) with a decrease of less than -5 letters It was determined that the disease activity was, -Fovear retinal thickness (CST) increased by 50 μm or more; and / or -Best corrected visual acuity (BCVA / ETDRS) with a decrease of 5 letters or more The method according to embodiment 33 or 34, as determined as follows.

[0222] 36. The absence of disease in a stable state is - Foveal region retinal thickness (CST) is less than approximately 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) It was determined that the disease activity was, - Foveal region retinal thickness (CST) exceeds approximately 300 μm (In one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Spectralis® instrument, it exceeds 325 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Cirrus® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using a Topcon® instrument, it exceeds 315 μm; in one embodiment, measured by spectral domain optical coherence tomography (SD-OCT) using an OptoVue® instrument, it exceeds 295 μm) The method according to embodiment 33 or 34, as determined as follows.

[0223] Examples Treatment of patients with ophthalmic vascular disease using bispecific antibodies that bind to human VEGF and human ANG2. Example 1A: Efficacy and duration of treatment for patients with diabetic macular edema (DME) the purpose First objective The primary objective of this clinical trial was as follows: To evaluate the efficacy of a bispecific antibody (VEGFang2-0016 and its production, as detailed in International Publication No. 2014 / 009465, incorporated by reference) that binds to human VEGF and human ANG2, containing the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, compared to an active drug in untreated patients with diabetic macular edema including the fovea (CI-DME). The names of this bispecific anti-VEGF / ANG2 antibody as used herein are RO6867461, RG7716, VEGFang2-0016, or falisimab. A sterile, colorless to brownish, preservative-free vial of RO6867461 for intravitreal administration was used at doses of either 1.5 mg or 6 mg every four weeks. The concentration of the bispecific antibody was approximately 120 mg / ml.

[0224] Second objective The second objective of this clinical trial was as follows: To investigate the pharmacokinetics and anatomical outcomes, to provide information on the mechanism of action of RO6867461, and 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 with the investigational product 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-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 study investigator.

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

[0229] Approximately 150 untreated patients and about 60 patients who had previously been treated intravitrealally with anti-VEGF antibodies participated in the clinical trial.

[0230] Approximately 50 untreated patients were randomized to one of the groups (1:1:1 randomization scheme), and approximately 30 patients who had previously been treated intravitrealally with anti-VEGF antibodies were randomized to groups A and C.

[0231] Target population Male and female patients aged 18 years or older with diabetic macular edema presenting with fovea.

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

[0233] Eye criteria for clinical trial eyes: Macular edema complicated with diabetic retinopathy, defined as macular thickening including the central part of the macula as measured by spectral domain optical coherence tomography (SD-OCT): at screening using Spectralis® (Heidelberg), a foveal retinal thickness (CST) of 325 μm or more (if Spectralis® is unavailable, the following instruments and foveal retinal thickness thresholds were acceptable: Cirrus® with a foveal retinal thickness of 315 μm or more, Topcon with a foveal retinal thickness of 315 μm or more, and Optov® with a foveal retinal thickness of 295 μm or more).

[0234] On day 1, the patient had a best corrected visual acuity (BCVA) letter score of 73-24 letters (including borderline values) on a chart similar to the Early Treatment Study of Diabetic Retinopathy (ETDRS) (equivalent to 20 / 40-20 / 320 on the Snellen scale), with reduced visual acuity primarily due to diabetic macular edema.

[0235] A transparent translucent material and appropriate pupil dilation enable the acquisition of high-quality retinal images for definitive diagnosis.

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

[0237] The patient must be prepared to provide written informed consent and have the ability and willingness to comply with the clinical trial protocol in accordance with the International Conference on Harmonisation 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 Harmonisation 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., have not received hormone replacement therapy, or have had 12 months or more of non-treatment-induced amenorrhea confirmed by follicle-stimulating hormone), or who have consented to sterilization (removal of ovaries and / or uterus) in order to maintain abstinence, or who use a combination contraceptive with an annual failure rate of less than 1% during the treatment period and for at least 4 weeks after the last dose.

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

[0241] Examples of contraceptive methods that are expected to have an annual failure rate of less than 1% include vasectomy, hormonal implants, the proper use of mixed oral or injectable hormonal contraceptives, and certain intrauterine devices. Alternatively, two methods (e.g., two barrier methods such as condoms and cervical caps) may be combined to achieve an annual failure rate of less than 1%, and the barrier methods must always be supplemented with the use of spermicides.

[0242] For men, they must agree to use barrier contraception for at least four weeks after the last dose of the investigational drug.

[0243] The patient must refrain from attempting to participate in any other clinical trials, including investigational medicinal products (IMPs) or devices, until the completion of the current clinical trial.

[0244] Exclusion Criteria Patients who met 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 corticosteroids into the vitreous or around the eyeball 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 condition in the study eye: History of rubeosis Any current or past eye condition other than diabetic macular edema that could interfere with the assessment of the macula or affect central visual acuity (e.g., age-related macular degeneration, retinal vein occlusion, uveitis, retinal pigment streaks, histoplasmosis, active or inactive cytomegalovirus, pathological myopia, retinal detachment, macular traction, macular hole, severe cataract). In the opinion of the principal investigator, vision loss will not improve with the recovery of macular edema, regardless of any current eye condition (e.g., foveal atrophy, pigment abnormalities, dense foveal hard exudates, or conditions other than retinal conditions). Any active eye infection on day 1. Any active intraocular inflammation (of very low or high grade) on day 1.

[0247] Characteristics of the other eye: Any anti-VEGF antibody treatment within 7 days prior to day 1. In the opinion of the principal investigator, any retinal condition requiring treatment with anti-VEGF antibodies within 7 days from day 1 is indicated.

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

[0249] Untreated diabetes, or initiation of oral antidiabetic drugs or insulin within four months prior to day 1, or anticipated changes in antidiabetic drugs during the study period. Renal failure requiring a kidney transplant, hemodialysis, or peritoneal dialysis within the six months prior to day 1, or expected to require hemodialysis or peritoneal dialysis at any point during the clinical trial. A history of other diseases, metabolic disorders, conditions that reasonably suggest the use of the investigational drug is contraindicated, or physical or laboratory findings that could affect the interpretation of the trial results, or that, in the opinion of the principal investigator, would place the patient at high risk in the management of complications. A woman of childbearing age tested positive for pregnancy via blood test. A woman who is breastfeeding. Systemic use of corticosteroids within one month prior to day 1. Any known hypersensitivity to the active drug, fluorescein, any component of the formulation used, mydriatic eye drops, or any anesthetic and antibacterial eye drops used. Any other restrictions arising from the use of the active drug. Any treatment using the investigational drug within three months prior to day 1.

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

[0251] End of clinical trial The end of the clinical trial was defined as the day the last patient's final observation (LPLO) took place. The LPLO was expected to occur 36 weeks after the last patient's enrollment.

[0252] Evaluation items for efficacy and pharmacokinetics The population used in the primary analysis consisted of untreated patients. Additional analyses may be performed in the entire population and in patients previously treated with intravitreal anti-VEGF antibodies.

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

[0254] Anatomical evaluation items using SD-OCT: Mean change in foveal thickness from baseline at week 24. Mean change from baseline in the mean foveal retinal thickness (1 mm diameter) at 24 weeks. The percentage of patients who showed dissipation of subretinal and intraretinal fluid at week 24. Anatomical evaluation items by fundus fluorescein angiography (FFA). Percentage of patients showing resolution of macula leakage at 24 weeks. Changes from baseline in the size of the foveal avascular area at 24 weeks.

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

[0256] Exploratory endpoints related to sustainability: Time to increase in foveal retinal thickness of 50 μm or more and / or decrease in best corrected visual acuity of 5 letters or more, compared to values ​​at 20 weeks, due to diabetic macular edema. Time until retreatment with 0.3 mg of ranibizumab after week 20.

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

[0258] The primary variable for efficacy was the change in best corrected visual acuity from baseline to week 24. The primary efficacy analysis was performed 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 before pupil dilation by a trained, certified visual acuity tester, with the assignment of the investigational drug group blinded.

[0260] The best corrected visual acuity was measured using a set of three Precision Vision® or Lighthouse Corporation distance visual acuity charts (Revised ETDRS Charts 1, 2, and R). A visual acuity manual was provided to the principal investigator. Certification of visual acuity examiners and visual acuity testing rooms was obtained before any visual acuity tests were performed.

[0261] The best-corrected visual acuity (BSC) examiner would be blinded to the assignment of the trial eye and procedure, and would only perform refractive and BSC assessments (e.g., visual acuity specification manuals). The BSC would also be blinded to the BSC letter scores from the patient's previous visits, and would only know the patient's refractive data from those previous visits. The BSC was not permitted to perform any other tasks, including direct patient care.

[0262] [Table 2]

[0263] The primary efficacy endpoint is shown in Figure 1. Figure 1 shows the primary efficacy endpoint: namely, the change in best corrected visual acuity from baseline to 24 weeks in previously untreated patients. VA2 refers to the bispecific anti-VEGF / ANG2 antibody RO6867461 (administered intravitreally at doses of 6.0 mg or 1.5 mg) containing the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, and RBZ refers to ranibizumab (Lucentis®) (administered intravitreally at doses of 0.3 mg).

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

[0265] Duration of effect / Time until retreatment Criteria for treatment with ranibizumab during the observation period At each follow-up visit following the final dose of medication for the clinical trial (week 20), the best corrected visual acuity was evaluated and SD-OCT angiography was performed (except at week 26).

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

[0267] If a patient met both of the following criteria, they received a single dose of 0.3 mg of ranibizumab and completed the trial: • Foveal retinal thickness increased by more than 50 μm. • Diabetic macular edema caused a decrease of 5 or more letters in maximum corrected visual acuity.

[0268] The results are shown in Figure 3: Figure 3 shows the results after discontinuation of medication (20 weeks or 6 weeks later), based on disease activity assessed by both the best corrected visual acuity (a decrease of 5 letters or more) and the foveal retinal thickness (an increase of 50 μm or more). Each month This indicates the time to retreatment after drug administration (the point in time after the last intravitreous (IVT) administration). The bispecific anti-VEGF / ANG2 antibody RO6867461 (administered intravitreally at doses of 6.0 mg or 1.5 mg), containing the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, was compared with ranibizumab (Lucentis®) (administered intravitreally at doses of 0.3 mg).

[0269] For an 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 for patients with diabetic macular edema (DME) In further clinical trials similar to those described above under Example 1A, patients with diabetic macular edema (e.g., diabetic macular edema including the fovea (CI-DME)) will be treated with bispecific antibodies that bind 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. The active agents used in the treatment will be, for example, aflibercept and / or ranibizumab and / or brolucizumab. Patients will include those who have not previously received treatment with anti-VEGF antibodies (i.e., those who have not been previously treated with anti-VEGF antibody monotherapy, e.g., aflibercept and / or ranibizumab and / or brolucizumab), as well as those who have previously received anti-VEGF antibody monotherapy. The names of the respective bispecific antibodies that bind to human VEGF and human ANG2 are RO6867461 and RG7716, respectively. Use a vial of a sterile, colorless to brownish, preservative-free solution of RO6867461 for intravitreal administration at a dose of either 1.5 mg or 6 mg.

[0271] Use one or more of the following medication plans: a) Patients suffering from diabetic macular edema should be treated (for example) 、 6 The first month of each term The treatment will be administered via injection with a consistent 8-week dosing schedule after the start of treatment. b) Patients with diabetic macular edema should be treated (for example) 、 6 The first month of each term Treatment will involve consistent medication every 12 weeks after the start of injections (one plan will initially include one cycle of medication every 8 weeks). c) Patients suffering from diabetic macular edema should be treated (for example) 、3 ~7 The first month of each term After initiation of injections, the medication plan will be adjusted to extend the injection interval if the disease is stable and absent, or to shorten the interval if disease activity is present. Such a plan may include, for example, the patient receiving medication at Q4W / Q8W / Q12W / Q16W depending on the patient's disease status.

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

[0273] In one embodiment, patients with diabetic macular edema are untreated (never previously treated with monotherapy using anti-VEGF antibodies, such as aflibercept and / or ranibizumab and / or brolucizumab).

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

[0275] In one embodiment, a patient suffering from diabetic macular edema undergoes treatment (for example) 、 6 The first month of each term After initiation of injections, treatment will be administered according to a fixed 8-week medication schedule.

[0276] In one embodiment, a patient suffering from diabetic macular edema undergoes treatment (for example) 、 6 The first month of each term After initiation of injections, treatment will be administered according to a fixed 12-week dosing schedule (in one embodiment, this initially includes one cycle of 8-week dosing).

[0277] In one embodiment, a patient suffering from diabetic macular edema undergoes treatment (for example) 、3 ~7 The first month of each term After initiation of injections, the medication prescription plan will be adjusted to either extend the injection interval if the disease is stable and absent, or shorten the interval if disease activity is present. In one embodiment, such a prescription plan includes the patient receiving medication at Q4W / Q8W / Q12W / Q16W depending on the patient's disease status.

[0278] In one embodiment, a patient suffering from age-related macular degeneration undergoes treatment (for example) 、3 ~4 The first month of each term After initiation of injections, the medication prescription plan will be adjusted to either extend the injection interval if the disease is stable and absent, or shorten the interval if disease activity is present. In one embodiment, such a prescription plan includes the patient receiving medication at Q4W / Q8W / Q12W / Q16W depending on the patient's disease status.

[0279] Example 2A: Efficacy and duration of treatment for patients with age-related macular degeneration (AMD) Objectives and evaluation items This clinical trial 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, containing the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 (this antibody VEGFang2-0016 and its production are also detailed in International Publication No. 2014 / 009465, which is incorporated by reference). The names used herein for this bispecific anti-VEGF / ANG2 antibody are RO6867461, RG7716, VEGFang2-0016, or falisimab.

[0280] The specific objectives for the clinical trial and an outline of the corresponding evaluation items are outlined below.

[0281] Objectives and corresponding evaluation items Main efficacy objectives • 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 the ETDRS chart.

[0283] Second effectiveness objective: 1) Evaluate the effectiveness of RO6867461 for additional visual acuity assessment items. Corresponding evaluation items • Average change from baseline in best corrected visual acuity over time, using ETDRS charts. • The percentage of patients whose best corrected visual acuity over time increased by 15 letters or more, 10 letters or more, 5 letters or more, or 0 letters or more from baseline. • The percentage of patients who avoided a decrease of 15 or more letters, 10 or more letters, 5 or more letters, or 0 or more letters from baseline in their best corrected visual acuity over time. • The percentage of patients with a best corrected visual acuity of 20 / 40 or better over time. • The percentage of patients with a best corrected visual acuity of 20 / 200 or worse over time.

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

[0285] 3) Evaluate the effectiveness of RO6867461 for anatomical evaluation items using fundus fluorescein angiography (FFA). Corresponding evaluation items • Mean change from baseline across the entire area of ​​choroidal angiogenesis (CNV) at weeks 40 and 52. • Mean change from baseline across the entire area of ​​choroidal neovascularization at weeks 40 and 52. • Mean change from baseline across the entire area of ​​leakage at weeks 40 and 52.

[0286] The purpose of exploring effectiveness • To investigate the incidence of disease activity at week 24. Corresponding evaluation items • The percentage of patients with disease activity at week 24.

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

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

[0289] The purpose of immunogenicity • To investigate the formation of anti-RO6867461 antibodies in plasma. Corresponding evaluation items • Incidence of anti-drug antibodies during clinical trials.

[0290] The purpose of searching for biomarkers To evaluate the response to RO6867461, explore the levels of candidate biomarkers for angiogenesis and inflammation in aqueous humor at baseline and additional time points. Corresponding evaluation items • Relationship between primary and secondary endpoints and aqueous humor concentrations of candidate biomarkers. Abbreviations used above: ADA = Anti-drug antibody; Ang-2 = Angiopoietin-2; BCVA = Best corrected visual acuity; CFT = Foveal thickness; CNV = Choroidal neovascularization; CST = Foveal region retinal thickness; ETDRS = Early treatment study for diabetic retinopathy; FFA = Fluorescein angiography of the fundus; IVT = Intravitreous; PK = Pharmacokinetics; SD-OCT = Spectral domain optical coherence tomography; VEGF-A = Vascular endothelial growth factor A.

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

[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 is administered intravitreally (IVT) every 4 weeks until week 12 (4 injections), and then 6 mg of RO6867461 is administered intravitreously every 12 weeks until week 48 (injections at weeks 24, 36, and 48; 3 injections). Group B (Q16W): 6 mg of RO6867461 intravitreously every 4 weeks (4 injections) until week 12, and then 6 mg of RO6867461 intravitreously every 16 weeks until week 48 (injections at weeks 28 and 44; 2 injections). Evaluation of disease activity at week 24 as defined by the protocol requires Group B patients with active disease (see criteria below) to switch to a prescription plan of 6 mg of RO6867461 every 12 weeks for the remainder of the trial, with injections initiated at week 24 and repeated at weeks 36 and 48. • Group C (comparison group): 0.5 mg of ranibizumab (intravitreal) every 4 weeks for 48 weeks (13 injections). Only one eye will be selected as the investigational 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. • Investigational procedure administration: From day 1 to week 48. Last visit: 52 weeks.

[0293] Patients underwent screening within four weeks prior to administration of the investigational treatment. The screening visit and the week 1 / day 1 (randomization) visit may be combined if all evaluations (except informed consent) were completed within 48 hours. Patient eligibility was assessed during screening (or the combined visit for screening / day 1) to ensure that choroidal neovascularization secondary to age-related macular degeneration met the pre-defined ocular criteria for the trial, including central review of fundus photography (FP), spectral domain optical coherence tomography (SD-OCT), and fundus fluorescein angiography (FFA). Patients deemed ineligible based on screening results for any of the following reasons were eligible for rescreening: Uncontrolled blood pressure • Administrative reasons (e.g., inability to schedule Day 1 within 28 days of the initial screening visit) • The patient does not meet the eligibility criteria for the trial eye (in such cases, the patient may be eligible to participate in a second eye after the initial screening period).

[0294] During the re-screening, an evaluation of all visits for the screening examinations was conducted (excluding the acquisition of images from fundus fluorescein angiography). However, eligible fundus fluorescein angiography (FFA) images from the Central Image Interpretation Center were acquired within four weeks prior to the new day 1 visit (randomization).

[0295] On day 1, eligible patients received their first intravitreal infusion of either RO6867461 or ranibizumab, in accordance with the randomization plan described above and the established standard dosing procedure. Patients returned to the ophthalmology clinic 7 days after the first intravitreal infusion, and then every 4 weeks thereafter, for investigational treatment administration and evaluation as outlined in the activity plan in the protocol. To maintain blinding throughout the study period, intravitreal infusions of placebo were delivered to patients randomized into groups A and B.

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

[0297] A diagnosis of active disease was made if any of the following criteria were met: Compared to the average foveal retinal thickness over the last two visits (weeks 16 and 20), there has been an increase in foveal retinal thickness (foveal retinal thickness exceeding 50 μm on Spectralis® OCT), or • An increase of 75 μm or more in foveal retinal thickness compared to the lowest foveal retinal thickness recorded at either week 16 or week 20, or • A decrease of at least 5 letters in best-corrected visual acuity (BCVA) compared to the mean best-corrected visual acuity over the two most recent visits (weeks 16 and 20) due to disease activity of neovascular age-related macular degeneration, or • A decrease of 10 letters or more in best-corrected visual acuity compared to the highest best-corrected visual acuity recorded at either week 16 or week 20 due to disease activity of neovascular age-related macular degeneration, or • The presence of new macular hemorrhages due to the activity of neovascular age-related macular degeneration.

[0298] The patient will return for a final visit at week 52. After the final visit, adverse events should be monitored as outlined in the protocol. Assessments performed in the case of unplanned visits will be determined by the principal investigator.

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

[0300] Target population Eligibility Criteria The patient met the following criteria for participation in the clinical trial: ocular criteria for the trial eye. Untreated choroidal neovascularization secondary to age-related macular degeneration (neovascular age-related macular degeneration). Subfoveal choroidal neovascularization or parafoveal choroidal neovascularization with a foveal component associated with choroidal neovascularization (as evidenced by subretinal fluid, subretinal hyperreflectivity, leakage, or hemorrhage) as determined by fundus fluorescein angiography or spectral domain optical coherence tomography. All types of choroidal neovascular lesions (overwhelmingly classic, minimally classic, or occult) accompanied by the following: total lesion size (including hematopoiesis, atrophy, fibrosis, and neovascularization) of 6 optic disc areas or less as measured by fundus fluorescein angiography, choroidal neovascularization area of ​​50% or more of total lesion size as measured by fundus fluorescein angiography, active choroidal neovascularization (evidence of leakage) as confirmed by fundus fluorescein angiography, and choroidal neovascular exudate (presence of fluid) as confirmed by spectral domain optical coherence tomography. • Transparent translucent media and appropriate pupil dilation enable the acquisition of high-quality retinal images for definitive diagnosis. general criteria • Signing the informed consent form. • The person must be 50 years of age or older on the first day. • The principal investigator must be able to determine that compliance with the clinical trial protocol is possible. • For women of childbearing age: They must agree to either maintain abstinence (refrain from sexual intercourse with the opposite sex) or to use a method of contraception with an annual failure rate of less than 1% throughout the treatment period and for at least 28 days after the last dose of the investigational treatment. Patients must not voluntarily participate in any other clinical trials, including those involving investigational drugs (IMPs) or devices, until the completion of the current clinical trial.

[0301] Exclusion criteria Patients who met any of the following criteria were excluded from participation in the clinical trial: Eye criteria for clinical trial eyes Choroidal neovascularization caused by factors other than age-related macular degeneration, such as ocular histoplasmosis, trauma, pathological myopia, retinal pigment streaks, choroidal rupture, or uveitis. • Central serous chorioretinopathy at the time of screening. • Retinal pigment epithelial tear, including the macula. Subretinal hemorrhage, including foveal fibrosis or atrophy, and / or including the fovea, in fundus fluorescein angiography, with a lesion area exceeding 50% of the total lesion area. Any pre-treatment or concomitant treatment for choroidal angiogenesis, including but not limited to intravitreal treatment (steroids, anti-vascular endothelial growth factor [VEGF] antibodies, tissue plasminogen activator, ocliplasmin, octafluoride propane gas, air), periorbital pharmacological interventions, argon laser photocoagulation, verteporfin photodynamic therapy, diode laser, transpupillary thermotherapy, or surgical interventions. • Cataract surgery performed within 3 months of baseline evaluation (Day 1). • All other intraocular surgeries (transciliary vitrectomy, glaucoma surgery, corneal transplantation, radiation therapy). Previous intravitreal treatments (including anti-VEGF antibody drugs), excluding management of cataract complications using intravitreal steroid treatments. • Previous periorbital pharmacological interventions for other retinal diseases. Current eye condition • Any concomitant ocular conditions in the investigational eye (e.g., amblyopia, aphakia, retinal detachment, cataract, diabetic retinopathy, or macular degeneration, or epiretinal membrane with traction) that, in the opinion of the principal investigator, reduce the likelihood of visual acuity improvement or may necessitate medical or surgical intervention during the course of the trial. • Active intraocular inflammation (very slight grade or higher) in the investigational eye on day 1 (pre-randomization). On day 1, the best corrected visual acuity letter score was 73-24 letters (including borderline values) on a chart similar to the Early Treatment Study of Diabetic Retinopathy (ETDRS) (equivalent to 20 / 40 to 20 / 320 on the Snellen scale). • Current intravitreous hemorrhage in trial eyes. • Uncontrolled glaucoma in the investigational eye (e.g., defined as progressive visual field loss or intraocular pressure [IOP] of 25 mmHg or greater despite treatment with glaucoma medication). • Equivalent spherical power of refractive error showing myopia exceeding 8 diopters in the trial eye. • A history of idiopathic or autoimmune uveitis in either eye. • Active, infectious conjunctivitis, keratitis, scleritis, or endophthalmitis in either eye on day 1 (before randomization). general criteria • Any major illness or major surgery within one month prior to screening. • Uncontrolled blood pressure (defined as systolic blood pressure greater than 180 mmHg and / or diastolic blood pressure greater than 100 mmHg at rest [blood pressure]). If the patient's initial decoding exceeds these values, a second decoding may be performed later on the same day or on another day during the screening period. If the patient's blood pressure is controlled by antihypertensive 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 the three months prior to day 1. • A history of other diseases, metabolic disorders, contraindications to the use of the investigational drug, or conditions that may affect the interpretation of the trial results, or conditions that reasonably suggest the patient is at high risk of complications from the procedure, as determined by the principal investigator, or any other physical examination or laboratory findings. • Pregnant, breastfeeding, or planning to become pregnant during the clinical trial. Women of childbearing age must have a negative pregnancy urine test result within 28 days prior to the start of the clinical trial procedure. If the pregnancy urine test is positive, it must be confirmed by a pregnancy serum test. • Known hypersensitivity to ranibizumab, fluorescein, any component of the formulation used, mydriatic eye drops, or any anesthetic and antibacterial eye drops used. • Treatment using the investigational therapy within three months prior to the start of the investigational treatment.

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

[0303] Clinical trial period The entire duration of the clinical trial, from the initial patient screening to the completion of the trial, was expected to be approximately 18 to 19 months.

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

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

[0306] Patients were given either a 50 μL intravitreal injection of RO6867461 or ranibizumab, or a placebo, to the investigational eye, in accordance with the randomization plan 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 trial eye.

[0308] Evaluation of clinical trials When several evaluations are conducted simultaneously, the following order may be suggested at the discretion of the principal investigator. The order may be adjusted to optimize on-site staffing and patient time management unless explicitly stated as mandatory (i.e., in italicized text). • Signs of life • Blood sample collection: In visits where fundus fluorescein angiography is performed, blood sample collection and angiography can be performed through the same intravenous cannula. The blood sample must be collected before angiography. • Evaluation and imaging of the eye. Best corrected visual acuity: Best corrected visual acuity must be measured before pupil dilation. At the time of the initial screening visit and on the first day visit, best corrected visual acuity may be measured before vital signs and blood sample collection in patients who may be screening failures based on the letter score of best corrected visual acuity, in order to avoid unnecessary investigations. Slit-lamp microscopy. Pupils dilated. SD-OCT. Fundus photography (+ infrared reflectance). Fundus fluorescein angiography. High-magnification fundus examination using binocular indirect ophthalmoscopy with dilated pupils. Intraocular pressure: This must be performed after all imaging assessments, and this method should be used throughout the entire clinical trial period.

[0309] • Collection of aqueous humor sample (optional) Disease-specific evaluation Unless otherwise specified in the activity plan (Addendum 1), all eye evaluations were performed binocularly.

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

[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, Inc. or Lighthouse Corporation. A visual acuity procedure manual was provided to the principal investigator. Accreditation of visual examiners and visual examination rooms was obtained before any visual acuity tests were performed.

[0312] The examiner for best-corrected visual acuity will be blinded to the assigned eye and procedure, and will perform the evaluation of refractive error and best-corrected visual acuity (e.g., visual acuity specification manual). The examiner for best-corrected visual acuity will also be blinded to the letter score of the patient's best-corrected visual acuity from previous visits, and may only know the patient's refractive data known from previous visits.

[0313] Evaluation of additional eyes Additional eye assessments performed during the clinical trial include: • Slit-lamp microscopy (The scale for grading the redness per cell and the density of intravitreous hemorrhage is detailed in Addendum 2) • High-magnification fundus examination using binocular indirect ophthalmoscopy with dilated pupils. • Intraocular pressure

[0314] The method used to measure intraocular pressure in patients remained consistent throughout the clinical trial. Intraocular pressure was measured in both eyes after all imaging was performed.

[0315] At the time of the clinical trial procedure visit, intraocular pressure (IOP) was measured before administration of the procedure and 30 (±15) minutes after administration to the investigational eye. If the IOP was 30 mmHg or higher, it should be re-evaluated 30 (±15) minutes later. If the IOP continued to rise, treatment was administered at the discretion of the principal investigator. • Visual acuity assessment using counting fingers In the investigational eye, the optic nerve head perfusion fluid after treatment was evaluated for each patient immediately after administration of the investigational procedure (within a maximum of 15 minutes after administration) by testing visual acuity by counting fingers, hand movement, or light perception as appropriate.

[0316] Eye imaging The Central Image Interpretation Center provided the site with the Central Image Interpretation Center Manual and training materials for ocular imaging required for the clinical trial. Prior to obtaining clinical trial images, site personnel and imaging systems (where applicable) were authorized by the Image Interpretation Center as specified in the Central Image Interpretation Center Manual. Ocular images of all clinical trial subjects were obtained only by trained and Central Image Interpretation Center certified personnel using equipment authorized / registered at the clinical trial site. Copies of all clinical trial subject ocular images were transferred to the Central Image Interpretation Center for archiving and independent analysis (including verification of eligibility to specified image-related criteria).

[0317] Assessment of disease activity at week 24 All patients were assessed for disease activity at week 24. Patients randomized to Group B who had active disease at week 24 (see criteria below) were switched to a Q12W dosing regimen of 6 mg of RO6867461 for the remainder of the trial, with injections initiated 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: Compared to the average foveal retinal thickness over the last two visits (weeks 16 and 20), an increase of more than 50 μm in foveal retinal thickness as measured by Spectralis OCT, or • An increase of 75 μm or more in foveal retinal thickness compared to the lowest foveal retinal thickness recorded at either week 16 or week 20, or • A decrease of at least 5 letters in best-corrected visual acuity compared to the average best-corrected visual acuity over the two most recent visits (weeks 16 and 20) due to disease activity of neovascular age-related macular degeneration, or • A decrease of 10 letters or more in best-corrected visual acuity compared to the highest best-corrected visual acuity recorded at either week 16 or week 20, due to disease activity of neovascular age-related macular degeneration, or • Presence of new macular hemorrhages due to the activity of neovascular age-related macular degeneration.

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

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

[0321] Example 2B: Efficacy and duration of treatment for patients with age-related macular degeneration (AMD) In further clinical trials similar to those described above under Example 2A, patients with age-related macular degeneration (e.g., exudative age-related macular degeneration (wAMD), particularly neovascular age-related macular degeneration) will be treated with bispecific antibodies that bind 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. The active agent used in the treatment will be, for example, aflibercept and / or ranibizumab and / or brolucizumab. Patients will include those who have not received treatment with an anti-VEGF antibody (i.e., those who have not been previously treated with anti-VEGF antibody monotherapy, e.g., aflibercept and / or ranibizumab and / or brolucizumab) and those who have been previously treated with anti-VEGF antibody monotherapy, e.g., aflibercept and / or ranibizumab and / or brolucizumab. The names of the bispecific antibodies that bind to human VEGF and human ANG2, respectively, are RO6867461 and RG7716. Use a sterile, colorless to brownish, preservative-free vial of RO6867461 for intravitreal administration at a dose of either 1.5 mg or 6 mg.

[0322] For example, use the following medication plan: Patients suffering from age-related macular degeneration may experience symptoms after the start of treatment (for example) 、3 ~7 The first month of each term Treatment will be managed using a medication schedule (using injections), which involves extending the injection interval when the disease is stable and absent, or shortening the interval when disease activity is present. Such a schedule might include, for example, the patient receiving medication at Q4W / Q8W / Q12W / Q16W, depending on the patient's disease status.

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

[0324] Example 3 Binding of anti-VEGF / ANG2 antibodies to VEGF, Ang2, FcγR, and FcRn kinetic affinity of VEGF isoforms, including evaluation of species cross-reactivity. A capture system with approximately 12,000 resonance units (RUs) (10 μg / ml goat anti-human F(ab)'2; order number: 28958325; GE Healthcare Biosciences, Sweden) was coupled to a CM5 tip (GE Healthcare BR-1005-30) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. The sample and system buffer was PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween® 20) at pH 7.4. The flow cell was set to 25°C (sample block was set to 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. The association was measured by injecting human hVEGF121, mouse mVEGF120, or rat rVEGF164 at various solution concentrations at a flow rate of 30 μl / min for 300 seconds, starting at 300 nM with a 1:3 dilution. The dissociation phase was monitored up to 1200 seconds and triggered by switching from the sample solution to the running buffer. The surface was regenerated by washing with 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 The Langmuir 1:1 model was used to calculate the and other dynamic parameters. The results are shown in Table 5.

[0325] Affinity of Ang2 solution, including evaluation of species cross-reactivity. Solution affinity measures the affinity of interactions by determining the concentration of free interaction pairs in an equilibrium mixture. Solution affinity assays are performed at constant concentrations. <vegf-ang-2>The procedure involves mixing a bispecific antibody with ligands (=Ang2) at various concentrations. The antibody with the largest 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 was HBS-P (pH 7.4). The flow cell was set to 25°C, the sample block to 12°C, and the mixture was primed twice with running buffer. To create a calibration curve, progressively increasing concentrations of Ang2 were used on the immobilized chip. <vegf-ang2>The bispecific antibody was injected into a ViaCore™ flow cell. The amount of bound Ang2 was determined as resonance units (RUs) and plotted against concentration. <vegf-ang-2>Solutions of bispecific antibodies at 11 concentrations (0–200 nM) were incubated with 10 nM Ang2 and allowed to reach equilibrium at room temperature. Free Ang2 concentration was determined from calibration curves created before and after measuring the response of a solution containing a known amount of Ang2. A 4-parameter fit was set using XLfit4 (IDBS software) with Model 201, using free Ang2 concentration as the y-axis and the concentration of the antibody used for inhibition as the x-axis. Affinity was calculated by determining the inflection point of this curve. The surface was regenerated by washing once with a 0.85% H3PO4 solution at a flow rate of 30 μl / min for 30 seconds. Differences in bulk refractive index were corrected by subtracting the response obtained from the bound surface of the blank. The results are shown in Table 6.

[0326] FcRn affinity in the steady state For FcRn measurement, bispecific antibodies against each other were compared using steady-state affinity. Human FcRn was diluted in coupling buffer (10 μg / ml, sodium acetate, pH 5.0) and immobilized on a C1 tip (GE Healthcare BR-1005-35) using a targeted immobilization procedure with the ViaCore® Wizard until a final response of 200 RU was obtained. The flow cell was set to 25°C and the sample block to 12°C, and both were primed twice with running buffer. The sample and system buffer was PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween® 20), pH 6.0. To evaluate various IgG concentrations for each antibody, concentrations of 62.5 nM, 125 nM, 250 nM, and 500 nM were prepared. The flow rate was set to 30 μl / min, and various samples were continuously injected onto the tip surface with a selected association time of 180 seconds. The surface was regenerated by injecting PBS-T (pH 8) at a flow rate of 30 μl / min for 60 seconds. Differences in bulk refractive index were corrected by subtracting the response obtained from the blank surface. Injection of buffer solution was also subtracted (= double control). The Bia-Evaluation software method was used to calculate steady-state affinity. Briefly, the numerical value of the resonance unit (maximum RU) was plotted against the analyzed concentration to obtain a dose-response curve. An upper asymptote was calculated based on a two-parameter fit, making it possible to determine the maximum half-value RU and, by extension, the affinity. The results are shown in Figure 5 and Table 7. Similarly, the affinity for FcRn in cynomolgus monkeys, mice, and rabbits can also be determined.

[0327] Measurement of FcγRIIIa A direct binding assay was used to measure FcγRIIIa. A capture system with approximately 3000 resonance units (RUs) (1 μg / ml penta-His; Qiagen) was coupled to a CM5 tip (GE Healthcare BR-1005-30) at pH 5.0 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 to 25°C (sample block was set to 12°C) and primed twice with running buffer. The FcγRIIIa-His receptor was captured by injecting a 100 nM solution at a flow rate of 5 μl / min for 60 seconds. Binding was measured by injecting a 100 nM bispecific antibody or a single specific control antibody (anti-Dig antibody for IgG1 and IgG4 subclass antibodies) at a flow rate of 30 μl / min for 180 seconds. The surface was regenerated by washing with a glycine (pH 2.5) solution at a flow rate of 30 μl / min for 120 seconds. Since the binding of FcγRIIIa differs from the Langmuir 1:1 model, binding only / absence 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 the introduction of the mutant P329G LALA prevents further detection of binding to FcγRIIIa.

[0328] <vegf-ang-2>Evaluation of independent binding of VEGF and Ang-2 to bispecific antibodies A capture system with approximately 3500 resonance units (RUs) (10 μg / ml goat anti-human IgG; GE Healthcare Biosciences, Sweden) was coupled to a CM4 tip (GE Healthcare BR-1005-34) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. The sample and system buffer was PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween® 20) at pH 7.4. The flow cell temperature was set to 25°C, and the sample block temperature was set to 12°C. Before 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 ability of each ligand added either sequentially or simultaneously (at a flow rate of 30 μl / min).

[0330] Human VEGF was injected at a concentration of 1,200 nM for 180 seconds (to identify single antigen binding). Human Ang2 was injected at a concentration of 2.100 nM for 180 seconds (to identify single antigen binding). 3. Human VEGF was injected at a concentration of 200 nM for 180 seconds, followed by a further injection of human Ang2 at a concentration of 100 nM for 180 seconds (to identify Ang2 binding in the presence of VEGF). 4. Human Ang2 was injected at a concentration of 100 nM for 180 seconds, followed by further injection of human VEGF at a concentration of 200 nM (to identify VEGF binding in the presence of Ang2). 5. Simultaneous injection of human VEGF at a concentration of 200 nM and human Ang2 at a concentration of 100 nM for 180 seconds (simultaneous identification of VEGF and Ang2 binding).

[0331] The surface was regenerated by washing with a 3 mM MgCl2 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 from approaches 1 and 2, then the bispecific antibodies can bind to both antigens independently of each other. The results are shown in the table below, which shows that VEGFang2-0016 (=RO6867461) can bind to VEGF and ANG2 independently of each other.

[0333] <vegf-ang-2>Evaluation of simultaneous binding of bispecific antibodies to VEGF and Ang2. First, VEGF (20 μg / ml) with approximately 1600 resonance units (RUs) was coupled onto a CM4 tip (GE Healthcare BR-1005-34) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. The sample and system buffer was PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween® 20) at pH 7.4. The flow cell was set to 25°C, and the sample block was set to 12°C and primed twice with running buffer. Secondly, a 50 nM bispecific antibody solution was injected at a flow rate of 30 μl / min for 180 seconds. Thirdly, hANG-2 was injected at a flow rate of 30 μl / min for 180 seconds. The binding response of hAng-2 was dependent on the amount of bispecific antibody bound to VEGF and showed co-binding. The surface was regenerated by washing with a 0.85% H3PO4 solution at a flow rate of 30 μl / min for 60 seconds. Co-binding occurs when VEGF is bound to the co-binding agent beforehand. <vegf-ang-2>This is indicated by the additional specific binding signal 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 falisimab, a bispecific antibody that binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), The initial dose of 6 mg of falisimab is administered intravitreally (IVT) 3 to 7 times every month, followed by 6 mg of falisimab administered IVT every 12 weeks. Pharmaceutical composition.

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