TW202344265A - Vegfr fusion protein pharmaceutical composition - Google Patents

Vegfr fusion protein pharmaceutical composition Download PDF

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TW202344265A
TW202344265A TW111118082A TW111118082A TW202344265A TW 202344265 A TW202344265 A TW 202344265A TW 111118082 A TW111118082 A TW 111118082A TW 111118082 A TW111118082 A TW 111118082A TW 202344265 A TW202344265 A TW 202344265A
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fusion protein
concentration
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TWI831202B (en
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譚 阮
吳佩姿
張永昇
馬渡 徹如庫瑞
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新源生物科技股份有限公司
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Abstract

The present invention relates to a biologic that inhibits angiogenesis. In particular, the present invention relates to fusion proteins that inhibit the integrin activated pathway and one other angiogenic factor-activated pathway as well as formulation compositions of such fusion proteins, as well as methods for producing and using the same.

Description

血管內皮生長因子受體融合蛋白醫藥組合物Vascular endothelial growth factor receptor fusion protein pharmaceutical composition

本發明係關於一種抑制血管性因子活化路徑（angiogenic factor-activated pathway）的融合蛋白醫藥組合物。具體而言，本發明係關於抑制血管性因子活化路徑的融合蛋白、該等融合蛋白的組合物、以及用於製造及使用彼等的方法。The present invention relates to a fusion protein pharmaceutical composition that inhibits the angiogenic factor-activated pathway. Specifically, the present invention relates to fusion proteins that inhibit vascular factor activation pathways, compositions of such fusion proteins, and methods for making and using the same.

血管新生係自既存血管形成新血管的過程，其在數種生理過程中皆扮演重要的角色，該生理過程包括胚胎發育以及組織及傷口修復（Folkman J等人，Angiogenic factors. Science 1987; 235:442-7）。血管新生之生理步驟已被清楚了解，包含胞外基質之分解、內皮細胞之增生、黏附、遷移、及重組成管道通路、壁細胞之募集及分化、以及胞外基質之產生（Carmeliet P等人，Nature. 2011; 473:298–307）。病理性的血管新生可發生在腫瘤形成、眼部病症（例如，糖尿病視網膜病變、糖尿病黃斑水腫、視網膜／脈絡膜新生血管、滲出性老年性黃斑退化、及新生血管性青光眼）、關節炎、牛皮癬、纖維化疾病、發炎性疾病、動脈粥狀硬化（atherosclerosis）及動脈硬化（arteriosclerosis）（Polverini PJ. Crit Rev Oral Biol Med. 1995; 6(3):230-47；Perrotta P et al. Vascular Pharmacology. 2019; 112:72–78）。Angiogenesis, the process of forming new blood vessels from existing blood vessels, plays an important role in several physiological processes, including embryonic development and tissue and wound repair (Folkman J et al., Angiogenic factors. Science 1987; 235: 442-7). The physiological steps of angiogenesis are well understood and include the breakdown of extracellular matrix, proliferation, adhesion, migration, and reorganization of endothelial cells into conduit pathways, recruitment and differentiation of mural cells, and production of extracellular matrix (Carmeliet P et al. , Nature. 2011;473:298–307). Pathological angiogenesis can occur in tumors, ocular disorders (eg, diabetic retinopathy, diabetic macular edema, retinal/choroidal neovascularization, exudative age-related macular degeneration, and neovascular glaucoma), arthritis, psoriasis, Fibrotic diseases, inflammatory diseases, atherosclerosis and arteriosclerosis (Polverini PJ. Crit Rev Oral Biol Med. 1995; 6(3):230-47; Perrotta P et al. Vascular Pharmacology. 2019;112:72–78).

病理性血管新生更加的多樣且混亂，通常呈現彎曲的血管組織、不同大小的低氧縫隙（hypoxic void），不均勻且不完整的血管壁及內壁（lining）、以及無效的灌流（Jain RK., Nat Med. 2003; 9(6):685–93）。疾病中之新血管形成的該些特殊表徵，使得靶向血管新生之治療具有挑戰性。雖然抗VEGF療法，例如：樂舒晴（Lucentis ®）、采視明（Eylea ®）、或藥品仿單標示外使用（off-label use）的癌思停（Avastin ®）一般可穩定或改善視覺功能，但在用抗VEGF治療兩年內，所有經治療之眼睛中大約半數會產生次視網膜結痂（纖維化），這被視為癒後不成功的其中一個原因（Daniel E等人，Ophthalmology. 2014;121(3):656-66）。在次視網膜纖維化中的許多關鍵參與者，很可能是參與在纖維化過程（細胞增生、遷移及ECM重塑）中的生長因子及基質細胞蛋白（matricellular protein）。儘管其具有複雜性，隨著我們對血管新生過程的知識增加，抑制血管新生藥物的發展依然是頗受關注的區域。 Pathological angiogenesis is more diverse and chaotic, usually showing curved vascular tissue, hypoxic voids of different sizes, uneven and incomplete vessel walls and lining, and ineffective perfusion (Jain RK ., Nat Med. 2003; 9(6):685–93). These unique features of neovascularization in disease make treatments targeting angiogenesis challenging. Although anti-VEGF therapies such as Lucentis ® , Eylea ® , or off-label use of Avastin ® generally stabilize or improve vision, function, but within two years of treatment with anti-VEGF, approximately half of all treated eyes develop subretinal scarring (fibrosis), which is considered to be one of the reasons for unsuccessful recovery (Daniel E et al., Ophthalmology . 2014;121(3):656-66). Many of the key players in subretinal fibrosis are likely to be growth factors and matricellular proteins involved in the fibrotic process (cell proliferation, migration, and ECM remodeling). Despite its complexity, the development of drugs that inhibit angiogenesis remains an area of considerable interest as our knowledge of the angiogenic process increases.

近年來，在新生血管（neovascularization）過程中的許多關鍵參與者已被確認，其中血管內皮生長因子（vascular endothelial growth factor，VEGF）家族扮演至為關鍵的角色。人類VEGF家族共有6個成員：VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E、及胎盤生長因子（placental growth factor，PlGF）。除此之外，VEGF-A、VEGF-B及PlGF之多種同功體係透過選擇性RNA剪接來產生（Sullivan等人，MAbs, 2002, 2(2):165-75）。VEGF-A係參與血管新生之主要因子；其與VEGFR-1及VEGFR-2二者結合。藉由阻礙VEGF-A訊息傳遞來抑制血管新生的策略已建立成功的治療方式，該治療方式係用於治療特定的癌症以及視網膜新生血管與缺血性疾病。（Major等人，J Pharmacol Exp Ther., 1997, 283(1):402-10；Willet等人，Nat. Med. 2004, 10:145-7；Papadopoulos等人，Angiogenesis, 2012, 15(2):171-85；Aiello等人，PNAS, 1995, 92:10457-61）。In recent years, many key players in the process of neovascularization have been identified, among which the vascular endothelial growth factor (VEGF) family plays a critical role. There are six members of the human VEGF family: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PlGF). In addition, various isoforms of VEGF-A, VEGF-B and PlGF are produced through alternative RNA splicing (Sullivan et al., MAbs, 2002, 2(2):165-75). VEGF-A is the main factor involved in angiogenesis; it binds to both VEGFR-1 and VEGFR-2. Strategies to inhibit angiogenesis by blocking VEGF-A signaling have established successful therapeutic modalities for the treatment of certain cancers as well as retinal neovascularization and ischemic diseases. (Major et al., J Pharmacol Exp Ther., 1997, 283(1):402-10; Willet et al., Nat. Med. 2004, 10:145-7; Papadopoulos et al., Angiogenesis, 2012, 15(2) :171-85; Aiello et al., PNAS, 1995, 92:10457-61).

其他生長因子、細胞激素（cytokine）、化學激活素（chemokine）包括：血小板衍生生長因子（Platelet-Derived Growth Factor，PDGF）、轉變生長因子-β（Transforming Growth Factor-β，TGF-β）、表皮生長因子（Epidermal Growth Factor，EGF）、神經生長因子（Nerve Growth Factor，NGF）、缺氧誘導因子（Hypoxia-Induced Factor，HIF）、鹼性成纖維母細胞生長因子或成纖維母細胞生長因子（bFGF或FGF-2）、結締組織生長因子（Connective-Tissue Growth Factor，CTGF）、顆粒球巨噬細胞株刺激因子（Granulocyte– Macrophage Colony-Stimulating Factor，GM-CSF）、類胰島素生長因子（Insulin-Like Growth Factor，IGF）、肝細胞生長因子／分散因子（Hepatocyte Growth Factors/Scatter Factor，HGF/SF）、腫瘤壞死因子-α（Tumor Necrosis Factor alpha，TNF-α）、基質細胞衍生因子-1（Stromal cell-derived factor-1，SDF-1）、介白素-1（Interleukin 1，IL-1）、介白素-6（IL-6）、介白素-8（IL-8）、介白素-17（IL-17）、介白素-18（IL-18）、介白素-20（IL-20）、介白素-23（IL-23）、化學誘質（例如C-C基序配體（CCL28、CCL21）及C-X-C基序配體（CXCL1、CXCL5））、巨噬細胞遷移抑制因子（Macrophage migration Inhibitory Factor，MIF）、及免疫細胞表面蛋白（例如分化簇（Clusters of Differentiation，CD））。該等因子在血管新生相關之疾病中已被報告會過度表現，並扮演關鍵性的角色（Elshabrawy等人，Angiogenesis (2015) 18:433–448; Somanath P R 等人，Cell Biochem Biophys. 2009; 53(2): 53–64，Eliceiri B P.，Circ Res. 2001 Dec 7; 89(12):1104-10）。靶向該等因子以減少其下游路徑的活化可降低血管新生相關之疾病。Other growth factors, cytokines, and chemokines include: Platelet-Derived Growth Factor (PDGF), Transforming Growth Factor-β (TGF-β), epidermal Growth factor (Epidermal Growth Factor, EGF), nerve growth factor (Nerve Growth Factor, NGF), hypoxia-induced factor (HIF), basic fibroblast growth factor or fibroblast growth factor ( bFGF or FGF-2), Connective-Tissue Growth Factor (CTGF), Granulocyte- Macrophage Colony-Stimulating Factor (GM-CSF), Insulin-like growth factor (Insulin- Like Growth Factor (IGF), Hepatocyte Growth Factors/Scatter Factor (HGF/SF), Tumor Necrosis Factor alpha (TNF-α), Stromal Cell Derived Factor-1 ( Stromal cell-derived factor-1 (SDF-1), interleukin 1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1 Interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-20 (IL-20), interleukin-23 (IL-23), chemical inducers (such as C-C based sequence ligands (CCL28, CCL21) and C-X-C motif ligands (CXCL1, CXCL5)), macrophage migration inhibitory factors (MIF), and immune cell surface proteins (such as Clusters of Differentiation, CD)). These factors have been reported to be overexpressed and play critical roles in angiogenesis-related diseases (Elshabrawy et al., Angiogenesis (2015) 18:433–448; Somanath P R et al., Cell Biochem Biophys. 2009; 53 (2): 53–64, Eliceiri B P., Circ Res. 2001 Dec 7; 89(12):1104-10). Targeting these factors to reduce activation of their downstream pathways may reduce angiogenesis-related diseases.

整合蛋白係細胞表面受體的一個家族，其亦被發現在內皮細胞表面過度表現，並被認為在血管新生期間會促進新形成之血管的生長及存活。整合蛋白係異二聚體型細胞表面受體，其與胞外基質蛋白交互作用，並對許多生物學的過程而言至關重要。在許多細胞型中整合蛋白的表現係參與在腫瘤發展，且其與生長因子受體交互溝通（crosstalk）的能力使其成為引人注目的治療標的（Staunton D E等人，Adv Immunol. 2006; 91:111-57；Avraamides, C. J.等人，Nat Rev Cancer 2008; 8:604-617；Somanath P R等人，Cell Biochem Biophys. 2009; 53(2): 53–64）。特別的是整合蛋白αvβ3在腫瘤細胞及血管新生的內皮細胞二者中皆會調升（upregulate），且其對腫瘤細胞遷移、血管新生、及細胞訊息失調而言係重要的。因此，整合蛋白αvβ3之拮抗劑正被廣泛地研究其抑制血管新生及抗腫瘤的性質（Desgrosellier JS等人，Nat Rev Cancer. 2010; 10:9-22）。Integrins are a family of cell surface receptors that are also found to be overexpressed on the surface of endothelial cells and are thought to promote the growth and survival of newly formed blood vessels during angiogenesis. Integrins are heterodimeric cell surface receptors that interact with extracellular matrix proteins and are critical for many biological processes. The expression of integrins in many cell types is involved in tumor development, and their ability to crosstalk with growth factor receptors makes them a compelling therapeutic target (Staunton D E et al., Adv Immunol. 2006; 91 :111-57; Avraamides, C. J. et al., Nat Rev Cancer 2008; 8:604-617; Somanath P R et al., Cell Biochem Biophys. 2009; 53(2): 53–64). In particular, integrin αvβ3 upregulates in both tumor cells and angiogenic endothelial cells, and is important for tumor cell migration, angiogenesis, and cell signaling dysregulation. Therefore, antagonists of integrin αvβ3 are being extensively studied for their inhibitory angiogenesis and anti-tumor properties (Desgrosellier JS et al., Nat Rev Cancer. 2010; 10:9-22).

去整合蛋白（disintegrin）係於蝮蛇家族之蛇毒中發現的胜肽，且其主要抑制β1-及β3-相關之整合蛋白。一開始去整合蛋白被辨識為整合蛋白αIIbβ3之抑制劑，接著被發現可與其他整合蛋白以高親合力結合，並阻斷整合蛋白與含RGD之蛋白的交互作用。去整合蛋白含有47至84個胺基酸、具有約4-7條雙硫鍵、並帶有相同的RGD模體（motif）（McLane MA等人，Proc Soc Exp Biol Med 1998 219: 109-119；Niewiarowski S等人， Semin Hematol 1994 31: 289-300；Calvete JJ., Curr Pharm Des. 2005 11: 829-835；Blobel CP等人，Curr Opin Cell Biol 1992 4: 760-765）。去整合蛋白家族中的保守性RGD序列，在辨認整合蛋白中扮演最重要的角色。已發現去整合蛋白與24種整合蛋白中的8種交互作用，並抑制整合蛋白調節（integrin-mediated）之細胞增殖、黏附、遷移、及血管新生（McLane MA等人，Front Biosci. 2008 13: 6617-6637；Swenson S等人，Curr Pharm Des. 2007 13: 2860-2871）。動物研究顯示去整合蛋白靶向新生血管內皮及轉移型腫瘤，說明其用於癌症治療的潛力。含RGD之蛋白與整合蛋白的專一性結合，係其構型及RGD模體周圍的序列二者的功能。許多研究已顯示在含RGD蛋白之RGD模體二側的殘基，會影響其對整合蛋白的結合專一性及親合力（Scarborough RM等人， J Biol Chem 1993; 268: 1058-1065；Rahman S等人，Biochem J 1998; 335: 247-257）。Disintegrin is a peptide found in the venom of snakes in the viper family, and it mainly inhibits β1- and β3-related integrins. Initially identified as an inhibitor of integrin αIIbβ3, disintegrin was subsequently found to bind with high affinity to other integrins and block the interaction of integrins with RGD-containing proteins. Disintegrin proteins contain 47 to 84 amino acids, have about 4-7 disulfide bonds, and carry the same RGD motif (McLane MA et al., Proc Soc Exp Biol Med 1998 219: 109-119 ; Niewiarowski S et al., Semin Hematol 1994 31: 289-300; Calvete JJ., Curr Pharm Des. 2005 11: 829-835; Blobel CP et al., Curr Opin Cell Biol 1992 4: 760-765). The conserved RGD sequence in the integrin family plays the most important role in identifying integrins. Disintegrin has been found to interact with 8 of 24 integrins and inhibit integrin-mediated cell proliferation, adhesion, migration, and angiogenesis (McLane MA et al., Front Biosci. 2008 13: 6617-6637; Swenson S et al., Curr Pharm Des. 2007 13: 2860-2871). Animal studies show that disintegrin targets neovascular endothelium and metastatic tumors, suggesting its potential for cancer treatment. The specific binding of RGD-containing proteins to integrins is a function of both their configuration and the sequences surrounding the RGD motif. Many studies have shown that the residues on both sides of the RGD motif of RGD-containing proteins affect its binding specificity and affinity for integrins (Scarborough RM et al., J Biol Chem 1993; 268: 1058-1065; Rahman S et al., Biochem J 1998; 335: 247-257).

血管新生係一複雜的生物學過程，牽涉多種生長因子及其訊息傳遞受體，且靶向在訊息傳遞路徑（signaling cascade）中的單一分子可能無法對疾病（例如癌症）中不受控之血管新生提供有效的臨床治療。因此，發展可協同結合數種關鍵之血管新生因子，以有效抑制血管新生及疾病進程之創新療法的需求將不斷的增加。Angiogenesis is a complex biological process involving multiple growth factors and their signaling receptors, and targeting a single molecule in the signaling cascade may not be able to control uncontrolled blood vessels in diseases such as cancer. Xinsheng provides effective clinical care. Therefore, there will be an increasing need to develop innovative therapies that synergistically combine several key angiogenic factors to effectively inhibit angiogenesis and disease progression.

本文係提供融合蛋白的醫藥配方、以及使用該等配方的方法。This article provides pharmaceutical formulations of fusion proteins and methods of using such formulations.

於一通用態樣中，本申請係關於一種醫藥配方，該配方包含： a)    一濃度為約0.5毫克／毫升（mg/mL）至約120 mg/mL之融合蛋白； b)    一濃度為約1%至約10%（重量／體積）之多元醇或醇類，該多元醇或醇類係選自由以下所組成之群組：蔗糖、海藻糖、甘露醇、山梨糖醇、苯甲醇、聚乙烯醇、及聚乙二醇（PEG）400-12000； c)    一濃度為約10毫莫耳（mM）至約50 mM之緩衝劑，該緩衝劑係選自由以下所組成之群組：磷酸鈉、組胺酸、檸檬酸鈉、醋酸鈉、碳酸氫鈉、及檸檬酸三鈉二水合物（trisodium citrate dihydrate）；以及 d)    一濃度為約0.01%至約4%（重量／體積）之界面活性劑， 其中，該配方的pH值為約5.5至7.5，且該配方視需要地更包含一選自由以下所組成之群組的多醣：羧甲基纖維素鈉、微晶纖維素、或透明質酸鈉。 In a general form, this application relates to a pharmaceutical formulation comprising: a) A fusion protein with a concentration of about 0.5 mg/mL to about 120 mg/mL; b) A polyol or alcohol at a concentration of about 1% to about 10% (weight/volume), the polyol or alcohol being selected from the group consisting of: sucrose, trehalose, mannitol, sorbose Alcohol, benzyl alcohol, polyvinyl alcohol, and polyethylene glycol (PEG) 400-12000; c) A buffer with a concentration of about 10 millimoles (mM) to about 50 mM, the buffer being selected from the group consisting of: sodium phosphate, histidine, sodium citrate, sodium acetate, bicarbonate Sodium, and trisodium citrate dihydrate; and d) A surfactant at a concentration of about 0.01% to about 4% (weight/volume), Wherein, the pH value of the formula is about 5.5 to 7.5, and the formula optionally further includes a polysaccharide selected from the group consisting of: sodium carboxymethyl cellulose, microcrystalline cellulose, or sodium hyaluronate .

根據本申請之實施態樣，該界面活性劑係選自由以下所組成之群組：聚山梨糖醇酯20、聚山梨糖醇酯80、及泊洛沙姆188，較佳為聚山梨糖醇酯20。According to the embodiment of the present application, the surfactant is selected from the group consisting of: polysorbate 20, polysorbate 80, and poloxamer 188, preferably polysorbate Ester 20.

根據本申請之實施態樣，該界面活性劑的濃度約為0.03%。According to an implementation aspect of the present application, the concentration of the surfactant is approximately 0.03%.

根據本申請之實施態樣，該融合蛋白的濃度為約1 mg/mL至約90 mg/mL，較佳為約20 mg/mL至約80 mg/mL，更佳地，該融合蛋白的濃度為約40 mg/mL。According to the embodiment of the present application, the concentration of the fusion protein is about 1 mg/mL to about 90 mg/mL, preferably about 20 mg/mL to about 80 mg/mL, and more preferably, the concentration of the fusion protein is is approximately 40 mg/mL.

根據本申請之實施態樣，該多元醇係海藻糖且濃度為約25 mM至250 mM，較佳為約190 mM。According to an embodiment of the present application, the polyol is trehalose and the concentration is about 25 mM to 250 mM, preferably about 190 mM.

根據本申請之實施態樣，該緩衝劑係組胺酸且濃度為約10 mM至約40 mM，較佳為約20 mM至約30 mM，更佳地，該組胺酸的濃度為約25 mM。According to an embodiment of the present application, the buffer is histidine and the concentration is about 10 mM to about 40 mM, preferably about 20 mM to about 30 mM, and more preferably, the concentration of the histidine is about 25 mM.

根據本申請之實施態樣，該融合蛋白係自N-端至C-端以如下順序包含： a)   一血管內皮生長因子受體（VEGFR）的細胞外結構域； b)   一人類免疫球蛋白G的Fc結構域；以及 c)    一結合整合蛋白之蛋白（integrin binding protein）或其片段。 According to an embodiment of the present application, the fusion protein includes in the following order from the N-terminus to the C-terminus: a) An extracellular domain of vascular endothelial growth factor receptor (VEGFR); b) an Fc domain of human immunoglobulin G; and c) An integrin binding protein or a fragment thereof.

根據本申請之實施態樣，該融合蛋白係包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO: 17、或SEQ ID NO: 18。According to an embodiment of the present application, the fusion protein includes SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

根據本申請之實施態樣，該pH值為約5.5至約7.0，較佳地，該pH值為約6.0。According to embodiments of the present application, the pH value is about 5.5 to about 7.0, preferably, the pH value is about 6.0.

根據本申請之實施態樣，該配方在-70℃、-20℃、及／或5℃下安定性維持至少24個月。According to the implementation aspect of the present application, the formula maintains stability at -70°C, -20°C, and/or 5°C for at least 24 months.

根據本申請之實施態樣，該配方在-70℃、-20℃、及／或2℃至8℃下（較佳在2℃至8℃下）至少9個月後係保持蛋白質的純度及活性（potency）。According to the implementation aspect of the present application, the formula maintains the purity of the protein after at least 9 months at -70°C, -20°C, and/or 2°C to 8°C (preferably at 2°C to 8°C). Potency.

根據本申請之實施態樣，該配方更包含一濃度為約10 mM至50 mM的鹽類。According to an embodiment of the present application, the formula further includes a salt at a concentration of about 10 mM to 50 mM.

根據本申請之實施態樣，該配方更包含至少一種胺基酸，該胺基酸的濃度為約10 mM至50 mM。According to an embodiment of the present application, the formula further includes at least one amino acid, and the concentration of the amino acid is about 10 mM to 50 mM.

根據本申請之實施態樣，該鹽類係選自氯化鈉、氯化鎂、氯化鈣、或氯化鉀。According to an embodiment of the present application, the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

根據本申請之實施態樣，該胺基酸係選自由以下所組成之群組：精胺酸、甲硫胺酸、脯胺酸、組胺酸、半胱胺酸、離胺酸、甘胺酸、天門冬胺酸、色胺酸、麩胺酸、及異白胺酸。According to an embodiment of the present application, the amino acid is selected from the group consisting of: arginine, methionine, proline, histidine, cysteine, lysine, and glyamine acid, aspartic acid, tryptophan, glutamic acid, and isoleucine.

根據本申請之實施態樣，該醫藥配方可用於一治療眼部疾病的方法。According to the implementation aspect of the present application, the pharmaceutical formula can be used in a method of treating eye diseases.

根據本申請之實施態樣，該眼部疾病係選自新生血管（neovascularization）或缺血性眼色素層炎、視網膜血管炎、血管樣痕、色素性視網膜炎、角膜新生血管、虹膜新生血管、新生血管性青光眼、青光眼術後纖維化、增殖性玻璃體視網膜病變（proliferative vitreoretinopathy，PVR）、脈絡膜新生血管（choroidal neovascularization，CNV）、視神經盤新生血管、視網膜新生血管、玻璃體新生血管（vitreal neovascularization）、血管翳（pannus）、翼狀贅肉（pterygium）、血管性視網膜病變（vascular retinopathy）、無糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、有糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、糖尿病黃斑水腫（DME）、滲出性（濕性）及非滲出性（乾性）之老年性黃斑退化（AMD）、黃斑水腫、視網膜靜脈阻塞後黃斑水腫（RVO）、視網膜靜脈阻塞（RVO）、中心性視網膜靜脈阻塞（CRVO）、分支性視網膜靜脈阻塞（BRVO）、視網膜血管瘤狀增殖（RAP）、息肉狀脈絡膜血管病變（PCV）、玻璃體黃斑沾黏（VMA）、及／或玻璃體黃斑牽引症（VMT）。According to an embodiment of the present application, the eye disease is selected from the group consisting of neovascularization or ischemic uveitis, retinal vasculitis, vascular scars, retinitis pigmentosa, corneal neovascularization, iris neovascularization, Neovascular glaucoma, postoperative glaucoma fibrosis, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, Pannus, pterygium, vascular retinopathy, diabetic retinopathy (DR, non-proliferative and proliferative DR) without diabetic macular edema (DME), diabetic macular edema ( DME) diabetic retinopathy (DR, non-proliferative and proliferative DR), diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, Postretinal vein occlusion macular edema (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branched retinal vein occlusion (BRVO), retinal angiomatous proliferation (RAP), polypoidal choroidal vasculopathy ( PCV), vitreomacular adhesion (VMA), and/or vitreomacular traction (VMT).

根據本申請之實施態樣，該配方係以每眼約0.03至10毫克（mg）的劑量投予，較佳為每眼約3.0至6.0 mg，更佳地，該配方係以每眼約4 mg的劑量投予。According to the implementation aspect of the present application, the formula is administered at a dosage of about 0.03 to 10 milligrams (mg) per eye, preferably about 3.0 to 6.0 mg per eye, and more preferably, the formula is administered at a dosage of about 4 mg per eye. mg doses are administered.

根據本申請之實施態樣，該配方係以每眼約4 mg的劑量投予。According to an aspect of the present application, the formulation is administered at a dose of approximately 4 mg per eye.

本申請之另一通用態樣係關於一種醫藥配方，該配方包含： a)    一濃度為40 mg/mL之融合蛋白； b)    25 mM之組胺酸； c)    190 mM之海藻糖、蔗糖、或甘露醇；以及 d)    0.03%之聚山梨糖醇酯20或聚山梨糖醇酯80， 其中，該配方的pH值為約6.0。 Another general aspect of the application relates to a pharmaceutical formulation comprising: a) A fusion protein with a concentration of 40 mg/mL; b) 25 mM histamine; c) 190 mM trehalose, sucrose, or mannitol; and d) 0.03% polysorbate 20 or polysorbate 80, Among them, the pH value of this formula is about 6.0.

本說明書足以使熟習此技術領域者能夠實行本發明。除了本文所示及描述外，由先前的描述，本發明之多種修飾對熟習此技術領域者而言係顯而易見的，並落入後附申請專利範圍內。本文所引用之所有出版品、專利、及專利申請案之全文倂於此處以供參考。This description is sufficient to enable those skilled in the art to practice the invention. In addition to what is shown and described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The full texts of all publications, patents, and patent applications cited herein are hereby incorporated by reference.

除非另外定義，此處所用之所有技術及科學術語係具有與本發明所屬技術領域中具通常知識者通常所了解之相同意義。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

於本文中，除非內文另外明確指出，否則單數型式之「一」、「該」係包括複數型式。因此，舉例而言，涉及「一結合結構域」係包括複數的結合結構域及其為熟習此項技術領域者所知的同等物。In this document, the singular forms "a", "the" and "the" include the plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a binding domain" includes the plural binding domains and their equivalents known to those skilled in the art.

於本文中，可交換使用術語「多胜肽」及「蛋白質」來表示長鏈胜肽，其具有天然蛋白質的胺基酸序列、或有一或多個突變（例如，一或多個胺基酸殘基的刪除（deletion）、添加（addition）、及／或取代（substitution））的胺基酸序列。As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to long-chain peptides that have the amino acid sequence of a native protein or one or more mutations (e.g., one or more amino acid Deletion, addition, and/or substitution of residues) in the amino acid sequence.

「融合蛋白」意指具有二或更多以共價連接之部分（portion）的蛋白，其中該各部分係衍生自不同的蛋白。"Fusion protein" means a protein having two or more covalently linked portions, where the portions are derived from different proteins.

本發明提供一種醫藥配方，其係包含一融合蛋白，該融合蛋白包含結合整合蛋白的胜肽、其他結合蛋白的胜肽以靶向血管新生因子、及Fc結構域，其中該結合整合蛋白的胜肽係選自由以下所組成之群組：去整合蛋白（參見美國專利號7,943,728及PCT申請號PCT/US 15/46322對胺基酸序列之描述，該些文獻之全文倂於此處以供參考）、抗整合蛋白αvβx抗體（參見美國專利號6,160, 099及8, 350, 010對胺基酸序列之描述，該些文獻之全文倂於此處以供參考）、抗整合蛋白α5β1抗體、靶向整合蛋白同功體αvβx或α5β1之纖維黏連蛋白（參見美國公開號2015/0218251對胺基酸序列之描述，該些文獻之全文倂於此處以供參考）以及其結合整合蛋白的片段；其中x係1、3、5、6或8。The present invention provides a pharmaceutical formula, which contains a fusion protein, the fusion protein includes a peptide that binds to integrin, other peptides that bind to proteins to target angiogenesis factors, and an Fc domain, wherein the peptide that binds to integrin The peptide is selected from the group consisting of: disintegrins (see U.S. Patent No. 7,943,728 and PCT Application No. PCT/US 15/46322 for descriptions of amino acid sequences, the full texts of which are hereby incorporated by reference) , anti-integrin αvβx antibody (see U.S. Patent Nos. 6,160,099 and 8,350,010 for descriptions of amino acid sequences, the full texts of which are hereby incorporated by reference), anti-integrin α5β1 antibody, targeted integration Fibronectin of the protein isoforms αvβx or α5β1 (see US Publication No. 2015/0218251 for a description of the amino acid sequence, the full text of which is incorporated herein by reference) and integrin-binding fragments thereof; where x Department 1, 3, 5, 6 or 8.

於本文中，術語「抗體」意指免疫球蛋白分子，其包含四條多胜肽鏈（二條重鏈及二條輕鏈，之間藉由雙硫鍵連結）。全長重鏈包括一個可變區結構域（variable region domain）VH以及三個恆定區結構域（constant region domain）CH1、CH2、及CH3。VH結構域係在多胜肽的胺基端，且CH3結構域係在羧基端。全長輕鏈包括一個可變區結構域VL、及一個恆定區結構域CL。抗原結合片段（antigen binding fragment，Fab）包含一條輕鏈、以及一條重鏈的CH1及可變區。Fab分子之重鏈不能與另一重鏈分子形成雙硫鍵。Fab’片段含有一輕鏈及一重鏈，該重鏈含有更多在CH1及CH2結構域之間的恆定區，致使二條重鏈之間可以形成鏈間雙硫鍵以形成雙體（diabody）。一可變片段（variable fragment，Fv）區係包含重鏈及輕鏈二者的可變區，但缺少恆定區。單鏈片段（single-chain fragment，scFv）係Fv分子，其中該重鏈及輕鏈可變區已藉由撓性連接子（flexible linker）連結以形成單多胜肽鏈（single polypeptide chain），其形成抗原結合區（antigen-binding region）。WO 88/01649、美國專利號4,946,778以及5,260,203中詳細討論單鏈抗體。於本文中，術語「抗體」係包括具有二條全長L-鏈及二條全長H-鏈的免疫球蛋白分子、及其片段（例如，抗原結合片段（Fab）、Fv區、scFv等）。As used herein, the term "antibody" refers to an immunoglobulin molecule that contains four polypeptide chains (two heavy chains and two light chains linked by disulfide bonds). The full-length heavy chain includes one variable region domain (VH) and three constant region domains (CH1, CH2, and CH3). The VH domain is located at the amino terminus of the polypeptide, and the CH3 domain is located at the carboxyl terminus. The full-length light chain includes a variable region domain VL, and a constant region domain CL. Antigen binding fragment (Fab) contains a light chain, and the CH1 and variable region of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The Fab' fragment contains a light chain and a heavy chain. The heavy chain contains more constant regions between the CH1 and CH2 domains, so that an interchain disulfide bond can be formed between the two heavy chains to form a diabody. A variable fragment (Fv) region contains the variable regions of both the heavy and light chains, but lacks the constant region. A single-chain fragment (scFv) is an Fv molecule in which the heavy chain and light chain variable regions have been linked by a flexible linker to form a single polypeptide chain. It forms the antigen-binding region. Single chain antibodies are discussed in detail in WO 88/01649, US Patent Nos. 4,946,778 and 5,260,203. As used herein, the term "antibody" includes immunoglobulin molecules having two full-length L-chains and two full-length H-chains, and fragments thereof (e.g., antigen-binding fragments (Fab), Fv regions, scFv, etc.).

術語「Fc結構域」意指單體或多聚體型式的分子或序列，其包含非抗原結合部分（non-antigen binding portion）的序列。Fc之原始免疫球蛋白來源較佳係源自人類，並可來自任意同功體，例如，IgG、IgA、IgM、IgE、或IgD。全長Fc由以下Ig重鏈區組成：介於CH1及CH2、CH2及CH3間的撓性鉸鏈區（flexible hinge region），其中該二鏈一般係藉由雙硫鍵於撓性鉸鏈區連結。The term "Fc domain" means a molecule or sequence in monomeric or multimeric form that contains a non-antigen binding portion of the sequence. The source of the original immunoglobulin for Fc is preferably of human origin and can be from any isoform, for example, IgG, IgA, IgM, IgE, or IgD. The full-length Fc consists of the following Ig heavy chain regions: a flexible hinge region between CH1 and CH2, CH2 and CH3, in which the two chains are generally connected by a disulfide bond in the flexible hinge region.

本發明提供融合蛋白，其係包含結合整合蛋白的胜肽、及其他結合蛋白的胜肽，其中該結合整合蛋白的胜肽包括去整合蛋白及其結合整合蛋白的片段；該其他結合蛋白的胜肽係包含VEGF受體之胞外結構域及Fc結構域；其中該結合整合蛋白的胜肽包含至少一在RGD模體上或相鄰處的突變。根據本發明之實施態樣，去整合蛋白及其結合整合蛋白的片段具有選自由以下所組成之群組的胺基酸序列：SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6及SEQ ID NO：7，或一與SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7具有至少85%序列同一性之胺基酸序列。The present invention provides a fusion protein, which includes a peptide that binds to integrin and a peptide that binds to other proteins, wherein the peptide that binds to integrin includes a disintegrin and a fragment that binds to integrin; the peptide of the other binding protein The peptide system includes the extracellular domain and the Fc domain of the VEGF receptor; wherein the integrin-binding peptide includes at least one mutation on or adjacent to the RGD motif. According to an embodiment of the present invention, the disintegrin and its integrin-binding fragment have an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 , SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, or together with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. The amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 having at least 85% sequence identity.

於本文中，「去整合蛋白」意指一類富含半胱胺酸的蛋白質或多胜肽，其係整合蛋白之有效的可溶性配體。RGD模體係三胜肽（tri-peptide）精胺酸- 甘胺酸- 天冬胺酸（Arg-Gly-Asp），其在大部分的單體去整合蛋白中係保守的，且位於整合蛋白結合環（integrin-binding loop）上。本文所述之去整合蛋白係分離自蛇毒、或衍生自野生型，並具有至少一在RGD模體上或相鄰處的突變，以選擇性地結合至或靶向多種整合蛋白同功體。於本文中，術語「RGD模體相鄰處」意謂在給定之胜肽、多胜肽、蛋白質序列中之任意突變係出現在任意自RGD模體算來第15-20個胺基酸內的胺基酸殘基。As used herein, "disintegrin" refers to a class of cysteine-rich proteins or polypeptides that are effective soluble ligands for integrins. The RGD template system tri-peptide arginine-glycine-aspartate (Arg-Gly-Asp) is conserved in most monomeric disintegrins and is located in integrins. On the integrin-binding loop. The disintegrins described herein are isolated from snake venom, or derived from wild type, and have at least one mutation on or adjacent to the RGD motif to selectively bind to or target multiple integrin isoforms. As used herein, the term "RGD motif adjacent" means that any mutation in a given peptide, polypeptide, or protein sequence occurs within any 15-20th amino acid sequence from the RGD motif. of amino acid residues.

在此亦考慮了去整合蛋白之其他胺基酸序列變異體。舉例而言，去整合蛋白的結合親合力及／或其他生物性質可藉由改變編碼該蛋白之胺基酸序列來改善。去整合蛋白突變體可藉由將適當的修飾引入編碼該蛋白的核酸序列中、或藉由胜肽合成引入修飾來製備。該等修飾包括突變，例如將該去整合蛋白之核酸或胺基酸序列進行刪除、***、及／或取代。去整合蛋白的最終胺基酸構建體（construct）可藉由進行刪除、***及取代之任意組合來達成，只要該最終構建體具有所需之特徵，例如結合至整合蛋白超家族成員及／或抑制整合蛋白活化之路徑。Other amino acid sequence variants of disintegrin proteins are also considered here. For example, the binding affinity and/or other biological properties of a disintegrin can be improved by altering the amino acid sequence encoding the protein. Disintegrin mutants can be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the protein, or by introducing modifications by peptide synthesis. Such modifications include mutations, such as deletion, insertion, and/or substitution of the nucleic acid or amino acid sequence of the disintegrating protein. The final amino acid construct that removes integrins can be achieved by making any combination of deletions, insertions, and substitutions, as long as the final construct has the desired characteristics, such as binding to integrin superfamily members and/or Inhibits the pathway of integrin activation.

對該蛋白質或多胜肽之生物性質的實質修飾係藉由選擇對維持以下各項有顯著不同效果的取代來實現：（a）取代區域之多胜肽骨架的構造，舉例而言，作為摺疊或螺旋構型、（b）分子在標靶位點的電荷或疏水性、或（c）支鏈之主體。Substantial modification of the biological properties of the protein or polypeptide is achieved by selecting substitutions that have significantly different effects on maintaining: (a) the structure of the polypeptide backbone in the substituted region, for example, as a fold or helical configuration, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the host of branches.

一種用於辨別融合蛋白之某些殘基或區之較佳突變作用位置的方法係稱為「丙胺酸掃描突變（alanine scanning mutagenesis）」，如Science, 1989, 244:1081-1085中所描述。舉例而言，將一殘基或一群標靶殘基辨別出來（例如，帶電荷之殘基，諸如，Arg、Asp、His、Lys、及Glu），並以中性（最佳係甘胺酸、丙胺酸或白胺酸）或相反帶電荷的胺基酸（從正電荷到負電荷，反之亦然）取代，以影響胺基酸與標靶結合伴侶之交互作用。接著，對取代呈現功能敏感性的該些胺基酸所在位置，藉由於該取代位點引入另一或其他變異體來改進。因此，雖然用於引入胺基酸序列變異的位點係預先決定的，但突變本身的性質不需要預先決定。舉例而言，為分析在給定位點之突變的性能，可在標靶密碼子或區實行隨機突變，並對表現出的融合多胜肽變異體進行所需活性篩選。舉例而言，可在融合蛋白或蛋白組分中添加半胱胺酸鍵以改善其安定性。One method used to identify preferred locations for mutations in certain residues or regions of a fusion protein is called alanine scanning mutagenesis, as described in Science, 1989, 244:1081-1085. For example, a residue or a group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and labeled as neutral (preferably glycine , alanine or leucine) or oppositely charged amino acids (from positive charge to negative charge or vice versa) to influence the interaction of the amino acid with the target binding partner. Next, the positions of those amino acids that exhibit functional sensitivity to substitution are improved by introducing another or other variants at that substitution site. Thus, while the site used to introduce amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of mutations at a given site, random mutations can be performed at target codons or regions, and fusion polypeptide variants that exhibit the desired activity can be screened. For example, cysteine bonds can be added to the fusion protein or protein components to improve its stability.

因此，本文提供之去整合蛋白突變體，可為本文所揭露之任意融合蛋白的組分。在一些實施態樣中，該去整合蛋白係包含與選自由以下所組成之群組的去整合蛋白胺基酸序列具有至少85%、至少90%、至少95%、或至少99%之序列同一性的胺基酸序列：馬來亞蝮素（Rhodostomin）（SEQ ID NO：1）、黃綠竹葉青素（Triflavin）（SEQ ID NO：3）、鉅鱗蝰素（Echistatin）（SEQ ID NO：4）、台灣龜殼花素（Trimucrin）（SEQ ID NO：5）、麗紋龜殼花蛇素（Elegantin）（SEQ ID NO：6）及赤尾鮐素（Trigramin）（SEQ ID NO：7）。在一些實施態樣中，去整合蛋白係包含一胺基酸序列，該胺基酸序列具有至少一在馬來亞蝮素（SEQ ID NO：1）、黃綠竹葉青素（SEQ ID NO：3）、鉅鱗蝰素（SEQ ID NO：4）、台灣龜殼花素（SEQ ID NO：5）、麗紋龜殼花蛇素（SEQ ID NO：6）或赤尾鮐素（SEQ ID NO：7）之RGD模體上或相鄰處的突變。在一些實施態樣中，去整合蛋白係包含與去整合蛋白突變體之胺基酸序列（SEQ ID NO：2）具有至少85%、至少90%、至少95%、或至少99%之序列同一性的胺基酸序列。在SEQ ID NO：2中的Xaa指出多個位置，該些位置可藉由***、取代或刪除來進行修飾，以生產與野生型去整合蛋白不同的胺基酸序列變異體。根據一些實施例，位於SEQ ID NO：2位置50之Xaa，其對應到野生型馬來亞蝮素（SEQ ID NO：1）之RGD模體中的甘胺酸（Gly），可用甘胺酸之外的天然產生胺基酸取代，以產生馬來亞蝮素變異體。在其他實施例中，SEQ ID NO：2中之一或多個Xaa亦可用原來在野生型馬來亞蝮素（SEQ ID NO：1）之對應位置上找到的胺基酸之外的天然產生胺基酸取代，以產生多種馬來亞蝮素變異體。還需說明的是，去整合蛋白變異體並不限於只包括SEQ ID NO：2中任意Xaa之單一突變，本發明之範圍亦包含在SEQ ID NO：2中Xaa的數個位置或在其他具一致序列的去整合蛋白（例如，SEQ ID NOs：3-7）中的對應位置產生多突變。Therefore, the deintegrin mutants provided herein can be components of any fusion protein disclosed herein. In some embodiments, the disintegrin comprises at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with a disintegrin amino acid sequence selected from the group consisting of: The specific amino acid sequence: Rhodostomin (SEQ ID NO: 1), Triflavin (SEQ ID NO: 3), Echistatin (SEQ ID NO: 4), Trimucrin (SEQ ID NO: 5), Elegantin (SEQ ID NO: 6) and Trigramin (SEQ ID NO: 7) . In some embodiments, the disintegrin protein system comprises an amino acid sequence, the amino acid sequence has at least one amino acid sequence in the malein (SEQ ID NO: 1), yellow green bamboo leaf cyanin (SEQ ID NO: 3 ), giant lepidopterin (SEQ ID NO: 4), Taiwanese leucophyllin (SEQ ID NO: 5), leucophyllin (SEQ ID NO: 6) or red tail leucophyllin (SEQ ID NO: 7) Mutation on or adjacent to the RGD motif. In some embodiments, the disintegrin protein comprises at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the amino acid sequence of the disintegrin mutant (SEQ ID NO: 2). sexual amino acid sequence. Xaa in SEQ ID NO: 2 indicates multiple positions that can be modified by insertion, substitution, or deletion to produce amino acid sequence variants that are different from the wild-type disintegrin. According to some embodiments, Xaa located at position 50 of SEQ ID NO: 2, which corresponds to glycine (Gly) in the RGD motif of wild-type malasin (SEQ ID NO: 1), can be used with glycine Amino acid substitutions other than naturally occurring amino acids were made to create malasin variants. In other embodiments, one or more Xaa's in SEQ ID NO: 2 can also be used with naturally occurring amino acids other than the amino acids originally found at the corresponding positions of wild-type malasin (SEQ ID NO: 1). Amino acid substitutions were made to produce multiple malayanin variants. It should also be noted that the deintegrin variant is not limited to include only a single mutation of any Xaa in SEQ ID NO: 2. The scope of the present invention also includes several positions of Xaa in SEQ ID NO: 2 or other specific mutations. Multiple mutations were generated at corresponding positions in the consensus sequence of the disintegrin (e.g., SEQ ID NOs: 3-7).

馬來亞蝮素變異體已被描述於美國專利號7,943,728及PCT申請號PCT/US15/46322，且其序列係併於此處以供參考。舉例言之，PCT/US15/46322係描述該去整合蛋白變異體包含一突變RGD環（具有選自由SEQ ID NO: 24至SEQ ID NO: 26所組成之群組的胺基酸序列）、以及至少一種突變連接子（具有選自由SEQ ID NO: 29至SEQ ID NO: 41所組成之群組的胺基酸序列）、以及一突變C末端（具有選自由SEQ ID NO: 42至SEQ ID NO: 47所組成之群組的胺基酸序列）。更佳地，該去整合蛋白變異體包含如本文中所描述之該突變RGD環、該突變連接子及該突變C末端。Malayan pyrin variants have been described in U.S. Patent No. 7,943,728 and PCT Application No. PCT/US15/46322, and their sequences are incorporated herein by reference. For example, PCT/US15/46322 describes that the disintegrin variant includes a mutated RGD loop (having an amino acid sequence selected from the group consisting of SEQ ID NO: 24 to SEQ ID NO: 26), and At least one mutated linker (having an amino acid sequence selected from the group consisting of SEQ ID NO: 29 to SEQ ID NO: 41), and a mutated C-terminus (having an amino acid sequence selected from the group consisting of SEQ ID NO: 42 to SEQ ID NO : Amino acid sequence of a group consisting of 47). More preferably, the disintegrin variant comprises the mutated RGD loop, the mutated linker and the mutated C-terminus as described herein.

具有一或多個除了RGD模體外（例如在連接子區域或C末端）之修飾的馬來亞蝮素變異體或去整合蛋白變異體展現結合至αvβ3、αvβ5、αvβ6、α5β1或αIIbβ3的能力與選擇性。舉例言之，具有連接子區域突變（39X40X41X42X43X）之馬來亞蝮素變異體，其中該SRAGK（SEQ ID NO: 50）係透過置換胺基酸KKKRT（SEQ ID NO: 51）、KKART（SEQ ID NO: 52）、MKKGT（SEQ ID NO: 53）、IEEGT（SEQ ID NO: 54）、LKEGT（SEQ ID NO: 55）、AKKRT（SEQ ID NO: 56）、KAKRT（SEQ ID NO: 57）、KKART（SEQ ID NO: 58）、KKKAT（SEQ ID NO: 59）、KKKRA（SEQ ID NO: 60）、KAKRA（SEQ ID NO: 61）、或SKAGT（SEQ ID NO: 62），且其對於整合蛋白的最大效果依序如下：αvβ3（約14倍）＞α5β1（約5倍）＞αIIbβ3（約2倍）。Malayanin variants or disintegrin variants with one or more modifications other than the RGD motif (e.g., in the linker region or the C-terminus) exhibit the ability to bind to αvβ3, αvβ5, αvβ6, α5β1, or αIIbβ3 and Selectivity. For example, a malayanin variant with a linker region mutation (39X40X41X42X43X), in which the SRAGK (SEQ ID NO: 50) is replaced by the amino acids KKKRT (SEQ ID NO: 51), KKART (SEQ ID NO: 52), MKKGT (SEQ ID NO: 53), IEEGT (SEQ ID NO: 54), LKEGT (SEQ ID NO: 55), AKKRT (SEQ ID NO: 56), KAKRT (SEQ ID NO: 57), KKART (SEQ ID NO: 58), KKKAT (SEQ ID NO: 59), KKKRA (SEQ ID NO: 60), KAKRA (SEQ ID NO: 61), or SKAGT (SEQ ID NO: 62), and for integration The maximum effects of the proteins are as follows: αvβ3 (approximately 14 times) > α5β1 (approximately 5 times) > αIIbβ3 (approximately 2 times).

具有C末端區域突變（66X67X68X69X70X）之馬來亞蝮素變異體，其中該RYH係透過置換胺基酸RYH（SEQ ID NO: 63）、RNGL（SEQ ID NO: 64）、RGLYG（SEQ ID NO: 65）、RGLY（SEQ ID NO: 66）、RDLYG（SEQ ID NO: 67）、RDLY（SEQ ID NO: 68）、RNGLYG（SEQ ID NO: 69）、或RNPWNG（SEQ ID NO: 70），且其對於整合蛋白的最大效果依序如下：αIIbβ3（約13倍）＞αvβ5（約8倍）＝αvβ6（約8倍）＞αvβ3（約4倍）＞α5β1（約2倍）。表1係顯示SEQ ID NOs: 24至49的序列，以及其在SEQ ID NO: 1上的對應位置。 表 1 SEQ ID NO 序列 在 SEQ ID NO: 1 上的位置 24 RIARGDNP 46-53 25 RRARGDNP 26 ARGRGDNP 27 ARGRGDDL 28 ARARGDNP 29 KKKRTIC 39-45 30 MKKGTIC 31 IEEGTIC 32 KGAGKIC 33 LKEGTIC 34 AKKRTIC 35 KAKRTIC 36 KKARTIC 37 KKKATIC 38 KKKRAIC 39 KAKRAIC 40 SKAGTIC 41 KKKRTIC 42 PRWNDL 65-68 43 PRNGLYG 44 PGLYG 45 PDLYG 46 PPLYG 47 PRLYG 48 PELYG 49 PYLYG Malayan pyrin variant with C-terminal region mutation (66X67X68X69X70X), in which the RYH is obtained by replacing the amino acids RYH (SEQ ID NO: 63), RNGL (SEQ ID NO: 64), RGLYG (SEQ ID NO: 65), RGLY (SEQ ID NO: 66), RDLYG (SEQ ID NO: 67), RDLY (SEQ ID NO: 68), RNGLYG (SEQ ID NO: 69), or RNPWNG (SEQ ID NO: 70), and Its maximum effect on integrins is as follows: αIIbβ3 (approximately 13 times) > αvβ5 (approximately 8 times) = αvβ6 (approximately 8 times) > αvβ3 (approximately 4 times) > α5β1 (approximately 2 times). Table 1 shows the sequences of SEQ ID NOs: 24 to 49, and their corresponding positions on SEQ ID NO: 1. Table 1 SEQ ID NO sequence Position on SEQ ID NO: 1 twenty four RIARGDNP 46-53 25 RRARGDNP 26 ARGRGDNP 27 ARGRGDDL 28 ARARGDNP 29 KKKRTIC 39-45 30 MKKGTIC 31 IEEGTIC 32 KGAGKIC 33 LKEGTIC 34 AKKRTIC 35 KAKRTIC 36 KKARTIC 37 KKKATIC 38 KKKRAIC 39 KAKRAIC 40 SKAGTIC 41 KKKRTIC 42 PRWNDL 65-68 43 PRNGLYG 44 PGLYG 45 PDLYG 46 PPLYG 47 PRLYG 48 PELYG 49 PYLYG

雖然去整合蛋白之變異體大多參考上述胺基酸序列來討論，編碼蛇毒蛋白之多胜肽序列或核苷酸序列、及其具有至少一RGD模體上或相鄰處之突變的變異體亦可被包含在本發明之範圍中，其中該蛇毒蛋白係例如白唇竹葉青蛇素（Albolabrin）、百步蛇素（Applagin）、墨西哥西海岸響尾蛇素（Basilicin）、矛頭蝮素（Batroxostatin）、鼓腹巨蛇素（Bitistatin）、西部亞種響尾蛇素（Cereberin）、角響尾蛇素（Cerastin）、西部菱背響尾蛇素（Crotatroxin）、南美響尾蛇素（Duressin）、黃綠龜殼花蛇素（Flavoridin）、黃綠龜殼花蛇毒解離素（Flavostatin）、日本蝮蛇素（Halysin）、西伯利亞蝮蛇毒解離素（Halystatin）、巴西蝮素（Jararacin）、巴西蝮蛇毒解離素（Jarastatin）、馬來亞紅口蝮素（Kistrin）、亞馬遜巨蝮素（Lachesin）、西部亞種響尾蛇素（Lutosin）、黑尾響尾蛇素（Molossin）、韓國亞種短尾蝮素（Salmosin）、白眉蝮素（Saxatilin）、北美侏儒響尾蛇素（Tergeminin）、黃綠龜殼花蛇毒解離素（Trimestatin）、台灣龜殼花去整合蛋白（Trimutase）、烏蘇里蝮素（Ussuristatin）、及草原響尾蛇素（Viridin）。Although variants of disintegrin proteins are mostly discussed with reference to the above amino acid sequences, polypeptide sequences or nucleotide sequences encoding snake venom proteins, and variants having at least one mutation on or adjacent to the RGD motif are also discussed. Can be included in the scope of the present invention, wherein the snake venom protein is, for example, Albolabrin, Applagin, Basilicin, Batroxostatin, Batroxostatin, Bitistatin, Cereberin, Cerastin, Crotatroxin, Duressin, Flavoridin, Yellow Flavostatin, Japanese Halysin, Siberian Halystatin, Jararacin, Brazilian Jarastatin, Malayan Red-mouthed Viper Kistrin, Lachesin, Lutosin, Molossin, Salmosin, Saxatilin, Saxatilin, North American pygmy Tergeminin, Trimestatin, Trimutase, Ussuristatin, and Viridin.

不受理論所限制，本文在此考慮了去整合蛋白藉由結合至整合蛋白超家族成員，以阻斷其與多價整合蛋白受體交互作用，來抑制整合蛋白活化之路徑。在一些方面，去整合蛋白結合至整合蛋白超家族成員，包括但不限於整合蛋白同功體：αvβ1、αvβ3、αvβ5、αvβ6、αvβ8、α5β1、及／或αvIIbβ3。Without being bound by theory, this article considers the pathway by which disintegrins inhibit integrin activation by binding to integrin superfamily members to block their interaction with multivalent integrin receptors. In some aspects, disintegrins bind to integrin superfamily members, including, but not limited to, integrin isoforms: αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, and/or αvIIbβ3.

根據本發明，融合蛋白之其他結合蛋白的胜肽可為受體蛋白，該受體蛋白結合至選自由以下所組成之群組的標靶：腫瘤抗原、腫瘤壞死因子（TNF）受體超家族成員、刺蝟因子（Hedgehog）家族成員、受體酪胺酸激酶、蛋白多醣相關之分子、腫瘤生長因子-β（TGF-β）超家族成員、Wnt相關之分子及血管新生標靶。According to the present invention, the peptide of the other binding protein of the fusion protein can be a receptor protein that binds to a target selected from the group consisting of: tumor antigens, tumor necrosis factor (TNF) receptor superfamily Members, Hedgehog family members, receptor tyrosine kinases, proteoglycan-related molecules, tumor growth factor-β (TGF-β) superfamily members, Wnt-related molecules and angiogenesis targets.

根據本發明之一些實施態樣，其他結合蛋白的胜肽可專一性地結合至血管新生標靶，其包括但不限於血管生成素（ANG）、酪胺酸蛋白激酶（Eph）、成纖維母細胞生長因子（FGF）、神經纖毛蛋白（NRP）、血纖維蛋白溶酶原活化物、血小板衍生生長因子（PDGF）、轉變生長因子-β（TGF-β）、血管內皮生長因子（VEGF）、血管內皮鈣黏蛋白（VE-cadherin）、腫瘤壞死因子-α（TNF-α）、類胰島素生長因子（IGF-1）及前述的受體。因此，根據本發明實施態樣，其他結合蛋白的胜肽可包括受體蛋白之胞外部分，其結合至並拮抗該血管新生標靶。在其他實施態樣中，其他結合蛋白的胜肽可結合至血管新生因子受體之胞外部分。According to some embodiments of the present invention, other protein-binding peptides can specifically bind to angiogenesis targets, including but not limited to angiopoietin (ANG), tyrosine protein kinase (Eph), fibroblast Cell growth factor (FGF), neuropilin (NRP), plasminogen activator, platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), Vascular endothelial cadherin (VE-cadherin), tumor necrosis factor-α (TNF-α), insulin-like growth factor (IGF-1) and the aforementioned receptors. Therefore, according to embodiments of the present invention, other protein-binding peptides may include extracellular portions of receptor proteins that bind to and antagonize the angiogenesis target. In other embodiments, other binding protein peptides can bind to the extracellular portion of the angiogenic factor receptor.

在一些實施態樣中，其他結合蛋白的胜肽可係結合至VEGF配體的抗VEGF抗體（參見WO2015/200905對胺基酸序列之描述，該文獻之全文倂於此處以供參考）、或結合至VEGF受體的抗VEGFR1或抗VEGFR2抗體（參見美國專利號5,874,542對胺基酸序列之描述，該文獻之全文倂於此處以供參考）。在其他實施態樣中，其他結合蛋白的胜肽亦可係結合至PDGF配體的抗PDGF抗體（參見美國專利號5,094,941對胺基酸序列之描述，該文獻之全文倂於此處以供參考）、或結合至PDGF受體的抗PDGFRβ抗體（參見美國專利號9,265,827對胺基酸序列之描述，該文獻之全文倂於此處以供參考）。In some embodiments, the other protein-binding peptide may be an anti-VEGF antibody that binds to a VEGF ligand (see WO2015/200905 for a description of the amino acid sequence, the full text of which is incorporated herein by reference), or Anti-VEGFR1 or anti-VEGFR2 antibodies that bind to the VEGF receptor (see US Pat. No. 5,874,542 for a description of the amino acid sequence, the entire text of which is incorporated herein by reference). In other embodiments, other protein-binding peptides may also be anti-PDGF antibodies that bind to PDGF ligands (see U.S. Pat. No. 5,094,941 for a description of the amino acid sequence, the full text of which is incorporated herein by reference) , or an anti-PDGFRβ antibody that binds to the PDGF receptor (see U.S. Patent No. 9,265,827 for a description of the amino acid sequence, the entire text of which is incorporated herein by reference).

在某些實施態樣中，其他結合蛋白的胜肽作為以下VEGF受體（VEGFR）之任意一者結合至相同的VEGF：VEGFR1、VEGFR2、及VEGFR3。在一些實施態樣中，其他結合蛋白的胜肽係包含VEGFR之至少一胞外部分，該VEGFR係本文所述之任意VEGFR。舉例而言，其他結合蛋白的胜肽係包含VEGFR1之至少一胞外部分、或VEGFR2之至少一胞外部分。在另一實施例中，其他結合蛋白的胜肽係包含VEGFR1之一胞外部分，例如類免疫球蛋白結構域2（D2）、及VEGFR2之一胞外部分，例如類免疫球蛋白結構域3（D3）。在一些方面，其他結合蛋白的胜肽係包含VEGFR1之一胞外部分（其包含SEQ ID NO：8之胺基酸序列）、及VEGFR2之一胞外部分（其包含SEQ ID NO：9之胺基酸序列）。在一些方面，其他結合蛋白的胜肽係包含VEGFR1及VEGFR2之胞外部分的融合體，其包含SEQ ID NO：10之胺基酸序列、或與SEQ ID NO：10具有至少85%序列同一性之胺基酸序列。In certain embodiments, peptides of other binding proteins bind to the same VEGF as any of the following VEGF receptors (VEGFR): VEGFRl, VEGFR2, and VEGFR3. In some embodiments, the peptide of the other binding protein includes at least an extracellular portion of a VEGFR, which is any VEGFR described herein. For example, peptides of other binding proteins include at least one extracellular portion of VEGFR1, or at least one extracellular portion of VEGFR2. In another embodiment, the peptides of other binding proteins include an extracellular portion of VEGFR1, such as immunoglobulin-like domain 2 (D2), and an extracellular portion of VEGFR2, such as immunoglobulin-like domain 3. (D3). In some aspects, the peptides of other binding proteins include an extracellular portion of VEGFR1 that includes the amino acid sequence of SEQ ID NO: 8, and an extracellular portion of VEGFR2 that includes the amine of SEQ ID NO: 9 amino acid sequence). In some aspects, the peptides of other binding proteins comprise a fusion of the extracellular portions of VEGFR1 and VEGFR2, which comprise the amino acid sequence of SEQ ID NO: 10, or have at least 85% sequence identity with SEQ ID NO: 10 The amino acid sequence.

在其他實施態樣中，其他結合蛋白的胜肽作為以下PDGF受體（PDGFR）之任一者結合至相同的PDGF：PDGFR-α、及PDGFR-β。在一些實施態樣中，其他結合蛋白的胜肽係包含PDGFR之至少一胞外部分，該PDGFR係本文所述之任意PDGFR。舉例而言，其他結合蛋白的胜肽係包含PDGFR-α之至少一胞外部分、或PDGFR-β之至少一胞外部分。在另一實施例中，其他結合蛋白的胜肽係包含PDGFR-β之一胞外部分，例如類免疫球蛋白結構域1至3。在一些態樣，其他結合蛋白的胜肽係包含PDGFR之胞外部分，其包含SEQ ID NO：11之胺基酸序列、或與SEQ ID NO：11具有至少85%序列同一性之胺基酸序列。In other embodiments, peptides of other binding proteins bind to the same PDGF as any of the following PDGF receptors (PDGFR): PDGFR-alpha, and PDGFR-beta. In some embodiments, the peptide of the other binding protein includes at least an extracellular portion of a PDGFR, which is any PDGFR described herein. For example, other binding protein peptides include at least one extracellular portion of PDGFR-α, or at least one extracellular portion of PDGFR-β. In another embodiment, other binding protein peptides comprise an extracellular portion of PDGFR-β, such as immunoglobulin-like domains 1 to 3. In some aspects, the peptide of the other binding protein includes an extracellular portion of PDGFR that includes the amino acid sequence of SEQ ID NO: 11, or an amino acid with at least 85% sequence identity to SEQ ID NO: 11 sequence.

根據其他實施態樣，本發明亦提供融合蛋白，其係包含結合整合蛋白之胜肽、人類或人源化恆定亞區、及其他結合蛋白的胜肽，其中該結合整合蛋白之胜肽係包括在RGD模體上或相鄰處具有至少一突變之SEQ ID NO：1的胺基酸序列、與SEQ ID NO：1具有至少85%序列同一性之胺基酸序列、或SEQ ID NO：2之胺基酸序列；該人類或人源化恆定亞區係包含免疫球蛋白CH2結構域及免疫球蛋白CH3結構域；該其他結合蛋白的胜肽具有VEGFR1之類免疫球蛋白D2及VEGFR2之類免疫球蛋白D3。在融合蛋白的另一實施態樣中，結合整合蛋白的胜肽具有與SEQ ID NO：2至少85%之序列同一性。According to other embodiments, the present invention also provides a fusion protein that includes an integrin-binding peptide, a human or humanized constant subregion, and other protein-binding peptides, wherein the integrin-binding peptide includes The amino acid sequence of SEQ ID NO: 1 having at least one mutation on or adjacent to the RGD motif, an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1, or SEQ ID NO: 2 The amino acid sequence; the human or humanized constant subregion includes an immunoglobulin CH2 domain and an immunoglobulin CH3 domain; the peptides of the other binding proteins have immunoglobulin D2 and VEGFR2 such as VEGFR1 Immunoglobulin D3. In another embodiment of the fusion protein, the integrin-binding peptide has at least 85% sequence identity with SEQ ID NO: 2.

就多胜肽或核酸序列而言，術語「序列同一性百分比（%）」係定義為：在比對序列並在需要時引入間隙（gap）以達到最大序列同一性百分比之後，在保守性置換（conservative substitution）不被視為序列同一性的一部分之情況下，候選序列中與參照的多胜肽或核酸序列中相同的胺基酸殘基或核苷酸的百分比。為了測定胺基酸或核酸序列同一性百分比所進行的比對，可用多種本技術領域中之方式達成，例如，使用公開之電腦軟體程式，舉例而言，如該等描述於Current Protocols in Molecular Biology（Ausubel等人，eds., 1987）中的電腦軟體程式，以及包括BLAST、BLAST-2、ALIGN、或Megalign（DNASTAR）軟體。熟習此項技術領域者可選定用於測量比對之適當參數，包括對欲比較序列之全長達到最大化比對所需的演算法。就本文之目的而言，給定胺基酸序列A對比給定胺基酸序列B之胺基酸序列同一性百分比，係如下計算：100乘以分數X／Y，其中X係藉由序列比對程式在程式比對A及B時，評分為相同的胺基酸殘基的數目，且其中Y係B中胺基酸殘基的總數。應了解，當胺基酸序列A之長度與胺基酸序列B之長度不相等時，A對B的胺基酸序列同一性百分比不會與B對A的胺基酸序列同一性百分比相等。With respect to polypeptide or nucleic acid sequences, the term "percent sequence identity (%)" is defined as: conservative substitutions after aligning the sequences and introducing gaps where necessary to achieve the maximum percent sequence identity. (conservative substitution) The percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the reference polypeptide or nucleic acid sequence without being considered part of the sequence identity. Alignment for determining percent amino acid or nucleic acid sequence identity can be accomplished in a variety of ways in the art, for example, using publicly available computer software programs, for example, as described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), and include BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can select appropriate parameters for measuring alignment, including algorithms required to maximize alignment over the full length of the sequences to be compared. For the purposes of this article, the percent amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B is calculated as follows: 100 multiplied by the fraction X/Y, where When the program compares A and B, the score is the number of identical amino acid residues, and Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not be equal to the percent amino acid sequence identity of B to A.

本發明提供二聚體融合蛋白，其係包含二融合蛋白，其中各融合蛋白包含本文所揭露的任意融合蛋白。在一實施態樣中，二聚體融合蛋白係包含二個完全相同的融合蛋白。在另一實施態樣中，二聚體融合蛋白可包含二個不同的融合蛋白。本文所揭露之融合蛋白可透過多聚化結構域形成二或更多個完全相同之融合蛋白多聚體或異種（heterologous）融合蛋白，該結構域包括人類或人源化抗體之恆定亞區。在一些實施態樣中，人類或人源化抗體之恆定亞區係選自由以下所組成之群組：IgG Fc區、IgA Fc區、IgM Fc區、IgD Fc區、及IgE Fc區。在另一實施態樣中，人類或人源化抗體之恆定亞區係選自由以下所組成之群組：IgG1 Fc區、IgG2 Fc區、IgG3 Fc區、及IgG4 Fc區。在一些態樣，亞區包含IgG1、IgG2、IgG3、或IgG4之CH2區及CH3區。編碼包含Fc區之免疫球蛋白的胺基酸序列，係為本技術領域所熟知。The present invention provides dimeric fusion proteins, which comprise two fusion proteins, wherein each fusion protein comprises any fusion protein disclosed herein. In one embodiment, the dimeric fusion protein contains two identical fusion proteins. In another embodiment, a dimeric fusion protein may comprise two different fusion proteins. The fusion proteins disclosed herein can form two or more identical fusion protein multimers or heterologous fusion proteins through a multimerization domain that includes the constant subregion of a human or humanized antibody. In some embodiments, the constant subregion of a human or humanized antibody is selected from the group consisting of: IgG Fc region, IgA Fc region, IgM Fc region, IgD Fc region, and IgE Fc region. In another embodiment, the constant subregion of the human or humanized antibody is selected from the group consisting of: IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, and IgG4 Fc region. In some aspects, the subregions include the CH2 and CH3 regions of IgGl, IgG2, IgG3, or IgG4. Amino acid sequences encoding immunoglobulins containing Fc regions are well known in the art.

融合蛋白的組分可互相直接連結，或透過連接子連結。一般而言，術語「連接子」意為一或多個分子（例如，核酸、胺基酸、或非胜肽部分（non-peptide moiety）），其可***於一或多個組分結構域之間。舉例而言，連接子可用於在組分之間提供所需之受關注位點，以便於操作。連接子亦可提供來使宿主細胞提升融合蛋白表現量、減少立體阻礙（steric hindrance）致使該組分可取得其最適的三級結構、及／或適當地與其標靶分子交互作用。連接子序列可包括一或多個天然連結至受體組分的胺基酸、或可係添加來用於提升融合蛋白表現量之序列，以提供特別所需之受關注位點、以使組分結構域形成最適的三級結構、及／或以提升組分與其標靶分子的交互作用。The components of the fusion protein can be linked to each other directly or through a linker. Generally speaking, the term "linker" means one or more molecules (e.g., nucleic acids, amino acids, or non-peptide moieties) that can be inserted into one or more component domains. between. For example, linkers can be used to provide desired sites of interest between components for ease of manipulation. Linkers can also be provided to enable the host cell to increase the expression of the fusion protein, reduce steric hindrance so that the component can obtain its optimal tertiary structure, and/or interact appropriately with its target molecule. The linker sequence may include one or more amino acids that are naturally linked to the receptor component, or may be a sequence added to enhance the expression of the fusion protein to provide specifically desired sites of interest to allow the assembly of The sub-domains form the optimal tertiary structure and/or enhance the interaction between the components and their target molecules.

較佳的情況為連接子增加融合蛋白組分之撓性，而不干擾在該融合蛋白中之各功能性組分的結構。在一些實施態樣中，連接子部分係長度為2至100個胺基酸之胜肽連接子。例示性連接子包括具有至少二個胺基酸殘基之線性胜肽，例如：Gly-Gly、Gly-Ala-Gly、Gly-Pro-Ala、Gly(G)n、及Gly-Ser（GS）連接子。本文所述之GS連接子包括但不限於(GS)n、(GSGSG)n、(G 2S)n、G 2S 2G、(G 2SG)n、(G 3S)n、(G 4S)n、(GGSGG)nGn、及GSG 4SG 4SG，其中n係1或1以上。(G)n連接子的一個例子包括G 9連接子。適合的線性胜肽包括聚甘胺酸（polyglycine）、聚絲胺酸（polyserine）、聚脯胺酸（polyproline）、聚丙胺酸（polyanaline）、及由丙胺醯基（alanyl）及／或絲胺醯基（serinyl）及／或脯胺醯基（prolinyl）及／或甘胺醯基（glycyl）之胺基酸殘基所組成的寡胜肽（oligopepetide）。連接子部分可用來連接本文所揭露之融合蛋白的任意組分。在一些實施態樣中，連接子係用在受體蛋白之胞外部分、及免疫球蛋白之恆定亞區之間。在其他實施態樣中，連接子係用在去整合蛋白或其變異體、及免疫球蛋白之恆定亞區之間。在某些實施態樣中，融合蛋白包含一位在受體蛋白之胞外部分、及去整合蛋白或其變異體之間的連接子；及一位在去整合蛋白或其變異體、及免疫球蛋白之恆定亞區之間的連接子。如本發明所體現，融合蛋白可包含至少一條但不多於四條之連接子。 Preferably, the linker increases the flexibility of the fusion protein components without disturbing the structure of the functional components in the fusion protein. In some embodiments, the linker portion is a peptide linker ranging from 2 to 100 amino acids in length. Exemplary linkers include linear peptides with at least two amino acid residues, such as: Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, Gly(G)n, and Gly-Ser(GS) connector. GS linkers described herein include, but are not limited to, (GS)n, (GSGSG)n, (G 2 S)n, G 2 S 2 G, (G 2 SG)n, (G 3 S)n, (G 4 S)n, (GGSGG)nGn, and GSG 4 SG 4 SG, where n is 1 or more. An example of a (G)n linker includes the G9 linker. Suitable linear peptides include polyglycine, polyserine, polyproline, polyanaline, and those composed of alanyl and/or serine Oligopepetide composed of amino acid residues of serinyl and/or prolinyl and/or glycyl groups. The linker moiety can be used to connect any of the components of the fusion proteins disclosed herein. In some embodiments, a linker is used between the extracellular portion of the receptor protein and the constant subregion of the immunoglobulin. In other embodiments, a linker is used between a disintegrin, or a variant thereof, and a constant subregion of an immunoglobulin. In some embodiments, the fusion protein includes a linker between the extracellular portion of the receptor protein and the disintegrin or a variant thereof; and a linker between the disintegrin or a variant thereof, and the immune The linker between the constant subregions of globulin. As embodied in the present invention, the fusion protein may comprise at least one but no more than four linkers.

本文所述之融合蛋白可包含或不包含使融合蛋白自宿主細胞分泌出來的訊息胜肽。編碼該訊息胜肽的核酸序列，能夠可操作地連接至編碼感興趣之蛋白的核酸序列。在一些實施態樣中，該融合蛋白包含訊息胜肽。在一些實施態樣中，融合蛋白並不包含訊息胜肽。The fusion proteins described herein may or may not contain a messaging peptide that causes the fusion protein to be secreted from the host cell. The nucleic acid sequence encoding the message peptide can be operably linked to the nucleic acid sequence encoding the protein of interest. In some embodiments, the fusion protein includes a message peptide. In some embodiments, the fusion protein does not include a message peptide.

此外，本發明所述之融合蛋白可包含結合蛋白的胜肽之經修飾型態。舉例而言，融合蛋白組分可具有轉譯後修飾，包括例如對任意結合蛋白的胜肽進行醣化、唾液酸化、乙醯化、及磷酸化。In addition, the fusion proteins of the present invention may comprise modified versions of peptides that bind the protein. For example, the fusion protein components may have post-translational modifications including, for example, glycation, sialylation, acetylation, and phosphorylation of any peptides that bind the protein.

雖然，實施態樣已大致描述包含於融合蛋白中的二條結合蛋白的胜肽，本發明亦包含併入二條以上結合蛋白的胜肽之融合蛋白，以在抑制血管新生過程之方面提供任何加成的或協同的效果。舉例而言，可有一額外的結合蛋白的胜肽，其結合至其他血管新生標靶、或作為血管新生因子拮抗劑而連接至現存之二條結合蛋白的胜肽。Although the embodiments have been generally described as containing peptides of two binding proteins in a fusion protein, the present invention also encompasses fusion proteins incorporating peptides of more than two binding proteins to provide any additive in inhibiting the angiogenesis process. or synergistic effect. For example, there can be an additional protein-binding peptide that binds to another angiogenesis target, or a peptide that acts as an angiogenesis factor antagonist and is linked to two existing protein-binding peptides.

用於本文中所揭露之醫藥配方的融合蛋白可使用一般所知的方法來純化及辨識，例如在免疫親合或離子交換管柱上進行分餾；乙醇沉澱法；反相高壓液相層析（HPLC）；在矽膠上或陽離子交換樹脂（例如，DEAE）上進行色層分析法；色層交集法（chromatofocusing）；SDS-PAGE；硫酸銨沉澱法；膠體過濾法（例如，葡萄糖凝膠G-75（Sephadex G-75））；疏水親合力樹脂，使用固化在基質上之適合之結合伴侶的配體親合力、離心、酵素免疫檢定法（Enzyme-Linked Immunosorbent Assay，ELISA）、BIACore、西方墨點法、胺基酸及核酸定序、及生物活性。在一些實施態樣中，融合蛋白係在宿主細胞內表現，並使用一種或多種標準純化技術之組合自其中純化，該等技術包括但不限於蛋白A親合力層析、蛋白G親合力層析、緩衝液置換、粒徑排阻層析、超過濾、及透析。因此，回收之融合蛋白實質上是純的。在另一實施態樣中，回收之融合蛋白係以下之任一者：純度至少為90%、95%、96%、97%、98%、或99%。Fusion proteins used in the pharmaceutical formulations disclosed herein can be purified and identified using commonly known methods, such as fractionation on immunoaffinity or ion exchange columns; ethanol precipitation; reversed-phase high-pressure liquid chromatography ( HPLC); chromatography on silica or cation exchange resin (e.g., DEAE); chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; colloidal filtration (e.g., glucose gel G- 75 (Sephadex G-75)); hydrophobic affinity resin, using ligand affinity of a suitable binding partner solidified on the matrix, centrifugation, enzyme immunoassay (Enzyme-Linked Immunosorbent Assay, ELISA), BIACore, Western Ink Spot method, amino acid and nucleic acid sequencing, and biological activity. In some embodiments, the fusion protein is expressed within a host cell and purified therefrom using one or a combination of standard purification techniques, including, but not limited to, Protein A affinity chromatography, Protein G affinity chromatography. , buffer exchange, particle size exclusion chromatography, ultrafiltration, and dialysis. Therefore, the recovered fusion protein is essentially pure. In another embodiment, the recovered fusion protein is any of the following: at least 90%, 95%, 96%, 97%, 98%, or 99% pure.

本文所揭露之融合蛋白或融合蛋白之組分，可對生物活性做定性或評估，該等生物活性包括但不限於對目標結合伴侶之親合力、競爭性結合、抑制性活性、細胞增生之抑制、腫瘤生長之抑制、及血管新生之抑制。在一些實施態樣中，本文所揭露之融合蛋白或融合蛋白之組分可在體外及體內進行生物活性評估。許多用於評估結合親合力之方法係為本技術領域所知，並透過滴定法可用於辨識融合蛋白或融合蛋白之組分對結合伴侶之結合親合力。結合動力學可以表示為穩態平衡結合常數，以解離常數（dissociation constant，K D）值或最大有效濃度之一半（half maximal effective concentration，EC 50）表示。 The fusion proteins or components of the fusion proteins disclosed herein can be used to characterize or evaluate biological activities. Such biological activities include but are not limited to affinity for target binding partners, competitive binding, inhibitory activity, and inhibition of cell proliferation. , inhibition of tumor growth, and inhibition of angiogenesis. In some embodiments, the fusion proteins or components of the fusion proteins disclosed herein can be evaluated for biological activity in vitro and in vivo. Many methods for assessing binding affinity are known in the art and can be used to identify the binding affinity of a fusion protein or components of a fusion protein for a binding partner through titration methods. Binding kinetics can be expressed as a steady-state equilibrium binding constant, expressed as a dissociation constant (K D ) value or half maximal effective concentration (EC 50 ).

在本文之某些實施態樣中，融合蛋白對活性之抑制（例如，對血管新生因子活性及／或整合蛋白活性之抑制）具有小於或等於1微莫耳（μM）、 100奈米莫耳（nM）、10 nM、1 nM、0.1 nM、0.01 nM、或0.001 nM的EC50。在本文之任意實施態樣中，融合蛋白對結合伴侶（血管新生因子及／或整合蛋白）具有小於約1.0 mM、500 μM、100 μM、50 μM、10 μM、5 μM、1 μM、500 nM、100 nM、50 nM、10 nM、5 nM、1 nM、500皮莫耳（pM）、100 pM、50 pM、10 pM、或5 pM的K D值，包括該些數字之間的任意值。 In certain embodiments herein, the fusion protein inhibits activity (e.g., inhibits angiogenic factor activity and/or integrin activity) with an activity less than or equal to 1 micromolar (μM), 100 nmol EC50 of (nM), 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM. In any embodiment herein, the fusion protein has a binding partner (angiogenic factor and/or integrin) of less than about 1.0 mM, 500 μM, 100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 500 nM , 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 picomoles (pM), 100 pM, 50 pM, 10 pM, or 5 pM, including any value in between. .

在某些實施態樣中，該融合蛋白的等電點（pI）為約4.0至9.0。在某些實施態樣中，該融合蛋白的等電點（pI）為約4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.7、4.8、4.9、5.0、5.1、5.2、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、9.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、或該些數字之間的任意值。較佳地，該融合蛋白的等電點（pI）為8.17。In certain embodiments, the fusion protein has an isoelectric point (pI) of about 4.0 to 9.0. In certain embodiments, the fusion protein has an isoelectric point (pI) of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 9.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any value in between. Preferably, the isoelectric point (pI) of the fusion protein is 8.17.

本發明亦提供一醫藥組合物，其係包含融合蛋白，該融合蛋白係包含結合整合蛋白的胜肽、其他結合蛋白的胜肽以靶向血管新生因子、及Fc結構域，其中該結合整合蛋白的胜肽係選自由以下所組成之群組：去整合蛋白、抗整合蛋白αvβx抗體、抗整合蛋白α5β1抗體、靶向整合蛋白αvβx或α5β1之纖維黏連蛋白、及其結合整合蛋白的片段，其中x係1、3、5、6或8。本發明組合物包含治療有效劑量之融合蛋白。The present invention also provides a pharmaceutical composition, which includes a fusion protein that includes an integrin-binding peptide, other protein-binding peptides to target angiogenesis factors, and an Fc domain, wherein the integrin-binding peptide The peptide is selected from the group consisting of: disintegrin, anti-integrin αvβx antibody, anti-integrin α5β1 antibody, fibronectin targeting integrin αvβx or α5β1, and fragments thereof that bind to integrin, where x is 1, 3, 5, 6 or 8. The compositions of the invention comprise a therapeutically effective dose of the fusion protein.

術語「治療有效劑量」意謂具治療活性之化合物引起所需之生物或臨床效果所需要之量。根據本發明之實施態樣，「治療有效劑量」係可達到有利影響或所需結果（包括臨床結果）之足夠的量。治療有效劑量可以一次或多次給藥中投予。依照疾病狀態，治療有效劑量係足夠改善、穩定、或延緩疾病發展的量。根據本發明之特定實施態樣，治療有效劑量係治療或預防不正常血管新生之病症（例如其表徵為新生血管、血管滲透性、水腫、發炎、視網膜病變、纖維化或癌症之疾病）所需之融合蛋白量。The term "therapeutically effective dose" means the amount of a therapeutically active compound required to produce the desired biological or clinical effect. According to aspects of the present invention, a "therapeutically effective dose" is an amount sufficient to achieve a beneficial effect or desired result (including clinical result). The therapeutically effective dose may be administered in one or more administrations. Depending on the disease state, a therapeutically effective dose is an amount sufficient to ameliorate, stabilize, or delay the progression of the disease. According to certain embodiments of the invention, a therapeutically effective dose is required to treat or prevent disorders of abnormal angiogenesis, such as disorders characterized by neovascularization, vascular permeability, edema, inflammation, retinopathy, fibrosis, or cancer. The amount of fusion protein.

術語「活性（potency）」係指該融合蛋白在臨床投予劑量下產生預期之功能的能力。The term "potency" refers to the ability of the fusion protein to produce the expected function at clinically administered doses.

本文中之術語「醫藥配方」係指包括醫藥上可接受之載劑的配方，該醫藥上可接受之載體係例如用以於治療／醫療用途中將VEGF受體融合蛋白（例如aflibercept或conbercept）投予至一個體。The term "pharmaceutical formulation" as used herein refers to a formulation including a pharmaceutically acceptable carrier, such as a VEGF receptor fusion protein (such as aflibercept or conbercept) for therapeutic/medical use. Invested in an individual.

本文中之術語「純度（purity）」係指該融合蛋白不存在汙染物。本文中所指汙染物包括除了所預期之融合蛋白分子以外的蛋白質種類，其係產生自化合物製造過程中的雜質及／或所製造之蛋白質化合物的降解。The term "purity" as used herein refers to the absence of contaminants in the fusion protein. Contaminants as referred to herein include proteinaceous species other than the intended fusion protein molecule that arise from impurities during the manufacturing process of the compound and/or degradation of the protein compound being manufactured.

在一些實施態樣中，包含融合蛋白之醫藥組合物係包含在緩衝液中調配的融合蛋白，其蛋白質濃度自約0.5至約100 mg/mL，較佳為約40至約80 mg/mL，例如約40、50、60、70或80 mg/mL，最佳為約40±約4 mg/mL。在其他較佳實施態樣中，融合蛋白係以高於約40 mg/mL之蛋白濃度在緩衝液中調配。In some embodiments, the pharmaceutical composition containing the fusion protein contains the fusion protein formulated in a buffer, and the protein concentration is from about 0.5 to about 100 mg/mL, preferably from about 40 to about 80 mg/mL. For example, about 40, 50, 60, 70 or 80 mg/mL, preferably about 40 ± about 4 mg/mL. In other preferred embodiments, the fusion protein is formulated in a buffer at a protein concentration greater than about 40 mg/mL.

在特定的實施態樣中，緩衝液係pH值為約5.5至7.0、更佳為約6.0至6.5、甚至更佳為約6.0的緩衝液。在特定的實施態樣中，該緩衝液為pH值為約6.5至8、更佳為約7至7.5、甚至更佳為約7.2的磷酸鹽緩衝液。該磷酸鹽緩衝液係包含約5至20 mM之磷酸鈉，例如5、10、15、或20 mM之磷酸鈉，更佳為約10 mM之磷酸鈉；約20至60 mM之氯化鈉，更佳為約40 mM之氯化鈉；約1至10%重量／體積（w/v）之蔗糖，更佳為約5%重量／體積之蔗糖；以及約0.01至0.05%重量／體積之界面活性劑，更佳為約0.03%重量／體積之聚山梨醇酯20。In a specific embodiment, the buffer solution has a pH value of about 5.5 to 7.0, more preferably about 6.0 to 6.5, and even more preferably about 6.0. In a specific embodiment, the buffer is a phosphate buffer with a pH value of about 6.5 to 8, more preferably about 7 to 7.5, even more preferably about 7.2. The phosphate buffer contains about 5 to 20 mM sodium phosphate, such as 5, 10, 15, or 20 mM sodium phosphate, more preferably about 10 mM sodium phosphate; about 20 to 60 mM sodium chloride, More preferably, about 40 mM sodium chloride; about 1 to 10% w/v sucrose, more preferably about 5% w/v sucrose; and about 0.01 to 0.05% w/v interface The active agent is preferably about 0.03% weight/volume polysorbate 20.

在特定的實施態樣中，該醫藥配方係包含一選自由以下所組成之群組的多元醇或醇類：蔗糖、海藻糖、甘露醇、山梨糖醇、苯甲醇、聚乙烯醇、及聚乙二醇（PEG）400-12000，且其濃度為約1%至約10%（重量／體積），例如約1%、2%、3%、4%、5%、6%、7%、8%、9%、或10%（重量／體積）。In a specific embodiment, the pharmaceutical formulation includes a polyol or alcohol selected from the group consisting of: sucrose, trehalose, mannitol, sorbitol, benzyl alcohol, polyvinyl alcohol, and polyvinyl alcohol. Ethylene glycol (PEG) 400-12000, and its concentration is about 1% to about 10% (weight/volume), such as about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (weight/volume).

在較佳的實施態樣中，該多元醇係濃度為約25 mM至約250 mM的海藻糖，例如約25 mM、50 mM、75 mM、100 mM、125 mM、150 mM、175 mM、200 mM、225 mM、或250 mM。在其他較佳的實施態樣中，該多元醇係濃度為約100至200 mM的海藻糖，例如約100 mM、110 mM、120 mM、130 mM、140 mM、150 mM、160 mM、170 mM、180 mM、190 mM、或200 mM。在其他較佳的實施態樣中，該多元醇係濃度為約190 mM的海藻糖。In a preferred embodiment, the polyol system has a trehalose concentration of about 25 mM to about 250 mM, such as about 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, or 250 mM. In other preferred embodiments, the polyol system has a trehalose concentration of about 100 to 200 mM, such as about 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM , 180 mM, 190 mM, or 200 mM. In other preferred embodiments, the polyol concentration is about 190 mM trehalose.

在特定的實施態樣中，該醫藥配方係包含一緩衝劑。在特定的實施態樣中，該緩衝劑係選自由以下所組成之群組：磷酸鈉、組胺酸、檸檬酸鈉、醋酸鈉、碳酸氫鈉、及檸檬酸三鈉二水合物（trisodium citrate dihydrate），且其濃度為約10 mM至約50 mM，例如約10 mM、20 mM、30 mM、40 mM、或50 mM。在較佳的實施態樣中，該緩衝劑的濃度為約10 mM至約40 mM，例如約10 mM、20 mM、30 mM、或40 mM。在其他較佳的實施態樣中，該緩衝劑的濃度為約20 mM至30 mM，例如約20 mM、21 mM、22 mM、23 mM、24 mM、25 mM、26 mM、27 mM、28 mM、29 mM、或30 mM。在其他較佳的實施態樣中，該緩衝劑的濃度為約25 mM。In certain embodiments, the pharmaceutical formulation includes a buffer. In a specific embodiment, the buffering agent is selected from the group consisting of sodium phosphate, histidine, sodium citrate, sodium acetate, sodium bicarbonate, and trisodium citrate dihydrate. dihydrate), and its concentration is about 10 mM to about 50 mM, such as about 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM. In a preferred embodiment, the concentration of the buffer is about 10 mM to about 40 mM, such as about 10 mM, 20 mM, 30 mM, or 40 mM. In other preferred embodiments, the concentration of the buffer is about 20 mM to 30 mM, such as about 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM. In other preferred embodiments, the buffer concentration is about 25 mM.

在特定的實施態樣中，該醫藥配方更包含一選自由以下所組成之群組的多醣：羧甲基纖維素鈉、微晶纖維素、或透明質酸鈉。In a specific embodiment, the pharmaceutical formula further includes a polysaccharide selected from the group consisting of sodium carboxymethyl cellulose, microcrystalline cellulose, or sodium hyaluronate.

在較佳的實施態樣中，該緩衝劑係濃度為約10 mM至約40 mM的組胺酸，例如約10 mM、20 mM、30 mM、或40 mM。在其他較佳的實施態樣中，該組胺酸的濃度為約20 mM至約30 mM，例如約20 mM、21 mM、22 mM、23 mM、24 mM、25 mM、26 mM、27 mM、28 mM、29 mM、或30 mM。在其他較佳的實施態樣中，該組胺酸的濃度為約25 mM。In a preferred embodiment, the buffer system has a concentration of histidine of about 10 mM to about 40 mM, such as about 10 mM, 20 mM, 30 mM, or 40 mM. In other preferred embodiments, the concentration of histidine is about 20 mM to about 30 mM, such as about 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM , 28 mM, 29 mM, or 30 mM. In other preferred embodiments, the concentration of histidine is about 25 mM.

在特定的實施態樣中，該醫藥配方係包含一界面活性劑。在較佳的實施態樣中，該界面活性劑的濃度為約0.01至約4%（重量／體積），例如約0.01%、0.5%、1.0%、1.5%、2.0%、2.5%、3.0%、3.5%、或4.0%。在其他較佳的實施態樣中，該界面活性劑的濃度為約0.01%至約1.0%（重量／體積），例如約0.01%、0.02%、0.03%、0.04%、0.05%、0.06%、0.07%、0.08%、0.09%、或1.0%（重量／體積）。在其他較佳的實施態樣中，該界面活性劑的濃度為約0.03%（重量／體積）。In certain embodiments, the pharmaceutical formulation includes a surfactant. In a preferred embodiment, the concentration of the surfactant is about 0.01 to about 4% (weight/volume), such as about 0.01%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0% , 3.5%, or 4.0%. In other preferred embodiments, the concentration of the surfactant is about 0.01% to about 1.0% (weight/volume), such as about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 1.0% (weight/volume). In other preferred embodiments, the concentration of the surfactant is about 0.03% (weight/volume).

在較佳的實施態樣中，該界面活性劑係選自由以下所組成之群組：聚山梨糖醇酯20、聚山梨糖醇酯80、及泊洛沙姆188，較佳為聚山梨糖醇酯20。在較佳的實施態樣中，該聚山梨糖醇酯20的濃度為約0.03%（重量／體積）。In a preferred embodiment, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188, preferably polysorbate. Alcohol ester 20. In a preferred embodiment, the concentration of polysorbate 20 is about 0.03% (weight/volume).

在一些實施態樣中，該融合蛋白係於一醫藥配方中，該醫藥配方在-70℃至5℃下安定性維持至少二年，例如在-70℃、-60℃、-50℃、-40℃、-30℃、-20℃、-10℃、0℃、或5℃下。在一些實施態樣中，該融合蛋白係於一醫藥配方中，該醫藥配方在-70℃、-20℃、及／或5℃下安定性維持至少6個月、7個月、8個月、9個月、10個月、11個月、或更長。在較佳的實施態樣中，該融合蛋白係於一醫藥配方中，該醫藥配方在-70℃、-20℃、及／或5℃下安定性維持至少一年、二年、三年、四年、五年、六年、七年、八年、九年、或十年或更多。在其他較佳的實施態樣中，該融合蛋白係於一醫藥配方中，該醫藥配方在-70℃、-20℃、及／或5℃下安定性維持至少二年。In some embodiments, the fusion protein is in a pharmaceutical formulation that is stable at -70°C to 5°C for at least two years, such as -70°C, -60°C, -50°C, - 40℃, -30℃, -20℃, -10℃, 0℃, or 5℃. In some embodiments, the fusion protein is in a pharmaceutical formulation that maintains stability at -70°C, -20°C, and/or 5°C for at least 6 months, 7 months, or 8 months , 9 months, 10 months, 11 months, or longer. In a preferred embodiment, the fusion protein is in a pharmaceutical formula, and the pharmaceutical formula maintains stability at -70°C, -20°C, and/or 5°C for at least one, two, three, Four years, five years, six years, seven years, eight years, nine years, or ten years or more. In other preferred embodiments, the fusion protein is in a pharmaceutical formulation, and the pharmaceutical formulation maintains stability at -70°C, -20°C, and/or 5°C for at least two years.

在一些實施態樣中，該配方在-70℃至25℃下至少6個月後係保持蛋白質的純度及活性，例如在-70℃、-60℃、-50℃、-40℃、-30℃、-20℃、-10℃、0℃、或5℃或25℃下。在一些實施態樣中，該配方在-70℃、-20℃、2℃至8℃、及／或25℃下至少6個月後係保持蛋白質的純度及活性，例如6個月、7個月、8個月、9個月、10個月、11個月、一年、二年、三年、四年、五年、六年、七年、八年、九年、或十年或更多。In some embodiments, the formulation maintains protein purity and activity after at least 6 months at -70°C to 25°C, such as -70°C, -60°C, -50°C, -40°C, -30°C. ℃, -20℃, -10℃, 0℃, or 5℃ or 25℃. In some embodiments, the formulation maintains protein purity and activity after at least 6 months at -70°C, -20°C, 2°C to 8°C, and/or 25°C, for example, 6 months, 7 months Months, 8 months, 9 months, 10 months, 11 months, one year, two years, three years, four years, five years, six years, seven years, eight years, nine years, or ten years or more many.

在一些實施態樣中，該配方更包含一鹽類。在一些實施態樣中，該鹽類係選自氯化鈉、氯化鎂、氯化鈣、或氯化鉀。In some embodiments, the formula further includes a salt. In some embodiments, the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

在特定的實施態樣中，該配方更包含至少一種胺基酸。在一些實施態樣中，該胺基酸係選自由以下所組成之群組：精胺酸、甲硫胺酸、脯胺酸、組胺酸、半胱胺酸、離胺酸、甘胺酸、天門冬胺酸、色胺酸、麩胺酸、及異白胺酸。In specific embodiments, the formulation further includes at least one amino acid. In some embodiments, the amino acid is selected from the group consisting of: arginine, methionine, proline, histidine, cysteine, lysine, and glycine. , aspartic acid, tryptophan, glutamic acid, and isoleucine.

在一些實施態樣中，該醫藥配方係包含濃度為40 mg/mL的融合蛋白、25 mM之組胺酸、190 mM之海藻糖、及0.03%之聚山梨糖醇酯20，其中該配方的pH值為約6.0。In some embodiments, the pharmaceutical formulation contains fusion protein at a concentration of 40 mg/mL, 25 mM histidine, 190 mM trehalose, and 0.03% polysorbate 20, wherein the formulation The pH value is about 6.0.

本發明亦提供一種製造本文中所列之任意配方的方法，其係包含將該配方中的各組分結合成一單一組合物的步驟。此一方法可包括將所獲配方添加至一小瓶或注射裝置中的步驟。該方法可額外包括無菌過濾步驟。此一方法之產物的任意組合物亦為本發明的一部分。舉例言之，本文中之實施態樣亦包括透過以下製備一配方的方法：將一基於組胺酸之緩衝液與一界面活性劑（例如聚山梨糖醇酯20）、一融合蛋白、海藻糖、以及視需要之一或多種其他組分（例如本文中所討論者）。The invention also provides a method of making any of the formulations listed herein, comprising the step of combining the components of the formulation into a single composition. Such a method may include the step of adding the resulting formulation to a vial or injection device. The method may additionally include a sterile filtration step. Any compositions of the products of this process are also part of the invention. For example, embodiments herein also include methods of preparing a formulation by combining a histidine-based buffer with a surfactant (such as polysorbate 20), a fusion protein, trehalose , and optionally one or more other components (such as those discussed herein).

本發明亦關於使用根據本發明之組合物，在一個體或受試者中治療或預防整合蛋白相關之疾病的用途。「個體」或「受試者」係哺乳類。哺乳類包括但不限於馴養動物（例如：牛、羊、貓、狗、及馬）、靈長類（例如：人類及非人類靈長類（non-human primates）（例如猴子））、兔子及囓齒類（例如：小鼠及大鼠）。在一些實施態樣中，用於治療或預防疾病之一或多種方面或症狀的方法係包含，對一個體投予一有效量之包含融合蛋白的組合物。The invention also relates to the use of a composition according to the invention for the treatment or prevention of integrin-related diseases in an individual or subject. The "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (such as cattle, sheep, cats, dogs, and horses), primates (such as humans and non-human primates (such as monkeys)), rabbits, and rodents categories (e.g. mice and rats). In some embodiments, methods for treating or preventing one or more aspects or symptoms of a disease comprise administering to a subject an effective amount of a composition comprising a fusion protein.

本文所述之方法可用於治療多種疾病，包括但不限於發炎性疾病、眼部疾病、自體免疫疾病、或癌症。在一些實施態樣中，欲治療之疾病包括但不限於類風濕性關節炎、炎症性關節炎、骨關節炎、癌症、組織／器官纖維化、色素性視網膜炎、眼色素層炎（例如：前眼色素層炎或後眼色素層炎）、及特徵係新生血管或缺血性眼部疾病（例如：脈絡膜新生血管、虹膜新生血管、新生血管性青光眼、青光眼術後纖維化、增殖性玻璃體視網膜病變（PVR）、脈絡膜新生血管（CNV）、視神經盤新生血管、視網膜新生血管、玻璃體新生血管、血管翳、翼狀贅肉、血管性視網膜病變、無糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、有糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、糖尿病黃斑水腫（DME）、滲出性（濕性）及非滲出性（乾性）之老年性黃斑退化（AMD）、黃斑水腫、視網膜靜脈阻塞後黃斑水腫（RVO）、視網膜靜脈阻塞（RVO）、中心性視網膜靜脈阻塞（CRVO）、分支性視網膜靜脈阻塞（BRVO）、視網膜血管瘤狀增殖（RAP）、息肉狀脈絡膜血管病變（PCV）、玻璃體黃斑沾黏（VMA）、及／或玻璃體黃斑牽引症（VMT））。The methods described herein can be used to treat a variety of diseases, including, but not limited to, inflammatory diseases, eye diseases, autoimmune diseases, or cancer. In some embodiments, the disease to be treated includes, but is not limited to, rheumatoid arthritis, inflammatory arthritis, osteoarthritis, cancer, tissue/organ fibrosis, retinitis pigmentosa, uveitis (e.g.: Anterior or posterior uveitis), and ocular diseases characterized by neovascularization or ischemia (e.g., choroidal neovascularization, iris neovascularization, neovascular glaucoma, postoperative glaucoma fibrosis, proliferative vitreous Retinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygoid, vascular retinopathy, diabetic retinopathy without diabetic macular edema (DME) ( DR, nonproliferative and proliferative DR), diabetic retinopathy with diabetic macular edema (DME) (DR, nonproliferative and proliferative DR), diabetic macular edema (DME), exudative (wet) and non-exudative Sexual (dry) age-related macular degeneration (AMD), macular edema, post-retinal vein occlusion macular edema (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branched retinal vein occlusion (BRVO) , retinal angiomatous proliferation (RAP), polypoidal choroidal vasculopathy (PCV), vitreomacular adhesion (VMA), and/or vitreomacular traction (VMT)).

本文所述之組合物可透過任意途徑投予至個體，途徑包括但不限於靜脈、腹膜、眼部、動脈、肺臟、口服、吸入、血管（intravesicular）、肌肉、氣管、皮下、脊髓（intrathecal）、經皮、經胸膜（transpleural）、局部、黏膜、胃腸、關節（intraarticular）、腦池內（intracisternal）、心室內、顱內、尿道內、肝內、及腫瘤內。在一些實施態樣中，組合物係系統性投予（例如藉由靜脈注射）。在一些實施態樣中，組合物係局部投予（例如藉由動脈注射或眼內注射）。The compositions described herein may be administered to an individual by any route, including, but not limited to, intravenous, peritoneal, ocular, arterial, pulmonary, oral, inhalational, intravesicular, intramuscular, tracheal, subcutaneous, intrathecal. , transcutaneous, transpleural, local, mucosal, gastrointestinal, intraarticular, intracisternal, intraventricular, intracranial, intraurethral, intrahepatic, and intratumoral. In some embodiments, the composition is administered systemically (eg, by intravenous injection). In some embodiments, the compositions are administered topically (eg, by intraarterial or intraocular injection).

在一些實施態樣中，組合物係直接投予至眼睛或眼睛組織。在一些實施態樣中，組合物係局部地投予至眼睛，舉例而言，眼藥水。在一些實施態樣中，組合物係藉由注射至眼睛（眼內注射）或至與眼部相連的組織來投予。組合物可被投予，舉例而言，藉由眼內注射、眼周注射、次視網膜注射、玻璃體內注射（intravitreal injection）、脈絡膜表面（superchoroidal）注射、經中膈（trans-septal）注射、鞏膜下（subscleral）注射、脈絡膜內注射、前房內（intracameral）注射、結膜下注射、坦諾囊下（sub-Tenon’s）注射、眼球後（retrobulbar）注射、眼球周邊（peribulbar）注射、或後鞏膜旁遞送（posterior juxtascleral delivery）。這些方法係為本技術領域所知。組合物可被投予至例如玻璃體、前房液、鞏膜、結膜、在鞏膜及結膜之間的部位、視網膜脈絡膜組織、黃斑、或其他在個體眼睛中或鄰近的部位。In some embodiments, the composition is administered directly to the eye or eye tissue. In some embodiments, the composition is administered topically to the eye, for example, as eye drops. In some embodiments, the compositions are administered by injection into the eye (intraocular injection) or into tissue associated with the eye. The compositions may be administered, for example, by intraocular injection, periocular injection, subretinal injection, intravitreal injection, superchoroidal injection, trans-septal injection, Subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or retrobulbar injection Posterior juxtascleral delivery. These methods are known in the art. The compositions may be administered, for example, to the vitreous humor, anterior chamber fluid, sclera, conjunctiva, the site between the sclera and the conjunctiva, retinal choroidal tissue, macula, or other sites in or adjacent to the eye of an individual.

在一些實施態樣中，該醫藥組合物係以每眼約0.03至10 mg的劑量投予至眼睛，例如0.03 mg、0.04 mg、0.05 mg、0.06 mg、0.07 mg、0.08 mg、0.09 mg、0.1 mg、0.2 mg、0.3 mg、0.4 mg、0.5 mg、0.6 mg、0.7 mg、0.8 mg、0.9 mg、1 mg、2 mg、3 mg、4 mg、5 mg、6 mg、7 mg、8 mg、9 mg、或10 mg。在較佳的實施態樣中，該醫藥組合物係以每眼約3.0至6.0 mg的劑量遞送至眼睛，例如3.0 mg、3.5 mg、4.0 mg、4.5 mg、5.0 mg、5.5 mg、或6 mg。在其他較佳的實施態樣中，該醫藥組合物係以每眼4 mg的劑量遞送至眼睛。In some embodiments, the pharmaceutical composition is administered to the eye at a dose of about 0.03 to 10 mg per eye, such as 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. In a preferred embodiment, the pharmaceutical composition is delivered to the eye at a dose of about 3.0 to 6.0 mg per eye, such as 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 5.5 mg, or 6 mg . In other preferred embodiments, the pharmaceutical composition is delivered to the eye at a dose of 4 mg per eye.

組合物之最適有效量可憑經驗決定，且將取決於疾病之型態及嚴重性、投予途徑、疾病進程及個體之健康狀況、重量及身體部位。該等決定係在本技術領域者之通常知識範圍內。包含融合蛋白之組合物亦可以一週六次、一週五次、一週四次、一週三次、一週二次、一週一次、二週一次、三週一次、一個月一次、二個月一次、三個月一次、六個月一次、九個月一次、或一年一次來投予。The optimal effective amount of the composition can be determined empirically and will depend on the type and severity of the disease, the route of administration, the course of the disease and the health, weight and body region of the individual. Such decisions are within the common knowledge of those skilled in the art. The composition containing the fusion protein can also be administered six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every two months, Give it once every three months, every six months, every nine months, or once a year.

除非另外定義，此處所用之所有技術及科學術語係具有與本發明所屬技術領域中具通常知識者通常所了解之相同意義。雖然任何與本文所述類似或同等之方法及材料可用來實施或測試本發明，但較佳的為現在描述的方法及材料。所有本文所提到之供參考的出版品係併於本文中，以描述與該些被引用之出版品相關的方法及／或材料。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods and materials now described are preferred. All publications mentioned herein by reference are herein incorporated by reference to describe the methods and/or materials associated with the cited publications.

實施態樣Implementation style

本申請包括但不限於以下所編列的實施態樣：This application includes but is not limited to the implementation forms listed below:

實施態樣1為一種醫藥配方，該醫藥配方包含： a)    一濃度為約0.5 mg/mL至約120 mg/mL之融合蛋白； b)    一濃度為約1%至約10%（重量／體積）之多元醇或醇類，該多元醇或醇類係選自由以下所組成之群組：蔗糖、海藻糖、甘露醇、山梨糖醇、苯甲醇、聚乙烯醇、及聚乙二醇（PEG）400-12000； c)    一濃度為約10 mM至約50 mM之緩衝劑，該緩衝劑係選自由以下所組成之群組：磷酸鈉、組胺酸、檸檬酸鈉、醋酸鈉、碳酸氫鈉、及檸檬酸三鈉二水合物（trisodium citrate dihydrate）；以及 d)    一濃度為約0.01%至約4%（重量／體積）之界面活性劑， 其中，該配方的pH值為約5.5至7.5，且該配方視需要地更包含一選自由以下所組成之群組的多醣：羧甲基纖維素鈉、微晶纖維素、或透明質酸鈉。 Implementation pattern 1 is a pharmaceutical formula, and the pharmaceutical formula includes: a) A fusion protein with a concentration of about 0.5 mg/mL to about 120 mg/mL; b) A polyol or alcohol at a concentration of about 1% to about 10% (weight/volume), the polyol or alcohol being selected from the group consisting of: sucrose, trehalose, mannitol, sorbose Alcohol, benzyl alcohol, polyvinyl alcohol, and polyethylene glycol (PEG) 400-12000; c) A buffer having a concentration of about 10 mM to about 50 mM, the buffer being selected from the group consisting of: sodium phosphate, histidine, sodium citrate, sodium acetate, sodium bicarbonate, and citric acid trisodium citrate dihydrate; and d) A surfactant at a concentration of about 0.01% to about 4% (weight/volume), Wherein, the pH value of the formula is about 5.5 to 7.5, and the formula optionally further includes a polysaccharide selected from the group consisting of: sodium carboxymethyl cellulose, microcrystalline cellulose, or sodium hyaluronate .

實施態樣2為如實施態樣1之醫藥配方，其中該界面活性劑係選自由以下所組成之群組：聚山梨糖醇酯20、聚山梨糖醇酯80、及泊洛沙姆188，較佳為聚山梨糖醇酯20。Embodiment 2 is a pharmaceutical formulation as in Embodiment 1, wherein the surfactant is selected from the group consisting of: polysorbate 20, polysorbate 80, and poloxamer 188, Polysorbate 20 is preferred.

實施態樣3為如實施態樣2之醫藥配方，其中該聚山梨糖醇酯為聚山梨糖醇酯20。Embodiment 3 is a pharmaceutical formulation as in Embodiment 2, wherein the polysorbate is polysorbate 20.

實施態樣4為如實施態樣1至3中任一者之醫藥配方，其中該界面活性劑的濃度約為0.03%（重量／體積）。Embodiment 4 is a pharmaceutical formulation according to any one of Embodiments 1 to 3, wherein the concentration of the surfactant is about 0.03% (weight/volume).

實施態樣5為如實施態樣1至4中任一者之醫藥配方，其中該融合蛋白的濃度為約1 mg/mL至約90 mg/mL，較佳為約20 mg/mL至約80 mg/mL，更佳地，該融合蛋白的濃度為約40 mg/mL。Embodiment 5 is a pharmaceutical formulation according to any one of embodiments 1 to 4, wherein the concentration of the fusion protein is about 1 mg/mL to about 90 mg/mL, preferably about 20 mg/mL to about 80 mg/mL, more preferably, the concentration of the fusion protein is about 40 mg/mL.

實施態樣6為如實施態樣5之醫藥配方，其中該融合蛋白的濃度為約40 mg/mL。Embodiment 6 is a pharmaceutical formulation as in Embodiment 5, wherein the concentration of the fusion protein is about 40 mg/mL.

實施態樣7為如實施態樣1至6中任一者之醫藥配方，其中該多元醇係海藻糖且濃度為約25 mM至250 mM，較佳為約190 mM。Embodiment 7 is a pharmaceutical formulation according to any one of embodiments 1 to 6, wherein the polyol is trehalose and the concentration is about 25 mM to 250 mM, preferably about 190 mM.

實施態樣8為如實施態樣7之醫藥配方，其中海藻糖的濃度為約190 mM。Embodiment 8 is a pharmaceutical formulation as in Embodiment 7, wherein the concentration of trehalose is about 190 mM.

實施態樣9為如實施態樣1至8中任一者之醫藥配方，其中該緩衝劑係組胺酸且濃度為約10 mM至約40 mM，較佳為約20 mM至約30 mM，更佳地，該組胺酸的濃度為約25 mM。Embodiment 9 is a pharmaceutical formulation according to any one of embodiments 1 to 8, wherein the buffer is histidine and the concentration is about 10 mM to about 40 mM, preferably about 20 mM to about 30 mM, More preferably, the concentration of histidine is about 25 mM.

實施態樣10為如實施態樣9之醫藥配方，其中該組胺酸的濃度為約25 mM。Embodiment 10 is a pharmaceutical formulation as in Embodiment 9, wherein the concentration of histamine is about 25 mM.

實施態樣11為如實施態樣1至10中任一者之醫藥配方，其中該融合蛋白係自N-端至C-端以如下順序包含： a)   一血管內皮生長因子受體（VEGFR）的細胞外結構域； b)   一人類免疫球蛋白G的Fc結構域；以及 c)    一結合整合蛋白之蛋白或其片段。 Embodiment 11 is a pharmaceutical formulation according to any one of embodiments 1 to 10, wherein the fusion protein includes in the following order from N-terminus to C-terminus: a) An extracellular domain of vascular endothelial growth factor receptor (VEGFR); b) an Fc domain of human immunoglobulin G; and c) A protein or fragment thereof that binds integrin.

實施態樣12為如實施態樣1至11中任一者之醫藥配方，其中該融合蛋白係包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO: 17、或SEQ ID NO: 18。Embodiment 12 is a pharmaceutical formulation as in any one of embodiments 1 to 11, wherein the fusion protein includes SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18 .

實施態樣13為如實施態樣1至12中任一者之醫藥配方，其中該pH值為約5.5至約7.0，較佳地，該pH值為約6.0。Embodiment 13 is a pharmaceutical formulation according to any one of embodiments 1 to 12, wherein the pH value is about 5.5 to about 7.0, preferably, the pH value is about 6.0.

實施態樣14為如實施態樣13之醫藥配方，其中該pH值為約6.0。Embodiment 14 is the pharmaceutical formulation of embodiment 13, wherein the pH value is about 6.0.

實施態樣15為如實施態樣1至14中任一者之醫藥配方，其中該配方在-70℃、-20℃、及／或2至8℃下安定性維持至少24個月。Embodiment 15 is a pharmaceutical formulation according to any one of embodiments 1 to 14, wherein the formulation maintains stability at -70°C, -20°C, and/or 2 to 8°C for at least 24 months.

實施態樣16為如實施態樣1至15中任一者之醫藥配方，其中該配方在-70℃、-20℃、及／或2℃至8℃下（較佳在2℃至8℃下）至少6個月後係保持蛋白質的純度及活性。Embodiment 16 is a pharmaceutical formula as in any one of embodiments 1 to 15, wherein the formula is at -70°C, -20°C, and/or 2°C to 8°C (preferably at 2°C to 8°C Bottom) The purity and activity of the protein will be maintained for at least 6 months.

實施態樣17為如實施態樣1至16中任一者之醫藥配方，其中該配方更包含一濃度為約10 mM至50 mM的鹽類。Embodiment 17 is a pharmaceutical formulation according to any one of embodiments 1 to 16, wherein the formulation further includes a salt at a concentration of about 10 mM to 50 mM.

實施態樣18為如實施態樣17之醫藥配方，其中該鹽類係選自氯化鈉、氯化鎂、氯化鈣、或氯化鉀。Embodiment 18 is a pharmaceutical formulation as in Embodiment 17, wherein the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

實施態樣19為如實施態樣1至18中任一者之醫藥配方，其中該配方更包含至少一種胺基酸，該胺基酸的濃度為約10 mM至50 mM。Embodiment 19 is a pharmaceutical formulation according to any one of embodiments 1 to 18, wherein the formulation further includes at least one amino acid, and the concentration of the amino acid is about 10 mM to 50 mM.

實施態樣20為如實施態樣19之醫藥配方，其中該胺基酸係選自由以下所組成之群組：精胺酸、甲硫胺酸、脯胺酸、組胺酸、半胱胺酸、離胺酸、甘胺酸、天門冬胺酸、色胺酸、麩胺酸、及異白胺酸。Embodiment 20 is a pharmaceutical formulation as in Embodiment 19, wherein the amino acid is selected from the group consisting of: arginine, methionine, proline, histidine, and cysteine , lysine, glycine, aspartic acid, tryptophan, glutamic acid, and isoleucine.

實施態樣21為如實施態樣1至20中任一者之醫藥配方，其中該配方係用於一治療眼部疾病的方法。Embodiment 21 is a pharmaceutical formulation according to any one of Embodiments 1 to 20, wherein the formulation is used for a method of treating eye diseases.

實施態樣22為如實施態樣21之醫藥配方，其中該眼部疾病係選自新生血管或缺血性眼色素層炎、視網膜血管炎、血管樣痕、色素性視網膜炎、角膜新生血管、虹膜新生血管、新生血管性青光眼、青光眼術後纖維化、增殖性玻璃體視網膜病變（PVR）、脈絡膜新生血管（CNV）、視神經盤新生血管、視網膜新生血管、玻璃體新生血管、血管翳、翼狀贅肉、血管性視網膜病變、無糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、有糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、糖尿病黃斑水腫（DME）、滲出性（濕性）及非滲出性（乾性）之老年性黃斑退化（AMD）、黃斑水腫、視網膜靜脈阻塞後黃斑水腫（RVO）、視網膜靜脈阻塞（RVO）、中心性視網膜靜脈阻塞（CRVO）、分支性視網膜靜脈阻塞（BRVO）、視網膜血管瘤狀增殖（RAP）、息肉狀脈絡膜血管病變（PCV）、玻璃體黃斑沾黏（VMA）、及／或玻璃體黃斑牽引症（VMT）。Embodiment 22 is a pharmaceutical formulation as in embodiment 21, wherein the eye disease is selected from the group consisting of neovascularization or ischemic uveitis, retinal vasculitis, vascular scars, retinitis pigmentosa, corneal neovascularization, Iris neovascularization, neovascular glaucoma, postoperative glaucoma fibrosis, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygoid cartilage , Vascular retinopathy, diabetic retinopathy (DR, non-proliferative and proliferative DR) without diabetic macular edema (DME), diabetic retinopathy (DR, non-proliferative and proliferative DR) with diabetic macular edema (DME) ), diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, post-retinal vein occlusion macular edema (RVO), retinal vein occlusion (RVO) , central retinal vein occlusion (CRVO), branched retinal vein occlusion (BRVO), retinal angiomatous proliferation (RAP), polypoidal choroidal vasculopathy (PCV), vitreomacular adhesion (VMA), and/or vitreous macula traction syndrome (VMT).

實施態樣23為如實施態樣1至22中任一者之醫藥配方，其中該配方係以每眼約0.03至10 mg的劑量投予，較佳為每眼約3.0至6.0 mg，更佳地，該配方係以每眼約4 mg的劑量投予。Embodiment 23 is a pharmaceutical formulation according to any one of embodiments 1 to 22, wherein the formulation is administered at a dose of about 0.03 to 10 mg per eye, preferably about 3.0 to 6.0 mg per eye, more preferably Typically, the formulation is administered at a dose of approximately 4 mg per eye.

實施態樣24為如實施態樣23之醫藥配方，其中該配方係以每眼約4 mg的劑量投予。Embodiment 24 is a pharmaceutical formulation as in Embodiment 23, wherein the formulation is administered at a dose of approximately 4 mg per eye.

實施態樣25為一種醫藥配方，該配方包含： a)   一濃度為40 mg/mL之融合蛋白； b)   25 mM之組胺酸； c)    190 mM之海藻酸、蔗糖、或甘露醇；以及 d)   0.03%之聚山梨糖醇酯20或聚山梨糖醇酯80， 其中該配方的pH值為約6.0。 Implementation aspect 25 is a pharmaceutical formula, which includes: a) A fusion protein with a concentration of 40 mg/mL; b) 25 mM histamine; c) 190 mM alginic acid, sucrose, or mannitol; and d) 0.03% polysorbate 20 or polysorbate 80, The pH of this formulation is approximately 6.0.

實施態樣26為如實施態樣25之醫藥配方，其中該融合蛋白係自N-端至C-端以如下順序包含： a)   一血管內皮生長因子受體（VEGFR）的細胞外結構域； b)   一人類免疫球蛋白G的Fc結構域；以及 c)    結合一整合蛋白之蛋白或其片段。 Embodiment 26 is a pharmaceutical formulation as in Embodiment 25, wherein the fusion protein includes in the following order from the N-terminus to the C-terminus: a) An extracellular domain of vascular endothelial growth factor receptor (VEGFR); b) an Fc domain of human immunoglobulin G; and c) Protein or fragment thereof that binds to an integrin.

實施態樣27為如實施態樣26之醫藥配方，其中該融合蛋白係包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO: 17、或SEQ ID NO: 18。Embodiment 27 is a pharmaceutical formulation as in Embodiment 26, wherein the fusion protein includes SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

實施態樣28為如實施態樣25至27中任一者之醫藥配方，其中該配方在-70℃、-20℃、及／或5℃下安定性維持至少24個月。Embodiment 28 is a pharmaceutical formulation according to any one of embodiments 25 to 27, wherein the formulation maintains stability at -70°C, -20°C, and/or 5°C for at least 24 months.

實施態樣29為如實施態樣25至28中任一者之醫藥配方，其中該配方在-70℃、-20℃、及／或2℃至8℃下（較佳在2℃至8℃下）至少6個月後係保持蛋白質的純度及活性。Embodiment 29 is a pharmaceutical formulation as in any one of embodiments 25 to 28, wherein the formulation is at -70°C, -20°C, and/or 2°C to 8°C (preferably at 2°C to 8°C Bottom) The purity and activity of the protein will be maintained for at least 6 months.

實施態樣30為如實施態樣25至29中任一者之醫藥配方，其中該配方更包含一濃度為約10 mM至50 mM的鹽類。Embodiment 30 is a pharmaceutical formulation according to any one of Embodiments 25 to 29, wherein the formulation further includes a salt at a concentration of about 10 mM to 50 mM.

實施態樣31為如實施態樣30之醫藥配方，其中該鹽類係選自氯化鈉、氯化鎂、氯化鈣、或氯化鉀。Embodiment 31 is a pharmaceutical formulation as in Embodiment 30, wherein the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

實施態樣32為如實施態樣25至31中任一者之醫藥配方，其中該配方更包含至少一種胺基酸，該胺基酸的濃度為約10 mM至50 mM。Embodiment 32 is a pharmaceutical formulation according to any one of embodiments 25 to 31, wherein the formulation further includes at least one amino acid, and the concentration of the amino acid is about 10 mM to 50 mM.

實施態樣33為如實施態樣32之醫藥配方，其中該胺基酸係選自由以下所組成之群組：精胺酸、甲硫胺酸、脯胺酸、組胺酸、半胱胺酸、離胺酸、甘胺酸、天門冬胺酸、色胺酸、麩胺酸、及異白胺酸。Embodiment 33 is a pharmaceutical formulation as in Embodiment 32, wherein the amino acid is selected from the group consisting of: arginine, methionine, proline, histidine, and cysteine. , lysine, glycine, aspartic acid, tryptophan, glutamic acid, and isoleucine.

實施態樣34為如實施態樣25至33中任一者之醫藥配方，其中該配方係用於一治療眼部疾病的方法。Embodiment 34 is a pharmaceutical formulation according to any one of Embodiments 25 to 33, wherein the formulation is used for a method of treating eye diseases.

實施態樣35為如實施態樣34之醫藥配方，其中該眼部疾病係選自新生血管或缺血性眼色素層炎、視網膜血管炎、血管樣痕、色素性視網膜炎、角膜新生血管、虹膜新生血管、新生血管性青光眼、青光眼術後纖維化、增殖性玻璃體視網膜病變（PVR）、脈絡膜新生血管（CNV）、視神經盤新生血管、視網膜新生血管、玻璃體新生血管、血管翳、翼狀贅肉、血管性視網膜病變、無糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、有糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、糖尿病黃斑水腫（DME）、滲出性（濕性）及非滲出性（乾性）之老年性黃斑退化（AMD）、黃斑水腫、視網膜靜脈阻塞後黃斑水腫（RVO）、視網膜靜脈阻塞（RVO）、中心性視網膜靜脈阻塞（CRVO）、分支性視網膜靜脈阻塞（BRVO）、視網膜血管瘤狀增殖（RAP）、息肉狀脈絡膜血管病變（PCV）、玻璃體黃斑沾黏（VMA）、及／或玻璃體黃斑牽引症（VMT）。Embodiment 35 is a pharmaceutical formulation as in Embodiment 34, wherein the eye disease is selected from the group consisting of neovascularization or ischemic uveitis, retinal vasculitis, vascular scars, retinitis pigmentosa, corneal neovascularization, Iris neovascularization, neovascular glaucoma, postoperative glaucoma fibrosis, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygoid cartilage , Vascular retinopathy, diabetic retinopathy (DR, non-proliferative and proliferative DR) without diabetic macular edema (DME), diabetic retinopathy (DR, non-proliferative and proliferative DR) with diabetic macular edema (DME) ), diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, post-retinal vein occlusion macular edema (RVO), retinal vein occlusion (RVO) , central retinal vein occlusion (CRVO), branched retinal vein occlusion (BRVO), retinal angiomatous proliferation (RAP), polypoidal choroidal vasculopathy (PCV), vitreomacular adhesion (VMA), and/or vitreous macula traction syndrome (VMT).

實施態樣36為如實施態樣25至35中任一者之醫藥配方，其中該配方係以每眼約0.03至10 mg的劑量投予，較佳為每眼約3.0至6.0 mg，更佳地，該配方係以每眼約4 mg的劑量投予。Embodiment 36 is a pharmaceutical formulation as in any one of Embodiments 25 to 35, wherein the formulation is administered at a dose of about 0.03 to 10 mg per eye, preferably about 3.0 to 6.0 mg per eye, more preferably Typically, the formulation is administered at a dose of approximately 4 mg per eye.

實施態樣37為如實施態樣36之醫藥配方，其中該配方係以每眼約4 mg的劑量投予Embodiment 37 is a pharmaceutical formulation as in Embodiment 36, wherein the formulation is administered at a dose of about 4 mg per eye

實施態樣38為一種治療一個體之眼部疾病的方法，該方法包含對該個體投予一包含以下的醫藥配方： a)    一濃度為約0.5 mg/mL至約120 mg/mL之融合蛋白； b)    一濃度為約1%至約10%（重量／體積）之多元醇或醇類，該多元醇或醇類係選自由以下所組成之群組：蔗糖、海藻糖、甘露醇、山梨糖醇、苯甲醇、聚乙烯醇、及聚乙二醇（PEG）400-12000； c)    一濃度為約10 mM至約50 mM之緩衝劑，該緩衝劑係選自由以下所組成之群組：磷酸鈉、組胺酸、檸檬酸鈉、醋酸鈉、碳酸氫鈉、及檸檬酸三鈉二水合物（trisodium citrate dihydrate）；以及 d)    一濃度為約0.01%至約4%（重量／體積）之界面活性劑， 其中，該配方的pH值為約5.5至7.5，且該配方視需要地更包含一選自由以下所組成之群組的多醣：羧甲基纖維素鈉、微晶纖維素、或透明質酸鈉。 Embodiment 38 is a method of treating an eye disease in an individual, the method comprising administering to the individual a pharmaceutical formulation including: a) A fusion protein with a concentration of about 0.5 mg/mL to about 120 mg/mL; b) A polyol or alcohol at a concentration of about 1% to about 10% (weight/volume), the polyol or alcohol being selected from the group consisting of: sucrose, trehalose, mannitol, sorbose Alcohol, benzyl alcohol, polyvinyl alcohol, and polyethylene glycol (PEG) 400-12000; c) A buffer having a concentration of about 10 mM to about 50 mM, the buffer being selected from the group consisting of: sodium phosphate, histidine, sodium citrate, sodium acetate, sodium bicarbonate, and citric acid trisodium citrate dihydrate; and d) A surfactant at a concentration of about 0.01% to about 4% (weight/volume), Wherein, the pH value of the formula is about 5.5 to 7.5, and the formula optionally further includes a polysaccharide selected from the group consisting of: sodium carboxymethyl cellulose, microcrystalline cellulose, or sodium hyaluronate .

實施態樣39為如實施態樣38之方法，其中該界面活性劑係選自由以下所組成之群組：聚山梨糖醇酯20、聚山梨糖醇酯80、及泊洛沙姆188，較佳為聚山梨糖醇酯20。Embodiment 39 is the method of embodiment 38, wherein the surfactant is selected from the group consisting of: polysorbate 20, polysorbate 80, and poloxamer 188, preferably Polysorbate 20 is preferred.

實施態樣40為如實施態樣39之方法，其中該界面活性劑係聚山梨糖醇酯20。Embodiment 40 is the method of embodiment 39, wherein the surfactant is polysorbate 20.

實施態樣41為如實施態樣38至40中任一者之方法，其中該界面活性劑的濃度約為0.03%（重量／體積）。Embodiment 41 is the method of any one of embodiments 38 to 40, wherein the concentration of the surfactant is about 0.03% (weight/volume).

實施態樣42為如實施態樣38至41中任一者之方法，其中該融合蛋白的濃度為約1 mg/mL至約90 mg/mL，較佳為約40 mg/mL至約80 mg/mL，更佳地，該融合蛋白的濃度為約40 mg/mL。Embodiment 42 is the method as in any one of embodiments 38 to 41, wherein the concentration of the fusion protein is about 1 mg/mL to about 90 mg/mL, preferably about 40 mg/mL to about 80 mg /mL, more preferably, the concentration of the fusion protein is about 40 mg/mL.

實施態樣43為如實施態樣42之方法，其中該融合蛋白的濃度為約40 mg/mL。Embodiment 43 is the method of embodiment 42, wherein the concentration of the fusion protein is about 40 mg/mL.

實施態樣44為如實施態樣38至43中任一者之方法，其中該多元醇係海藻糖且濃度為約150 mM至230 mM，較佳為約190 mM。Embodiment 44 is the method of any one of embodiments 38 to 43, wherein the polyol is trehalose and the concentration is about 150 mM to 230 mM, preferably about 190 mM.

實施態樣45為如實施態樣44之方法，其中該海藻糖的濃度為約190 mM。Embodiment 45 is the method of embodiment 44, wherein the concentration of trehalose is about 190 mM.

實施態樣46為如實施態樣38至45中任一者之方法，其中該緩衝劑係組胺酸且濃度為約20 mM至約40 mM，較佳為約20 mM至約30 mM，更佳地，該組胺酸的濃度為約25 mM。Embodiment 46 is a method as in any one of embodiments 38 to 45, wherein the buffer is histidine and the concentration is about 20 mM to about 40 mM, preferably about 20 mM to about 30 mM, more preferably Preferably, the histidine concentration is about 25 mM.

實施態樣47為如實施態樣46之方法，其中該組胺酸的濃度為約25 mM。Embodiment 47 is the method of embodiment 46, wherein the concentration of histidine is about 25 mM.

實施態樣48為如實施態樣38至47中任一者之方法，其中該融合蛋白係自N-端至C-端以如下順序包含： a)   一血管內皮生長因子受體（VEGFR）的細胞外結構域； b)   一人類免疫球蛋白G的Fc結構域；以及 c)    一結合整合蛋白之蛋白（integrin binding protein）或其片段。 Embodiment 48 is a method as in any one of embodiments 38 to 47, wherein the fusion protein includes in the following order from N-terminus to C-terminus: a) An extracellular domain of vascular endothelial growth factor receptor (VEGFR); b) an Fc domain of human immunoglobulin G; and c) An integrin binding protein or a fragment thereof.

實施態樣49為如實施態樣38至48中任一者之方法，其中該融合蛋白係包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO: 17、或SEQ ID NO: 18。Embodiment 49 is the method of any one of embodiments 38 to 48, wherein the fusion protein comprises SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

實施態樣50為如實施態樣38至49中任一者之方法，其中該pH值為約6.0至約6.5，較佳地，該pH值為約6.0。Embodiment 50 is the method of any one of embodiments 38 to 49, wherein the pH value is about 6.0 to about 6.5, preferably, the pH value is about 6.0.

實施態樣51為如實施態樣50之方法，其中該pH值為約6.0。Embodiment 51 is the method of embodiment 50, wherein the pH value is about 6.0.

實施態樣52為如實施態樣38至51中任一者之方法，其中該配方在-70℃、-20℃、及／或2至8℃下安定性維持至少24個月。Embodiment 52 is the method of any one of embodiments 38 to 51, wherein the formulation maintains stability at -70°C, -20°C, and/or 2 to 8°C for at least 24 months.

實施態樣53為如實施態樣38至52中任一者之方法，其中該配方在-70℃、-20℃、及／或2℃至8℃下（較佳在2℃至8℃下）至少6個月後係保持蛋白質的純度及活性。Embodiment 53 is a method as in any one of embodiments 38 to 52, wherein the formulation is at -70°C, -20°C, and/or 2°C to 8°C (preferably at 2°C to 8°C ) will maintain the purity and activity of the protein for at least 6 months.

實施態樣54為如實施態樣38至53中任一者之方法，其中該配方更包含一濃度為約10 mM至50 mM的鹽類。Embodiment 54 is the method of any one of embodiments 38 to 53, wherein the formulation further includes a salt at a concentration of about 10 mM to 50 mM.

實施態樣55為如實施態樣54之方法，其中該鹽類係選自氯化鈉、氯化鎂、氯化鈣、或氯化鉀。Embodiment 55 is the method of embodiment 54, wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

實施態樣56為如實施態樣38至55中任一者之方法，其中該配方更包含至少一種胺基酸，該胺基酸的濃度為約10 mM至50 mM。Embodiment 56 is the method of any one of embodiments 38 to 55, wherein the formulation further includes at least one amino acid, and the concentration of the amino acid is about 10 mM to 50 mM.

實施態樣57為如實施態樣56之方法，其中該胺基酸係選自由以下所組成之群組：精胺酸、甲硫胺酸、脯胺酸、組胺酸、半胱胺酸、離胺酸、甘胺酸、天門冬胺酸、色胺酸、麩胺酸、及異白胺酸。Embodiment 57 is the method of embodiment 56, wherein the amino acid is selected from the group consisting of: arginine, methionine, proline, histidine, cysteine, Lysine, glycine, aspartic acid, tryptophan, glutamic acid, and isoleucine.

實施態樣58為如實施態樣38至57中任一者之方法，其中該眼部疾病係選自新生血管或缺血性眼色素層炎、視網膜血管炎、血管樣痕、色素性視網膜炎、角膜新生血管、虹膜新生血管、新生血管性青光眼、青光眼術後纖維化、增殖性玻璃體視網膜病變（PVR）、脈絡膜新生血管（CNV）、視神經盤新生血管、視網膜新生血管、玻璃體新生血管、血管翳、翼狀贅肉、血管性視網膜病變、無糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、有糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、糖尿病黃斑水腫（DME）、滲出性（濕性）及非滲出性（乾性）之老年性黃斑退化（AMD）、黃斑水腫、視網膜靜脈阻塞後黃斑水腫（RVO）、視網膜靜脈阻塞（RVO）、中心性視網膜靜脈阻塞（CRVO）、分支性視網膜靜脈阻塞（BRVO）、視網膜血管瘤狀增殖（RAP）、息肉狀脈絡膜血管病變（PCV）、玻璃體黃斑沾黏（VMA）、及／或玻璃體黃斑牽引症（VMT）。Embodiment 58 is the method of any one of embodiments 38 to 57, wherein the ocular disease is selected from the group consisting of neovascular or ischemic uveitis, retinal vasculitis, vascular scars, and retinitis pigmentosa. , corneal neovascularization, iris neovascularization, neovascular glaucoma, postoperative glaucoma fibrosis, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, blood vessels Pannus, pterygoid, vascular retinopathy, diabetic retinopathy (DR, non-proliferative and proliferative DR) without diabetic macular edema (DME), diabetic retinopathy (DR, non-proliferative) with diabetic macular edema (DME) and proliferative DR), diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, post-retinal vein occlusion macular edema (RVO), retina Venous occlusion (RVO), central retinal vein occlusion (CRVO), branched retinal vein occlusion (BRVO), retinal angiomatous proliferation (RAP), polypoidal choroidal vasculopathy (PCV), vitreomacular adhesion (VMA), and/or vitreomacular traction (VMT).

實施態樣59為如實施態樣38至58中任一者之方法，其中該配方係以每眼約0.03至10 mg的劑量投予，較佳為每眼約3.0至6.0 mg，更佳地，該配方係以每眼約4 mg的劑量投予。Embodiment 59 is the method of any one of embodiments 38 to 58, wherein the formulation is administered at a dose of about 0.03 to 10 mg per eye, preferably about 3.0 to 6.0 mg per eye, more preferably , this formulation is administered at a dose of approximately 4 mg per eye.

實施態樣60為如實施態樣59之方法，其中該配方係以每眼約4 mg的劑量投予。Embodiment 60 is the method of embodiment 59, wherein the formulation is administered at a dose of about 4 mg per eye.

實施態樣61為一種治療一個體之眼部疾病的方法，該方法包含對該個體投予一包含以下之醫藥配方： a)   一濃度為40 mg/mL之融合蛋白； b)   25 mM之組胺酸； c)    190 mM之海藻酸、蔗糖、或甘露醇；以及 d)   0.03%之聚山梨糖醇酯20或聚山梨糖醇酯80， 其中該配方的pH值為約6.0。 Embodiment 61 is a method of treating an eye disease in an individual, the method comprising administering to the individual a pharmaceutical formulation including: a) A fusion protein with a concentration of 40 mg/mL; b) 25 mM histamine; c) 190 mM alginic acid, sucrose, or mannitol; and d) 0.03% polysorbate 20 or polysorbate 80, The pH of this formulation is approximately 6.0.

實施態樣62為如實施態樣61之方法，其中該融合蛋白係自N-端至C-端以如下順序包含： a)   一血管內皮生長因子受體（VEGFR）的細胞外結構域； b)   一人類免疫球蛋白G的Fc結構域；以及 c)    一結合整合蛋白之蛋白（integrin binding protein）或其片段。 Embodiment 62 is the method of embodiment 61, wherein the fusion protein includes in the following order from N-terminus to C-terminus: a) An extracellular domain of vascular endothelial growth factor receptor (VEGFR); b) an Fc domain of human immunoglobulin G; and c) An integrin binding protein or a fragment thereof.

實施態樣63為如實施態樣62之方法，其中該融合蛋白係包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO: 17、或SEQ ID NO: 18。Embodiment 63 is the method of embodiment 62, wherein the fusion protein comprises SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

實施態樣64為如實施態樣61至63中任一者之方法，其中該配方在-70℃、-20℃、及／或5℃下安定性維持至少24個月。Embodiment 64 is the method of any one of embodiments 61 to 63, wherein the formulation maintains stability at -70°C, -20°C, and/or 5°C for at least 24 months.

實施態樣65為如實施態樣61至64中任一者之方法，其中該配方在-70℃、-20℃、及／或2℃至8℃下（較佳在2℃至8℃下）至少6個月後係保持蛋白質的純度及活性。Embodiment 65 is a method as in any one of embodiments 61 to 64, wherein the formulation is at -70°C, -20°C, and/or 2°C to 8°C (preferably at 2°C to 8°C ) will maintain the purity and activity of the protein for at least 6 months.

實施態樣66為如實施態樣61至65中任一者之方法，其中該配方更包含一濃度為約10 mM至50 mM的鹽類。Embodiment 66 is the method of any one of embodiments 61 to 65, wherein the formulation further includes a salt at a concentration of about 10 mM to 50 mM.

實施態樣67為如實施態樣66之方法，其中該鹽類係選自氯化鈉、氯化鎂、氯化鈣、或氯化鉀。Embodiment 67 is the method of embodiment 66, wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, or potassium chloride.

實施態樣68為如實施態樣61至67中任一者之方法，其中該配方更包含至少一種胺基酸，該胺基酸的濃度為約10 mM至50 mM。Embodiment 68 is the method of any one of embodiments 61 to 67, wherein the formulation further includes at least one amino acid, and the concentration of the amino acid is about 10 mM to 50 mM.

實施態樣69為如實施態樣68之方法，其中該胺基酸係選自由以下所組成之群組：精胺酸、甲硫胺酸、脯胺酸、組胺酸、半胱胺酸、離胺酸、甘胺酸、天門冬胺酸、色胺酸、麩胺酸、及異白胺酸。Embodiment 69 is the method of embodiment 68, wherein the amino acid is selected from the group consisting of: arginine, methionine, proline, histidine, cysteine, Lysine, glycine, aspartic acid, tryptophan, glutamic acid, and isoleucine.

實施態樣70為如實施態樣61至69中任一者之方法，其中該眼部疾病係選自新生血管或缺血性眼色素層炎、視網膜血管炎、血管樣痕、色素性視網膜炎、角膜新生血管、虹膜新生血管、新生血管性青光眼、青光眼術後纖維化、增殖性玻璃體視網膜病變（PVR）、脈絡膜新生血管（CNV）、視神經盤新生血管、視網膜新生血管、玻璃體新生血管、血管翳、翼狀贅肉、血管性視網膜病變、無糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、有糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、糖尿病黃斑水腫（DME）、滲出性（濕性）及非滲出性（乾性）之老年性黃斑退化（AMD）、黃斑水腫、視網膜靜脈阻塞後黃斑水腫（RVO）、視網膜靜脈阻塞（RVO）、中心性視網膜靜脈阻塞（CRVO）、分支性視網膜靜脈阻塞（BRVO）、視網膜血管瘤狀增殖（RAP）、息肉狀脈絡膜血管病變（PCV）、玻璃體黃斑沾黏（VMA）、及／或玻璃體黃斑牽引症（VMT）。Embodiment 70 is the method of any one of embodiments 61 to 69, wherein the ocular disease is selected from the group consisting of neovascular or ischemic uveitis, retinal vasculitis, vascular scars, and retinitis pigmentosa. , corneal neovascularization, iris neovascularization, neovascular glaucoma, postoperative glaucoma fibrosis, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, blood vessels Pannus, pterygoid, vascular retinopathy, diabetic retinopathy (DR, non-proliferative and proliferative DR) without diabetic macular edema (DME), diabetic retinopathy (DR, non-proliferative) with diabetic macular edema (DME) and proliferative DR), diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, post-retinal vein occlusion macular edema (RVO), retina Venous occlusion (RVO), central retinal vein occlusion (CRVO), branched retinal vein occlusion (BRVO), retinal angiomatous proliferation (RAP), polypoidal choroidal vasculopathy (PCV), vitreomacular adhesion (VMA), and/or vitreomacular traction (VMT).

實施態樣71為如實施態樣61至70中任一者之方法，其中該配方係以每眼約0.03至10 mg的劑量投予，較佳為每眼約3.0至6.0 mg，更佳地，該配方係以每眼約4 mg的劑量投予。Embodiment 71 is the method of any one of embodiments 61 to 70, wherein the formulation is administered at a dose of about 0.03 to 10 mg per eye, preferably about 3.0 to 6.0 mg per eye, more preferably , this formulation is administered at a dose of approximately 4 mg per eye.

實施態樣72為如實施態樣71之方法，其中該配方係以每眼約4 mg的劑量投予。 實施例 Embodiment 72 is the method of embodiment 71, wherein the formulation is administered at a dose of about 4 mg per eye. Example

提供以下實施例來說明本發明，但本發明並不以此為限。本領域技藝人士將明瞭以下步驟可使用本領域具有通常知識者所知的方法進行修飾。 實施例 1. 融合蛋白 1 於恆河猴之雷射誘導脈絡膜新生血管模型的評估 The following examples are provided to illustrate the present invention, but the present invention is not limited thereto. It will be apparent to those skilled in the art that the following steps may be modified using methods known to those of ordinary skill in the art. Example 1. Evaluation of Fusion Protein 1 in the Laser-Induced Choroidal Neovascularization Model of Rhesus Monkeys

於脈絡膜新生血管（CNV）之猴子模型的眼睛中評估融合蛋白1（一種具有多重標的之免疫球蛋白G1 Fc–融合蛋白）。Fusion protein 1, a multitargeted immunoglobulin G1 Fc–fusion protein, was evaluated in the eyes of a monkey model of choroidal neovascularization (CNV).

於該CNV模型中，恆河猴（每組n＝4隻眼睛）係使用光凝固術（photocoagulation）對視網膜進行雷射灼傷，並且使脈絡膜新生血管發展21天（第-21天至第0天）。在第0天時，藉由單次玻璃體內（IVT）注射將空白載劑（vehicle）、每眼0.5mg之Lucentis®（0.5 mg／眼）、或融合蛋白1（0.6 mg、1.0 mg、1.9 mg／眼）注射至該猴子的眼睛。療效指標（efficacy endpoint）係於給藥前之基線（前2天（Day -2））、及注射後之第14天與第28天對所有組別進行評估。In this CNV model, rhesus monkeys (n = 4 eyes per group) were subjected to laser burns of the retina using photocoagulation, and choroidal neovascularization was allowed to develop for 21 days (day -21 to day 0 ). On day 0, vehicle, 0.5 mg of Lucentis® per eye (0.5 mg/eye), or fusion protein 1 (0.6 mg, 1.0 mg, 1.9 mg/eye) was injected into the monkey's eye. Efficacy endpoints were evaluated for all groups at baseline before dosing (Day -2), and on days 14 and 28 after injection.

於使用0.5mg之Lucentis®處理的眼睛中，平均滲漏區域分數相較於基線係分別在第14天與第28天下降了91%與92%（圖1）。相較於基線，平均視網膜厚度亦下降了117%與120%（圖2）。相較於使用空白對照處理之眼睛，Lucentis®係顯示統計上顯著的改善。In eyes treated with 0.5 mg of Lucentis®, the mean leakage area fraction decreased by 91% and 92% compared to baseline at days 14 and 28, respectively (Figure 1). Compared with baseline, mean retinal thickness also decreased by 117% and 120% (Figure 2). The Lucentis® system showed a statistically significant improvement compared to eyes treated with placebo.

相較於基線及試驗前的數值，融合蛋白1之全部三個劑量組別皆展現出雷射誘導視網膜厚度、及滲漏區域的恢復。平均滲漏區域在第14天與第28天分別下降了87%至89%與79%至90%（圖1）。平均視網膜厚度亦分別下降了98%至121%與104%至129%（圖2）。相較於使用空白對照處理之眼睛，融合蛋白1係於0.6 mg及1.0 mg的劑量在第14天與第28天時顯示出統計上顯著的改善。由於全部的劑量皆顯示相似的療效，並未觀察到融合蛋白1的劑量依存作用。All three dose groups of Fusion Protein 1 demonstrated laser-induced recovery of retinal thickness and leakage areas compared to baseline and pre-test values. The average leakage area decreased by 87% to 89% and 79% to 90% on days 14 and 28, respectively (Figure 1). The average retinal thickness also decreased by 98% to 121% and 104% to 129% respectively (Figure 2). Fusion protein 1 showed statistically significant improvement at doses of 0.6 mg and 1.0 mg on days 14 and 28 compared to placebo-treated eyes. As all doses showed similar efficacy, no dose-dependent effect of fusion protein 1 was observed.

使用Masson染色膠原蛋白（一種纖維化的前驅物）進行眼睛的組織學檢查，其係顯示融合蛋白1對於膠原蛋白厚度（眼纖維化）具有劑量依存性的減少，但此並未存在於空白載劑或Lucentis®處理的組別（圖3）。Histological examination of eyes using Masson staining for collagen, a precursor to fibrosis, showed a dose-dependent reduction in collagen thickness (ocular fibrosis) with fusion protein 1, but this was not present in the blank. agent or Lucentis® treated groups (Figure 3).

因此，每眼0.6、1.0、及1.9 mg之融合蛋白1的單次IVT注射係有效地在恆河猴之雷射誘導CNV模型中抑制血管滲漏、視網膜厚度、及纖維化。基於人類（4 mL）與猴子（2 mL）之間的玻璃體體積比，該些劑量相當於每眼1.2 mg、2 mg、及4 mg的人類同等劑量。此可作為融合蛋白1於治療視網膜疾病（例如糖尿病黃斑水腫（DME）及新生血管老年性黃斑退化（nAMD））之建議劑量的支持。此外，該數據係意味著相較於傳統的抗VEGF單一療法，融合蛋白1具有使病患受益的加乘作用。 實施例 2. 融合蛋白 1 於博萊黴素誘導之 C57BL/6 小鼠肺纖維化模型中的評估 Therefore, a single IVT injection of 0.6, 1.0, and 1.9 mg of fusion protein 1 per eye was effective in inhibiting vascular leakage, retinal thickness, and fibrosis in a laser-induced CNV model in rhesus monkeys. Based on the vitreous volume ratio between humans (4 mL) and monkeys (2 mL), these doses are equivalent to human equivalent doses of 1.2 mg, 2 mg, and 4 mg per eye. This supports the recommended dosage of fusion protein 1 for the treatment of retinal diseases such as diabetic macular edema (DME) and neovascular age-related macular degeneration (nAMD). Furthermore, the data imply that Fusion Protein 1 has an additive effect in benefiting patients compared to traditional anti-VEGF monotherapy. Example 2. Evaluation of Fusion Protein 1 in the Bleomycin-Induced C57BL/6 Mouse Pulmonary Fibrosis Model

於博萊黴素誘導之肺纖維化小鼠模型中評估融合蛋白1（靜脈投予39毫克／公斤（mg/kg））可能的抗纖維化活性。該些小鼠係使用PENNCENTURY™肺內噴霧器在第一天時以1.5 U/kg之博萊黴素處理。該些小鼠係自第1天至第21天每日靜脈投予融合蛋白1、尼達尼布乙磺酸鹽（Nintedanib ethanesulfonate，Nintedanib）或空白載劑（配方緩衝劑），且於第22天時進行犧牲。用50%三氯乙酸處理收集的肺組織放在冰上20分鐘從中沉澱出全部的蛋白質。樣品進行離心，並將沉澱物與1 mL之12N的氫氯酸（HCl）混合並在110℃下烘焙14至18小時，直到樣品燒焦且乾燥。藉由在室溫下溫育72小時將該樣品再次懸浮於2 mL之去離子水中，同時間歇地進行震盪。將反-4-羥基-L-脯胺酸（trans-4-hydroxy-L-proline；來源：美國Sigma）之標準品自0.5 mg/mL開始製備其序列稀釋。將500微升（µL）之1.4%氯氨T（於0.5M之醋酸鈉／10%之異丙醇中；來源：美國Fisher Sci）添加至200 µL之經震盪的樣品（或標準品）中，並於室溫下溫育20分鐘。然後，添加500 µL之Ehrlich溶液（於70%異丙醇／30%高氯酸中之1M的對二甲胺基苯甲醛（p-dimethylaminobenzaldehyde）；美國Fisher Sci），並混合及在65℃下溫育15分鐘。樣品達到室溫後，在550 nm下量測各樣品及標準品的吸光度，以及自羥脯胺酸（hydroxyproline）標準曲線計算肺羥脯胺酸的濃度（μg／肺）。脯胺酸為膠原蛋白的主要組分，其係用於確定纖維化形成的標誌物之一。The possible anti-fibrotic activity of fusion protein 1 (39 mg/kg administered intravenously) was evaluated in a mouse model of bleomycin-induced pulmonary fibrosis. The mice were treated with 1.5 U/kg bleomycin on day 1 using a PENNCENTURY™ intrapulmonary nebulizer. These mice were intravenously administered fusion protein 1, nintedanib ethanesulfonate (Nintedanib) or blank vehicle (formulation buffer) daily from day 1 to day 21, and on day 22 time to sacrifice. The collected lung tissue was treated with 50% trichloroacetic acid and placed on ice for 20 minutes to precipitate all proteins. The sample was centrifuged and the pellet was mixed with 1 mL of 12N hydrochloric acid (HCl) and baked at 110°C for 14 to 18 hours until the sample was charred and dry. The sample was resuspended in 2 mL of deionized water by incubating at room temperature for 72 hours while shaking intermittently. Prepare serial dilutions of trans-4-hydroxy-L-proline (trans-4-hydroxy-L-proline; source: Sigma, USA) starting from 0.5 mg/mL. Add 500 microliters (µL) of 1.4% Chloramine T (in 0.5M sodium acetate/10% isopropyl alcohol; source: Fisher Sci, USA) to 200 µL of shaken sample (or standard) , and incubate at room temperature for 20 minutes. Then, add 500 µL of Ehrlich's solution (1M p-dimethylaminobenzaldehyde in 70% isopropanol/30% perchloric acid; Fisher Sci, USA), mix and incubate at 65°C. Incubate for 15 minutes. After the samples reached room temperature, the absorbance of each sample and standard was measured at 550 nm, and the lung hydroxyproline concentration (μg/lung) was calculated from the hydroxyproline standard curve. Proline is a major component of collagen and is one of the markers used to determine the formation of fibrosis.

肺羥脯胺酸係在空白對照組中顯著地上升（p＜0.05）（圖4），此意味著成功誘導肺纖維化。相較於空白對照，融合蛋白1的多次投予係明顯地抑制了羥脯胺酸水平（p＜0.05），此表示融合蛋白1注射對於該些標誌物的加乘作用（圖4）。Nintedanib之連續口服暴露對於減少羥脯胺酸水平具有輕微影響（圖4）。Pulmonary hydroxyproline increased significantly in the blank control group (p<0.05) (Figure 4), which means that pulmonary fibrosis was successfully induced. Compared with the blank control, multiple administrations of fusion protein 1 significantly inhibited hydroxyproline levels (p<0.05), which indicated the additive effect of fusion protein 1 injection on these markers (Figure 4). Continuous oral exposure to nintedanib had a slight effect on reducing hydroxyproline levels (Figure 4).

因此，39 mg/kg之融合蛋白1每日一次靜脈投予21天係與肺羥脯胺酸的顯著下降相關，其中該肺羥脯胺酸為小鼠博萊黴素誘導肺纖維化的一種主要肺纖維化標誌物。 實施例 3. 用於玻璃體內注射之融合蛋白 1 的預調配 Thus, once-daily intravenous administration of fusion protein 1 at 39 mg/kg for 21 days was associated with a significant decrease in lung hydroxyproline, a component of bleomycin-induced pulmonary fibrosis in mice. Major Pulmonary Fibrosis Markers. Example 3. Preformulation of Fusion Protein 1 for Intravitreal Injection

進行融合蛋白1的預調配試驗以產生融合蛋白1的合適液體配方。該試驗之目的如下： 1.   評估使用所選緩衝劑之各種濃度的融合蛋白1（40 mg/mL及80 mg/mL）相容性及膠體安定性； 2.   評估短期儲存後在加速的條件（40℃、25℃、2至8℃）下品質屬性的改變； 3.   賦形劑選擇：滲壓劑、氯化鈉、多元醇（即，海藻糖、蔗糖、甘露醇、及山梨糖醇）及其他穩定劑（例如甲硫胺酸、精胺酸）； 4.   透過實驗設計（DoE）計畫來評估所選緩衝劑與潛在的穩定劑的相容性；以及 5.   確定凍乾保護劑的效果。 Preformulation trials of Fusion Protein 1 were performed to generate a suitable liquid formulation of Fusion Protein 1. The purpose of this test is as follows: 1. Evaluate the compatibility and colloidal stability of various concentrations of fusion protein 1 (40 mg/mL and 80 mg/mL) using the selected buffer; 2. Evaluate the changes in quality attributes under accelerated conditions (40°C, 25°C, 2 to 8°C) after short-term storage; 3. Excipient selection: osmotic agents, sodium chloride, polyols (i.e., trehalose, sucrose, mannitol, and sorbitol) and other stabilizers (e.g., methionine, arginine); 4. Evaluate the compatibility of selected buffers with potential stabilizers through a Design of Experiments (DoE) plan; and 5. Determine the effect of freeze-drying protective agent.

使用一實驗設計手段開始針對原料藥物的配方開發工作，以鑑定合適的緩衝劑、多元醇、界面活性劑、及其他穩定劑。在配方開發期間測試二種濃度（40及80 mg/mL）的融合蛋白1。在配方開發期間使用熱壓力及搖動與冷凍／解凍壓力。表2係統整在後續試驗中的成分及相關功效。 表2. 探討之配方統整（所篩選的成分） 項目 成分 功效 測試濃度 緩衝劑 檸檬酸 緩衝劑 25 mM，pH 5.5及pH 6.0 組胺酸 25 mM，pH 6.0及pH 6.5 磷酸鈉 25 mM，pH 6.5及pH 7.0 胺基酸 甲硫胺酸 緩衝劑 40 mM 精胺酸 40 mM 鹽類 氯化鈉 滲壓劑及緩衝用劑 40 mM 多元醇 蔗糖 滲壓劑及穩定劑 190 mM 甘露醇 190 mM 海藻糖 190 mM 山梨糖醇 190 mM 界面活性劑 聚山梨糖醇酯20 穩定劑 0.03% 聚山梨糖醇酯80 0.03% 於各種緩衝劑及 pH 值下之融合蛋白 1 的膠體安定性及相容性 Begin formulation development work on the drug substance using a design of experiments approach to identify appropriate buffers, polyols, surfactants, and other stabilizers. Two concentrations (40 and 80 mg/mL) of fusion protein 1 were tested during formulation development. Use heat pressure as well as shaking and freeze/thaw pressure during recipe development. Table 2. System components and related efficacy in subsequent trials. Table 2. Formula integration discussed (selected ingredients) Project Element effect Test concentration Buffer citric acid Buffer 25 mM, pH 5.5 and pH 6.0 Histidine 25 mM, pH 6.0 and pH 6.5 sodium phosphate 25 mM, pH 6.5 and pH 7.0 amino acids methionine Buffer 40mM Arginine 40mM Salts sodium chloride Osmotic agents and buffering agents 40mM Polyol sucrose Osmotic agents and stabilizers 190mM Mannitol 190mM Trehalose 190mM Sorbitol 190mM surfactant Polysorbate 20 Stabilizer 0.03% Polysorbate 80 0.03% Colloidal stability and compatibility of fusion protein 1 under various buffers and pH values

通常使用溶解度及擴散係數（D）來評估蛋白質–蛋白質交互作用，且亦可用來證明蛋白質的膠體安定性。膠體安定性為表示在分散後分子之長期完整性及其在溶液中對沉澱／析出之抗性的重要參數。此外，此方法可快速獲取緩衝劑對該分子的相容性。因此，對融合蛋白1使用可緩衝pH值之三種常用的眼科溶液緩衝劑，磷酸鈉（NaPi）、組胺酸（His）、及檸檬酸（Cit）（表3）。藉由粒徑排阻層析儀串聯高效液相層析儀（SEC-HPLC）（表4）、動態光散射（DLS）（表5）、及濁度（圖6）分析融合蛋白1的屬性。 表3. 探討之緩衝系統及對應的pH值範圍 緩衝劑 測試之緩衝劑 pH 值 25 mM磷酸鈉（磷酸鹽緩衝劑） 7.0 6.5 25 mM L-組胺酸／L-組胺酸鹽酸鹽（組胺酸緩衝劑） 6.5 6.0 25 mM檸檬酸／檸檬酸鈉（檸檬酸緩衝劑） 6.0 5.5 Solubility and diffusion coefficient (D) are commonly used to evaluate protein-protein interactions and can also be used to demonstrate the colloidal stability of proteins. Colloidal stability is an important parameter indicating the long-term integrity of molecules after dispersion and their resistance to precipitation/segregation in solution. In addition, this method allows rapid access to buffer compatibility for this molecule. Therefore, three common ophthalmic solution buffers that can buffer the pH, sodium phosphate (NaPi), histidine (His), and citric acid (Cit), were used for fusion protein 1 (Table 3). The properties of fusion protein 1 were analyzed by size exclusion chromatography tandem high performance liquid chromatography (SEC-HPLC) (Table 4), dynamic light scattering (DLS) (Table 5), and turbidity (Figure 6) . Table 3. Buffer systems discussed and corresponding pH ranges Buffer Test buffer pH value 25 mM sodium phosphate (phosphate buffer) 7.0 6.5 25 mM L-histamine/L-histamine hydrochloride (histamine buffer) 6.5 6.0 25 mM citric acid/sodium citrate (citrate buffer) 6.0 5.5

在磷酸鹽及檸檬酸緩衝劑中之融合蛋白1的擴散係數展現出對上升濃度的負相關，此表示蛋白質–蛋白質交互作用提升的可能性（圖5A、5B、5E、及5F）。相反地，觀察到在pH值為6.5及6.0之組胺酸緩衝劑中的融合蛋白1為排斥的蛋白質–蛋白質交互作用，且在擴散係數與融合蛋白1的各種濃度之間具有正相關（圖5C及5D）。因此，在組胺酸緩衝劑中融合蛋白1之排斥的蛋白質–蛋白質交互作用係表示融合蛋白1之有利的膠體安定性。The diffusion coefficient of fusion protein 1 in phosphate and citrate buffers exhibited an inverse correlation with increasing concentration, indicating an increased likelihood of protein–protein interactions (Figures 5A, 5B, 5E, and 5F). In contrast, an exclusive protein–protein interaction was observed for fusion protein 1 in histidine buffer at pH 6.5 and 6.0, with a positive correlation between the diffusion coefficient and various concentrations of fusion protein 1 (Fig. 5C and 5D). Therefore, the exclusive protein-protein interaction of Fusion Protein 1 in histidine buffer indicates the favorable colloidal stability of Fusion Protein 1.

在將融合蛋白1的濃度提升到超過40 mg/mL至60及80 mg/mL時，所有的配方（即使是組胺酸緩衝劑配方）皆檢測到下降的擴散係數。對於組胺酸緩衝劑配方，此表示一旦濃度提升到超過40 mg/mL就有可能發生初步聚合。基於SEC-HPLC的結果，對於所有的緩衝劑及pH值（除了pH值為6.5之組胺酸緩衝劑以外）而言，調配為80 mg/mL的蛋白質顯示在緩衝劑中較低的純度、以及較大的高分子量聚合物（表4）。相較於40 mg/mL的樣品，在所有的80 mg/mL配方中皆檢測到在顯微鏡下可見的粒子（subvisible particle）增加（100至1,000 nm，透過DLS檢測）（表5）。在40及80 mg/mL樣品之間的濁度沒有顯著差異（圖6A及6B）。綜合前述，透過DLS分析之粒子大小顯示在自40至80 mg/mL的濃度提升時，所有緩衝劑的在顯微鏡下可見的粒子皆提升，透過SEC-HPLC檢測到所有的配方（除了pH 6.5的組胺酸緩衝劑以外）其高分子量物質的聚合物（HMW）皆有輕微的增加，以及觀察到任意配方的濁度都沒有顯著提升。 表4. 在不同緩衝劑中融合蛋白1的SEC-HPLC純度及UV濃度結果 樣品 A280 的濃度（ mg/mL ） SEC-HPLC （ % ） 預期 實際測量 HMW a 主要波峰 b 對照組1 # 40 37.7 0.66 99.34 25 mM檸檬酸緩衝劑（pH 5.5） 40 45.2 0.55 99.45 80 76.9 0.77 99.23 25 mM檸檬酸緩衝劑（pH 6.0） 40 43.0 0.61 99.39 80 73.8 0.79 99.21 25 mM組胺酸緩衝劑（pH 6.0） 40 42.8 0.48 99.52 80 79.7 0.52 99.48 25 mM組胺酸緩衝劑（pH 6.5） 40 42.6 0.55 99.45 80 74.1 0.44 99.56 25 mM磷酸鹽緩衝劑（pH 6.5） 40 38.5 0.60 99.40 80 81.3 1.01 98.99 25 mM磷酸鹽緩衝劑（pH 7.0） 40 44.7 0.69 99.31 80 83.4 0.98 99.02 #對照組1係以25 mM組胺酸、6%蔗糖、20 mM 氯化鈉、0.03% 聚山梨糖醇酯20（pH 6.0）調配。 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 表5. 藉由動態光散射分析在不同緩衝劑中在顯微鏡下可見的粒子分布 緩衝劑 pH 值 濃度 (mg/mL) 第一粒子的平均半徑 (0.1-10 nm) %Pd 第二粒子的平均半徑 (10-100 nm) %Pd 第三粒子的平均半徑 (100-1000 nm) %Pd 磷酸鹽 7.0 40 8.86 22.50 - - - - 80 8.93 13.09 - - 364.12 10.88 6.5 40 7.85 15.14 - - - - 80 9.35 18.08 - - 222.78 16.76 組胺酸 6.5 40 4.86 54.30 - - - - 80 4.52 27.13 25.6 18.32 475.84 19.79 6.0 40 3.68 7.64 - - 213.06 6.28 80 3.40 5.40 52.19 8.50 799.23 11.84 檸檬酸 6.0 40 8.80 21.22 - - - - 80 9.78 17.64 - - 322.11 11.93 5.5 40 8.60 16.05 - - - - 80 10.16 14.83 - - 305.76 13.83 %Pd＝多分散性的百分比（percent polydispersity） 緩衝劑的選擇與融合蛋白 1 屬性的確定 As the concentration of fusion protein 1 was increased beyond 40 mg/mL to 60 and 80 mg/mL, a decrease in diffusion coefficient was detected in all formulations, even the histidine buffer formulation. For histidine buffer formulations, this means that initial polymerization may occur once the concentration is increased above 40 mg/mL. Based on the SEC-HPLC results, for all buffers and pH values (except histidine buffer at pH 6.5), the protein formulated at 80 mg/mL showed lower purity in the buffer, and larger high molecular weight polymers (Table 4). An increase in microscopically visible particles (100 to 1,000 nm, detected by DLS) was detected in all 80 mg/mL formulations compared to the 40 mg/mL sample (Table 5). There was no significant difference in turbidity between the 40 and 80 mg/mL samples (Figures 6A and 6B). Based on the above, particle size analysis by DLS showed that microscopically visible particles increased for all buffers as the concentration increased from 40 to 80 mg/mL, as detected by SEC-HPLC for all formulations (except pH 6.5). There was a slight increase in the polymer of high molecular weight substances (HMW) except for histidine buffer, and no significant increase in turbidity was observed in any formulation. Table 4. SEC-HPLC purity and UV concentration results of fusion protein 1 in different buffers sample Concentration of A280 ( mg/mL ) SEC-HPLC ( % ) expected actual measurement hMW main peak b Control group 1 # 40 37.7 0.66 99.34 25 mM citrate buffer (pH 5.5) 40 45.2 0.55 99.45 80 76.9 0.77 99.23 25 mM citrate buffer (pH 6.0) 40 43.0 0.61 99.39 80 73.8 0.79 99.21 25 mM Histidine Buffer (pH 6.0) 40 42.8 0.48 99.52 80 79.7 0.52 99.48 25 mM Histidine Buffer (pH 6.5) 40 42.6 0.55 99.45 80 74.1 0.44 99.56 25 mM phosphate buffer (pH 6.5) 40 38.5 0.60 99.40 80 81.3 1.01 98.99 25 mM phosphate buffer (pH 7.0) 40 44.7 0.69 99.31 80 83.4 0.98 99.02 #Control 1 is prepared with 25 mM histidine, 6% sucrose, 20 mM sodium chloride, and 0.03% polysorbate 20 (pH 6.0). a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. Table 5. Microscopic particle distribution in different buffers analyzed by dynamic light scattering Buffer pH value Concentration (mg/mL) Average radius of the first particle (0.1-10 nm) %Pd Average radius of the second particle (10-100 nm) %Pd The average radius of the third particle (100-1000 nm) %Pd Phosphate 7.0 40 8.86 22.50 - - - - 80 8.93 13.09 - - 364.12 10.88 6.5 40 7.85 15.14 - - - - 80 9.35 18.08 - - 222.78 16.76 Histidine 6.5 40 4.86 54.30 - - - - 80 4.52 27.13 25.6 18.32 475.84 19.79 6.0 40 3.68 7.64 - - 213.06 6.28 80 3.40 5.40 52.19 8.50 799.23 11.84 citric acid 6.0 40 8.80 21.22 - - - - 80 9.78 17.64 - - 322.11 11.93 5.5 40 8.60 16.05 - - - - 80 10.16 14.83 - - 305.76 13.83 %Pd=percent polydispersity Selection of buffers and determination of properties of fusion protein 1

測試調配於25 mM檸檬酸或組胺酸緩衝劑（pH 6.0）中之40及80 mg/mL融合蛋白1的可行性與加速的安定性。對於40及80 mg/mL二種濃度而言，儲存在2℃至8℃下於檸檬酸及組胺酸中之融合蛋白1的非聚合（二聚體形式）的量顯示在儲存時無明顯變化（表6）。然而，於40℃的加速條件下，80 mg/mL融合蛋白1的主要波峰係在28天後於檸檬酸緩衝劑中下降了約70%（表7）。相比而言，當以組胺酸緩衝劑調配時，融合蛋白1的含量係下降約50%。而且，相較於沒有下降的組胺酸緩衝劑，80 mg/mL融合蛋白1的蛋白質濃度係於檸檬酸緩衝劑中下降29%。因此，在組胺酸緩衝劑中的融合蛋白1在pH 6.0下係比在檸檬酸緩衝劑中更加安定。 表6. 在2℃至8℃下儲存28天之融合蛋白1的蛋白質含量、純度、及聚合物變化 緩衝劑（ pH 值） 預期濃度 （ mg/mL ） 時間點 ( 天數 ) 實際測量濃度（ mg/mL ） SEC-HPLC （ % ） HMW a 主要波峰 b 25 mM 檸檬酸 （pH 6.0） 40 0 41.2 4.15 95.85 4 33.5 4.22 95.78 7 36.0 4.43 95.57 14 36.3 4.46 95.54 28 35.5 4.62 95.35 80 0 79.3 4.32 95.69 4 59.1 4.51 95.49 7 65.7 4.61 95.39 14 72.4 4.89 95.11 28 61.7 5.13 94.87 25 mM 組胺酸 （pH 6.0） 40 0 38.5 4.07 95.93 4 35.3 4.28 95.72 7 35.7 4.39 95.61 14 37.6 4.59 95.41 28 37.0 4.92 95.08 80 0 78.5 4.22 95.79 4 51.3 4.47 95.53 7 53.1 4.73 95.27 14 63.8 5.00 95.00 28 60.5 5.40 94.60 藉由在A 280下的UV吸光值（濃度）及SEC-HPLC分析樣品。 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 表7. 在40℃下28天之融合蛋白1的蛋白質含量、純度、及聚合物變化 樣品 預期濃度 (mg/mL) 時間點 ( 天數 ) 實際測量 濃度 (mg/mL) SEC-HPLC （ % ） HHMW # HMW a 主要波峰 b LMW* 25 mM 檸檬酸 (pH 6.0) 40 0 41.2 - 4.15 95.85 - 4 33.9 10.02 14.94 75.04 - 7 30.3 15.60 20.25 63.02 1.13 14 37.4 28.78 21.34 47.67 2.21 28 39.4 35.26 22.87 38.89 2.98 80 0 79.3 - 4.32 95.69 - 4 56.4 18.36 18.30 62.63 0.71 7 62.2 28.11 20.03 50.56 1.30 14 69.3 40.47 21.45 36.11 1.96 28 56.2 48.56 19.34 29.37 2.73 25 mM 組胺酸 (pH 6.0) 40 0 38.5 - 4.07 95.93 - 4 35.3 1.82 7.84 90.34 - 7 35.7 1.44 11.04 87.53 - 14 38.9 4.68 13.35 80.87 1.10 28 36.8 9.01 18.79 70.90 1.31 80 0 78.5 - 4.22 95.79 - 4 61.7 3.85 11.22 84.93 - 7 67.8 5.73 14.42 79.33 0.52 14 81.2 11.32 20.12 67.46 1.10 28 78.5 25.02 22.69 50.32 1.97 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 #HHMW：更高分子量物質的聚合物，其係指具有滯留時間為7.6±0.1分鐘的波峰。 *LMW：低分子量物質，滯留時間為13.5±0.1分鐘 「-」：未偵測到 The feasibility and accelerated stability of 40 and 80 mg/mL fusion protein 1 in 25 mM citric acid or histidine buffer (pH 6.0) were tested. For both concentrations of 40 and 80 mg/mL, the amount of non-polymerized (dimeric form) of fusion protein 1 stored in citric acid and histidine at 2°C to 8°C showed no significant change during storage. changes (Table 6). However, under accelerated conditions at 40°C, the main peak of 80 mg/mL fusion protein 1 decreased by approximately 70% in citrate buffer after 28 days (Table 7). In comparison, when formulated with histidine buffer, the content of fusion protein 1 decreased by approximately 50%. Furthermore, the protein concentration of 80 mg/mL fusion protein 1 decreased by 29% in citrate buffer compared to no decrease in histamine buffer. Therefore, fusion protein 1 in histidine buffer is more stable at pH 6.0 than in citrate buffer. Table 6. Protein content, purity, and polymer changes of fusion protein 1 stored at 2°C to 8°C for 28 days Buffer ( pH value) Expected concentration ( mg/mL ) Time point ( number of days ) Actual measured concentration ( mg/mL ) SEC-HPLC ( % ) hMW main peak b 25 mM citric acid (pH 6.0) 40 0 41.2 4.15 95.85 4 33.5 4.22 95.78 7 36.0 4.43 95.57 14 36.3 4.46 95.54 28 35.5 4.62 95.35 80 0 79.3 4.32 95.69 4 59.1 4.51 95.49 7 65.7 4.61 95.39 14 72.4 4.89 95.11 28 61.7 5.13 94.87 25 mM histidine (pH 6.0) 40 0 38.5 4.07 95.93 4 35.3 4.28 95.72 7 35.7 4.39 95.61 14 37.6 4.59 95.41 28 37.0 4.92 95.08 80 0 78.5 4.22 95.79 4 51.3 4.47 95.53 7 53.1 4.73 95.27 14 63.8 5.00 95.00 28 60.5 5.40 94.60 Samples were analyzed by UV absorbance (concentration) at A 280 and SEC-HPLC. a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. Table 7. Protein content, purity, and polymer changes of fusion protein 1 at 40°C for 28 days sample Expected concentration (mg/mL) Time point ( number of days ) Actual measured concentration (mg/mL) SEC-HPLC ( % ) HHMW # hMW main peak b LMW* 25 mM citric acid (pH 6.0) 40 0 41.2 - 4.15 95.85 - 4 33.9 10.02 14.94 75.04 - 7 30.3 15.60 20.25 63.02 1.13 14 37.4 28.78 21.34 47.67 2.21 28 39.4 35.26 22.87 38.89 2.98 80 0 79.3 - 4.32 95.69 - 4 56.4 18.36 18.30 62.63 0.71 7 62.2 28.11 20.03 50.56 1.30 14 69.3 40.47 21.45 36.11 1.96 28 56.2 48.56 19.34 29.37 2.73 25 mM histidine (pH 6.0) 40 0 38.5 - 4.07 95.93 - 4 35.3 1.82 7.84 90.34 - 7 35.7 1.44 11.04 87.53 - 14 38.9 4.68 13.35 80.87 1.10 28 36.8 9.01 18.79 70.90 1.31 80 0 78.5 - 4.22 95.79 - 4 61.7 3.85 11.22 84.93 - 7 67.8 5.73 14.42 79.33 0.52 14 81.2 11.32 20.12 67.46 1.10 28 78.5 25.02 22.69 50.32 1.97 a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. # HHMW: Polymer of higher molecular weight species, which refers to a wave peak with a retention time of 7.6 ± 0.1 minutes. * LMW: low molecular weight substance, retention time is 13.5±0.1 minutes "-": not detected

此外，在40℃溫育後，於檸檬酸緩衝劑中的融合蛋白1係在非還原SDS-PAGE中約250 kDa的位置顯示出主要條帶（band）強度減少較多（圖7A至7E）。融合蛋白1的蛋白質降解特性使得產物降解片段及高分子量聚合物的條帶隨著時間增加。透過還原SDS-PAGE的分析係顯示在約75 kDa之主要條帶強度減少且片段的條帶增加（圖8A至8E）。總體而言，蛋白質純度及完整性探討係表示相較於檸檬酸緩衝劑，融合蛋白1在pH 6.0之25 mM組胺酸緩衝劑中展現出較好的安定性。In addition, after incubation at 40°C, the fusion protein 1 line in citrate buffer showed a greater reduction in the intensity of the main band at approximately 250 kDa in non-reducing SDS-PAGE (Figures 7A to 7E) . The protein degradation characteristics of fusion protein 1 cause the bands of product degradation fragments and high molecular weight polymers to increase over time. Analysis by reducing SDS-PAGE showed a decrease in intensity of the major band at approximately 75 kDa and an increase in fragmented bands (Figures 8A to 8E). Overall, the protein purity and integrity studies indicate that fusion protein 1 exhibits better stability in 25 mM histidine buffer at pH 6.0 compared to citrate buffer.

亦對短期儲存的加速冷凍／解凍條件進行評估，以測試融合蛋白1樣品的品質屬性變化。將樣品在40℃下溫育4天。一個-70℃與室溫（RT）之冷凍–解凍循環測試顯示在沒有多元醇保護下融合蛋白1仍為安定的（表8）。 表8. 在一個冷凍–解凍循環後融合蛋白1的蛋白質純度及聚合物變化 樣品 預期濃度 （ mg/mL ） 處理 SEC-HPLC （ % ） HMW a 主要波峰 b 25 mM檸檬酸 （pH 6.0） 40 前 4.15 95.85 後 4.13 95.76 80 前 4.32 95.69 後 4.14 95.56 25 mM組胺酸 （pH 6.0） 40 前 4.07 95.93 後 4.24 95.87 80 前 4.22 95.79 後 4.44 95.86 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 賦形劑篩選 Accelerated freezing/thawing conditions for short-term storage were also evaluated to test changes in quality attributes of Fusion Protein 1 samples. Samples were incubated at 40°C for 4 days. A freeze-thaw cycle test at -70°C and room temperature (RT) showed that fusion protein 1 was stable without polyol protection (Table 8). Table 8. Protein purity and polymer changes of fusion protein 1 after one freeze–thaw cycle sample Expected concentration ( mg/mL ) handle SEC-HPLC ( % ) hMW main peak b 25 mM citric acid (pH 6.0) 40 forward 4.15 95.85 back 4.13 95.76 80 forward 4.32 95.69 back 4.14 95.56 25 mM histidine (pH 6.0) 40 forward 4.07 95.93 back 4.24 95.87 80 forward 4.22 95.79 back 4.44 95.86 a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. Excipient screening

為了探討賦形劑的適用性，進行一前導研究以藉由SEC-HPLC評估經調配之融合蛋白1樣品的主要波峰純度變化。基於上述研究，選擇25 mM組胺酸緩衝劑（pH 6.0）進行後續的賦形劑篩選測試。所包括之三種型態的添加劑為多元醇、鹽類、及胺基酸。評估係經設計，以七種條件並透過在40℃的加速條件下4天進行測試（表9）。 表9. 所篩選的賦形劑 # 測試 所測試的穩定劑（ 150 mM ） 40 mM 氯化鈉 胺基酸（ 40 mM ） 海藻糖 甘露醇 蔗糖 山梨 糖醇 精胺酸 甲硫胺酸 0 -- -- -- -- -- -- -- 1 + -- -- -- -- -- -- 2 -- + -- -- -- -- -- 3 -- -- + -- -- -- -- 4 -- -- -- + -- -- -- 5 -- -- -- -- + -- -- 6 + -- -- -- + -- -- 7 -- + -- -- + -- -- 8 -- -- + -- + -- -- 9 -- -- -- + + -- -- 10 -- -- + -- -- + -- 11 -- -- + -- -- -- + 註：融合蛋白1係經調配於25 mM組胺酸緩衝劑（pH 6.0）中，並使用各種不同的多元醇（#1至#4）測試、或與40 mM 氯化鈉（#6至#9）組合。測試#5＝在沒有多元醇或胺基酸情況下的40 mM 氯化鈉。測試#10及#11＝蔗糖與2種胺基酸的組合。 To investigate the suitability of the excipients, a pilot study was conducted to evaluate the major peak purity changes of formulated Fusion Protein 1 samples by SEC-HPLC. Based on the above studies, 25 mM histidine buffer (pH 6.0) was selected for subsequent excipient screening tests. The three types of additives included are polyols, salts, and amino acids. The evaluation system was designed to be tested under seven conditions and through accelerated conditions at 40°C for 4 days (Table 9). Table 9. Screened excipients #test _ Stabilizer tested ( 150 mM ) 40 mM sodium chloride Amino acids ( 40 mM ) Trehalose Mannitol sucrose Sorbitol _ Arginine methionine 0 -- -- -- -- -- -- -- 1 + -- -- -- -- -- -- 2 -- + -- -- -- -- -- 3 -- -- + -- -- -- -- 4 -- -- -- + -- -- -- 5 -- -- -- -- + -- -- 6 + -- -- -- + -- -- 7 -- + -- -- + -- -- 8 -- -- + -- + -- -- 9 -- -- -- + + -- -- 10 -- -- + -- -- + -- 11 -- -- + -- -- -- + NOTE: Fusion protein 1 was formulated in 25 mM histidine buffer (pH 6.0) and tested with various polyols (#1 to #4) or with 40 mM sodium chloride (#6 to # 9) Combination. Test #5 = 40 mM sodium chloride without polyol or amino acid. Tests #10 and #11 = Combination of sucrose and 2 amino acids.

在使用各種多元醇（表10，測試#1至#4）、海藻糖與氯化鈉的組合（測試#6）、及蔗糖與甲硫胺酸的組合（測試#11）進行調配時，主要波峰純度≧90%。 表10. 在40℃下4天後添加有各種賦形劑之融合蛋白1的純度 # 測試 SEC-HPLC （ % ） HHMW # HMW a 主要波峰 b LMW * 處理前 - 4.07 95.93 - 0 2.11 8.35 89.21 0.33 1 1.59 6.81 91.23 0.37 2 1.26 5.91 92.66 0.18 3 1.74 7.21 90.74 0.31 4 1.84 7.48 90.31 0.37 5 2.09 7.99 89.63 0.30 6 1.64 6.89 90.93 0.54 7 2.44 8.23 88.94 0.40 8 2.56 8.71 88.43 0.30 9 2.26 8.30 89.08 0.36 10 9.57 16.38 73.88 0.17 11 1.52 6.32 91.81 0.36 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 #HHMW：更高分子量物質的聚合物，其係指具有滯留時間為7.6±0.1分鐘的波峰。 *LMW：低分子量物質，滯留時間為13.5±0.1分鐘 「-」：未偵測到 When formulating with various polyols (Table 10, Tests #1 to #4), the combination of trehalose and sodium chloride (Test #6), and the combination of sucrose and methionine (Test #11), the main Peak purity ≧90%. Table 10. Purity of fusion protein 1 with various excipients added after 4 days at 40°C #test _ SEC-HPLC ( % ) HHMW # hMW main peak b LMW * Before processing - 4.07 95.93 - 0 2.11 8.35 89.21 0.33 1 1.59 6.81 91.23 0.37 2 1.26 5.91 92.66 0.18 3 1.74 7.21 90.74 0.31 4 1.84 7.48 90.31 0.37 5 2.09 7.99 89.63 0.30 6 1.64 6.89 90.93 0.54 7 2.44 8.23 88.94 0.40 8 2.56 8.71 88.43 0.30 9 2.26 8.30 89.08 0.36 10 9.57 16.38 73.88 0.17 11 1.52 6.32 91.81 0.36 a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. # HHMW: Polymer of higher molecular weight species, which refers to a wave peak with a retention time of 7.6 ± 0.1 minutes. * LMW: low molecular weight substance, retention time is 13.5±0.1 minutes "-": not detected

相較於沒有氯化鈉的測試，留有多元醇與氯化鈉的組合輕微提升聚合物的%（表10，測試#6、#7、#8、#9）。於精胺酸與蔗糖的組合中，在加速條件下的儲存後，純度下降至73.88%且聚合物超過16%（表10，測試#10）。因此，選擇海藻糖、蔗糖、及甘露醇進行實驗設計的探討。 透過 DoE 最佳化組合物的候選配方 The combination of leaving polyol and sodium chloride slightly increased the % of polymer compared to the tests without sodium chloride (Table 10, tests #6, #7, #8, #9). In the combination of arginine and sucrose, after storage under accelerated conditions, the purity dropped to 73.88% and the polymer exceeded 16% (Table 10, Test #10). Therefore, trehalose, sucrose, and mannitol were selected for discussion of experimental design. Candidate formulations of compositions optimized through DoE

為了探討源自候選穩定劑的顯著性及明確氯化鈉與各個多元醇的交叉作用，藉由如表11所示之計畫的DoE測試所選的多元醇及各種濃度的氯化鈉。將加速試驗條件與分析統整於表12。根據SEC-HPLC的結果（表13），具有200 mM多元醇／糖類（尤其是海藻糖及蔗糖）的25 mM組胺酸（His）緩衝劑（pH 6.0）提供了融合蛋白1對於熱壓力的最佳保護，以維持融合蛋白1的高百分比純度。 表11. 使用各種濃度之成分測試80 mg/mL融合蛋白1的DoE計畫 測試 # 多元醇 * （ mM ） 氯化鈉（ mM ） 1 112.5 25 2 200 50 3 112.5 25 4 200 0 5 200 50 6 200 0 7 25 0 8 112.5 25 9 25 0 10 25 50 11 25 50 *表示多元醇為海藻糖、蔗糖、或甘露醇，且各測試係個別地具有或不具有氯化鈉。 表12. 加速條件測試 儲存條件 時間點 分析 40 ± 2 ℃／75 ± 5% RH 第0、4、及7天 純度 滲透度 25 ± 2 ℃／60 ± 5% RH 第0、4、及7天 5 ± 3°C 第0、4、及7天 In order to explore the significance derived from the candidate stabilizers and to clarify the cross-talk between sodium chloride and each polyol, the selected polyols and various concentrations of sodium chloride were tested through a planned DoE as shown in Table 11. The accelerated test conditions and analysis are summarized in Table 12. According to the results of SEC-HPLC (Table 13), 25 mM histidine (His) buffer (pH 6.0) with 200 mM polyols/saccharides (especially trehalose and sucrose) provided fusion protein 1 resistance to thermal stress. Optimal protection to maintain high percent purity of fusion protein 1. Table 11. DoE plan for testing 80 mg/mL fusion protein 1 using various concentrations of ingredients. test # Polyol * ( mM ) Sodium chloride ( mM ) 1 112.5 25 2 200 50 3 112.5 25 4 200 0 5 200 50 6 200 0 7 25 0 8 112.5 25 9 25 0 10 25 50 11 25 50 *Indicates the polyol was trehalose, sucrose, or mannitol, and each test system was individually with or without sodium chloride. Table 12. Accelerated condition test Storage conditions time point analyze 40 ± 2 ℃ / 75 ± 5% RH Days 0, 4, and 7 Purity Penetration 25 ± 2 ℃ / 60 ± 5% RH Days 0, 4, and 7 5±3°C Days 0, 4, and 7

在80 mg/mL融合蛋白1於40°C下儲存4及7天（表12）後，調配有200 mM多元醇之25 mM組胺酸緩衝劑（pH 6.0）使融合蛋白1對於熱壓力係安定的，維持相對純度＞80%，且提供融合蛋白1之最佳的結果純度（表13之測試#4及#6）。具有多元醇與50mM 氯化鈉的樣品（測試#2及#5）皆顯示低於僅具有多元醇之樣品的純度（測試#4及#6）。相較於具有25 mM多元醇及50 mM 氯化鈉的樣品（測試#10及#11），亦在具有25 mM多元醇且不具有氯化鈉的樣品（測試#7及#9）觀察到此效果。該些組合之間的差異在4°C及25°C下並無不同（未揭示數據）。 表13. 於40°C下儲存之融合蛋白1的純度及滲透度分析 樣品 SEC-HPLC 分析之主要波峰的 # 相對純度（ % ） 滲透度 （ mOsm/kg ） 4 天 7 天 海藻糖 測試1 86.70 78.32 256 測試2 86.83 80.97 451 測試3 86.49 79.29 247 測試 4 90.23 85.48 312 測試5 87.30 81.05 432 測試 6 90.31 85.41 304 測試7 86.50 79.85 77 測試8 86.51 79.30 253 測試9 86.51 79.83 73 測試10 82.22 72.47 195 測試11 81.87 72.32 201 甘露醇 測試1 85.30 77.47 246 測試2 85.92 78.27 415 測試3 86.60 78.98 231 測試 4 89.01 83.62 300 測試5 85.95 78.49 417 測試 6 89.41 83.93 295 測試7 86.34 79.15 77 測試8 85.49 77.51 239 測試9 86.70 79.88 75 測試10 81.22 71.36 193 測試11 81.42 71.74 186 蔗糖 測試1 86.92 79.71 256 測試2 87.51 80.38 431 測試3 86.64 78.89 249 測試 4 90.10 85.21 301 測試5 87.62 81.60 431 測試 6 90.43 85.59 325 測試7 86.88 79.64 77 測試8 86.84 79.17 248 測試9 87.12 79.89 75 測試10 82.13 72.52 194 測試11 82.43 72.61 195 #表示使用T0樣品（基線）標準化之在40°C下樣品純度的相對純度。 After storage of 80 mg/mL fusion protein 1 at 40°C for 4 and 7 days (Table 12), 25 mM histidine buffer (pH 6.0) with 200 mM polyol was prepared to render fusion protein 1 resistant to thermal stress. Stable, maintaining relative purity >80%, and providing the best resulting purity of Fusion Protein 1 (Tests #4 and #6 in Table 13). Samples with polyol and 50mM sodium chloride (tests #2 and #5) both showed lower purity than samples with polyol alone (tests #4 and #6). Also observed in samples with 25 mM polyol and no sodium chloride (tests #7 and #9) compared to samples with 25 mM polyol and 50 mM sodium chloride (tests #10 and #11) This effect. The differences between the combinations were not different at 4°C and 25°C (data not disclosed). Table 13. Purity and Permeability Analysis of Fusion Protein 1 Stored at 40°C sample #Relative purity of main peaks in SEC-HPLC analysis ( % ) Penetration ( mOsm/kg ) 4 days 7 days Trehalose Test 1 86.70 78.32 256 Test 2 86.83 80.97 451 Test 3 86.49 79.29 247 Test 4 90.23 85.48 312 Test 5 87.30 81.05 432 Test 6 90.31 85.41 304 Test 7 86.50 79.85 77 Test 8 86.51 79.30 253 Test 9 86.51 79.83 73 Test 10 82.22 72.47 195 Test 11 81.87 72.32 201 Mannitol Test 1 85.30 77.47 246 Test 2 85.92 78.27 415 Test 3 86.60 78.98 231 Test 4 89.01 83.62 300 Test 5 85.95 78.49 417 Test 6 89.41 83.93 295 Test 7 86.34 79.15 77 Test 8 85.49 77.51 239 Test 9 86.70 79.88 75 Test 10 81.22 71.36 193 Test 11 81.42 71.74 186 sucrose Test 1 86.92 79.71 256 Test 2 87.51 80.38 431 Test 3 86.64 78.89 249 Test 4 90.10 85.21 301 Test 5 87.62 81.60 431 Test 6 90.43 85.59 325 Test 7 86.88 79.64 77 Test 8 86.84 79.17 248 Test 9 87.12 79.89 75 Test 10 82.13 72.52 194 Test 11 82.43 72.61 195 # represents the relative purity of the sample at 40°C normalized using the T0 sample (baseline).

雖然具有200 mM多元醇之較高組胺酸緩衝劑維持了蛋白質的品質屬性，必須要將滲透度維持在280-310 mOsm/kg的眼部生理範圍。因此，設定300 mOsm/kg作為目標DoE統計計算。選擇與80 mg/mL融合蛋白調配之成分組合物為25 mM組胺酸緩衝劑（pH 6.0）、190 mM的海藻糖、蔗糖、或海藻糖與蔗糖的組合。 確定配方可能的賦形劑組成 Although the higher histidine buffer with 200 mM polyol maintains the quality attributes of the protein, it is necessary to maintain permeability within the physiological ocular range of 280-310 mOsm/kg. Therefore, 300 mOsm/kg was set as the target DoE statistics calculation. The ingredient composition selected to be formulated with 80 mg/mL fusion protein is 25 mM histidine buffer (pH 6.0), 190 mM trehalose, sucrose, or a combination of trehalose and sucrose. Determine the possible excipient composition of the formulation

為了評估在組合配方中之合適成分的相容性，進一步測試將80 mg/mL融合蛋白1用於4種候選配方中。於此試驗中，一併添加0.03%之聚山梨糖醇酯20或聚山梨糖醇酯80與海藻糖或蔗糖，以確定在使用加速條件處理一週及／或一個月、各種介於-20°C及室溫（RT）間之冷凍–解凍循環、以及搖動24及48小時之後（表14A至14B）融合蛋白1的屬性。並行測試了該容器的短期相容性。 表14A. 在4°C及40°C下28天進行分析且添加有凍乾保護劑的候選配方 測試 容器 屬性 時間點 條件 T0 D4 D7 D14 D28 熱 2R第I型硼矽酸玻璃瓶 ․純度 ․活性 ․在顯微鏡下可見的粒子 ․分子完整性 ․分子含量 4°C 基線 - - V V 40°C V V - - V＝測試時間點；T＝時間；D＝天。 表14B. 在冷凍／解凍或搖動下進行測試且添加有凍乾保護劑的候選配方 測試 容器 屬性 處理方式 循環次數／時間點 冷凍–解凍 0.2 mL 冷凍瓶 ․純度 ․活性 ․在顯微鏡下可見的粒子 ․分子完整性 ․分子含量 ․冷凍：放置-20°C下23小時 ․解凍：放置25°C下1小時 3次 6次 搖動 2R第I型硼矽酸玻璃瓶 ․搖動：在25°C下以220 rpm 速度搖晃 ․空白對照：靜置在25°C下 24小時 48小時 To evaluate the compatibility of suitable ingredients in combination formulations, 80 mg/mL Fusion Protein 1 was further tested in 4 candidate formulations. In this test, 0.03% polysorbate 20 or polysorbate 80 was added together with trehalose or sucrose to determine the effects of accelerated treatment at -20° for one week and/or one month. Properties of fusion protein 1 after freeze-thaw cycles between C and room temperature (RT), and after 24 and 48 hours of shaking (Tables 14A to 14B). The container was tested in parallel for short-term compatibility. Table 14A. Candidate formulations with added lyoprotectant analyzed at 4°C and 40°C for 28 days test container Properties time point conditions T0 D4 D7 D14 D28 hot 2R Type I borosilicate glass bottle ․ Purity․ active. Particles visible under a microscope․ Molecular Integrity․ molecular content 4°C baseline - - V V 40°C V V - - V = test time point; T = time; D = day. Table 14B. Candidate formulations tested under freeze/thaw or shaking with added lyoprotectant test container Properties Processing method Number of cycles/time points Freeze – Thaw 0.2 mL cryovial ․ Purity․ active. Particles visible under a microscope․ Molecular Integrity․ molecular content ․ Freezing: Place at -20°C for 23 hours․ Thaw: place at 25°C for 1 hour 3 times 6 times shake 2R Type I borosilicate glass bottle ․ Shake: Shake at 220 rpm at 25°C․ Blank control: let stand at 25°C 24 hours 48 hours

該4個候選配方組合物對於熱壓力試驗之屬性改變顯示出相似的觀察結果。80 mg/mL濃度之融合蛋白1對40°C溫度敏感，此造成在7天時主要波峰減少至小於95%且超過5%的聚合物。此現象未在4°C儲存至少一個月時觀察到（表15及16）。此外，相較於對照組，VEGF及／或整合蛋白αvβ3結合的相對活性係在目標範圍（70至130%）內，且顯示出在加速條件下儲存後，對於VEGF結合的輕微改變、以及對αvβ3結合的無顯著變化。The four candidate formulation compositions showed similar observations regarding property changes in the thermal stress test. Fusion protein 1 at a concentration of 80 mg/mL was temperature sensitive to 40°C, resulting in a reduction of the major peak to less than 95% and more than 5% polymer at 7 days. This phenomenon was not observed when stored at 4°C for at least one month (Tables 15 and 16). Additionally, the relative activity of VEGF and/or integrin αvβ3 binding was within the target range (70 to 130%) compared to controls and showed slight changes in VEGF binding after storage under accelerated conditions, as well as No significant changes in αvβ3 binding.

具體而言，粒徑排阻層析法的試驗表示相較於其他配方，25 mM組胺酸、190 mM海藻糖、及0.03%聚山梨醇酯20（pH 6.0）的配方在4°C及40°C下都可實現融合蛋白1的較佳安定性。 表15. 在4°C下熱試驗的安定性結果 候選組別 成分 時間點 ( 天數 ) 實際測試濃度 (mg/mL) SEC-HPLC (%) 相對活性 * (%) HMW a 主要波峰 b VEGF 結合 αvβ3 結合 對照組1# 25 Mm組胺酸、6%蔗糖、20 mM 氯化鈉、 0.03%聚山梨醇酯20 （pH 6.0） NA 37.7 NA NA 100 100 1 25 mM 組胺酸、190 mM海藻糖、 0.03%聚山梨醇酯20 0 73.5 0.94 99.06 86.2 96.9 14 73.1 1.31 98.69 87.7 91.8 28 74.5 1.78 98.22 88.3 119.7 2 25 mM組胺酸、190 mM蔗糖、 0.03%聚山梨醇酯20 0 78.7 0.83 99.17 93.3 85.9 14 83.5 1.47 98.53 90.9 96.4 28 85.8 1.99 98.01 87.8 131.5 3 25 mM組胺酸、190 mM海藻糖、 0.03%聚山梨醇酯80 0 81.9 0.85 99.15 86.6 141.8 14 83.0 1.46 98.54 88.6 107.3 28 85.4 2.04 97.96 91.3 134.9 4 25 mM組胺酸、190 mM蔗糖、 0.03%聚山梨醇酯80 0 80.6 0.74 99.26 90.0 118.5 14 81.6 1.41 98.59 95.6 109.9 28 81.9 1.99 98.02 89.7 114.7 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 #對照組1係以25 mM組胺酸、6%蔗糖、20 mM 氯化鈉、0.03%聚山梨醇酯20（pH 6.0）調配。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 *係表示相較於對照組，預期在70至130%內的相對活性。 NA係表示不適用。 表16. 在40°C下熱試驗的安定性結果 候選組別 成分 時間點 ( 天數 ) 濃度 (mg/mL) SEC-HPLC (%) 相對活性 * (%) HMW a 主要波峰 b VEGF 結合 αvβ3 結合 對照組1 # 25 mM組胺酸、6%蔗糖、20 mM 氯化鈉、 0.03%聚山梨醇酯20 （pH 6.0） NA 37.7 NA NA 100 100 1 25 mM組胺酸、190 mM海藻糖、 0.03%聚山梨醇酯20 0 73.5 0.94 99.06 86.2 96.9 4 74.2 4.18 95.82 80.8 122.5 7 72.9 6.43 93.58 82.1 95.5 2 25 mM組胺酸、190 mM蔗糖、 0.03%聚山梨醇酯20 0 78.7 0.83 99.17 93.3 85.9 4 82.6 5.21 94.79 83.8 92.7 7 79.5 7.24 92.76 87.7 94.1 3 25 mM組胺酸、190 mM海藻糖、 0.03%聚山梨醇酯80 0 81.9 0.85 99.15 86.6 141.8 4 83.6 5.09 94.91 82.9 87.4 7 82.6 7.34 92.66 78.2 102.7 4 25 mM組胺酸、190 mM蔗糖、 0.03%聚山梨醇酯80 0 80.6 0.74 99.26 90.0 118.5 4 80.3 4.85 95.15 86.5 70.2 7 81.7 7.38 92.63 77.7 112.7 #對照組1係以25 mM組胺酸、6%蔗糖、20 mM 氯化鈉、0.03%聚山梨醇酯20（pH 6.0）調配。 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 *係表示相較於參考，預期在70至130%內的目標相對活性。 NA係表示不適用。 Specifically, particle size exclusion chromatography experiments showed that compared to other formulations, the formulation of 25 mM histidine, 190 mM trehalose, and 0.03% polysorbate 20 (pH 6.0) performed better at 4°C and The best stability of fusion protein 1 can be achieved at 40°C. Table 15. Stability results of thermal testing at 4°C Candidate group Element Time point ( number of days ) Actual test concentration (mg/mL) SEC-HPLC (%) Relative activity * (%) hMW main peak b VEGF binding αvβ3 binding Control group 1# 25 mM histidine, 6% sucrose, 20 mM sodium chloride, 0.03% polysorbate 20 (pH 6.0) NA 37.7 NA NA 100 100 1 25 mM Histidine, 190 mM Trehalose, 0.03% Polysorbate 20 0 73.5 0.94 99.06 86.2 96.9 14 73.1 1.31 98.69 87.7 91.8 28 74.5 1.78 98.22 88.3 119.7 2 25 mM histidine, 190 mM sucrose, 0.03% polysorbate 20 0 78.7 0.83 99.17 93.3 85.9 14 83.5 1.47 98.53 90.9 96.4 28 85.8 1.99 98.01 87.8 131.5 3 25 mM Histidine, 190 mM Trehalose, 0.03% Polysorbate 80 0 81.9 0.85 99.15 86.6 141.8 14 83.0 1.46 98.54 88.6 107.3 28 85.4 2.04 97.96 91.3 134.9 4 25 mM histidine, 190 mM sucrose, 0.03% polysorbate 80 0 80.6 0.74 99.26 90.0 118.5 14 81.6 1.41 98.59 95.6 109.9 28 81.9 1.99 98.02 89.7 114.7 a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. #Control 1 is prepared with 25 mM histidine, 6% sucrose, 20 mM sodium chloride, and 0.03% polysorbate 20 (pH 6.0). b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. * stands for relative activity expected to be in the range of 70 to 130% compared to the control group. NA means not applicable. Table 16. Stability results of thermal testing at 40°C Candidate group Element Time point ( number of days ) Concentration (mg/mL) SEC-HPLC (%) Relative activity * (%) hMW main peak b VEGF binding αvβ3 binding Control group 1 # 25 mM histidine, 6% sucrose, 20 mM sodium chloride, 0.03% polysorbate 20 (pH 6.0) NA 37.7 NA NA 100 100 1 25 mM Histidine, 190 mM Trehalose, 0.03% Polysorbate 20 0 73.5 0.94 99.06 86.2 96.9 4 74.2 4.18 95.82 80.8 122.5 7 72.9 6.43 93.58 82.1 95.5 2 25 mM histidine, 190 mM sucrose, 0.03% polysorbate 20 0 78.7 0.83 99.17 93.3 85.9 4 82.6 5.21 94.79 83.8 92.7 7 79.5 7.24 92.76 87.7 94.1 3 25 mM Histidine, 190 mM Trehalose, 0.03% Polysorbate 80 0 81.9 0.85 99.15 86.6 141.8 4 83.6 5.09 94.91 82.9 87.4 7 82.6 7.34 92.66 78.2 102.7 4 25 mM histidine, 190 mM sucrose, 0.03% polysorbate 80 0 80.6 0.74 99.26 90.0 118.5 4 80.3 4.85 95.15 86.5 70.2 7 81.7 7.38 92.63 77.7 112.7 #Control 1 is prepared with 25 mM histidine, 6% sucrose, 20 mM sodium chloride, and 0.03% polysorbate 20 (pH 6.0). a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. * stands for target relative activity expected to be within 70 to 130% compared to the reference. NA means not applicable.

以候選配方調配之80 mg/mL融合蛋白1係於進行以下操作3及6個循環後測試品質屬性：在-20°C下冷凍約23小時且在25°C下解凍至少1小時。透過SEC-HPLC之分析沒有在融合蛋白1之主要波峰及其聚合物的百分比中檢測到任何顯著的變化（表17）。殘留的蛋白質係大於95%且該聚合物係少於5%。此外，相較於對照組，融合蛋白1結合至VEGF及整合蛋白αvβ3的活性於候選組別#3及#4中下降至少於70%（表17）。 表17. 經過3至6次冷凍–解凍（F/T）循環後的壓力安定性測試 候選組別 組成 冷凍 / 解凍循環數 A280 之濃度 (mg/mL) SEC-HPLC (%) 相對活性 * (%) HMW a 主要波峰 b VEGF 結合 αvβ3 結合 對照組1# 25 mM組胺酸、6%蔗糖、20 mM 氯化鈉、0.03% 聚山梨醇酯20 （pH 6.0） 4 37.7 NA NA 100 100 1 25 mM組胺酸、190 mM海藻糖、 0.03% 聚山梨醇酯20 0 73.5 0.94 99.06 86.2 96.9 3 77.6 1.34 98.66 76.9 100.3 6 80.1 1.11 98.90 83.2 106.4 2 25 mM組胺酸、190 mM蔗糖、 0.03% 聚山梨醇酯20 0 78.7 0.83 99.17 93.3 85.9 3 82.3 1.32 98.68 83.0 96.7 6 84.9 1.15 98.85 93.2 153.0 3 25 mM組胺酸、190 mM海藻糖、 0.03% 聚山梨醇酯80 0 81.9 0.85 99.15 86.6 141.8 3 85.5 1.35 98.65 77.0 100.7 6 86.8 0.99 99.01 82.8 54.8 * 4 25 mM組胺酸、190 mM蔗糖、 0.03% 聚山梨醇酯80 0 80.6 0.74 99.26 90.0 118.5 3 87.4 1.32 98.68 57.5 * 99.2 6 88.2 1.09 98.91 87.5 99.5 #對照組1係以25 mM組胺酸、6%蔗糖、20 mM 氯化鈉、0.03%聚山梨醇酯20（pH 6.0）調配。 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 *係表示相較於參考，預期在70至130%內的目標相對活性。 NA係表示不適用。 The quality attributes of 80 mg/mL Fusion Protein 1 formulated in the candidate formulation were tested after 3 and 6 cycles of freezing at -20°C for approximately 23 hours and thawing at 25°C for at least 1 hour. Analysis by SEC-HPLC did not detect any significant changes in the major peaks of fusion protein 1 and its polymer percentage (Table 17). The remaining protein system is greater than 95% and the polymer system is less than 5%. In addition, compared to the control group, the activity of fusion protein 1 in binding to VEGF and integrin αvβ3 decreased to less than 70% in candidate groups #3 and #4 (Table 17). Table 17. Pressure Stability Testing After 3 to 6 Freeze–Thaw (F/T) Cycles Candidate group composition Number of freeze / thaw cycles Concentration of A280 (mg/mL) SEC-HPLC (%) Relative activity * (%) hMW main peak b VEGF binding αvβ3 binding Control group 1# 25 mM histidine, 6% sucrose, 20 mM sodium chloride, 0.03% polysorbate 20 (pH 6.0) 4 37.7 NA NA 100 100 1 25 mM Histidine, 190 mM Trehalose, 0.03% Polysorbate 20 0 73.5 0.94 99.06 86.2 96.9 3 77.6 1.34 98.66 76.9 100.3 6 80.1 1.11 98.90 83.2 106.4 2 25 mM Histidine, 190 mM Sucrose, 0.03% Polysorbate 20 0 78.7 0.83 99.17 93.3 85.9 3 82.3 1.32 98.68 83.0 96.7 6 84.9 1.15 98.85 93.2 153.0 3 25 mM Histidine, 190 mM Trehalose, 0.03% Polysorbate 80 0 81.9 0.85 99.15 86.6 141.8 3 85.5 1.35 98.65 77.0 100.7 6 86.8 0.99 99.01 82.8 54.8 * 4 25 mM Histidine, 190 mM Sucrose, 0.03% Polysorbate 80 0 80.6 0.74 99.26 90.0 118.5 3 87.4 1.32 98.68 57.5 * 99.2 6 88.2 1.09 98.91 87.5 99.5 #Control 1 is prepared with 25 mM histidine, 6% sucrose, 20 mM sodium chloride, and 0.03% polysorbate 20 (pH 6.0). a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. * stands for target relative activity expected to be within 70 to 130% compared to the reference. NA means not applicable.

搖動試驗係測試蛋白質安定性，以模擬融合蛋白1在液體形式中可能的處理及運輸。透過在25°C環境下以每分鐘220轉（rpm）搖晃小瓶24及48小時來加速條件。相較於處於靜置條件及基線的樣品，在此一嚴格條件下處理蛋白質樣品係衝擊蛋白質純度及與主要目標的結合（表18）。 表18. 24及48小時的搖動壓力安定性測試 候選組別 成分 測試 期間 (小時) 測試 濃度 (mg/mL) SEC-HPLC (%) 相對活性* (%) HMW a 主要波峰 b VEGF結合 αvβ3 結合 對照組1# 25 mM組胺酸、6%蔗糖、20 mM 氯化鈉、0.03% 聚山梨醇酯20 (pH 6.0) NA NA 37.73 NA NA 100 100 1 25 mM組胺酸、190 mM海藻糖、 0.03% 聚山梨醇酯20 基線 0 73.5 0.94 99.06 86.2 96.9 靜置 24 72.5 1.30 98.7 81.3 105.5 搖動 72.1 1.44 98.56 82.3 118.7 靜置 48 71.5 1.74 98.26 103.4 118.7 搖動 77.8 1.53 98.47 76.3 111.0 2 25 mM組胺酸、190 mM蔗糖、 0.03% 聚山梨醇酯20 基線 0 78.7 0.83 99.17 93.3 85.9 靜置 24 83.8 1.43 98.57 79.1 98.6 搖動 87.7 1.58 98.42 76.5 89.3 靜置 48 82.9 1.86 98.14 108.0 119.0 搖動 85.3 1.79 98.21 84.5 78.7 3 25 mM組胺酸、190 mM海藻糖、 0.03% 聚山梨醇酯80 基線 0 81.9 0.85 99.15 86.6 141.8 靜置 24 83.1 1.50 98.5 75.8 88.9 搖動 83.3 1.61 98.39 78.8 103.0 靜置 48 85.9 1.88 98.12 97.7 103.3 搖動 82.3 1.72 98.28 81.5 94.2 4 25 mM組胺酸、190 mM蔗糖、 0.03% 聚山梨醇酯80 基線 0 80.6 0.74 99.26 90.0 118.5 靜置 24 81.3 1.50 98.5 80.6 131.8 搖動 80.3 1.58 98.42 82.7 123.4 靜置 48 86.4 1.89 98.11 105.4 183.1 搖動 78.5 1.71 98.29 85.7 134.4 #對照組1係以25 mM組胺酸、6%蔗糖、20 mM 氯化鈉、0.03%聚山梨醇酯20（pH 6.0）調配。 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 *係表示相較於參考，預期在70至130%內的目標相對活性。 NA係表示不適用的。 The shaking test tests protein stability to simulate possible handling and transport of Fusion Protein 1 in liquid form. Accelerate conditions by shaking vials at 220 revolutions per minute (rpm) for 24 and 48 hours at 25°C. Processing protein samples under these stringent conditions impacts protein purity and binding to primary targets compared to samples under standing conditions and baseline (Table 18). Table 18. 24 and 48 hours shaking pressure stability test Candidate group Element test period (hours) Test concentration (mg/mL) SEC-HPLC (%) Relative activity* (%) hMW main peak b VEGF binding αvβ3 binding Control group 1# 25 mM histidine, 6% sucrose, 20 mM sodium chloride, 0.03% polysorbate 20 (pH 6.0) NA NA 37.73 NA NA 100 100 1 25 mM Histidine, 190 mM Trehalose, 0.03% Polysorbate 20 baseline 0 73.5 0.94 99.06 86.2 96.9 Let stand twenty four 72.5 1.30 98.7 81.3 105.5 shake 72.1 1.44 98.56 82.3 118.7 Let stand 48 71.5 1.74 98.26 103.4 118.7 shake 77.8 1.53 98.47 76.3 111.0 2 25 mM Histidine, 190 mM Sucrose, 0.03% Polysorbate 20 baseline 0 78.7 0.83 99.17 93.3 85.9 Let stand twenty four 83.8 1.43 98.57 79.1 98.6 shake 87.7 1.58 98.42 76.5 89.3 Let stand 48 82.9 1.86 98.14 108.0 119.0 shake 85.3 1.79 98.21 84.5 78.7 3 25 mM Histidine, 190 mM Trehalose, 0.03% Polysorbate 80 baseline 0 81.9 0.85 99.15 86.6 141.8 Let stand twenty four 83.1 1.50 98.5 75.8 88.9 shake 83.3 1.61 98.39 78.8 103.0 Let stand 48 85.9 1.88 98.12 97.7 103.3 shake 82.3 1.72 98.28 81.5 94.2 4 25 mM Histidine, 190 mM Sucrose, 0.03% Polysorbate 80 baseline 0 80.6 0.74 99.26 90.0 118.5 Let stand twenty four 81.3 1.50 98.5 80.6 131.8 shake 80.3 1.58 98.42 82.7 123.4 Let stand 48 86.4 1.89 98.11 105.4 183.1 shake 78.5 1.71 98.29 85.7 134.4 #Control 1 is prepared with 25 mM histidine, 6% sucrose, 20 mM sodium chloride, and 0.03% polysorbate 20 (pH 6.0). a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. * stands for target relative activity expected to be within 70 to 130% compared to the reference. NA means not applicable.

除了關鍵的品質屬性（例如：含量、純度及活性），在壓力條件後在顯微鏡下可見的粒子的形成及分布也被檢視。根據動態光散射（DLS）分析，將觀察到的在顯微鏡下可見的粒子以尺寸、%多分散性、及粒子不同尺寸的比例進行分類。該結果表示無法對獲得所欲濃度80 mg/mL之濃縮融合蛋白1的過程進行合適的監測，於此，在基線（D0）時，不僅檢測到有半徑約5 nm之主要的融合蛋白1分子，還有可被視為較大尺寸粒子（即，10-100 nm或100-1000 nm）的聚合體。無法透過DLS總結出何種候選配方緩衝劑具有最佳作用。In addition to key quality attributes (such as content, purity and activity), the formation and distribution of particles visible under the microscope after stress conditions were also examined. Based on dynamic light scattering (DLS) analysis, the observed particles visible under the microscope are classified by size, % polydispersity, and the proportion of different particle sizes. This result indicates that the process of obtaining concentrated fusion protein 1 at the desired concentration of 80 mg/mL cannot be properly monitored. Here, at the baseline (D0), not only the main fusion protein 1 molecule with a radius of about 5 nm was detected , there are also aggregates that can be considered larger size particles (i.e., 10-100 nm or 100-1000 nm). It is not possible to conclude through DLS which candidate formulation buffer will work best.

此外，非還原及還原SDS-PAGE分析無法於壓力條件處理後分辨出融合蛋白1之完整性的變化。該些測試的候選配方的滲透度（表19）係顯示除了候選配方#4（所示滲透度較低）以外，其他的配方係有相似的範圍。 表19. 候選配方的滲透度 配方 組成 滲透度 （ mOsm/kg ） 1 25 mM組胺酸、190 mM海藻糖、0.03%聚山梨醇酯20（pH 6.0） 248 2 25 mM組胺酸、190 mM蔗糖、0.03%聚山梨醇酯20（pH 6.0） 257 3 25 mM組胺酸、190 mM海藻糖、0.03%聚山梨醇酯80（pH 6.0） 250 4 25 mM組胺酸、190 mM蔗糖、0.03%聚山梨醇酯80（pH 6.0） 209 結論 In addition, non-reducing and reducing SDS-PAGE analysis cannot distinguish changes in the integrity of fusion protein 1 after treatment under pressure conditions. The penetrations of the candidate formulations tested (Table 19) show that with the exception of candidate formulation #4 (showing lower penetration), the other formulations have similar ranges. Table 19. Penetration of candidate formulations formula composition Penetration ( mOsm/kg ) 1 25 mM histidine, 190 mM trehalose, 0.03% polysorbate 20 (pH 6.0) 248 2 25 mM histidine, 190 mM sucrose, 0.03% polysorbate 20 (pH 6.0) 257 3 25 mM histidine, 190 mM trehalose, 0.03% polysorbate 80 (pH 6.0) 250 4 25 mM histidine, 190 mM sucrose, 0.03% polysorbate 80 (pH 6.0) 209 Conclusion

基於該些結果，係使用具有25 mM組胺酸（His）且含有190 mM海藻糖及0.03%聚山梨醇酯20（pH 6.0）的配方1，以進行一前導藥物產物之12個月安定性試驗的測試。 實施例 4. 玻璃體內注射之融合蛋白 1 配方 Based on these results, a 12-month stability study of a lead drug product was performed using Formulation 1 with 25 mM histidine (His) and 190 mM trehalose and 0.03% polysorbate 20 (pH 6.0). Test of tests. Example 4. Fusion protein 1 formula for intravitreal injection

進行安定性試驗以開發融合蛋白1的最終配方。 原料藥濃度測試（ Bulk drug substance concentration testing ） Stability testing was performed to develop the final formulation of Fusion Protein 1. Bulk drug substance concentration testing

為了透過製造過程選擇合適濃度以支持融合蛋白1的最終原料藥，將40及80 mg/mL的融合蛋白1調配於主要配方組成（25 mM組胺酸、190 mM海藻糖、及0.03%聚山梨醇酯20，pH 6.0）中。具有二種濃度之融合蛋白1的配方係在短期加速條件下測試，以透過SEC-HPLC、DLS、及SDS-PAGE分析來確定關鍵品質屬性的任何變化。In order to select the appropriate concentration to support the final API of Fusion Protein 1 through the manufacturing process, 40 and 80 mg/mL of Fusion Protein 1 were formulated in the main formulation composition (25 mM histidine, 190 mM trehalose, and 0.03% polysorbate alcohol ester 20, pH 6.0). Formulations with two concentrations of Fusion Protein 1 were tested under short-term accelerated conditions to determine any changes in critical quality attributes through SEC-HPLC, DLS, and SDS-PAGE analysis.

蛋白質的量係在40°C短期儲存7天的40 mg/mL樣品及80 mg/mL樣品中展現出不同（表20）。觀察到在80 mg/mL時聚合物的量些微較多。儲存於40°C之樣品的結果表示，在第4天及第7天時，相較於80 mg/mL，40 mg/mL之融合蛋白1形成較少的高分子量聚合物。該測試#2蛋白質樣品（40 mg/mL，具有氯化鈉）展現出＞5%的檢測聚合物。冷凍／解凍循環測試未顯示出40及80 mg/mL樣品之聚合物%或純度有顯著變化（數據未示出）。 表20. 於具有及不具有氯化鈉之配方緩衝劑中的40及80 mg/mL融合蛋白1在40°C下的安定性 樣品 組成 期間 ( 天數 ) 測試濃度 (mg/mL) SEC-HPLC (%) HMW a 主要波峰 b 測試1 40 mg/mL融合蛋白1、 25 mM組胺酸、190 mM海藻糖、0.03%聚山梨醇酯20 （pH 6.0） 0 42.8 1.52 98.48 4 48.3 2.83 97.17 49.9 2.93 97.07 7 42.2 3.69 96.31 43.3 3.74 96.26 測試2 40 mg/mL融合蛋白1、 25 mM組胺酸、190 mM海藻糖、 10 mM 氯化鈉、0.03%聚山梨醇酯20 （pH 6.0） 0 42.9 1.63 98.38 4 45.4 4.22 95.78 46.3 4.26 95.74 7 42.5 5.20 94.02 42.4 5.21 94.07 測試3 80 mg/mL融合蛋白1 25 mM組胺酸、190 mM海藻糖、0.03%聚山梨醇酯20 （pH 6.0） 0 87.3 1.63 98.37 4 90.8 4.75 94.67 90.0 4.73 94.64 7 86.2 6.55 92.34 86.2 6.52 92.49 測試4 80 mg/mL融合蛋白1 25 mM組胺酸、190 mM海藻糖、 10 mM 氯化鈉、0.03%聚山梨醇酯20 （pH 6.0） 0 85.6 1.66 98.34 4 88.1 5.08 94.22 92.9 5.08 94.22 7 84.3 6.95 91.78 84.4 6.98 91.90 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 The amount of protein differed between the 40 mg/mL sample and the 80 mg/mL sample stored short-term at 40°C for 7 days (Table 20). A slightly higher amount of polymer was observed at 80 mg/mL. Results for samples stored at 40°C showed that 40 mg/mL of Fusion Protein 1 formed less high molecular weight polymers compared to 80 mg/mL on days 4 and 7. The Test #2 protein sample (40 mg/mL with sodium chloride) exhibited >5% detected polymer. Freeze/thaw cycle testing did not show significant changes in polymer % or purity for the 40 and 80 mg/mL samples (data not shown). Table 20. Stability of Fusion Protein 1 at 40°C at 40 and 80 mg/mL in formulation buffer with and without sodium chloride sample composition Period ( number of days ) Test concentration (mg/mL) SEC-HPLC (%) hMW _ main peak b Test 1 40 mg/mL Fusion Protein 1, 25 mM Histidine, 190 mM Trehalose, 0.03% Polysorbate 20 (pH 6.0) 0 42.8 1.52 98.48 4 48.3 2.83 97.17 49.9 2.93 97.07 7 42.2 3.69 96.31 43.3 3.74 96.26 Test 2 40 mg/mL Fusion Protein 1, 25 mM Histidine, 190 mM Trehalose, 10 mM Sodium Chloride , 0.03% Polysorbate 20 (pH 6.0) 0 42.9 1.63 98.38 4 45.4 4.22 95.78 46.3 4.26 95.74 7 42.5 5.20 94.02 42.4 5.21 94.07 Test 3 80 mg/mL Fusion Protein 1 25 mM Histidine, 190 mM Trehalose, 0.03% Polysorbate 20 (pH 6.0) 0 87.3 1.63 98.37 4 90.8 4.75 94.67 90.0 4.73 94.64 7 86.2 6.55 92.34 86.2 6.52 92.49 Test 4 80 mg/mL Fusion Protein 1 25 mM Histidine, 190 mM Trehalose, 10 mM Sodium Chloride , 0.03% Polysorbate 20 (pH 6.0) 0 85.6 1.66 98.34 4 88.1 5.08 94.22 92.9 5.08 94.22 7 84.3 6.95 91.78 84.4 6.98 91.90 a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1.

使用DLS以分析在顯微鏡下可見的粒子及其分布，且所有蛋白質樣品皆含有基線之尺寸範圍在10 nm至100 nm及／或100至1000 nm間的粒子，其可能會使得壓力測試後無法分辨變化。 藥物前導安定性測試 DLS is used to analyze particles and their distribution visible under the microscope, and all protein samples contain particles with a baseline size range of 10 nm to 100 nm and/or 100 to 1000 nm, which may make them indistinguishable after stress testing. change. Drug lead stability testing

基於上述試驗且融合蛋白1維持基於SEC-HPLC之最一致的純度，融合蛋白1之所選配方組成如下：40 mg/mL融合蛋白1、25 mM組胺酸、190 mM海藻糖及0.03%聚山梨醇酯20（pH 6.0）。為了驗證且評估容器系統的相容性、以及為了收集初始安定性，以此配方製備40 mg/mL的融合蛋白1，並填充於硼矽酸第I型玻璃瓶（以橡膠堵塞及翻蓋密封）且在以下三種條件下溫育：-25°C至-15°C、2°C至8°C、及25°C／60%之相對溼度（RH）（表21）。 表21. 配方開發試驗計畫 條件 測試時間點 T=0 2wks 1M 3M 6M 9M 12M 25°C ／ 60% RH X X X X X -- -- 4°C X X X X X X -20°C （於 2R 小瓶中） X X X X -- -- X＝確認蛋白質品質屬性的時間點 Wks＝週；M＝月；T＝時間 Based on the above experiments and that fusion protein 1 maintains the most consistent purity based on SEC-HPLC, the selected formula of fusion protein 1 is as follows: 40 mg/mL fusion protein 1, 25 mM histidine, 190 mM trehalose and 0.03% poly Sorbitol Ester 20 (pH 6.0). In order to verify and evaluate the compatibility of the container system and to collect the initial stability, 40 mg/mL of fusion protein 1 was prepared according to this formula and filled into borosilicate type I glass bottles (sealed with rubber plugs and flip caps) and incubated under the following three conditions: -25°C to -15°C, 2°C to 8°C, and 25°C/60% relative humidity (RH) (Table 21). Table 21. Formulation development pilot plan condition Test time point T=0 2wks 1M 3M 6M 9M 12M 25°C ／ 60%RH X X X X X -- -- 4°C X X X X X X -20°C (in 2R vial) X X X X -- -- X = time point to confirm protein quality attributes Wks = weeks; M = months; T = time

試驗的結果表示，40 mg/mL融合蛋白1在-20°C下儲存至少12個月時係安定的（表22）。在4°C之儲存條件下6個月，純度有輕微的下降且聚合物提升（表23）。於25°C之加速儲存期間，SEC-HPLC檢測到經過6個月聚合物%提升，但主要波峰仍維持≧95%，以及該分子特異性活性及完整性（即，十二烷基硫酸鈉毛細管電泳（CE-SDS）的數據）係維持最小變化（表24）。 表22. 儲存在-20°C下之融合蛋白1藥物前導安定性試驗 測試 物品 時間點（月） A280 之濃度 (mg/mL) SEC-HPLC (%) VEGF 結合 CE-SDS HMW a 主要波峰 b 相對活性 @ %/EC 50 非還原 (%) 還原 (%) Z19003 # 0 41 0.8 99.20 92/0.408 100 99.3 1 41.6 0.94 99.06 96/0.368 100 100 3 42.3 1.14 98.86 98/0.393 100 99.5 6 41.0 0.72 99.28 97/0.397 100 99.3 9 42.2 0.95 99.05 95/0.407 100 99.6 12 43.4 0.71 99.29 93/0.370 100 99.4 Z19003 # (1x 冷凍 / 解凍循環 ) 0 41 0.87 99.13 92/0.408 100 99.3 1 41.8 0.87 99.13 95/0.374 100 99.3 3 42.3 0.93 99.07 102/0.380 100 99.1 6 42.0 0.76 99.24 96/0.401 100 98.8 9 41.7 0.99 99.01 95/0.409 100 99.5 12 42.3 0.72 99.28 93/0.372 100 99.5 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 @：相較於基線（D0），基於EC50計算的相對活性。 #：Z19003為該測試物品的批號（Lot No.）。 表23. 在4°C下之融合蛋白1藥物前導安定性試驗 測試 物品 時間點 （月） A280 之濃度 (mg/mL) SEC-HPLC VEGF 結合 CE-SDS HMW a 主要波峰 b 相對活性 @ %/EC 50 非還原 (%) 還原 (%) Z19003 # 0 41.0 0.8 99.20 92/0.408 100 99.3 0.5 41.7 0.97 99.04 98/0.250 100 99.5 1 42.4 1.15 98.85 93/0.381 100 99.8 3 41.8 1.60 98.41 93/0.461 100 99.2 6 41.0 1.41 98.59 89/0.431 100 99.4 9 41.3 2.18 97.83 101/0.377 100 99.4 12 42.0 1.80 98.20 95/0.365 100 99.4 Z19003 # (1x 冷凍 / 解凍循環 ) 0 41.0 0.87 99.13 92/0.408 100 99.3 0.5 42.1 0.93 99.07 97/0.254 100 99.2 1 42.3 1.12 98.88 91/0.389 100 99.4 3 44.7 1.47 98.53 86/0.499 100 99.4 6 41.6 1.43 98.57 89/0.432 100 99.3 9 41.5 2.21 97.79 95/0.398 100 99.6 12 41.4 1.94 98.06 97/0.357 100 99.4 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 @：相較於基線（D0），基於EC50計算的相對活性。 #：Z19003為該測試物品的批號（Lot No.）。 表24. 在25°C下之融合蛋白1藥物前導安定性試驗 測試 物品 時間點 （月） A280 之濃度 (mg/mL) SEC-HPLC VEGF 結合 CE-SDS HHMW # HMW a 主要波峰 b 相對 活性 @ %/EC 50 非還原 (%) 還原 (%) Z19003 # 0 41.0 - 0.8 99.20 92/0.408 100 99.3 0.5 42.9 - 1.40 98.60 94/0.263 100 99.3 1 42.6 - 1.85 98.15 99/0.370 100 99.1 3 42.8 0.365 3.21 96.43 92/0.421 100 98.7 6 41.8 - 3.30 96.70 78/0.494 100 98.7 Z19003 # (1x 冷凍 / 解凍循環 ) 0 41.0 - 0.87 99.13 92/0.408 100 99.3 0.5 43.8 - 1.42 98.59 91/0.271 100 99.2 1 40.9 - 1.89 98.11 95/0.373 100 99.3 3 44.5 0.214 2.90 96.88 91/0.424 100 98.7 6 41.3 - 3.49 96.51 80/0.480 100 86.8 aHMW：高分子量物質的聚合物，滯留時間為8.3±0.1分鐘。 b主要波峰的滯留時間為9.5±0.1分鐘，此表示融合蛋白1的非聚合二聚體形式。 #HHMW：更高分子量物質的聚合物，其係指具有滯留時間為7.6±0.1分鐘的波峰。 @：相較於基線（D0），基於EC50計算的相對活性。 #：Z19003為該測試物品的批號（Lot No.）。 The results of the test showed that 40 mg/mL fusion protein 1 was stable when stored at -20°C for at least 12 months (Table 22). After 6 months of storage at 4°C, there was a slight decrease in purity and an increase in polymer (Table 23). During accelerated storage at 25°C, SEC-HPLC detected an increase in polymer % after 6 months, but the main peak still maintained ≧95%, as well as the specific activity and integrity of the molecule (i.e., sodium lauryl sulfate Capillary electrophoresis (CE-SDS) data) maintained minimal changes (Table 24). Table 22. Stability test of fusion protein 1 drug lead stored at -20°C Test items Time point (month) Concentration of A280 (mg/mL) SEC-HPLC (%) VEGF binding CE-SDS hMW _ main peak b Relative activity @ %/EC 50 Non-restore (%) Restore (%) Z19003 # 0 41 0.8 99.20 92/0.408 100 99.3 1 41.6 0.94 99.06 96/0.368 100 100 3 42.3 1.14 98.86 98/0.393 100 99.5 6 41.0 0.72 99.28 97/0.397 100 99.3 9 42.2 0.95 99.05 95/0.407 100 99.6 12 43.4 0.71 99.29 93/0.370 100 99.4 Z19003 # (1x freeze / thaw cycle ) 0 41 0.87 99.13 92/0.408 100 99.3 1 41.8 0.87 99.13 95/0.374 100 99.3 3 42.3 0.93 99.07 102/0.380 100 99.1 6 42.0 0.76 99.24 96/0.401 100 98.8 9 41.7 0.99 99.01 95/0.409 100 99.5 12 42.3 0.72 99.28 93/0.372 100 99.5 a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. @ : Relative activity calculated based on EC50 compared to baseline (D0). # : Z19003 is the lot number (Lot No.) of the test item. Table 23. Fusion protein 1 drug lead stability test at 4°C Test items Time point (month) Concentration of A280 (mg/mL) SEC-HPLC VEGF binding CE-SDS hMW main peak b Relative activity @ %/EC 50 Non-restore (%) Restore (%) Z19003 # 0 41.0 0.8 99.20 92/0.408 100 99.3 0.5 41.7 0.97 99.04 98/0.250 100 99.5 1 42.4 1.15 98.85 93/0.381 100 99.8 3 41.8 1.60 98.41 93/0.461 100 99.2 6 41.0 1.41 98.59 89/0.431 100 99.4 9 41.3 2.18 97.83 101/0.377 100 99.4 12 42.0 1.80 98.20 95/0.365 100 99.4 Z19003 # (1x freeze / thaw cycle ) 0 41.0 0.87 99.13 92/0.408 100 99.3 0.5 42.1 0.93 99.07 97/0.254 100 99.2 1 42.3 1.12 98.88 91/0.389 100 99.4 3 44.7 1.47 98.53 86/0.499 100 99.4 6 41.6 1.43 98.57 89/0.432 100 99.3 9 41.5 2.21 97.79 95/0.398 100 99.6 12 41.4 1.94 98.06 97/0.357 100 99.4 a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. @ : Relative activity calculated based on EC50 compared to baseline (D0). # : Z19003 is the lot number (Lot No.) of the test item. Table 24. Fusion protein 1 drug lead stability test at 25°C Test items Time point (month) Concentration of A280 (mg/mL) SEC-HPLC VEGF binding CE-SDS HHMW # hMW main peak b Relative activity @ %/EC 50 Non-restore (%) Restore (%) Z19003 # 0 41.0 - 0.8 99.20 92/0.408 100 99.3 0.5 42.9 - 1.40 98.60 94/0.263 100 99.3 1 42.6 - 1.85 98.15 99/0.370 100 99.1 3 42.8 0.365 3.21 96.43 92/0.421 100 98.7 6 41.8 - 3.30 96.70 78/0.494 100 98.7 Z19003 # (1x freeze / thaw cycle ) 0 41.0 - 0.87 99.13 92/0.408 100 99.3 0.5 43.8 - 1.42 98.59 91/0.271 100 99.2 1 40.9 - 1.89 98.11 95/0.373 100 99.3 3 44.5 0.214 2.90 96.88 91/0.424 100 98.7 6 41.3 - 3.49 96.51 80/0.480 100 86.8 a HMW: Polymer of high molecular weight substances, residence time is 8.3±0.1 minutes. b The retention time of the main peak is 9.5 ± 0.1 minutes, which represents the non-polymerized dimer form of fusion protein 1. # HHMW: Polymer of higher molecular weight species, which refers to a wave peak with a residence time of 7.6 ± 0.1 minutes. @ : Relative activity calculated based on EC50 compared to baseline (D0). # : Z19003 is the lot number (Lot No.) of the test item.

根據DLS分析，將物品儲存在-20°C及4°C下至少12個月未檢測到在顯微鏡下可見的粒子，除了一樣品係儲存在4°C下的樣品係於12個月的時間點進行一冷凍／解凍循環並形成微量（2%）的較大尺寸的粒子（半徑2208 nm）。在25°C儲存下於3及6個月時檢測到較大尺寸的粒子（半徑2195及2264 nm）。According to DLS analysis, no microscopically visible particles were detected in items stored at -20°C and 4°C for at least 12 months, except for one strain where samples were stored at 4°C for 12 months. The site underwent a freeze/thaw cycle and formed trace amounts (2%) of larger sized particles (radius 2208 nm). Larger size particles (radii 2195 and 2264 nm) were detected at 3 and 6 months after storage at 25°C.

分析儲存在4°C之備份樣品，以確認在12個月時觀察到的在顯微鏡下可見的粒子。亦將此樣品儲存12個月且經歷一冷凍／解凍循環。在試驗的備份樣品中觀察到在顯微鏡下可見的粒子。因此，融合蛋白1在4°C下儲存12個月係對冷凍／解凍壓力敏感。此即，在沒有冷凍／解凍循環的情況下，融合蛋白1可在4°C下儲存至少12個月。然而，若融合蛋白1將儲存超過6個月，則建議儲存在-20°C下。Backup samples stored at 4°C were analyzed to confirm microscopic particles observed at 12 months. This sample was also stored for 12 months and subjected to a freeze/thaw cycle. Microscopically visible particles were observed in backup samples of the test. Therefore, fusion protein 1 is sensitive to freezing/thawing stress when stored at 4°C for 12 months. That is, Fusion Protein 1 can be stored at 4°C for at least 12 months without freeze/thaw cycles. However, if Fusion Protein 1 will be stored for more than 6 months, storage at -20°C is recommended.

配方的黏度測量係使用一黏度計而獲得。該受測配方（於具有所選成分之25 mM組胺酸緩衝劑溶液中的40 mg/mL融合蛋白1）具有5.402 cP（釐泊，centipoise）的低黏度（表25）。所選配方具有好的蛋白質熱特性，如圖9之代表性的差示掃描熱分析儀（DSC）熱譜圖所示。反摺積熱譜圖（deconvoluted thermogram）具有4個熱傳導波峰，Tm值為61.47°C、66.51°C、81.55°C、及84.21°C。 表25. 在4°C下溶液中之融合蛋白1及配方溶液的黏度 測試樣品 配方溶液（0 mg/mL融合蛋白1、25 mM組胺酸、190 mM海藻糖、0.03%聚山梨糖醇酯20，pH 6.0） 蛋白質批號 Z19003（40 mg/mL融合蛋白1、25 mM組胺酸、190 mM海藻糖、0.03%聚山梨糖醇酯20，pH 6.0） 平均值 (n=3 或 4)* 4.495 cP 5.402 cP 標準差 0.274 0.884 *：自三或四位個體測試結果獲取平均值。 結論 The viscosity measurement of the formulation was obtained using a viscometer. The formulation tested (40 mg/mL Fusion Protein 1 in 25 mM histidine buffer solution with selected ingredients) had a low viscosity of 5.402 cP (centipoise) (Table 25). The selected formulation has good protein thermal properties, as shown in the representative differential scanning calorimeter (DSC) thermogram in Figure 9. The deconvoluted thermogram has four heat conduction peaks, with Tm values of 61.47°C, 66.51°C, 81.55°C, and 84.21°C. Table 25. Viscosity of Fusion Protein 1 and Formulated Solutions in Solution at 4°C test sample Formulated solution (0 mg/mL fusion protein 1, 25 mM histidine, 190 mM trehalose, 0.03% polysorbate 20, pH 6.0) Protein Lot Z19003 (40 mg/mL Fusion Protein 1, 25 mM Histidine, 190 mM Trehalose, 0.03% Polysorbate 20, pH 6.0) Average (n=3 or 4)* 4.495cP 5.402cP standard deviation 0.274 0.884 *: Average obtained from three or four individual test results. Conclusion

鑒於此試驗的結果，於25 mM組胺酸、190 mM海藻糖及0.03%聚山梨醇酯20（pH 6.0）中調配的40 mg/mL融合蛋白1可與所選容器封蓋系統（container closure system）相容，並在-20°C 及2°C至8°C下儲存至少12個月仍保持安定。 實施例 5. 融合蛋白 1 的長期安定性試驗 Based on the results of this experiment, 40 mg/mL fusion protein 1 formulated in 25 mM histidine, 190 mM trehalose, and 0.03% polysorbate 20 (pH 6.0) can be used with the container closure system of choice. system) and remain stable when stored at -20°C and 2°C to 8°C for at least 12 months. Example 5. Long-term stability test of fusion protein 1

進行一試驗以測試於25 mM組胺酸、190 mM海藻糖及0.03%聚山梨醇酯20（pH 6.0）中調配的40 mg/mL融合蛋白1在各種溫度下的長期安定性。在儲存於-70°C下，融合蛋白1係展現出超過二年的安定性（表26）。觀察到αvβ3及α5β1結合在第9、18、及24個月（T9、T18、及T24）與第9、12、及24個月（T9、T12、及T24）時αvβ3及α5β1的EC 50差異性（variation）。對此差異進行探討並認為，這可能是源於測試試劑失效。 表26. 在-70°C±10°C下融合蛋白1之安定性試驗 測試項目 T 0 T1 T3 T6 T9 T12 T18 T24 外觀 BY6 BY6 BY6 BY6 BY6 BY6 BY5 BY5 pH值 6.2 6.2 6.2 6.2 6.1 6.1 6.1 6.1 A280 (mg/mL) 41 41 41 41 40 39 41 40 SEC-HPLC/UV， 主要波峰 (%) 98.8 99.2 99.1 99.2 99.1 99.2 99.1 99.2 SEC-HPLC/UV，聚合物(%) 1.2 0.8 0.9 0.8 0.9 0.8 0.9 0.8 非還原CE-SDS (%) 100 100 100 100 100 100 100 100 還原CE-SDS (%) 99.3 99.3 99.4 99.4 99.3 99.3 99.3 99.3 毛細管等電聚焦(cIEF) (%) 99.78 99.68 99.82 99.78 99.82 99.66 99.55 99.74 VEGF-A 165ELISA 結合親和力(EC 50) 0.408 0.370 0.350 0.478 0.385 0.404 0.427 0.489 αvβ3 ELISA 結合親和力(EC 50) 0.012 0.014 0.015 0.014 0.032 0.006 0.023 0.085 α5β1 ELISA 結合親和力(EC 50) 0.251 0.086 0.088 0.090 0.180 0.031 0.047 0.112 T＝材料儲存的月安定性 BY＝棕黃色 *α5β1結合ELISA在0個月後進一步優化，T1數據係用於α5β1結合ELISA安定性的趨勢分析。 An experiment was conducted to test the long-term stability of 40 mg/mL fusion protein 1 formulated in 25 mM histidine, 190 mM trehalose, and 0.03% polysorbate 20 (pH 6.0) at various temperatures. Fusion protein line 1 demonstrated stability for over two years when stored at -70°C (Table 26). Differences in the EC 50 of αvβ3 and α5β1 binding were observed at 9, 18, and 24 months (T9, T18, and T24) and at 9, 12, and 24 months (T9, T12, and T24). Variation. This discrepancy was discussed and believed to be due to the failure of the test reagent. Table 26. Stability test of fusion protein 1 at -70°C±10°C test items T 0 T1 T3 T6 T9 T12 T18 T24 Appearance BY6 BY6 BY6 BY6 BY6 BY6 BY5 BY5 pH value 6.2 6.2 6.2 6.2 6.1 6.1 6.1 6.1 A280 (mg/mL) 41 41 41 41 40 39 41 40 SEC-HPLC/UV, main peaks (%) 98.8 99.2 99.1 99.2 99.1 99.2 99.1 99.2 SEC-HPLC/UV, polymer (%) 1.2 0.8 0.9 0.8 0.9 0.8 0.9 0.8 Non-reducing CE-SDS (%) 100 100 100 100 100 100 100 100 Restore CE-SDS (%) 99.3 99.3 99.4 99.4 99.3 99.3 99.3 99.3 Capillary Isoelectric Focusing (cIEF) (%) 99.78 99.68 99.82 99.78 99.82 99.66 99.55 99.74 VEGF-A 165 ELISA binding affinity (EC 50 ) 0.408 0.370 0.350 0.478 0.385 0.404 0.427 0.489 αvβ3 ELISA binding affinity (EC 50 ) 0.012 0.014 0.015 0.014 0.032 0.006 0.023 0.085 α5β1 ELISA binding affinity (EC 50 ) 0.251 0.086 0.088 0.090 0.180 0.031 0.047 0.112 T = monthly stability of material storage BY = brown * α5β1 binding ELISA was further optimized after 0 months, and T1 data was used for trend analysis of α5β1 binding ELISA stability.

對在25 mM組胺酸、190 mM海藻糖及0.03%聚山梨醇酯20（pH 6.0）中調配的40 mg/mL且接著儲存於-20°C的融合蛋白1進行檢視研究持續36個月。儲存於-20°C下9個月後的安定性結果統整係如表27所示。 表27. 在-20°C±5°C之融合蛋白1的安定性試驗，正立（Upright） 測試項目 T0 T1 T3 T6 T9 T12 T18 T24 T36 外觀 BY6，基本上不存在可見顆粒 BY5，基本上不存在可見顆粒 BY5，基本上不存在可見顆粒 BY6，基本上不存在可見顆粒 介於BY5及BY6間，基本上不存在可見顆粒 TBD TBD TBD TBD pH值 6.1 6.1 6.1 6.1 6.1 TBD TBD TBD TBD 滲透度(Osmo/kg) 268 267 267 255 261.7* TBD TBD TBD TBD cIEF (%)  99.97  99.89 99.92 99.78  99.85 TBD TBD TBD TBD A280 (mg/mL) 38 39 38 37 37 TBD TBD TBD TBD SEC-HPLC/UV，主要波峰(%) 99.2 99.2 97.8 99.2 99.1 TBD TBD TBD TBD SEC-HPLC/UV，聚合物(%) 0.8 0.8 2.2 0.8  0.9 TBD TBD TBD TBD 非還原CE-SDS (%) 100 100 100 100 100 TBD TBD TBD TBD 還原CE-SDS (%) 99.3 99.3 99.4 99.4 99.2 TBD TBD TBD TBD VEGF-A 165結合相對活性(%) 109 99 104 96 106 TBD TBD TBD TBD αvβ3結合相對活性(%) 129 114 125 88 101 TBD TBD TBD TBD α5β1結合相對活性(%) 129 100 112 113 104 TBD TBD TBD TBD 在顯微鏡下可見的粒子 10 µm 1 NT NT 17 NT TBD NT TBD TBD 25 µm 0 NT NT 0 NT TBD NT TBD TBD 50 µm 0 NT NT 0 NT TBD NT TBD TBD T＝材料儲存的月安定性；BY＝棕黃色；NT＝未測試；TBD＝待確定；TO＝省略測試；*自滲透度探討收集的數據。 Fusion protein 1 was studied at 40 mg/mL formulated in 25 mM histidine, 190 mM trehalose, and 0.03% polysorbate 20 (pH 6.0) and then stored at -20°C for 36 months. . The stability results after 9 months of storage at -20°C are summarized in Table 27. Table 27. Stability test of fusion protein 1 at -20°C±5°C, Upright test items T0 T1 T3 T6 T9 T12 T18 T24 T36 Appearance BY6, basically no visible particles BY5, basically no visible particles BY5, basically no visible particles BY6, basically no visible particles Between BY5 and BY6, there are basically no visible particles. TBD TBD TBD TBD pH value 6.1 6.1 6.1 6.1 6.1 TBD TBD TBD TBD Penetration(Osmo/kg) 268 267 267 255 261.7* TBD TBD TBD TBD cIEF (%) 99.97 99.89 99.92 99.78 99.85 TBD TBD TBD TBD A280 (mg/mL) 38 39 38 37 37 TBD TBD TBD TBD SEC-HPLC/UV, main peaks (%) 99.2 99.2 97.8 99.2 99.1 TBD TBD TBD TBD SEC-HPLC/UV, polymer (%) 0.8 0.8 2.2 0.8 0.9 TBD TBD TBD TBD Non-reducing CE-SDS (%) 100 100 100 100 100 TBD TBD TBD TBD Restore CE-SDS (%) 99.3 99.3 99.4 99.4 99.2 TBD TBD TBD TBD VEGF-A 165 binding relative activity (%) 109 99 104 96 106 TBD TBD TBD TBD αvβ3 binding relative activity (%) 129 114 125 88 101 TBD TBD TBD TBD α5β1 binding relative activity (%) 129 100 112 113 104 TBD TBD TBD TBD particles visible under a microscope 10 µm 1 NT NT 17 NT TBD NT TBD TBD 25 µm 0 NT NT 0 NT TBD NT TBD TBD 50 µm 0 NT NT 0 NT TBD NT TBD TBD T = Monthly stability of material in storage; BY = Brownish yellow; NT = Not tested; TBD = To be determined; TO = Testing omitted; * Data collected from permeability study.

此外，對在25 mM組胺酸、190 mM海藻糖及0.03%聚山梨醇酯20（pH 6.0）中調配的40 mg/mL且接著儲存於5°C的融合蛋白1進行檢視研究持續36個月。正立（upright）或倒立（inverted）儲存於5°C下9個月後的安定性結果統整係分別如表28及29所示。 表28. 在5°C±3°C之融合蛋白1的安定性試驗，正立 測試項目 T0 T1 T3 T6 T9 T12 T18 T24 T36 外觀 BY6，基本上不存在可見顆粒 BY5，基本上不存在可見顆粒 BY5，基本上不存在可見顆粒 BY6，基本上不存在可見顆粒 介於BY5及BY6間，基本上不存在可見顆粒 TBD TBD TBD TBD pH值 6.1 6.1 6.1 6.1 6.1 TBD TBD TBD TBD 滲透度(mOsmo/kg) 268 267 267 267 266 TBD TBD TBD TBD cIEF (%) 99.97 99.93 99.78 99.89 99.92 TBD TBD TBD TBD A280 (mg/mL) 38 38 38 38 38 TBD TBD TBD TBD SEC-HPLC/UV， 主要波峰(%) 99.2 99.0 99.3 98.7 98.5 TBD TBD TBD TBD SEC-HPLC/UV， 聚合物(%) 0.8 1.0 0.7 1.3 1.5 TBD TBD TBD TBD 非還原CE-SDS (%) 100 100 100 100 100 TBD TBD TBD TBD 還原CE-SDS (%) 99.3 99.3 99.3 99.3 99.1 TBD TBD TBD TBD VEGF-A 165結合相對活性(%) 109 108 102 102 102 TBD TBD TBD TBD αvβ3結合相對活性(%) 129 89 100 104 95 TBD TBD TBD TBD α5β1結合相對活性(%) 129 103 103 105 102 TBD TBD TBD TBD T＝材料儲存的月安定性；BY＝棕黃色；TBD＝待確定。 表29. 在5°C±3°C之融合蛋白1的安定性試驗，倒立（inverted） 測試項目 T0 T1 T3 T6 T9 T12 T18 T24 T36 外觀 BY6，基本上不存在可見顆粒 BY5，基本上不存在可見顆粒 BY5，基本上不存在可見顆粒 BY6，基本上不存在可見顆粒 介於BY5及BY6間，基本上不存在可見顆粒 TBD TBD TBD TBD pH值 6.1 6.1 6.1 6.1 6.1 TBD TBD TBD TBD 滲透度(mOsmo/kg) 268 268 267 267 267 TBD TBD TBD TBD cIEF (%)  99.97 99.89 99.80  99.85 99.98 TBD TBD TBD TBD A280 (mg/mL) 38 38 38 38 38 TBD TBD TBD TBD SEC-HPLC/UV， 主要波峰(%) 99.2 99.0 98.8 98.6 98.5 TBD TBD TBD TBD SEC-HPLC/UV， 聚合物(%) 0.8  1.0 1.2  1.4 1.5 TBD TBD TBD TBD 非還原CE-SDS (%) 100 100 100 100 100 TBD TBD TBD TBD 還原CE-SDS (%) 99.3 99.4 99.3 99.3 99.2 TBD TBD TBD TBD VEGF-A 165結合相對活性(%) 109 103 103 92 92 TBD TBD TBD TBD αvβ3結合相對活性(%) 129 122 100 90 107 TBD TBD TBD TBD α5β1結合相對活性(%) 129 101 127 94 99 TBD TBD TBD TBD 在顯微鏡下可見的粒子 10 µm 1 NT NT 23 NT TBD NT TBD TBD 25 µm 0 NT NT 4 NT TBD NT TBD TBD 50 µm 0 NT NT 0 NT TBD NT TBD TBD T＝材料儲存的月安定性；BY＝棕黃色；NT＝未測試；TBD＝待確定 In addition, fusion protein 1 was examined at 40 mg/mL formulated in 25 mM histidine, 190 mM trehalose, and 0.03% polysorbate 20 (pH 6.0) and then stored at 5°C for 36 days. moon. The stability results after being stored upright or inverted at 5°C for 9 months are shown in Tables 28 and 29 respectively. Table 28. Stability test of fusion protein 1 at 5°C±3°C, upright test items T0 T1 T3 T6 T9 T12 T18 T24 T36 Appearance BY6, basically no visible particles BY5, basically no visible particles BY5, basically no visible particles BY6, basically no visible particles Between BY5 and BY6, there are basically no visible particles. TBD TBD TBD TBD pH value 6.1 6.1 6.1 6.1 6.1 TBD TBD TBD TBD Penetration (mOsmo/kg) 268 267 267 267 266 TBD TBD TBD TBD cIEF (%) 99.97 99.93 99.78 99.89 99.92 TBD TBD TBD TBD A280 (mg/mL) 38 38 38 38 38 TBD TBD TBD TBD SEC-HPLC/UV, main peaks (%) 99.2 99.0 99.3 98.7 98.5 TBD TBD TBD TBD SEC-HPLC/UV, polymer (%) 0.8 1.0 0.7 1.3 1.5 TBD TBD TBD TBD Non-reducing CE-SDS (%) 100 100 100 100 100 TBD TBD TBD TBD Restore CE-SDS (%) 99.3 99.3 99.3 99.3 99.1 TBD TBD TBD TBD VEGF-A 165 binding relative activity (%) 109 108 102 102 102 TBD TBD TBD TBD αvβ3 binding relative activity (%) 129 89 100 104 95 TBD TBD TBD TBD α5β1 binding relative activity (%) 129 103 103 105 102 TBD TBD TBD TBD T=monthly stability of material storage; BY=brown; TBD=to be determined. Table 29. Stability test of fusion protein 1 at 5°C±3°C, inverted test items T0 T1 T3 T6 T9 T12 T18 T24 T36 Appearance BY6, basically no visible particles BY5, basically no visible particles BY5, basically no visible particles BY6, basically no visible particles Between BY5 and BY6, there are basically no visible particles. TBD TBD TBD TBD pH value 6.1 6.1 6.1 6.1 6.1 TBD TBD TBD TBD Penetration (mOsmo/kg) 268 268 267 267 267 TBD TBD TBD TBD cIEF (%) 99.97 99.89 99.80 99.85 99.98 TBD TBD TBD TBD A280 (mg/mL) 38 38 38 38 38 TBD TBD TBD TBD SEC-HPLC/UV, main peaks (%) 99.2 99.0 98.8 98.6 98.5 TBD TBD TBD TBD SEC-HPLC/UV, polymer (%) 0.8 1.0 1.2 1.4 1.5 TBD TBD TBD TBD Non-reducing CE-SDS (%) 100 100 100 100 100 TBD TBD TBD TBD Restore CE-SDS (%) 99.3 99.4 99.3 99.3 99.2 TBD TBD TBD TBD VEGF-A 165 binding relative activity (%) 109 103 103 92 92 TBD TBD TBD TBD αvβ3 binding relative activity (%) 129 122 100 90 107 TBD TBD TBD TBD α5β1 binding relative activity (%) 129 101 127 94 99 TBD TBD TBD TBD particles visible under a microscope 10 µm 1 NT NT twenty three NT TBD NT TBD TBD 25 µm 0 NT NT 4 NT TBD NT TBD TBD 50 µm 0 NT NT 0 NT TBD NT TBD TBD T=monthly stability of material in storage; BY=brown; NT=not tested; TBD=to be determined

此外，對在25 mM組胺酸、190 mM海藻糖及0.03%聚山梨醇酯20（pH 6.0）中調配的40 mg/mL且後續儲存於25°C的融合蛋白1安定性進行檢視研究持續6個月。在25°C下儲存6個月後的安定性結果統整係如表30所示。 表30. 在25°C±2°C／60%±5% RH之融合蛋白1的安定性試驗，正立 測試項目 T0 T1 T3 T6 外觀 BY6，基本上不存在可見顆粒 BY5，基本上不存在可見顆粒 BY5，基本上不存在可見顆粒 BY5，基本上不存在可見顆粒 pH值 6.1 6.1 6.1 6.1 滲透度(mOsm/kg) 268 268 269 271 cIEF (%)  99.97  99.91  99.85 99.83 A280 (mg/mL) 38 38 38 38 SEC-HPLC/UV， 主要波峰(%) 99.2 98.3 98.8 96.9 SEC-HPLC/UV 聚合物(%) 0.8  1.7  1.2 3.1 非還原CE-SDS (%) 100 100 100 100 還原CE-SDS (%) 99.3 99.4 98.9 98.5 VEGF-A 165結合相對活性(%) 109 103 98 89 αvβ3結合相對活性(%) 129 116 125 111 α5β1結合ELISA (%) 129 108 126 89 在顯微鏡下可見的粒子 10 µm 1 NT NT 18 25 µm 0 NT NT 2 50 µm 0 NT NT 2 T＝材料儲存的月安定性；BY＝棕黃色；NT＝未測試 實施例 6. 以融合蛋白 1 配方處理之兔子在使用 VEGF-A 165 刺激後的血管滲漏分數 In addition, the stability of fusion protein 1 formulated at 40 mg/mL in 25 mM histidine, 190 mM trehalose, and 0.03% polysorbate 20 (pH 6.0) and subsequently stored at 25°C was examined. 6 months. The stability results after 6 months of storage at 25°C are summarized in Table 30. Table 30. Stability test of fusion protein 1 at 25°C±2°C/60%±5% RH, upright test items T0 T1 T3 T6 Appearance BY6, basically no visible particles BY5, basically no visible particles BY5, basically no visible particles BY5, basically no visible particles pH value 6.1 6.1 6.1 6.1 Penetration (mOsm/kg) 268 268 269 271 cIEF (%) 99.97 99.91 99.85 99.83 A280 (mg/mL) 38 38 38 38 SEC-HPLC/UV, main peaks (%) 99.2 98.3 98.8 96.9 SEC-HPLC/UV polymer(%) 0.8 1.7 1.2 3.1 Non-reducing CE-SDS (%) 100 100 100 100 Restore CE-SDS (%) 99.3 99.4 98.9 98.5 VEGF-A 165 binding relative activity (%) 109 103 98 89 αvβ3 binding relative activity (%) 129 116 125 111 α5β1 binding ELISA (%) 129 108 126 89 particles visible under a microscope 10 µm 1 NT NT 18 25 µm 0 NT NT 2 50 µm 0 NT NT 2 T = monthly stability of material storage; BY = brownish yellow; NT = not tested Example 6. Vascular leakage fraction of rabbits treated with fusion protein 1 formula after stimulation with VEGF-A 165

進行一體內試驗以測試調配於上述感興趣之配方（於25 mM組胺酸、190 mM海藻糖及0.03%聚山梨醇酯20（pH 6.0）之40 mg/mL融合蛋白1）中的融合蛋白1的各種劑量的效果。人類VEGF誘導視網膜滲漏係透過經定義之滲漏分數（基於標準化之螢光血管攝影（FA）評分影像）於荷蘭黑帶兔中測試。將滲漏分數自0至4進行分類（0：主要血管非常直，有一些彎曲的小血管；1：主要血管彎曲增加及／或血管擴張；2：主要血管間產生滲漏；3：主要血管及次要血管間產生滲漏，次要血管仍然可分辨；4：主要及次要血管間產生滲漏，次要血管無法分辨）。An in vivo assay was performed to test the fusion protein formulated in the above formulation of interest (40 mg/mL fusion protein 1 in 25 mM histidine, 190 mM trehalose, and 0.03% polysorbate 20 (pH 6.0)) 1 Effect of various doses. Human VEGF-induced retinal leakage was tested in Dutch black-banded rabbits via defined leakage fractions based on standardized fluorescein angiography (FA) scoring images. Leakage scores were classified from 0 to 4 (0: main blood vessels are very straight with some small curved blood vessels; 1: main blood vessels have increased curvature and/or blood vessel dilation; 2: leakage occurs between major blood vessels; 3: main blood vessels Leakage occurs between the primary and secondary blood vessels, and the secondary blood vessels can still be distinguished; 4: Leakage occurs between the primary and secondary blood vessels, and the secondary blood vessels cannot be distinguished).

兔子皆各自以50 µL進行雙側的單次玻璃體內注射，每組具有一共3隻兔子及6隻眼睛。在第0天時，對兔子投予50 µL的空白對照緩衝液、Avastin®（每眼1.25 mg）、Eylea®（每眼0.625 mg）、以及遞增濃度的融合蛋白1（即，每眼0.03 mg、0.1 mg、0.3 mg及1 mg）。然後，使用1000 ng的人類VEGF-A 165在第2天時對兔子進行刺激，並且在第5天時使用FA確定滲漏分數。 Rabbits each received a single intravitreal injection of 50 µL bilaterally, with a total of 3 rabbits and 6 eyes in each group. On day 0, rabbits were administered 50 µL of blank control buffer, Avastin® (1.25 mg per eye), Eylea® (0.625 mg per eye), and increasing concentrations of Fusion Protein 1 (i.e., 0.03 mg per eye). , 0.1 mg, 0.3 mg and 1 mg). Rabbits were then stimulated with 1000 ng of human VEGF-A 165 on day 2, and leakage fractions were determined on day 5 using FA.

使用低至0.03 mg之融合蛋白1治療的兔子係展現出下降的血管滲漏分數（圖10）。此即，低至0.03 mg之融合蛋白1可有效減少血管滲漏。Rabbit lines treated with as little as 0.03 mg of Fusion Protein 1 exhibited reduced fractional vascular leakage (Figure 10). That is, as little as 0.03 mg of fusion protein 1 can effectively reduce vascular leakage.

融合蛋白1的該等劑量係在抑制VEGF誘導之視網膜滲漏的能力與Avastin®及Eylea®相當。這些數據係支持融合蛋白1適用於治療血管性視網膜疾病（例如：DME、nAMD、及RVO） 電子遞交之序列表的參考 These doses of Fusion Protein 1 are comparable to Avastin® and Eylea® in their ability to inhibit VEGF-induced retinal leakage. These data are a reference for the electronic submission of sequence listings supporting the suitability of fusion protein 1 for the treatment of vascular retinal diseases (e.g., DME, nAMD, and RVO).

本申請係含有一序列表，其係透過EFS-Web以ASCII格式之序列表進行電子遞交（檔名為「SequenceListing7WO1」；創建日期：2022年5月12日；且檔案大小為45.0 kb）。該透過EFS-Web遞交之序列表為本說明書的一部分且其全文係藉由引用而併入本文中。This application contains a sequence listing, which was submitted electronically through EFS-Web as a sequence listing in ASCII format (file name "SequenceListing7WO1"; creation date: May 12, 2022; and file size is 45.0 kb). The sequence listing submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety.

例示性實施態樣之前述及其他目的、方面、特徵及優點，將因參照下列描述及配合附圖，而變得更顯而易見並更易於理解。The aforementioned and other objects, aspects, features and advantages of the exemplary embodiments will become more apparent and easier to understand with reference to the following description and accompanying drawings.

圖1為自血管滲漏區域之基線的平均百分比變化圖表，其係對恆河猴（rhesus monkey）之雷射誘導的脈絡膜新生血管模型進行每眼0.6、1.0、及1.9 mg之融合蛋白1的單次玻璃體內注射（intravitreal injection）。數據以平均值±標準誤差呈現。所有的劑量皆在第0天時以單次玻璃體內劑量投予。Lucentis®（0.5 mg）係作為系統適用性（system suitability）及陽性對照。經分析的所有雷射斑點皆為第III／IV級（Grade III/IV）。％血管滲漏＝（基線滲漏區域－治療滲漏區域）÷（基線滲漏區域）×100％；統計分析係透過Mann-Whitney U檢定而進行空白組（vehicle group）及治療組的比較，*為p＜0.05，在1.9 mg之組別中的 a係表示已移除會影響統計顯著性（p＝0.057）之來自一罹患數種眼部發炎之猴子的數據。n＝每組4隻眼睛。 Figure 1 is a graph of the mean percentage change from baseline in the area of vascular leakage in a laser-induced choroidal neovascularization model in rhesus monkeys with 0.6, 1.0, and 1.9 mg of fusion protein 1 per eye. Single intravitreal injection. Data are presented as mean ± standard error. All doses were administered as a single intravitreal dose on Day 0. Lucentis® (0.5 mg) was used as system suitability and positive control. All analyzed laser spots are Grade III/IV. % vascular leakage = (baseline leakage area - treatment leakage area) ÷ (baseline leakage area) × 100%; statistical analysis was performed through the Mann-Whitney U test to compare the vehicle group and the treatment group. * is p<0.05, a in the 1.9 mg group indicates that data from a monkey suffering from several types of ocular inflammation have been removed that would affect statistical significance (p=0.057). n = 4 eyes per group.

圖2為透過光同調斷層掃描（optical coherence tomography）確定之平均視網膜厚度的圖表，其係對恆河猴之雷射誘導的脈絡膜新生血管模型進行每眼0.6、1.0、及1.9 mg之融合蛋白1的單次玻璃體內注射。數據以平均值±標準誤差呈現。所有的劑量皆在第0天時以單次玻璃體內劑量投予。Lucentis®（0.5 mg）係作為系統適用性及陽性對照。經分析的所有雷射斑點皆為第III／IV級。％視網膜厚度＝（基線視網膜厚度－治療視網膜厚度）÷（基線視網膜厚度－試驗前視網膜厚度）×100％；統計分析係透過Mann-Whitney U檢定而進行空白組及治療組的比較，*為p＜0.05，在1.9 mg之組別中的 a係表示已移除會影響統計顯著性（p＝0.057）之來自一罹患數種眼部發炎之猴子的數據。n＝每組4隻眼睛。 Figure 2 is a graph of mean retinal thickness determined by optical coherence tomography in a laser-induced choroidal neovascularization model of rhesus monkeys treated with 0.6, 1.0, and 1.9 mg of fusion protein 1 per eye. of a single intravitreal injection. Data are presented as mean ± standard error. All doses were administered as a single intravitreal dose on Day 0. Lucentis® (0.5 mg) was used as system suitability and positive control. All laser spots analyzed were Class III/IV. % retinal thickness = (baseline retinal thickness - treatment retinal thickness) ÷ (baseline retinal thickness - pre-test retinal thickness) × 100%; statistical analysis was performed to compare the blank group and the treatment group through the Mann-Whitney U test, * is p <0.05, a in the 1.9 mg group indicates that data from a monkey suffering from several types of ocular inflammation have been removed that would affect statistical significance (p=0.057). n = 4 eyes per group.

圖3為Masson三色染色法的圖表，其係對經所述處理之恆河猴雷射誘導脈絡膜新生血管模型的第III／IV級病灶進行染色。數據以平均值±標準誤差呈現。在第29天時進行摘出術（Enucleation）。所選擇之第4級病灶的雷射斑點係透過Masson三色染色法進行分析。使用學生t檢定分析來比較空白組與治療組，*為p＜0.05，n＝每組4隻眼睛。Figure 3 is a diagram of Masson's trichrome staining method, which stains grade III/IV lesions in the laser-induced choroidal neovascularization model of rhesus monkeys. Data are presented as mean ± standard error. Enucleation was performed on day 29. The laser spots of selected grade 4 lesions were analyzed by Masson's trichrome staining. Student's t-test analysis was used to compare blank and treatment groups, * is p<0.05, n=4 eyes per group.

圖4為博萊黴素（bleomycin）誘導之C57BL/6小鼠肺纖維化模型的肺羥脯胺酸水平圖表，其係如所述般地進行治療（每一治療組n＝8隻動物且該sham對照組n＝4）。$為p＜0.05，治療組與sham對照組比較，未配對學生t檢定；*為p＜0.05，治療組與空白組比較，單因子變異數分析（One-way ANOVA）及Dunnett檢定。在融合蛋白1的組別中，一隻動物的數據因為在第3天時不明原因的提早死亡而未採用。Figure 4 is a graph of lung hydroxyproline levels in the bleomycin-induced C57BL/6 mouse model of pulmonary fibrosis, which were treated as described (n=8 animals per treatment group and The sham control group n = 4). $ means p<0.05, comparison between the treatment group and the sham control group, unpaired Student's t test; * means p<0.05, comparison between the treatment group and the blank group, one-way ANOVA and Dunnett's test. In the fusion protein 1 group, data from one animal were not used due to unexplained premature death on day 3.

圖5A至圖5F為融合蛋白1分別在pH7.0之磷酸鹽緩衝液（圖5A）、pH6.5之磷酸鹽緩衝液（圖5B）、pH6.5之組胺酸緩衝液（圖5C）、pH6.0之組胺酸緩衝液（圖5D）、pH6.0之檸檬酸緩衝液（圖5E）、及pH5.5之檸檬酸緩衝液（圖5F）中之擴散係數的點圖。該擴散係數是透過動態光散射（DLS）確定。Figures 5A to 5F show fusion protein 1 in phosphate buffer at pH 7.0 (Figure 5A), phosphate buffer at pH 6.5 (Figure 5B), and histidine buffer at pH 6.5 (Figure 5C), respectively. , pH 6.0 histidine buffer (Figure 5D), pH 6.0 citrate buffer (Figure 5E), and pH 5.5 citrate buffer (Figure 5F). The diffusion coefficient is determined by dynamic light scattering (DLS).

圖6A至圖6B為對40 mg/mL及80 mg/mL之融合蛋白1在二種波長下分析的濁度測量圖表。圖6A係描繪波長660奈米（nm）及圖6B係描繪波長320 nm。NaPi＝磷酸鈉，Cit＝檸檬酸，及His＝組胺酸。Figure 6A to Figure 6B are turbidity measurement charts of 40 mg/mL and 80 mg/mL fusion protein 1 analyzed at two wavelengths. Figure 6A depicts a wavelength of 660 nanometers (nm) and Figure 6B depicts a wavelength of 320 nm. NaPi = sodium phosphate, Cit = citric acid, and His = histidine.

圖7A至圖7E為融合蛋白1之以考馬斯藍（Coomassie Blue）染色膠體的非還原聚丙烯醯氨凝膠電泳（SDS-PAGE）。圖7A及圖7B包括在4℃或40℃下於檸檬酸緩衝液中溫育一段時間之40 mg/mL及80 mg/mL的融合蛋白1，起始（泳道2及3）、第4天（泳道4及5）、第7天（泳道6及7）、以及第14天（泳道8及9）。圖7C及圖7D包括在4℃或40℃下於組胺酸緩衝液中溫育一段時間之40 mg/mL及80 mg/mL的融合蛋白1，起始（泳道2及3）、第4天（泳道4及5）、第7天（泳道6及7）、以及第14天（泳道8及9）。圖7E包括在第28天時在4℃或40℃下於組胺酸緩衝液或檸檬酸緩衝液中溫育一段時間之40 mg/mL及80 mg/mL的融合蛋白1。NaPi＝磷酸鈉，Cit＝檸檬酸，及His＝組胺酸。每一個樣品孔槽中皆置入3微克（μg）的蛋白。Figures 7A to 7E show non-reducing polyacrylamide gel electrophoresis (SDS-PAGE) of fusion protein 1 on a colloid stained with Coomassie Blue. Figures 7A and 7B include 40 mg/mL and 80 mg/mL of fusion protein 1 in citrate buffer incubated at 4°C or 40°C for a period of time, starting (lanes 2 and 3), day 4 (lanes 4 and 5), day 7 (lanes 6 and 7), and day 14 (lanes 8 and 9). Figures 7C and 7D include 40 mg/mL and 80 mg/mL of fusion protein 1, starting (lanes 2 and 3), 4 day (lanes 4 and 5), day 7 (lanes 6 and 7), and day 14 (lanes 8 and 9). Figure 7E includes 40 mg/mL and 80 mg/mL of Fusion Protein 1 incubated in histidine buffer or citrate buffer for a period of time at 4°C or 40°C on day 28. NaPi = sodium phosphate, Cit = citric acid, and His = histidine. Place 3 micrograms (μg) of protein into each sample well.

圖8A至圖8E為融合蛋白1之以考馬斯藍染色膠體的還原聚丙烯醯氨凝膠電泳。圖8A及圖8B包括在4℃或40℃下於檸檬酸緩衝液中溫育一段時間之40 mg/mL及80 mg/mL的融合蛋白1，起始（泳道2及3）、第4天（泳道4及5）、第7天（泳道6及7）、以及第14天（泳道8及9）。圖8C及圖8D包括在4℃或40℃下於組胺酸緩衝液中溫育一段時間之40 mg/mL及80 mg/mL的融合蛋白1，起始（泳道2及3）、第4天（泳道4及5）、第7天（泳道6及7）、以及第14天（泳道8及9）。圖8E包括在第28天時在4℃或40℃下於組胺酸緩衝液或檸檬酸緩衝液中溫育一段時間之40 mg/mL及80 mg/mL的融合蛋白1。NaPi＝磷酸鈉，Cit＝檸檬酸，及His＝組胺酸。每一個樣品孔槽中皆置入3 μg的蛋白。Figures 8A to 8E show reducing polyacrylamide gel electrophoresis of fusion protein 1 using Coomassie blue-stained colloid. Figures 8A and 8B include 40 mg/mL and 80 mg/mL of fusion protein 1 in citrate buffer incubated at 4°C or 40°C for a period of time, starting (lanes 2 and 3), day 4 (lanes 4 and 5), day 7 (lanes 6 and 7), and day 14 (lanes 8 and 9). Figures 8C and 8D include 40 mg/mL and 80 mg/mL of fusion protein 1, starting (lanes 2 and 3), 4 day (lanes 4 and 5), day 7 (lanes 6 and 7), and day 14 (lanes 8 and 9). Figure 8E includes 40 mg/mL and 80 mg/mL of Fusion Protein 1 incubated in histidine buffer or citrate buffer at 4°C or 40°C for a period of time on day 28. NaPi = sodium phosphate, Cit = citric acid, and His = histidine. Place 3 μg of protein into each sample well.

圖9為1 mg/mL之融合蛋白1的熱安定性圖表，其係調配於25 mM之組胺酸緩衝液、190 mM之海藻糖、0.03%之PS20（pH 6.0）中並透過差示掃描熱分析儀（Differential Scanning Calorimeter，DSC）測定。Figure 9 is a thermal stability chart of fusion protein 1 at 1 mg/mL, which was formulated in 25 mM histidine buffer, 190 mM trehalose, 0.03% PS20 (pH 6.0) and subjected to differential scanning. Thermal analyzer (Differential Scanning Calorimeter, DSC) measurement.

圖10為對荷蘭黑帶兔使用如所述濃度之融合蛋白1的單次劑量投予圖表，其係抑制人類VEGF-A 165所誘導的視網膜血管滲透性。 Figure 10 is a graph of the inhibition of human VEGF-A 165 -induced retinal vascular permeability in Dutch black belt rabbits following single dose administration of Fusion Protein 1 at the concentrations indicated.

Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0001

Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0002

Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0003

Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0004

Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0005

Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0006

Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0007

Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0008

Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0009

Figure 12_A0101_SEQ_0010
Figure 12_A0101_SEQ_0010

Figure 12_A0101_SEQ_0011
Figure 12_A0101_SEQ_0011

Figure 12_A0101_SEQ_0012
Figure 12_A0101_SEQ_0012

Figure 12_A0101_SEQ_0013
Figure 12_A0101_SEQ_0013

Figure 12_A0101_SEQ_0014
Figure 12_A0101_SEQ_0014

Figure 12_A0101_SEQ_0015
Figure 12_A0101_SEQ_0015

Figure 12_A0101_SEQ_0016
Figure 12_A0101_SEQ_0016

Figure 12_A0101_SEQ_0017
Figure 12_A0101_SEQ_0017

Figure 12_A0101_SEQ_0018
Figure 12_A0101_SEQ_0018

Figure 12_A0101_SEQ_0019
Figure 12_A0101_SEQ_0019

Figure 12_A0101_SEQ_0020
Figure 12_A0101_SEQ_0020

Figure 12_A0101_SEQ_0021
Figure 12_A0101_SEQ_0021

Figure 12_A0101_SEQ_0022
Figure 12_A0101_SEQ_0022

Figure 12_A0101_SEQ_0023
Figure 12_A0101_SEQ_0023

Figure 12_A0101_SEQ_0024
Figure 12_A0101_SEQ_0024

Figure 12_A0101_SEQ_0025
Figure 12_A0101_SEQ_0025

Figure 12_A0101_SEQ_0026
Figure 12_A0101_SEQ_0026

Figure 12_A0101_SEQ_0027
Figure 12_A0101_SEQ_0027

Figure 12_A0101_SEQ_0028
Figure 12_A0101_SEQ_0028

Figure 12_A0101_SEQ_0029
Figure 12_A0101_SEQ_0029

Figure 12_A0101_SEQ_0030
Figure 12_A0101_SEQ_0030

Figure 12_A0101_SEQ_0031
Figure 12_A0101_SEQ_0031

Figure 12_A0101_SEQ_0032
Figure 12_A0101_SEQ_0032

Figure 12_A0101_SEQ_0033
Figure 12_A0101_SEQ_0033

Figure 12_A0101_SEQ_0034
Figure 12_A0101_SEQ_0034

Figure 12_A0101_SEQ_0035
Figure 12_A0101_SEQ_0035

Figure 12_A0101_SEQ_0036
Figure 12_A0101_SEQ_0036

Figure 12_A0101_SEQ_0037
Figure 12_A0101_SEQ_0037

Figure 12_A0101_SEQ_0038
Figure 12_A0101_SEQ_0038

Figure 12_A0101_SEQ_0039
Figure 12_A0101_SEQ_0039

Figure 12_A0101_SEQ_0040
Figure 12_A0101_SEQ_0040

Figure 12_A0101_SEQ_0041
Figure 12_A0101_SEQ_0041

Figure 12_A0101_SEQ_0042
Figure 12_A0101_SEQ_0042

Figure 12_A0101_SEQ_0043
Figure 12_A0101_SEQ_0043

Figure 12_A0101_SEQ_0044
Figure 12_A0101_SEQ_0044

Figure 12_A0101_SEQ_0045
Figure 12_A0101_SEQ_0045

Figure 12_A0101_SEQ_0046
Figure 12_A0101_SEQ_0046

Figure 12_A0101_SEQ_0047
Figure 12_A0101_SEQ_0047

Figure 12_A0101_SEQ_0048
Figure 12_A0101_SEQ_0048

Figure 12_A0101_SEQ_0049
Figure 12_A0101_SEQ_0049

Figure 12_A0101_SEQ_0050
Figure 12_A0101_SEQ_0050

Figure 12_A0101_SEQ_0051
Figure 12_A0101_SEQ_0051

Figure 12_A0101_SEQ_0052
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Figure 12_A0101_SEQ_0053
Figure 12_A0101_SEQ_0053

Claims (50)

一種醫藥配方，該配方包含： a)    一濃度為約0.5毫克／毫升（mg/mL）至約120 mg/mL之融合蛋白； b)    一濃度為約1%至約10%（重量／體積）之多元醇或醇類，該多元醇或醇類係選自由以下所組成之群組：蔗糖、海藻糖、甘露醇、山梨糖醇、苯甲醇、聚乙烯醇、及聚乙二醇（PEG）400-12000； c)    一濃度為約10毫莫耳（mM）至約50 mM之緩衝劑，該緩衝劑係選自由以下所組成之群組：磷酸鈉、組胺酸、檸檬酸鈉、醋酸鈉、碳酸氫鈉、及檸檬酸三鈉二水合物（trisodium citrate dihydrate）；以及 d)    一濃度為約0.01%至約4%（重量／體積）之界面活性劑， 其中，該配方的pH值為約5.5至7.5，且該配方視需要地更包含一選自由以下所組成之群組的多醣：羧甲基纖維素鈉、微晶纖維素、或透明質酸鈉。 A pharmaceutical formula containing: a) A fusion protein with a concentration of about 0.5 mg/mL to about 120 mg/mL; b) A polyol or alcohol at a concentration of about 1% to about 10% (weight/volume), the polyol or alcohol being selected from the group consisting of: sucrose, trehalose, mannitol, sorbose Alcohol, benzyl alcohol, polyvinyl alcohol, and polyethylene glycol (PEG) 400-12000; c) A buffer with a concentration of about 10 millimoles (mM) to about 50 mM, the buffer being selected from the group consisting of: sodium phosphate, histidine, sodium citrate, sodium acetate, bicarbonate Sodium, and trisodium citrate dihydrate; and d) A surfactant at a concentration of about 0.01% to about 4% (weight/volume), Wherein, the pH value of the formula is about 5.5 to 7.5, and the formula optionally further includes a polysaccharide selected from the group consisting of: sodium carboxymethyl cellulose, microcrystalline cellulose, or sodium hyaluronate . 如請求項1所述之醫藥配方，其中該界面活性劑係選自由以下所組成之群組：聚山梨糖醇酯20、聚山梨糖醇酯80、及泊洛沙姆188。The pharmaceutical formulation of claim 1, wherein the surfactant is selected from the group consisting of: polysorbate 20, polysorbate 80, and poloxamer 188. 如請求項1或2所述之醫藥配方，其中該界面活性劑的濃度約為0.03%（重量／體積）。The pharmaceutical formulation as described in claim 1 or 2, wherein the concentration of the surfactant is approximately 0.03% (weight/volume). 如請求項1或2所述之醫藥配方，其中該融合蛋白的濃度為約1 mg/mL至約90 mg/mL。The pharmaceutical formulation as described in claim 1 or 2, wherein the concentration of the fusion protein is about 1 mg/mL to about 90 mg/mL. 如請求項1或2所述之醫藥配方，其中該多元醇係海藻糖且濃度為約25 mM至250 mM。The pharmaceutical formulation as claimed in claim 1 or 2, wherein the polyol is trehalose and the concentration is about 25 mM to 250 mM. 如請求項1或2所述之醫藥配方，其中該緩衝劑係組胺酸且濃度為約10 mM至約40 mM。The pharmaceutical formulation as claimed in claim 1 or 2, wherein the buffer is histidine and the concentration is from about 10 mM to about 40 mM. 如請求項1或2所述之醫藥配方，其中該融合蛋白係自N-端至C-端以如下順序包含： a)   一血管內皮生長因子受體（VEGFR）的細胞外結構域； b)   一人類免疫球蛋白G的Fc結構域；以及 c)    一結合整合蛋白之蛋白（integrin binding protein）或其片段。 The pharmaceutical formula as described in claim 1 or 2, wherein the fusion protein includes in the following order from N-terminus to C-terminus: a) An extracellular domain of vascular endothelial growth factor receptor (VEGFR); b) an Fc domain of human immunoglobulin G; and c) An integrin binding protein or a fragment thereof. 如請求項7所述之醫藥配方，其中該融合蛋白係包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO: 17、或SEQ ID NO: 18。The pharmaceutical formulation of claim 7, wherein the fusion protein contains SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. 如請求項1或2所述之醫藥配方，其中該pH值為約5.5至約7.0。The pharmaceutical formulation as claimed in claim 1 or 2, wherein the pH value is from about 5.5 to about 7.0. 如請求項1或2所述之醫藥配方，其中該配方在-70℃、-20℃、及／或5℃下安定性維持至少24個月。The pharmaceutical formula as described in claim 1 or 2, wherein the formula maintains stability at -70°C, -20°C, and/or 5°C for at least 24 months. 如請求項1或2所述之醫藥配方，其中該配方在-70℃、-20℃、2℃至8℃、及／或25℃下至少6個月後係保持蛋白質的純度及活性（potency）。The pharmaceutical formula as described in claim 1 or 2, wherein the formula maintains the purity and activity (potency) of the protein after at least 6 months at -70°C, -20°C, 2°C to 8°C, and/or 25°C. ). 如請求項1或2所述之醫藥配方，其中該配方更包含一濃度為約10 mM至50 mM的鹽類。The pharmaceutical formula as claimed in claim 1 or 2, wherein the formula further contains a salt at a concentration of about 10 mM to 50 mM. 如請求項1或2所述之醫藥配方，其中該配方更包含至少一種胺基酸，該胺基酸的濃度為約10 mM至50 mM。The pharmaceutical formula as claimed in claim 1 or 2, wherein the formula further contains at least one amino acid, and the concentration of the amino acid is about 10 mM to 50 mM. 如請求項12所述之醫藥配方，其中該鹽類係選自氯化鈉、氯化鎂、氯化鈣、或氯化鉀。The pharmaceutical formula as claimed in claim 12, wherein the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride. 如請求項13所述之醫藥配方，其中該胺基酸係選自由以下所組成之群組：精胺酸、甲硫胺酸、脯胺酸、組胺酸、半胱胺酸、離胺酸、甘胺酸、天門冬胺酸、色胺酸、麩胺酸、及異白胺酸。The pharmaceutical formula as claimed in claim 13, wherein the amino acid is selected from the group consisting of: arginine, methionine, proline, histidine, cysteine, lysine , glycine, aspartic acid, tryptophan, glutamic acid, and isoleucine. 一種使用如請求項1至15中任一項所述之醫藥配方於製備一醫藥組合物的用途，其中該醫藥組合物係用於治療一個體之眼部疾病。A method of using the pharmaceutical formulation as described in any one of claims 1 to 15 to prepare a pharmaceutical composition, wherein the pharmaceutical composition is used to treat an eye disease in an individual. 如請求項16所述之用途，其中該眼部疾病係選自新生血管（neovascularization）或缺血性眼色素層炎、視網膜血管炎、血管樣痕、色素性視網膜炎、角膜新生血管、虹膜新生血管、新生血管性青光眼、青光眼術後纖維化、增殖性玻璃體視網膜病變（proliferative vitreoretinopathy，PVR）、脈絡膜新生血管（choroidal neovascularization，CNV）、視神經盤新生血管、視網膜新生血管、玻璃體新生血管（vitreal neovascularization）、血管翳（pannus）、翼狀贅肉（pterygium）、血管性視網膜病變（vascular retinopathy）、無糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、有糖尿病黃斑水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、糖尿病黃斑水腫（DME）、滲出性（濕性）及非滲出性（乾性）之老年性黃斑退化（AMD）、黃斑水腫、視網膜靜脈阻塞後黃斑水腫（RVO）、視網膜靜脈阻塞（RVO）、中心性視網膜靜脈阻塞（CRVO）、分支性視網膜靜脈阻塞（BRVO）、視網膜血管瘤狀增殖（RAP）、息肉狀脈絡膜血管病變（polypoidal choroidal vasculopathy，PCV）、玻璃體黃斑沾黏（VMA）、及／或玻璃體黃斑牽引症（VMT）。The use as described in claim 16, wherein the eye disease is selected from the group consisting of neovascularization or ischemic uveitis, retinal vasculitis, vascular scars, retinitis pigmentosa, corneal neovascularization, and iris neoplasia Blood vessels, neovascular glaucoma, post-glaucoma fibrosis, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization ), pannus, pterygium, vascular retinopathy, diabetic retinopathy (DR, non-proliferative and proliferative DR) without diabetic macular edema (DME), diabetic macula Diabetic retinopathy (DR, non-proliferative and proliferative DR) with edema (DME), diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular Edema, macular edema after retinal vein occlusion (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branched retinal vein occlusion (BRVO), retinal angiomatous proliferation (RAP), polypoidal choroidal vessels Lesion (polypoidal choroidal vasculopathy, PCV), vitreomacular adhesion (VMA), and/or vitreomacular traction (VMT). 如請求項16或17所述之用途，其中該配方係以每眼約0.5至10毫克（mg）的劑量投予。The use of claim 16 or 17, wherein the formulation is administered at a dose of about 0.5 to 10 milligrams (mg) per eye. 如請求項18所述之用途，其中該配方係以每眼約4 mg的劑量投予。The use of claim 18, wherein the formulation is administered at a dose of about 4 mg per eye. 一種醫藥配方，該配方包含： a)   一濃度為40 mg/mL之融合蛋白； b)   25 mM之組胺酸； c)    190 mM之海藻酸、蔗糖、或甘露醇；以及 d)   0.03%之聚山梨糖醇酯20或聚山梨糖醇酯80， 其中該配方的pH值為約6.0。 A pharmaceutical formula containing: a) A fusion protein with a concentration of 40 mg/mL; b) 25 mM histamine; c) 190 mM alginic acid, sucrose, or mannitol; and d) 0.03% polysorbate 20 or polysorbate 80, The pH of this formulation is approximately 6.0. 如請求項20所述之醫藥配方，其中該融合蛋白係自N-端至C-端以如下順序包含： a)   一血管內皮生長因子受體（VEGFR）的細胞外結構域； b)   一人類免疫球蛋白G的Fc結構域；以及 c)    結合一整合蛋白之蛋白或其片段。 The pharmaceutical formula as described in claim 20, wherein the fusion protein includes in the following order from N-terminus to C-terminus: a) An extracellular domain of vascular endothelial growth factor receptor (VEGFR); b) an Fc domain of human immunoglobulin G; and c) Protein or fragment thereof that binds to an integrin. 如請求項21所述之醫藥配方，其中該融合蛋白係包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO: 17、或SEQ ID NO: 18。The pharmaceutical formulation of claim 21, wherein the fusion protein contains SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. 如請求項20至22中任一項所述之醫藥配方，其中該配方在-70℃、-20℃、及／或5℃下安定性維持至少24個月。The pharmaceutical formula as described in any one of claims 20 to 22, wherein the formula maintains stability at -70°C, -20°C, and/or 5°C for at least 24 months. 如請求項20至22中任一項所述之醫藥配方，其中該配方在-70℃、-20℃、及／或2℃至8℃下至少6個月後係保持蛋白質的純度及活性。The pharmaceutical formulation according to any one of claims 20 to 22, wherein the formulation maintains protein purity and activity after at least 6 months at -70°C, -20°C, and/or 2°C to 8°C. 如請求項20至22中任一項所述之醫藥配方，其中該配方更包含一濃度為約10 mM至50 mM的鹽類。The pharmaceutical formula according to any one of claims 20 to 22, wherein the formula further contains a salt at a concentration of about 10 mM to 50 mM. 如請求項20至22中任一項所述之醫藥配方，其中該配方更包含至少一種胺基酸，該胺基酸的濃度為約10 mM至50 mM。The pharmaceutical formulation according to any one of claims 20 to 22, wherein the formulation further contains at least one amino acid, and the concentration of the amino acid is about 10 mM to 50 mM. 如請求項25所述之醫藥配方，其中該鹽類係選自氯化鈉、氯化鎂、氯化鈣、或氯化鉀。The pharmaceutical formula as claimed in claim 25, wherein the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride. 如請求項26所述之醫藥配方，其中該胺基酸係選自由以下所組成的群組：精胺酸、甲硫胺酸、脯胺酸、組胺酸、半胱胺酸、離胺酸、甘胺酸、天門冬胺酸、色胺酸、麩胺酸、及異白胺酸。The pharmaceutical formulation of claim 26, wherein the amino acid is selected from the group consisting of: arginine, methionine, proline, histidine, cysteine, lysine , glycine, aspartic acid, tryptophan, glutamic acid, and isoleucine. 一種使用如請求項20至28中任一項所述之醫藥配方於製備一醫藥組合物的用途，其中該醫藥組合物係用於治療一個體之眼部疾病。A method of using the pharmaceutical formulation as described in any one of claims 20 to 28 to prepare a pharmaceutical composition, wherein the pharmaceutical composition is used to treat an eye disease in an individual. 如請求項29所述之用途，其中該眼部疾病係選自新生血管或缺血性眼色素層炎、視網膜血管炎、血管狀痕、色素性視網膜炎、角膜新生血管、虹膜新生血管、新生血管性青光眼、青光眼術後纖維化、增殖性玻璃體視網膜病變（PVR）、脈絡膜新生血管（CNV）、視神經盤新生血管、視網膜新生血管、玻璃體新生血管、血管翳、翼狀贅肉、血管性視網膜病變、無糖尿病黃斑部水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、有糖尿病黃斑部水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、糖尿病黃斑部水腫（DME）、滲出性（濕性）及非滲出性（乾性）之年齡相關性黃斑部退化（AMD）、黃斑部水腫、視網膜靜脈阻塞後黃斑部水腫（RVO）、視網膜靜脈阻塞（RVO）、中心性視網膜靜脈阻塞（CRVO）、視網膜分支靜脈阻塞（BRVO）、視網膜血管瘤狀增殖（RAP）、息肉狀脈絡膜血管病變（PCV）、玻璃體黃斑部沾黏（VMA）及／或玻璃體黃斑部牽引症（VMT）。The use as claimed in claim 29, wherein the eye disease is selected from the group consisting of neovascular or ischemic uveitis, retinal vasculitis, vascular scars, retinitis pigmentosa, corneal neovascularization, iris neovascularization, neovascularization, Vascular glaucoma, postoperative glaucoma fibrosis, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy , diabetic retinopathy (DR, non-proliferative and proliferative DR) without diabetic macular edema (DME), diabetic retinopathy (DR, non-proliferative and proliferative DR) with diabetic macular edema (DME), diabetes Macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, post-retinal vein occlusion macular edema (RVO), retinal vein occlusion ( RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), retinal angiomatous proliferation (RAP), polypoidal choroidal vasculopathy (PCV), vitreomacular adhesion (VMA) and/or vitreous Macular Traction (VMT). 如請求項29或30所述之用途，其中該配方係以每眼約0.5至10 mg的劑量投予。The use as claimed in claim 29 or 30, wherein the formulation is administered at a dose of about 0.5 to 10 mg per eye. 如請求項31所述之用途，其中該配方係以每眼約4 mg的劑量投予。The use as claimed in claim 31, wherein the formulation is administered at a dose of about 4 mg per eye. 一種使用一醫藥配方於製備一醫藥組合物的用途，其中該醫藥組合物係用於治療一個體之眼部疾病，該醫藥配方係包含： a)    一濃度為約0.5 mg/mL至約120 mg/mL之融合蛋白； b)   一濃度為約1%至約10%（重量／體積）之多元醇或醇類，該多元醇或醇類係選自由以下所組成之群組：蔗糖、海藻糖、甘露醇、山梨糖醇、苯甲醇、聚乙烯醇、及聚乙二醇（PEG）400-12000； c)    一濃度為約10 mM至約50 mM之緩衝劑，該緩衝劑係選自由以下所組成之群組：磷酸鈉、組胺酸、檸檬酸鈉、醋酸鈉、碳酸氫鈉、及檸檬酸三鈉二水合物；以及 d)   一濃度為約0.01%至約4%（重量／體積）之界面活性劑， 其中，該配方的pH值為約5.5至7.5，且該配方視需要地更包含一選自由以下所組成之群組的多醣：羧甲基纖維素鈉、微晶纖維素、或透明質酸鈉。 A method of using a pharmaceutical formula to prepare a pharmaceutical composition, wherein the pharmaceutical composition is used to treat an eye disease in an individual, and the pharmaceutical formula contains: a) A fusion protein with a concentration of about 0.5 mg/mL to about 120 mg/mL; b) A polyol or alcohol at a concentration of about 1% to about 10% (weight/volume), the polyol or alcohol being selected from the group consisting of: sucrose, trehalose, mannitol, sorbose Alcohol, benzyl alcohol, polyvinyl alcohol, and polyethylene glycol (PEG) 400-12000; c) A buffer having a concentration of about 10 mM to about 50 mM, the buffer being selected from the group consisting of: sodium phosphate, histidine, sodium citrate, sodium acetate, sodium bicarbonate, and citric acid Trisodium dihydrate; and d) A surfactant at a concentration of about 0.01% to about 4% (weight/volume), Wherein, the pH value of the formula is about 5.5 to 7.5, and the formula optionally further includes a polysaccharide selected from the group consisting of: sodium carboxymethylcellulose, microcrystalline cellulose, or sodium hyaluronate . 如請求項33所述之用途，其中該界面活性劑係選自由以下所組成之群組：聚山梨糖醇酯20、聚山梨糖醇酯80、及泊洛沙姆188。The use as claimed in claim 33, wherein the surfactant is selected from the group consisting of: polysorbate 20, polysorbate 80, and poloxamer 188. 如請求項33或34所述之用途，其中該界面活性劑的濃度為約0.03%。The use as claimed in claim 33 or 34, wherein the concentration of the surfactant is about 0.03%. 如請求項33或34所述之用途，其中該融合蛋白的濃度為約20 mg/mL至約90 mg/mL。The use as claimed in claim 33 or 34, wherein the concentration of the fusion protein is about 20 mg/mL to about 90 mg/mL. 如請求項33或34所述之用途，其中該多元醇係海藻糖且濃度為約150 mM至230 mM。The use as claimed in claim 33 or 34, wherein the polyol is trehalose and the concentration is about 150 mM to 230 mM. 如請求項33或34所述之用途，其中該緩衝劑係組胺酸且濃度為約20 mM至約40 mM。The use as claimed in claim 33 or 34, wherein the buffer is histidine and the concentration is from about 20 mM to about 40 mM. 如請求項33或34所述之用途，其中該融合蛋白自N-端至C-端係以如下順序包含： a)   一血管內皮生長因子受體（VEGFR）的細胞外結構域； b)   一人類免疫球蛋白G的Fc結構域；以及 c)    一結合整合蛋白之蛋白或其片段。 The use as described in claim 33 or 34, wherein the fusion protein includes in the following order from N-terminus to C-terminus: a) An extracellular domain of vascular endothelial growth factor receptor (VEGFR); b) an Fc domain of human immunoglobulin G; and c) A protein or fragment thereof that binds integrin. 如請求項39所述之用途，其中該融合蛋白係包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO: 17、或SEQ ID NO: 18。The use as claimed in claim 39, wherein the fusion protein contains SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. 如請求項33或34所述之用途，其中該pH值為約6.0至約6.5。The use as claimed in claim 33 or 34, wherein the pH value is about 6.0 to about 6.5. 如請求項33或34所述之用途，其中該配方在-70℃、-20℃、及／或5℃下安定性維持至少24個月。The use as described in claim 33 or 34, wherein the formulation maintains stability at -70°C, -20°C, and/or 5°C for at least 24 months. 如請求項33或34所述之用途，其中該配方在-70℃、-20℃、及／或2℃至8℃下至少6個月後係保持蛋白質的純度及活性。The use as described in claim 33 or 34, wherein the formulation maintains the purity and activity of the protein after at least 6 months at -70°C, -20°C, and/or 2°C to 8°C. 如請求項33或34所述之用途，其中該配方更包含一濃度為約10 mM至50 mM的鹽類。The use as claimed in claim 33 or 34, wherein the formulation further contains a salt at a concentration of about 10 mM to 50 mM. 如請求項33或34所述之用途，其中該配方更包含至少一種胺基酸，該胺基酸的濃度為約10 mM至50 mM。The use as claimed in claim 33 or 34, wherein the formulation further contains at least one amino acid, and the concentration of the amino acid is about 10 mM to 50 mM. 如請求項44所述之用途，其中該鹽類係選自氯化鈉、氯化鎂、氯化鈣、或氯化鉀。The use as claimed in claim 44, wherein the salt is selected from sodium chloride, magnesium chloride, calcium chloride, or potassium chloride. 如請求項45所述之用途，其中該胺基酸係選自由以下所組成之群組：精胺酸、甲硫胺酸、脯胺酸、組胺酸、半胱胺酸、離胺酸、甘胺酸、天門冬胺酸、色胺酸、麩胺酸、及異白胺酸。The use as claimed in claim 45, wherein the amino acid is selected from the group consisting of: arginine, methionine, proline, histidine, cysteine, lysine, Glycine, aspartic acid, tryptophan, glutamic acid, and isoleucine. 如請求項33或34所述之用途，其中該眼部疾病係包含新生血管或缺血性眼色素層炎、視網膜血管炎、血管狀痕、色素性視網膜炎、角膜新生血管、虹膜新生血管、新生血管性青光眼、青光眼術後纖維化、增殖性玻璃體視網膜病變（PVR）、脈絡膜新生血管（CNV）、視神經盤新生血管、視網膜新生血管、玻璃體新生血管、血管翳、翼狀贅肉、血管性視網膜病變、無糖尿病黃斑部水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、有糖尿病黃斑部水腫（DME）之糖尿病視網膜病變（DR，非增生性及增生性DR）、糖尿病黃斑部水腫（DME）、滲出性（濕性）及非滲出性（乾性）之年齡相關性黃斑部退化（AMD）、黃斑部水腫、視網膜靜脈阻塞後黃斑部水腫（RVO）、視網膜靜脈阻塞（RVO）、中心性視網膜靜脈阻塞（CRVO）、視網膜分支靜脈阻塞（BRVO）、視網膜血管瘤狀增殖（RAP）、息肉狀脈絡膜血管病變（PCV）、玻璃體黃斑部沾黏（VMA）及／或玻璃體黃斑部牽引症（VMT）。The use as described in claim 33 or 34, wherein the eye disease includes neovascular or ischemic uveitis, retinal vasculitis, vascular scars, retinitis pigmentosa, corneal neovascularization, iris neovascularization, Neovascular glaucoma, postoperative glaucoma fibrosis, proliferative vitreoretinopathy (PVR), choroidal neovascularization (CNV), optic disc neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retina Lesions, diabetic retinopathy (DR, non-proliferative and proliferative DR) without diabetic macular edema (DME), diabetic retinopathy (DR, non-proliferative and proliferative DR) with diabetic macular edema (DME), Diabetic macular edema (DME), exudative (wet) and non-exudative (dry) age-related macular degeneration (AMD), macular edema, post-retinal vein occlusion macular edema (RVO), retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), retinal angiomatous proliferation (RAP), polypoidal choroidal vasculopathy (PCV), vitreomacular adhesion (VMA) and/or Vitreomacular traction (VMT). 如請求項33或34所述之用途，其中該配方係以每眼約0.03至10 mg的劑量投予。The use as claimed in claim 33 or 34, wherein the formulation is administered at a dose of about 0.03 to 10 mg per eye. 如請求項49所述之用途，其中該配方係以每眼約4 mg的劑量投予。The use of claim 49, wherein the formulation is administered at a dose of about 4 mg per eye.
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