TW577896B

TW577896B - Hybrid with interferon-alpha and an immunoglobulin Fc linked through a non-immunogenic peptide

Info

Publication number: TW577896B
Application number: TW86110733A
Authority: TW
Inventors: Liming Yu; Tse-Wen Chang
Original assignee: Tanox Biosystems Inc
Priority date: 1996-09-25
Filing date: 1997-07-28
Publication date: 2004-03-01

Abstract

Disclosed is a hybrid recombinant protein consisting of human interferon, preferably interferon-alpha (IFNalpha), and human immunoglobulin Fc fragment, preferably gamma4 chain, joined by a peptide linker comprising a T cell inert sequence, for example, the sequence Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser.

Description

577896 經濟部中央標準局員工消費合作社印製 A7 B7 1、發明説明（1 ) 發明背景干擾素- a (“IFΝα”）是以重組體DNA技術所產生的第一種細胞動素，且已示出在如發炎，病毒及惡性疾病等狀況下具有治療價値。許多種IFNcx製劑，包括純化自天然來源及由重組DNA技術所產生者已被應用，且於各種惡性及病毒疾病中被測試。IFNa可造成某些已發展腫瘤之消退，且在某些病毒感染中謗導正面反應。目前爲止， IFNa已於許多國家中被許可或試驗於以下適應症：如卡波西氏肉瘤；髮細胞白血病；惡性黑色素瘤；基礎細胞癌；多發性骨髓瘤；腎細胞癌；B型肝炎；C型肝炎；花柳疣；I/II疱疹，水痘/輸狀疱疹；及蕈樣肉芽腫。大多數的細胞動素，包括IFNa，由於於活體内產生以局邵及暫時作用，因此有相當短的循環半衰期。j F N a之血清半衰期僅約2至8小時（Roche Labs. Referon A，577896 A7 B7 printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 1. Description of the invention (1) Background of the invention Interferon-a ("IFNα") is the first cytokine produced by recombinant DNA technology and has been shown It has therapeutic value in conditions such as inflammation, viruses and malignant diseases. Many IFNcx preparations, including those purified from natural sources and those produced by recombinant DNA technology, have been used and tested in a variety of malignant and viral diseases. IFNa can cause regression of some developed tumors and defame positive reactions in certain viral infections. So far, IFNa has been licensed or tested in many countries for the following indications: such as Kaposi's sarcoma; hairy cell leukemia; malignant melanoma; basal cell carcinoma; multiple myeloma; renal cell carcinoma; hepatitis B; Hepatitis C; willow warts; I / II herpes, chicken pox / herpes zoster; and mycosis fungoides. Most cytokines, including IFNa, have relatively short circulating half-lives due to their local and transient effects in vivo. j F N a has a serum half-life of only about 2 to 8 hours (Roche Labs. Referon A,

Schering Intron A，Physicians，Desk Reference， 47th edition， 1 993，pp· 2006-2008 ^ 2194- 2201)。爲運用IFNa爲有效率之全身性治療劑，其需以極大劑量且經常投藥。例如，對AID S -相關之卡波西氏肉瘤的建議療程之一是始自每天36百萬IU之謗導劑量歷1〇至1 2週，以肌内或皮下注射方式投予，繼以3 6百萬iu,之維持劑量，一週 3 次。（R0che Labs. Referon a， Physicians, Desk Reference，47th edition，1 993, pp. 2 006-2 008)。此種經常性腸外投藥是不方便且令人疼痛的。再者可能由高劑量引起之毒性作用，爲某些患者 -4- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） --------- (請先閱讀背面之注意事項再填寫本頁) 、訂 577896 A7Schering Intron A, Physicians, Desk Reference, 47th edition, 1 993, pp. 2006-2008 ^ 2194- 2201). In order to use IFNa as an effective systemic therapeutic agent, it needs to be administered in extremely large doses and often. For example, one of the recommended courses of treatment for AID S-associated Kaposi's sarcoma is a defamatory dose starting from 36 million IU per day for 10 to 12 weeks, administered by intramuscular or subcutaneous injection followed by A maintenance dose of 36 million iu, 3 times a week. (R0che Labs. Referon a, Physicians, Desk Reference, 47th edition, 1 993, pp. 2 006-2 008). Such regular parenteral administration is inconvenient and painful. Furthermore, the toxic effect may be caused by high doses for some patients. -4- This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) --------- (Please read the back (Please fill in this page again), order 577896 A7

之難處所在。據報的有皮膚，神經學，内分泌及免产主性。爲克服這些缺點，吾等可修飾分子以増加其循期或改變藥物之調和物以延長其釋出時間。劑量及投藥頻率可減少而仍增加其效力。據報，低於9百萬單位之劑量是可耐受的，而高於36百萬單位之劑量常會謗生嚴重的母性及顯著地改變病人狀況。（Quesada，J r et ai j Clin· 〇nc〇l·，4 ·· 234-43，1 986)。經由產生新型 IFNa也可能實質地減少毒性作用，其係可在循環中更穩定且需較少之劑量。目前已致力於製造滞留時間有所延長之重組體IFNa-明膠共輕物（Tabata，Y. et ai Cancer Res· 5 1 : 5 5 3 2-8, 1 99 1 )。於動物中也已試驗以脂質爲基礎之經包膠之IFNa調和物，並達成蛋白質在腹膜中延長之釋放（Bonetti，A. and Kim，S. Caneer Chemother Pharmacol. 3 3 : 2 5 8-26 1，1 993) 〇經濟部中央標準局員工消費合作社印製 I L-1 (請先閱讀背面之注意事項再填寫本頁)The difficulty is. Skin, neurology, endocrinology and immunity are reported. To overcome these shortcomings, we can modify the molecule to increase its period or change the blend of the drug to extend its release time. Dose and frequency of administration can be reduced while still increasing its effectiveness. It has been reported that doses below 9 million units are tolerable, while dosages above 36 million units often stigmatize severe motherhood and significantly alter the condition of the patient. (Quesada, Jr et ai j Clin. OncOl., 4..234-43, 1 986). It is also possible to substantially reduce toxic effects by producing novel IFNa, which can be more stable in circulation and requires fewer doses. At present, efforts have been made to produce recombinant IFNa-gelatin co-lights with extended residence time (Tabata, Y. et ai Cancer Res · 5 1: 5 5 3 2-8, 1 99 1). Lipid-based encapsulated IFNa mediators have also been tested in animals and have achieved prolonged release of proteins in the peritoneum (Bonetti, A. and Kim, S. Caneer Chemother Pharmacol. 3 3: 2 5 8-26 1,1 993) 〇 I L-1 printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page)

IgG及IgM類免疫球蛋白爲人體血液中含量最豐之一類。其循環半衰期由數天至21天。當應用於形成重組融合體時’頃發現IgG可增加許多配體結合蛋白質（受體）之半衰期，包括可溶性CD4分子，LHR及IFN-γ受體 (Mordenti J. et al.，Nature, 3 3 7 : 5 2 5 -3 1，1 989 ; Capon， D. J. and Lasky， L. A.，美國專利案 5，1 1 6,964 ; Kurschner，C. et al·，J. Immunol. 149 : 4096-4100，1992)。然而，此種融合體出現難題，即在活性部份C-末端之肽，及在Fc部份N-末端之肽，在融合完處會生成新的肽序列，其是一種新抗原，且 -5- 本紙張尺度適用中國國家標準（CNS ) A4規格（2丨0'〆297公釐） 577896 、發明説明（3 經濟部中央標準局員工消費合作社印製具免疫原性。本發明是有關一種IFNa_Fc融合體，其經過設計可克服此問題並延長IFNa之半衰期。發明要點本發明疋有關由二個亞單位組成之融合體重組蛋白質。各亞單位包括一個人類干擾素，較好是IFNa，由一個肽連接子聯結，其主要由τ細胞惰性序列所組成，聯結至人類免疫球蛋白Fc片段上，較好是丫4鏈。γ4鏈優於口鏈，因爲前者之補體活化能力較少或無此能力。 IFNa之C_末端聯結至Fc片段之Ν_末端。額外的 IFNa(或其他細胞動素）可黏附至pc片段其他任何未結合 Fc鏈之N-末端上，造成γ4鏈之同二體。若所選擇的1^片段是另一鏈，如μ鏈，則由於卜片段可與1〇個可能的結合位置形成五聚體，此可生成有干擾素或其他細胞動素在 1 0個結合位置各自上聯結之分子。融合體的二個部份經由Τ細胞免疫學上惰性的肽而聯結，如Gly Gly Ser Gly (Hy Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser。此肽本身是免疫學上無活性的。在融合點處將此肽嵌入，可將由於二個肽部份聯結所生成之新抗原性消除。連接子肽也可增加這些部份之撓性，且可保留生物活性。此相當長的連接子肽有助於克服由融合體F c邵份來的可能的立體位阻，後者可干擾融合體之活性。融合體有較天然IFNa還長之半衰期。由於連接子，可設計以將產生新的免疫原性表位（新抗原）之可能性也減 ---------·裝 ί (請先聞讀背面之注意事項再填寫本頁) 、11 J# -6 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X297公董） 577896 A7 -----B7 五、發明説明（4 ) ' '~ ~ -一低，此點是IFNa及免疫球蛋白以片段之融合點。細胞動素通常是有相當短半衰期之小蛋白質，其可在各種組織上，W非欲求位置，快速消1。咸信少量的某些細胞動素可穿越血腦障壁，並進入中樞神經系，由是造成嚴重的神經毒性。IFNa聯結至本發明Fey，尤其可適用於治療B或C型肝炎，因爲這些產物在血管内（一旦經靜脈内投藥）有長的滯留時間，且不會穿透非欲求位置。此中所述的特異融合體也可充作設計及構築其他細胞動素-Fc融合體之模式。可使用相同或類似的連接子以減少產生新的免疫原性表位之可能性，同時保留生物活性。利用相同的技術可製成其中間白素是細胞動素之細胞動素 -Fc融合體，或包括有其他細胞動素之融合體。製備及使用本發明之詳細説明經濟部中央標準局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁} 本發明的融合體分子包括一個干擾素部份，經由獨特的連接子聯結至免疫球蛋白FC部份。較好，二個干擾素部份的C -末端係分別地黏附至重鏈γ4 F c片段二個N•末端各自上，而生成一個同二體結構。獨特的連接子肽，Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser，其核甞酸序列示於附錄“A”，可生成以聯結二部份。較佳的γ4融合體之完全的核芬酸序列（包括連接子及F c部份）示於附錄“ Β，，中，且連接子位在i 8 9 至204號胺基酸殘基。融合體甚於天然細胞動素之優點在於活體内之半衰期更長。包括干擾素及γ4鏈Fc同二體之融合體較天然干擾素本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 577896 經濟部中央標準局員工消費合作社印製 A7 _______B7_·__ 五、發明説明（5 ) 還大。由於肝中血管之孔經較大，此大分子更適用於治療肝炎’在此病毒主要是負責侵襲肝臟。連接子肽經過設計可增加二部份之撓性，且因此維持其生物活性。雖然干擾素及免疫球蛋白均源自人體，在二分子融合點處始終有產生新的免疫原性表位之可能性。因此，本發明連接子（其主要由T細胞惰性序列所組成）的其他優點在於可在融合點減低免疫原性。於附錄“B，，中可見，若連接子（189-204號殘基）不存在，可生成由第189 號前之殘基及2 0 4號後之殘基所組成之新序列。此新序列對人體而言將是一個新抗原。人類IFNa衍生自許多不同基因族。自基因及蛋白質序列資料中，目前已鑑定出24種以上。其到處有相異處，由一些至最多35個胺基酸。大多數有一個23個胺基酸殘基之訊號肽序列，及166個胺基酸殘基之成熟胺基酸序列。(Goeddel，D.V· et al·，Nature，290 : 20_26, 1 9 8 1: Wei s smann, C. and Weber, H.? Prog. Nuc. Acid Res. Mol. Biol. 3 3 : 25 1 -3 00，1 986 ; Zoon， K.C·，Interferon，9 : 1-12，1 987) 0 INFot2(也稱爲IFNaA)爲最被充份研究之干擾素之一。IFNa2之重組體型式已充作治療劑使用達數年。目前買得到的二種IFNa2重組體產物爲IFNa2a及 IFNa2b，其相異處僅在23位置之一個胺基酸，且二者生物活性間並無顯著不同（von Gabain，A.，et al.，Eur. J. Biochem. 1 90 : 257-61，1 990) ° -8- 本紙張尺度適用中國國家標準（CNS ) A4規格（21 OX297公釐） ---------^^裝 j------訂------ (請先閱讀背面之注意事項再填寫本頁) 577896 經濟部中央標準局員工消費合作社印製 A7 ____B7_ 五、發明説明（6 ) IFNa2a被選爲本發明干擾素融合體之融合配對，雖然 IFNcx2b或其他的干擾素（包括IFNp)也可使用。以其他細胞動素也可製備類似的構體，如間白素_ i或間白素_ 2。可使用相同的連接子，或不具有免疫原性且可保有構體生物活性者也可替代之。融合體中以γ4鏈爲Fc部份的一個優點在於其在人體循環中是穩定的。γ4鏈（和γ 1鏈不同）可避免大範園的二次生物特性，如補體固定及抗體一依賴性細胞調介之胞毒性 (ADCC)，其可能是非欲求之特性。 IFNa2a之cDNA可由反轉錄作用及PCR獲得，利用可表現IFNa之白血球萃取RNA而進行。此種細胞株之一， KG_1可得自美國標準菌種收集所（ATCC)，於 Rockville，Maryland，其編號爲 CCL 246。在製備本發明融合體之步驟中，於RNA萃取之前，細胞以仙台病毒挑戰之以增加干擾素之轉錄作用（Cantell，K. et al.， Methods in Enzymology，78A : 29-3 8，Adacemic Press， 1981)。如上述，IFNa爲IFN之集合，且各細胞可在相同時間表現許多不同的IFNa亞型。在這些各型中DNA序歹J之同質性如此高以致RT-PCR可能可擴大一群而非特殊的一種。爲特異地獲得IFNa2a cDNA，要設計PCR引子如此二個引子的最後一個核甞酸可在IFNa2a獨特有之胺基酸密碼處結束。其各別位置S22及161(看Zoon，K.C. Interferon，9 : 1-12，1 987)。利用重疊的 PCR 技術（Daugherty，B.L. et al.， •9- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐) '" (請先閱讀背面之注意事項再填寫本頁) I# 項再填· 裝· -訂經濟部中央標準局員工消費合作社印製 577896 Α7 Β7 五、發明説明（7 )IgG and IgM immunoglobulins are among the most abundant in human blood. Its cyclic half-life ranges from several days to 21 days. When applied to the formation of recombinant fusions, 'IgG was found to increase the half-life of many ligand-binding proteins (receptors), including soluble CD4 molecules, LHR and IFN-γ receptors (Mordenti J. et al., Nature, 3 3 7: 5 2 5 -3 1,1 989; Capon, DJ and Lasky, LA, U.S. Patent No. 5,1 1 6,964; Kurschner, C. et al., J. Immunol. 149: 4096-4100, 1992). However, such fusions have problems, that is, the peptide at the C-terminus of the active part and the peptide at the N-terminus of the Fc part will generate a new peptide sequence at the end of fusion, which is a new antigen, and- 5- This paper size applies to Chinese National Standard (CNS) A4 specifications (2 丨 0'〆297 mm) 577896, invention description (3 Employees' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, printed with immunogenicity. The present invention relates to a IFNa_Fc fusion, which is designed to overcome this problem and prolong the half-life of IFNa. Summary of the invention The invention relates to a recombinant recombinant protein composed of two subunits. Each subunit includes a human interferon, preferably IFNa, and consists of A peptide linker is mainly composed of τ cell inert sequences and is connected to the human immunoglobulin Fc fragment, preferably the y4 chain. The γ4 chain is better than the mouth chain because the former has less or no complement activation capacity This ability. The C-terminus of IFNa is linked to the N-terminus of the Fc fragment. Additional IFNa (or other cytokines) can be attached to the N-terminus of any other unbound Fc chain of the pc fragment, resulting in the same γ4 chain. If the selected fragment is another chain, such as the μ chain, since the fragment can form a pentamer with 10 possible binding positions, this can generate interferon or other cytokines in 10 Molecules linked to each binding site. The two parts of the fusion are linked via T-cell immunologically inactive peptides, such as Gly Gly Ser Gly (Hy Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser. The peptide itself is Immunologically inactive. Embedding this peptide at the fusion point can eliminate the new antigenicity generated by the connection of the two peptide parts. The linker peptide can also increase the flexibility of these parts and retain biological Activity. This rather long linker peptide helps to overcome possible steric hindrance from the fusion F c, which can interfere with the activity of the fusion. The fusion has a half-life longer than that of natural IFNa. Because of the linker , Can be designed to reduce the possibility of generating new immunogenic epitopes (neoantigens) --------- · installed (please read the precautions on the back before filling out this page), 11 J # -6 This paper size applies to China National Standard (CNS) Α4 specifications (210X297 public director) 577896 A7 ----- B7 V. Description of the invention (4) '~~-A low, this point is the fusion point of IFNa and immunoglobulin in fragments. Cytokines are usually quite short A half-life small protein, which can be quickly removed in various tissues, where it is not desired. 1. A small amount of certain cytokines can cross the blood-brain barrier and enter the central nervous system, causing severe neurotoxicity. IFNa linked to Fey of the present invention is particularly suitable for the treatment of hepatitis B or C, because these products have a long residence time in blood vessels (once intravenously) and do not penetrate non-desired sites. The specific fusions described herein can also be used as a model for designing and constructing other cytokinin-Fc fusions. The same or similar linkers can be used to reduce the possibility of generating new immunogenic epitopes while retaining biological activity. Using the same technique, a cytokinin-Fc fusion in which interleukin is cytokinin, or a fusion including other cytokinins can be made. Detailed description of the preparation and use of the present invention Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) The fusion molecule of the present invention includes an interferon moiety and is connected by a unique linker To the immunoglobulin FC part. Preferably, the C-terminus of the two interferon moieties are respectively attached to the two N • termini of the heavy chain γ4 F c fragment to form a homodimeric structure. Unique Linker peptide, Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly G Ser, whose nucleotide sequence is shown in Appendix "A", can be generated to link the two parts. The complete γ4 fusion is better Nucleic acid sequences (including the linker and F c part) are shown in the appendix "B ,, and the linker is located at amino acid residues i 8 9 to 204. The advantages of fusions over natural cytokines It has a longer half-life in the living body. It includes fusions of interferon and γ4 chain Fc homodimers than natural interferons. This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm). Cooperation A7 _______ B7_ · __ printed by the agency V. Description of the invention (5) is still large. Due to the large pore diameter of the blood vessels in the liver, this macromolecule is more suitable for treating hepatitis. Here the virus is mainly responsible for invading the liver. The linker peptide is designed Can increase the flexibility of the two parts, and therefore maintain their biological activity. Although both interferon and immunoglobulin are derived from the human body, there is always the possibility of generating new immunogenic epitopes at the fusion point of the two molecules. Therefore, Another advantage of the linker of the present invention, which is mainly composed of inert sequences of T cells, is that it can reduce immunogenicity at the fusion point. As can be seen in Appendix "B ,, if the linker (residues 189-204) does not exist , Can generate a new sequence consisting of residues before 189 and residues after 204. This new sequence will be a new antigen for the human body. Human IFNa is derived from many different gene families. From genes And protein sequence data, more than 24 species have been identified. There are differences everywhere, from some to a maximum of 35 amino acids. Most have a signal peptide sequence of 23 amino acid residues, and 166 Amino acid residues Mature amino acid sequence. (Goeddel, DV · et al ·, Nature, 290: 20-26, 1 9 8 1: Wei s smann, C. and Weber, H.? Prog. Nuc. Acid Res. Mol. Biol. 3 3: 25 1 -3 00, 1 986; Zoon, KC ·, Interferon, 9: 1-12, 1 987) 0 INFot2 (also known as IFNaA) is one of the most studied interferons. The recombinant form of IFNa2 has been used as a therapeutic agent for several years. The two currently available IFNa2 recombinant products are IFNa2a and IFNa2b, which differ in only one amino acid at the 23 position, and there is no significant difference in biological activity between the two (von Gabain, A., et al., Eur. J. Biochem. 1 90: 257-61, 1 990) ° -8- This paper size applies to China National Standard (CNS) A4 (21 OX297 mm) --------- ^^ j ------ Order ------ (Please read the notes on the back before filling in this page) 577896 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 ____B7_ V. Description of Invention (6) IFNa2a was selected This is a fusion pairing of the interferon fusions of the invention, although IFNcx2b or other interferons (including IFNp) can also be used. Similar constructs can also be prepared with other cytokines, such as interleukin_i or interleukin_2. The same linker can be used, or it can be substituted if it is not immunogenic and can retain the biological activity of the construct. One advantage of using the γ4 chain as the Fc portion of the fusion is that it is stable in human circulation. The γ4 chain (different from the γ1 chain) can avoid the secondary biological properties of Dafanyuan, such as complement fixation and antibody-dependent cell-mediated cytotoxicity (ADCC), which may be undesired properties. The IFNa2a cDNA can be obtained by reverse transcription and PCR, and extracted from white blood cells expressing IFNa using RNA. One such cell line, KG_1, is available from the American Standard Strain Collection (ATCC) in Rockville, Maryland, under the number CCL 246. In the step of preparing the fusion of the present invention, before RNA extraction, cells are challenged with Sendai virus to increase the transcription of interferon (Cantell, K. et al., Methods in Enzymology, 78A: 29-3 8, Adacemic Press , 1981). As mentioned above, IFNa is a collection of IFN, and each cell can express many different IFNa subtypes at the same time. The homology of the DNA sequence 歹 J among these types is so high that RT-PCR may expand a group rather than a specific one. To specifically obtain IFNa2a cDNA, PCR primers should be designed so that the last nucleotide of the two primers can end at the amino acid code unique to IFNa2a. Their respective positions S22 and 161 (see Zooon, K.C. Interferon, 9: 1-12, 1 987). Use of overlapping PCR technology (Daugherty, BL et al., • 9- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) '" (Please read the notes on the back before filling this page) I # Items are refilled · Equipment ·-Ordered by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 577896 Α7 Β7 V. Description of the Invention (7)

Nucleic Acids Res. 1 9 : 2471 -6，1991)，吾等可在依欲求之任何位置處容易地連接二個基因片段。然而， PCR擴大作用的缺點是有相當高的突變比例（Saiki，R.K. e t al., Science, 2 39:487，1988) 〇因此也進行 DNA 定序以檢查由PCR獲得之每一個DNA片段有否突變作用。當片段大小超過1 kb時，定序是冗長且費時的，就如全長的IFNa-Fc cDNA。然而，可將限制酶-BamH I，納入連接子核甞酸序列中，而不致改變其胺基酸序列。此位置位在核甞酸15及16號間，見附錄“A”。來自PCR的二個基因片段可分別選殖至選殖載體内。此使DNA更易且更快被定序，因爲二片段僅數百鹼基對長而已。一旦鑑定出有正確DN A序列之純系，二個基因片段可經由BamH I位置聯結在一起。勿需予二輪的重疊 PCR及接下來全長片段之〇ΝΑ定序工作。Nucleic Acids Res. 1 9: 2471 -6, 1991), we can easily connect two gene fragments at any position as desired. However, the disadvantage of PCR amplification is that it has a relatively high proportion of mutations (Saiki, RK et al., Science, 2 39: 487, 1988). Therefore, DNA sequencing is also performed to check whether each DNA fragment obtained by PCR is available. Mutation effect. When the fragment size exceeds 1 kb, sequencing is lengthy and time-consuming, such as the full-length IFNa-Fc cDNA. However, the restriction enzyme-BamH I can be incorporated into the linker nucleotide sequence without altering its amino acid sequence. This position is between nucleotides 15 and 16, see Appendix "A". The two gene fragments from the PCR can be individually selected into a selection vector. This makes DNA easier and faster to sequence because the two fragments are only a few hundred base pairs long. Once a pure line with the correct DNA sequence is identified, the two gene fragments can be linked together via the BamH I position. It is not necessary to perform two rounds of overlapping PCR and subsequent 〇NA sequencing of full length fragments.

於試管内表現重組體蛋白質之方式有許多種，包括於大腸桿菌’杆病毒，酵母，哺乳動物細胞或其他表現系。原核細胞系統一大腸桿菌，無法行轉譯後修飾作用，如糖基化作用。但此點對IF N a - F c融合體而言並非嚴重問題，因爲天然的IFNa-及免疫球蛋白γ4分子並未嚴重糖基化再者已有報告指出，無任何糖基化作用之重組體 IFNa可保有其生物活性（Bar〇n，& and Narula SThere are many ways to express recombinant proteins in test tubes, including coliform ' baculovirus, yeast, mammalian cells or other expression lines. The prokaryotic cell system, E. coli, cannot perform post-translational modifications such as glycosylation. However, this is not a serious problem for IF N a-F c fusions, because the natural IFNa- and immunoglobulin γ4 molecules are not severely glycosylated. Moreover, it has been reported that there is no glycosylation recombination IFNa can retain its biological activity (BarOn, & and Narula S

Bio/technology，i〇 : 179_19〇，199〇)。然而，自大腸桿菌溶胞產物中純化重組體蛋白質是十分困難的。由大腸桿菌表現之外來蛋白質常會聚集且形成不溶的包涵體。 -10 _ 本紙i尺度lit國國家標準（CNS) 公釐)------ ---------^w^ — — (請先閲讀背面之注意事項再填寫本頁) 、11 經濟部中央標準局員工消費合作社印製 577896 A7Bio / technology, i0: 179-19, 199). However, purification of recombinant proteins from E. coli lysates is very difficult. Foreign proteins expressed by E. coli often aggregate and form insoluble inclusion bodies. -10 _ This paper is a national standard (CNS) mm (lit.) ------ --------- ^ w ^ — — (Please read the precautions on the back before filling out this page), 11 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 577896 A7

因此常需要溶解作用及包涵體接下來之再折疊（Schein， C.H· and Noteborn，Η·Μ·，Bio/technology，6 : 291 -294，1 9 8 8 ; Wilkinson，D.L. and Harrison， R G·，Bio/technology，9 : 443-448，1991)。酵母表現系Pichia past〇ris-巴斯德畢赤酵母 (Invitrogen，San Dieg〇，ca)可克服利用細菌系統時所遭遇到的某些問題。其通常可生成高產率，且有能力進行各種轉澤後處理修飾作用。所表現之外來蛋白質可分泌至培養物上清液中，其中並無其他許多蛋白質存留，而得蛋白質之純化作用及過程之擴大更爲容易。此系統先可表現IFNa_Fc融合體或野生型117>^23。不幸地，IFNa_ F c分泌出後頃發現在s d S - P A GE上會部份降解，而單獨的IFNa2a則否。咸信降解作用係由酵母表現系中存在之蛋白酶活性所致，此由S c 〇 r er，c . A. e t al.，G en e， 1 3 6 : 1 1 1 - 9，1 9 9 3中所報告。在樞鈕區中相當弱的點可能是蛋白酶之目標。也嗜試IF N a - F c融合體之哺乳動物細胞表現系統。哺乳動物表現載體 ’ pCDNA3(Invitrogen，San Diego, CA)其含有一個CMV啓動子及一個ΝΕΟ抗性基因，應用於此中。宿主細胞，NSO細胞，以pCDNA3/IFNot-Fc表現載體利用電泳脈動法轉感。細胞以0 8毫克/毫升濃度之 G418選擇。以ELISA鑑定可表現純系之IFNa_Fc。於此系統融合體可成功地表現，且無降解現象。在此哺乳動物表現系中有許多優點。首先，重組體蛋白 -11 - 本紙張尺度適用中關家標準（CNS ) A4規格（210X297公釐） ' --- (請先閱讀背面之注意事項再填寫本頁)Therefore, lysis and subsequent refolding of inclusion bodies are often required (Schein, CH · and Noteborn, ΗM ·, Bio / technology, 6: 291-294, 198 8; Wilkinson, DL and Harrison, RG ·, Bio / technology, 9: 443-448, 1991). The yeast expression line Pichia pastoris-Pichia pastoris (Invitrogen, San Dieg0, ca) can overcome some of the problems encountered when using bacterial systems. It usually produces high yields and has the ability to perform various post-transformation modification effects. The expressed foreign protein can be secreted into the culture supernatant, and there are no many other proteins remaining, and the purification effect of the protein and the expansion of the process are easier. This system can first express IFNa_Fc fusion or wild type 117 > ^ 23. Unfortunately, IFNa_Fc was secreted and found to be partially degraded on SD-PAGE, while IFNa2a alone was not. Xianxin degradation is caused by the protease activity present in yeast expression lines, which is caused by Scoor, c. A. et al., Gene, 1 36: 1 1 1-9, 1 9 9 Reported in 3. A rather weak spot in the hub area may be the target of a protease. The mammalian cell expression system of IF N a-F c fusions has also been tested. The mammalian expression vector 'pCDNA3 (Invitrogen, San Diego, CA), which contains a CMV promoter and a NEO resistance gene, is used herein. The host cells, NSO cells, were transduced by electrophoretic pulsation using the pCDNA3 / IFNot-Fc expression vector. Cells were selected at G418 at a concentration of 0.8 mg / ml. ELISA was used to identify IFNa_Fc that could express pure lines. In this system, the fusion can be successfully performed without degradation. There are many advantages in this mammalian expression line. First of all, recombinant protein -11-This paper size applies the Zhongguanjia Standard (CNS) A4 specification (210X297 mm) '--- (Please read the precautions on the back before filling this page)

經濟部中央標準局員工消費合作社印製 577896 A7 ____________________ B7 五、發明説明（9 ) 質分泌至培養物上清液中，且在此無聚集作用，由是可簡化純化作用。利用蛋白質A管柱的一個層析步驟可生成經純化之IFN_a-Fc蛋白質。同時以此系統產生的蛋白質，其糖基化作用型式和天然分子極相似，因其以哺乳動物細胞表現。再者，天然的IFNa2a訊號肽序列包括於表現載體中。因此由細胞分泌的蛋白質具有眞實的N·末端，然而，在大腸样菌或酵母表現系統中，或是無訊號肽或是使用非IFNa訊號肽。不論何者，可在重組體IFNa-pciN_ 末端帶來額外的人工胺基酸殘基。如上述，自培号物上青液中純化IFN-aFc重組體蛋白質是相當直接的。經由蛋白質A管柱的單步驟親和力層析，可容易地獲得純度在9 0 %以上之蛋白質，以s D S PAGE判斷。偵測IFNa生物活性有許多分析方法可應用。利用抗病毒分析法，已證明在附錄“ B ”之融合體，具有約5至1 0倍高於相關IFNa-Fc融合體之比活性，其中連接子分子具有Gly Gly Ser Gly Gly Ser序列，且融合體之Fc部份係衍生自人類I g G 1而非I g G 4。然而，雖然示於附錄‘‘ B，，中之融合體之生物活性有實質地改進，其仍較天然IFNa 爲低。然而可預期，此融合體有更長之活體内半衰期。此期望以下列證明結果爲基礎，即在一個老鼠模式中，具有 Gly Gly Ser Gly Gly Ser連接子序列及IgGl Fc部份之相關IFNa融合體有轉天然IFNcx更長之半衰期。因爲附錄“B”之融合體預期有較天然IFNa更長之活體 -12- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） alw tmemat ·ϋ ϋ II (請先閲讀背面之注意事項再填寫本I) 訂 577896 A7 B7 五、發明説明（11 ) GGTGGCCCTC CTGGTGCTCA GCTGCAAGTC AAGCTGCTCT G。V引子含有可指導合成部份連接子附錄“A”之序列及IFNa2a後5個胺基酸，且在連接子序列中整合有一個BamHI位置CTCTGCGGAT CCACCTGAGC CACCTTCCTT ACTTCTTAAA 。 PCR 緩衝溶液含有 50mM KC1，1 OmMTris-Hcl (ρΗ8·3)，1.5mM MgCl2，0.01% 明膠，0.1 毫莫耳各 dNTP，0.5微莫耳各引子，5微升RT反應混合物，及1單位Taq DNA聚合酶，於共50微升體積中。PCR條件爲94 °C(1分鐘），55°C(2分鐘）及72°C(2分鐘），40個循環於 GeneAmp PCR 系統 9600 (Perkin Elmer, Norwalk， CT)。經濟部中央標準局員工消費合作社印製 (請先閱讀背面之注意事項再填寫本頁) 以逆轉錄作用及PCR得到人類免疫球蛋白γ4 Fc之 cDNA，如上述方式進行。RNA萃取自人類扁桃腺B細胞。5f引子有以下序列：AATGGATCCG GTGGAGGCGG AAGCGGCGGT GGAGGATCAG AGTCCAAATA TGGTCCCC。V引子有以下序列： ATCGAATTCT ATTTACCCAG AGACAGGGAG AGGCTCTTCT GT。二個PCR擴大的DNA片段選殖至pUC18中，分別在 Hind III/BamH I 位置或 BamH I/EcoR I位置。於其 DNA序列以DNA是序利用USB套組證實後（Cleveland， Ohio) ’二個片段經由BamH I位置以第二輪選殖連接在一起。全長之IFNa_Fc cDNA再嵌入哺乳動物表現載體 -14- 本紙張尺度適用中關家標準（CNS ) A4規格（210X297公釐) 經濟部中央標準局員工消費合作社印製 577896 A7 _ _B7 五、發明説明（12 ) pCDNA3 内（Invitrogen，San Diego, CA)、經由 Hind III及EcoR I位置嵌入。實例2 : IF N α _ F c於哺乳動物細胞中之表現將107 NSO細胞與10微克線化的pCDNA3/IFNcx-Fc 質體混合於0·8毫升PBS中，並置冰中5分鐘。在200v， 960μΡ 下利用 Gene Pulser (BioRad，Hircules，CA) 進行電泳脈動。細胞再置回冰上2 0分鐘，並轉移至加有 10毫升DMEM(並添加2%FCS)之100毫米組織培養盤内。經在3 7°C下培育2天後，細胞洗滌並再懸浮於相同培養基中。加入0.6毫克/毫升G418開始篩選。細胞塗佈在 8個96孔洞之微量盤上，並在37 °C下培育。一週後出現集落’其可在2週内容易地篩選。各孔洞之上清液，若有單一集落生長者則予以收集。在上清液中iIFNa_Fc以 ELISA分析法定量決定，此中應用山羊抗人類IgG及抗_ 人類Fc共軛有辣根過氧化酶。選出具較高ELISA讀値及較小集洛尺寸之純系進一步繼代選殖。這些集落轉移至 24孔洞盤内，並供應以含有G418之培養基。將具最高分泌水平之純系擴大，並使其在s p i n n e r中生長。於大規模製備時，收集培養物上清液，並通過以平衡的蛋白質 A瓊脂糖管柱。結合至蛋白質八之蛋白質以5〇檸檬酸鹽（pH 3.0)溶離，並以冷凍乾燥法濃縮。實例3 :鑑定IFNa-Fc融合體分離自NSO培養基之重組蛋白質，其純度以sds_ PAGE及西方墨點法檢查。在針對全蛋白質，以麗替紅s -15 - 本紙張尺度適用中國國家標準（CNS ) A4規格（2ι〇ϋ7公羡）-------- ---------— (請先閲讀背面之注意事項再填寫本頁) 、11 577896 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（13 ) 染色的吸潰膜上可見僅-個蛋白質帶，顯示蛋白質製劑之同質性。此蛋白質的表現分子量在還原條件下爲約 55kd，在非還原條件下爲u〇kd，此確實是合體之預期大小。在非還原條件下，其表現分子量之加倍可建議此融合體係呈二'聚體型式。重組體蛋白質可由抗: Fc及抗-IFNa抗體所確認，證實其由二個部份，汀以以及 F c片段所組成。Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 577896 A7 ____________________ B7 V. Description of the invention (9) The mass is secreted into the culture supernatant, and there is no aggregation effect, which can simplify the purification effect. A chromatographic step using a protein A column can produce purified IFN_a-Fc protein. At the same time, the glycosylation pattern of proteins produced by this system is very similar to that of natural molecules, because they are expressed in mammalian cells. Furthermore, the natural IFNa2a signal peptide sequence is included in the expression vector. Therefore, the protein secreted by the cell has a solid N-terminus. However, in the coliform or yeast expression system, either a signal-free peptide or a non-IFNa signal peptide is used. Either way, additional artificial amino acid residues can be brought to the recombinant IFNa-pciN_ terminus. As described above, the purification of recombinant IFN-aFc protein from the green liquor of the culture medium is quite straightforward. Through a single-step affinity chromatography of a protein A column, a protein with a purity of more than 90% can be easily obtained and judged by s D PAGE. There are many analytical methods for detecting IFNa biological activity. Using an antiviral analysis method, it has been shown that the fusion in Appendix "B" has a specific activity about 5 to 10 times higher than that of related IFNa-Fc fusions, in which the linker molecule has a Gly Gly Ser Gly Gly Ser sequence, and The Fc portion of the fusion is derived from human IgG1 and not IgG4. However, despite the substantial improvement in the biological activity of the fusion shown in the appendix ‘’ B, it is still lower than natural IFNa. However, it is expected that this fusion will have a longer half-life in vivo. This expectation is based on the results of the proof that in a mouse model, the relevant IFNa fusions with Gly Gly Ser Gly Gly Ser linker sequence and IgG1 Fc portion have a longer half-life of transgenic IFNcx. Because the fusion of appendix "B" is expected to have a longer living body than natural IFNa-12- This paper size applies Chinese National Standard (CNS) A4 specification (210 × 297 mm) alw tmemat · ϋ ϋ II (Please read the note on the back first Matters refill this I) Order 577896 A7 B7 V. Description of the invention (11) GGTGGCCCTC CTGGTGCTCA GCTGCAAGTC AAGCTGCTCT G. The V primer contains the sequence of appendix "A" which can guide the synthesis of some linkers and the five amino acids after IFNa2a, and integrates a BamHI position CTCTGCGGAT CCACCTGAGC CACCTTCCTT ACTTCTTAAA in the linker sequence. The PCR buffer solution contains 50 mM KC1, 1 OmM Tris-Hcl (ρΗ8.3), 1.5 mM MgCl2, 0.01% gelatin, 0.1 mM dNTPs, 0.5 μM primers, 5 μL RT reaction mixture, and 1 unit Taq DNA polymerase in a total volume of 50 μl. PCR conditions were 94 ° C (1 minute), 55 ° C (2 minutes), and 72 ° C (2 minutes). 40 cycles were performed on the GeneAmp PCR System 9600 (Perkin Elmer, Norwalk, CT). Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). Obtain the human immunoglobulin γ4 Fc cDNA by reverse transcription and PCR, as described above. RNA was extracted from human tonsil B cells. The 5f primer has the following sequence: AATGGATCCG GTGGAGGCGG AAGCGGCGGT GGAGGATCAG AGTCCAAATA TGGTCCCC. The V primer has the following sequence: ATCGAATTCT ATTTACCCAG AGACAGGGAG AGGCTCTTCT GT. Two PCR amplified DNA fragments were cloned into pUC18, at the Hind III / BamH I position or the BamH I / EcoR I position, respectively. After the DNA sequence was confirmed in the DNA sequence using a USB kit (Cleveland, Ohio), the two fragments were linked together via the BamH I position in a second round of breeding. Full-length IFNa_Fc cDNA is re-embedded in mammalian expression vectors-14- This paper is scaled to the Zhongguanjia Standard (CNS) A4 (210X297 mm) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economy 577896 A7 _ _B7 V. Description of the invention ( 12) Embedded in pCDNA3 (Invitrogen, San Diego, CA) via Hind III and EcoR I positions. Example 2: Expression of IF N α _ F c in mammalian cells 107 NSO cells and 10 μg of linearized pCDNA3 / IFNcx-Fc plastids were mixed in 0.8 ml of PBS and placed in ice for 5 minutes. Electrophoretic pulsation was performed using Gene Pulser (BioRad, Hircules, CA) at 200v, 960μP. The cells were placed back on ice for 20 minutes, and transferred to a 100 mm tissue culture plate containing 10 ml of DMEM (with 2% FCS). After 2 days of incubation at 37 ° C, the cells were washed and resuspended in the same medium. Screening was started by adding 0.6 mg / ml G418. Cells were plated on 8 96-well microplates and incubated at 37 ° C. Colonies appeared after one week 'which can be easily screened in two weeks. Supernatants from each hole were collected if there was a single colony grower. The iIFNa_Fc in the supernatant was determined quantitatively by ELISA analysis. Here, goat anti-human IgG and anti-human Fc conjugated horseradish peroxidase were used. Pure clones with higher ELISA readings and smaller chilo sizes were selected for further sub-generation. These colonies were transferred to 24-well plates and supplied with G418-containing medium. The pure line with the highest secretion level was expanded and allowed to grow in spini ner. For large-scale preparations, the culture supernatant was collected and passed through an equilibrated protein A agarose column. The protein bound to protein eight was isolated with 50 citrate (pH 3.0) and concentrated by freeze-drying. Example 3: Identification of IFNa-Fc fusion recombinant protein isolated from NSO medium, its purity was checked by sds_PAGE and Western blot method. For full protein, Li Tihong s -15-This paper size applies Chinese National Standard (CNS) A4 specification (2ι〇7 public envy) -------- ---------— (Please read the precautions on the back before filling out this page), 11 577896 A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (13) Only one protein band can be seen on the stained suction film, showing protein The homogeneity of the preparation. The apparent molecular weight of this protein is about 55kd under reducing conditions and uOkd under non-reducing conditions, which is indeed the expected size of the complex. Under non-reducing conditions, the doubling of its displayed molecular weight may suggest that this fusion system is a di'mer type. The recombinant protein can be confirmed by anti: Fc and anti-IFNa antibodies, and it is confirmed that it consists of two parts, Ting and Fc fragments.

對IFNa-Fc之生物活性分析是一種抗病毒分析法。特言之，所使用之分析方法爲Robert M Friedman et U 所述策略之修飾（Measurement of antivirai activity induced by interferons α，β and γ，CurrentAnalysis of the biological activity of IFNa-Fc is an antiviral assay. In particular, the analysis method used is a modification of the strategy described by Robert M Friedman et U (Measurement of antivirai activity induced by interferons α, β and γ, Current

Protocols in Immunology， 1 994, pp. 6.9.1-6.9.8) 0間吕之，人類肺癌細胞（A549， 1 8 5 )以4 0，0 0 0細胞/孔洞之密度種入9 6孔洞盤内，並在 37°C下培育24小時。加入1 : 2連續稀釋的ifnoc-fc融合Protocols in Immunology, 1 994, pp. 6.9.1-6.9.8) Lu Zhizhi, human lung cancer cells (A549, 1 8 5) were implanted into 96-well plate at a density of 4 0,0 0 cells / holes. And incubate at 37 ° C for 24 hours. Add 1: 2 serial dilution of ifnoc-fc fusion

體或天然的 IFNa(NIH# Gxa01-901-535)，並在 37°C 下培育2 4小時。每一樣品進行三次。培養基以含有濃度約0·1 MOI/細胞腦心肌炎病毒（ATCC #VR 129B)之新鮮培養基替換’並在37 C下再培養48小時。死細胞吸出洗去，並以P B S充份洗滌。已黏附的細胞以2 %甲酸固定，再以gi em s a染料染色。盤以自來水潤洗再令其乾燥。經染色的細胞以甲醇溶解，樣品在5 9 5毫微米下分光光度地讀取。IFNa-Fc融合體之抗病毒活性與IFNa標準品比較而評估，且發現是IFNa標準品活性之約30- -16 - 本紙張尺度適用中國國家標準（CNS )八4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) -訂 577896 A7 B7 五、發明説明（14 ) 60% ° 應了解，此中所使用之術語及表示僅供示範但不予以P艮制，且本發明範圍僅由其後之申請專利範圍所定義，並包括這些申請專利範圍主題中的所有同等事件。附錄“A” GGT GGC TCA GGT GGA TCC GGT GGA GGC GGA AGC GGC 36 GLY GLY SER GLY GLY SER GLY GLY GLY GLY SER GLY 12 GGT GGA GGA TCA 48 GLY GLY GLY SER 16 (請先閲讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 -17- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 577896 A7 ____B7五、發明説明（15 ) 經濟部中央標準局員工消費合作社印製附錄“ B ” ATG GCC HG ACC TTT GCT TTA CTG GTG GCC CTC CTG GTG 39 MET ALA LEU THR PHE AU LEU LEU VAL ALA LEU LEU VAL 13 CTC AGC TGC AAG TCA AGC TGC TCT CTG GGC TGT GAT CTG 78 LEU SER CYS LYS SER SER CYS SER LEU GLY CYS ASP LEU 26 CCT CAA ACC CAC AGC CTG GGT AGC AGG AGG ACC HG ATG 117 PRO GLN THR HIS SER LEU GLY SER ARG ARG THR LEU MET 39 CTC CTG GCA CAG ATG AGG AAA ATC TCT CTT TTC TCC TGC 156 LEU LEU ALA GLN MET ARG LYS HE SER LEU PHE SER CYS 52 HG AAG GAC AG A CAT GAC TTT GGA TTT CCC CAG GAG GAG 195 LEU LYS ASP ARG HIS ASP PHE GLY PHE PRO GLN GLU GLU 65 TTT GGC AAC CAG TTC CAA AAG GCT GAA ACC ATC CCT GTC 234 PHE GLY ASN GLN PHE GLN LYS ALA GLU THR ILE PHE VAL 78 CTC CAT GAG ATG ATC CAG CAG ATC TTC AAT CTC TTC AGC 273 LEU HIS GLU MET ILE GLU GLU ILE PHE ASN LEU PHE SER 91 ACA AAG GAC TCA TCT GCT GCT TGG GAT GAG ACC CTC CTA 312 THR LYS ASP SER SER ALA ALA TRP ASP GLU THR LEU LEU 104 GAC AAA TTC TAC ACT GAA CTC TAC CAG CAG CTG AAT GAC 351 ASP LYS PHE TYR THR GLU LEU TYR GLN GLN LEU ASN ASP 117 CTG GAA GCC TGT GTG ATA CAG GGG GTG GGG GTG ACA GAG 390 LEU GLU ALA CYS VAL ILE GLN GLY VAL GLY VAL THR GLU 130 ACT CCC CTG ATG AAG GAG GAC TCC ATT CTG GCT GTG AGG 429 THR PRO LEU MET LYS GLU ASP SER HE LEU ALA VAL ARG 143 AAA TAC TTC CAA AGA ATC ACT CTC TAT CTG AAA GAG AAG 468 LYS TYR PHE GLN ARG ILE THR LEU TYR LEU LYS GLU LYS 156 AAA TAC AGC CCT TGT GCC TGG GAG GTT GTC AGA GCA GAA 507 LYS TYR SER PHE CYS ALA TRP GLU VAL VAL ARG ALA GLU 169 ATC ATG AGA TCT TTT TCT TTG TCA ACA AAC TTG CAA GAA 546 ILE MET ARG SER PHE SER LEU SER THR ASN LEU GLN GLU 182 ------IT------0 (請先閲讀背面之注意事項再填寫本頁) -18- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 577896 A7 B7 五、發明説明（16 ) AGT TTA AGA AGT AAG GAA GGT GGC TCA GGT GGA TCC GGT 585 SER LEU ARG SER LYS GLU GLY GLY SER GLY GLY SER GLY 195 GGA GGC GGA AGC GGC GGT GGA GGA TCA GAG TCC AAA TAT 624 GLY GLY GLY SER GLY GLY GLY GLY SER GLU SER LYS TYR 208 GGT CCC CCG TGC CCA TCA TGC CCA GCA CCT GAG TTC CTG 663 GLY PRO PRO CYS PRO SER CYS PRO ALA PRO GLU PHE LEU 221 GGG GGA CCA TCA GTC TTC CTG TTC CCC CCA AAA CCC AAG 702 GLY GLY PRO SER VAL PHE LEU PHE PRO PRO LYS PRO LYS 234 GAC ACT CTC ATG ATC TCC CGG ACC CCT GAG GTC ACG TGC 741 ASP THR LEU MET HE SER ARG THR PRO GLU VAL THR CYS 247 GTG GTG GTG GAC GTG AGC CAG GAA GAC CCC GAG GTC CAG 780 VAL VAL VAL ASP VAL SER GLN GLU ASP PRO GLU VAL GLN 260 TTC AAC TGG TAC GTG GAT GGC GTG GAG GTG CAT AAT GCC 819 PHE ASM TRP TYR VAL ASP GLY VAL GLU VAL HIS ASM ALA 273 AAG ACA AAG CCG CGG GAG GAG CAG TTC AAC AGC ACG TAC 858 LYS THR LYS PRO ARG GLU GLU GLN PHE ASN SER THR TYR 286 CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG 897 ARG VAL VAL SER VAL LEU THR VAL LEU HIS GLN ASP TRP 299 CTG AAC GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA 936 LEU ASN GLY LYS GLU TYR LYS CYS LYS VAL SER ASN LYS 312 ---------— — (請先閲讀背面之注意事項再填寫本頁) 訂經濟部中央標準局員工消費合作社印製 GGC CTC CCG TCC TCC ATC GAG AAA ACC ATC TCC AAA GCC 975 GLY LEU PRO SER SER HE GLU LYS THR ILE SER LYS ALA 325 AAA GGG CAG CCC CGA GAG CCA CAG GTG TAC ACC CTG CCC 1014 LYS GLY GLN PRO ARG GLU PRO GLN VAL TYR THR LEU PRO 338 CCA TCC CAG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG 1053 PRO SER GLN GLU GLU MET THR LYS ASN GLN VAL SER LEU 351 -19- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X 297公釐） 577896 A7 B7 五、發明説明（17 ) ACC TGC CTG GTC AAA GGC TTC TAC CCC AGC GAC-ATC GCC 1092 THR CYS LEU VAL LYS GLY PHE TYR PRO SER ASP HE ALA 364 GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC 1131 VAL GLU TRP GLU SER ASN GLY GLN PRO GLU ASM ASN TYR 377 AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC 1170 LYS THR THR PRO PRO VAL LEU ASP SER ASP GLY SER PHE 390 TTC CTC TAC AGC AGG CTA ACC GTG GAC AAG AGC AGG TGG 1209 PHE LYS TYR SER ARG LEU THR VAL ASP LYS SER ARG TRP 403 CAG GAG GGG AAT GTC TTC TCA TGC TCC GTG ATG CAT GAG 1248 GLN GLU GLY ASN VAL PHE SER CYS SER VAL MET HIS GLU 416 GCT CTG CAC AAC CAC TAC AC A CAG AAG AGC CTC TCC CTG 1287 ALA LEU HIS ASN HIS TYR THR GLN LYS SER LEU SER LEU 429 (請先閲讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消費合作社印製 TCT CTG GGT AAA TAG 1302 SER LEU GLY LYS 433 -20- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）Or natural IFNa (NIH # Gxa01-901-535) and incubate at 37 ° C for 24 hours. Each sample was performed in triplicate. The medium was replaced with a fresh medium containing MOI / cell encephalomyocarditis virus (ATCC #VR 129B) at a concentration of about 0.1 and cultured at 37 C for an additional 48 hours. Dead cells were aspirated and washed away, and washed thoroughly with P B S. The adhered cells were fixed with 2% formic acid and then stained with gi em sa dye. Rinse the dishes with tap water and let them dry. The stained cells were lysed with methanol and the samples were read spectrophotometrically at 595 nm. The antiviral activity of the IFNa-Fc fusion was evaluated by comparison with the IFNa standard, and it was found to be about 30- -16 of the activity of the IFNa standard.-This paper size is applicable to the Chinese National Standard (CNS) 8-4 (210X297 mm) ( (Please read the precautions on the back before filling this page)-Order 577896 A7 B7 V. Description of the invention (14) 60% ° It should be understood that the terms and expressions used herein are for demonstration purposes only, and are not intended to be used in the system. The scope of the invention is defined solely by the scope of subsequent patent applications and includes all equivalent events in the subject matter of those patent applications. Appendix "A" GGT GGC TCA GGT GGA TCC GGT GGA GGC GGA AGC GGC 36 GLY GLY SER GLY GLY SER GLY GLY GLY GLY SER GLY 12 GGT GGA GGA TCA 48 GLY GLY GLY SER 16 (Please read the notes on the back before filling out (This page) Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs -17- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) 577896 A7 ____B7 V. Description of the invention (15) Staff consumption of the Central Bureau of Standards of the Ministry of Economic Affairs Cooperative printed "B" ATG GCC HG ACC TTT GCT TTA CTG GTG GCC CTC CTG GTG 39 MET ALA LEU THR PHE AU LEU LEU VAL ALA LEU LEU VAL 13 CTC AGC TGC AAG TCA AGC TGC TCT CTG GGC TGT GAT CTG 78 LEU SER CYS LYS SER SER CYS SER LEU GLY CYS ASP LEU 26 CCT CAA ACC CAC AGC CTG GGT AGC AGG AGG ACC HG ATG 117 PRO GLN THR HIS SER LEU GLY SER ARG ARG THR LEU MET 39 CTC CTG GCA CAG ATG AGG AAA CTT TTC TCC TGC 156 LEU LEU ALA GLN MET ARG LYS HE SER LEU PHE SER CYS 52 HG AAG GAC AG A CAT GAC TTT GGA TTT CCC CAG GAG GAG 195 LEU LYS ASP ARG HIS ASP PHE GLY PHE PRO GLN GLU GLU 65 TTT GGC AAC CAG TTC CAA AAG GCT GAA ACC ATC CCT GTC 234 PHE GLY ASN GLN PHE GLN LYS ALA GLU THR ILE PHE VAL 78 CTC CAT GAG ATG ATC CAG CAG ATC TTC AAT CTC TTC AGC 273 LEU HIS GLU MET ILE GLU GLU ASN LEU PHE SER 91 ACA AAG GAC TCA TCT GCT GCT TGG GAT GAG ACC CTC CTA 312 THR LYS ASP SER SER ALA ALA TRP ASP GLU THR LEU LEU 104 GAC AAA TTC TAC ACT GAA CTC TAC CAG CAG CTG AAT GAC 351 ASP LYSHE TYR THR GLU LEU TYR GLN GLN LEU ASN ASP 117 CTG GAA GCC TGT GTG ATA CAG GGG GTG GGG GTG ACA GAG 390 LEU GLU ALA CYS VAL ILE GLN GLY VAL GLY VAL THR GLU 130 ACT CCC CTG ATG AAG GAG TCG GTG AGG 429 THR PRO LEU MET LYS GLU ASP SER HE LEU ALA VAL ARG 143 AAA TAC TTC CAA AGA ATC ACT CTC TAT CTG AAA GAG AAG 468 LYS TYR PHE GLN ARG ILE THR LEU TYR LEU LYS GLU LYC 156 TAAA GCC TGG GAG GTT GTC AGA GCA GAA 507 LYS TYR SER PHE CYS ALA TRP GLU VAL VAL ARG ALA GLU 169 ATC ATG AGA TCT TTT TCT TTG TCA ACA AAC TTG CAA GAA 546 ILE MET ARG SER PHE SER LEU SER THR ASN 18 2 ------ IT ------ 0 (Please read the precautions on the back before filling out this page) -18- This paper size applies to China National Standard (CNS) A4 (210X297 mm) 577896 A7 B7 V. Invention Description (16) AGT TTA AGA AGT AAG GAA GGT GGC TCA GGT GGA TCC GGT 585 SER LEU ARG SER LYS GLU GLY GLY SER GLY GLY SER GLY 195 GGA GGC GGA AGC GGC GGT GGA GGA TCA GAG TCC AAA TAT 624 GLY GLY GLY SER GLY GLY GLY GLY SER GLU SER LYS TYR 208 GGT CCC CCG TGC CCA TCA TGC CCA GCA CCT GAG TTC CTG 663 GLY PRO PRO CYS PRO SER CYS PRO ALA PRO GLU PHE LEU 221 GGG GGA CCA TCA GTC TTC CTG CCC CCA AAA CCC AAG 702 GLY GLY PRO SER VAL PHE LEU PHE PRO PRO LYS PRO LYS 234 GAC ACT CTC ATG ATC TCC CGG ACC CCT GAG GTC ACG TGC 741 ASP THR LEU MET HE SER ARG THR PRO GLU VAL THR CYS 247 GTG GTG GTG GAC GTG AGC CAG GAA GAC CCC GAG GTC CAG 780 VAL VAL VAL ASP VAL SER GLN GLU ASP PRO GLU VAL GLN 260 TTC AAC TGG TAC GTG GAT GGC GTG GAG GTG CAT AAT GCC 819 PHE ASM TRP TYR VAL ASP GLY VALLU HIS ASM ALA 273 AAG ACA AAG CCG CGG GAG GAG CAG TTC AAC AGC ACG TAC 858 LYS THR LYS PRO ARG GLU GLU GLN PHE ASN SER THR TYR 286 CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG 897 ARG VAL VAL SER VAL LEU THR VAL LEU HIS GLN ASP 299 AAC GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA 936 LEU ASN GLY LYS GLU TYR LYS CYS LYS VAL SER ASN LYS 312 ----------- — (Please read the notes on the back before filling this page ) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. Printed by GGC CTC CCG TCC TCC ATC GAG AAA ACC ATC TCC AAA GCC 975 GLY LEU PRO SER SER HE GLU LYS THR ILE SER LYS ALA 325 AAA GGG CAG CCC CGA GAG CCA CAG GTG TAC ACC CTG CCC 1014 LYS GLY GLN PRO ARG GLU PRO GLN VAL TYR THR LEU PRO 338 CCA TCC CAG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG 1053 PRO SER GLN GLU GLU MET THR LYS ASN GLN VAL SER LEU 351 351 -19- Paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 577896 A7 B7 V. Description of invention (17) ACC TGC CTG GTC AAA GGC TTC TAC CCC AGC GAC-ATC GCC 1092 THR CYS LEU VAL LYS GLY PHE TYR PRO SER ASP HE ALA 36 4 GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC 1131 VAL GLU TRP GLU SER ASN GLY GLN PRO GLU ASM ASN TYR 377 AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC 1170 LYS THR THR PRO VAL ASP SER ASP GLY SER PHE 390 TTC CTC TAC AGC AGG CTA ACC GTG GAC AAG AGC AGG TGG 1209 PHE LYS TYR SER ARG LEU THR VAL ASP LYS SER ARG TRP 403 CAG GAG GGG AAT GTC TTC TCA TGC TCC GTG ATG CAT GAG 12 GLU GLY ASN VAL PHE SER CYS SER VAL MET HIS GLU 416 GCT CTG CAC AAC CAC TAC AC A CAG AAG AGC CTC TCC CTG 1287 ALA LEU HIS ASN HIS TYR THR GLN LYS SER LEU SER LEU 429 (Please read the precautions on the back first (Fill in this page again) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs TCT CTG GGT AAA TAG 1302 SER LEU GLY LYS 433 -20- This paper size applies to China National Standard (CNS) A4 (210X297 mm)

Claims

577896 A8 B8 C8 D8 第086110733號專利申請案中文申請專利範圍修正本（91年10月）六：Jit專利範圍577896 A8 B8 C8 D8 Patent Application No. 086110733 Patent Amendment of Chinese Patent Application Range (October 91) Six: Jit Patent Scope

公告本 θ—種融合體分于，其中含有干擾素分子IFNa2a， IFNoc2b或IFN/5並在其C-末端經由一個肽連接子聯結至免疫球蛋白Fc片段之N-末端。 2. 根據申請專利範圍第1項之融合體分子，其中的肽連接子具有 Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser序列。 3. 根據申請專利範圍第1至2項任一項之融合體分子，其中另一個干擾素分子在其C-末端經由另一肽連接子聯結至免疫球蛋白Fc片段鏈之N-末端，由是形成一個同 4.根據申請專利範圍第1至2項之融合體分子，其中的Fc 片段是γ4鏈Fc片段。本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 第86110733號專利申請案中文補充說明書(89年3月）Announcement θ-type fusion is divided into, which contains the interferon molecule IFNa2a, IFNoc2b or IFN / 5 and is linked at its C-terminus to the N-terminus of the immunoglobulin Fc fragment via a peptide linker. 2. The fusion molecule according to item 1 of the patent application, wherein the peptide linker has a Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser sequence. 3. The fusion molecule according to any one of claims 1 to 2, wherein the other interferon molecule is linked to the N-terminus of the immunoglobulin Fc fragment chain at the C-terminus of the peptide via another peptide linker. It forms a fusion molecule according to item 1 to 2 of the scope of patent application, wherein the Fc fragment is a γ4 chain Fc fragment. This paper size applies to China National Standard (CNS) A4 (210X 297 mm) Patent Application No. 86110733 Supplementary Manual in Chinese (March 89)

修正1 . 抛實例I : INFa_Fc融合體之半衰期較天然娜α顯示大幅增加美國專利第 5,723,25之揭示（併入參考)描述以具下列序列之連接子：吻卿 Ser Gly Gly Ser (序列辨識號碼：1〇)製造_a_Fc (ri)融合體。該融合體之比活性在Daudi細胞之體外分析為7 7x1〇s單位/微莫耳。在稍後之細胞病變效應抑制分析，融合體顯示22χ1〇8 IU/pmole之抗病毒比活性，其低於未修飾干擾素μ之3 8χι〇9 IU/fimole。為嘗試增加融合體之比活性，連接子被延伸以增加柔靭性並減少立體障礙。具序列·· Gly Gly ser (Jly (5ly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (序列辨識號碼：之連接子被使用。新融合體之另一差兴為其Fc邵份為r4Fc，而不是rlFc。在體外之病毒細胞病變效應抑制分析結果顯示新融合體具有1K 2.2X10 IU/jimole之抗病毒比活性較舊有者增加倍。但是，其仍較未修飾干擾素-α少2-3倍，其在相同分析中具3 8><1〇9 IU/pmole之比活性。然而，在靈長類之體内藥理動力學研究中，本發明所請新融合體之血清半衰期較未修飾干擾素長約40倍。皮下投藥融合體後之清除半衰期大約較120倍長。當皮下投藥後，該融合體亦有被良好吸收。新融合體在AUC (曲線下面積)之大幅增加表示其較天然干擾素-α較有效，與其較低比活性無關。 U:\TYPE\LUI\SYC-2.DOC 1 577896 接著進行下列實驗以測定使用在細胞病變活性上之不同長度之連接子。實例Π :各種連接子在IFN-Fc細胞病變活性上之效果研究 1. EFN-α (16)Fc 及 IFN-a-Ala-Fc 之比較連接子肤之作用係藉比較IFN-a (16) Fc及IFNa-Ala-Fc而測試。IFN-a (16) Fc含有經由16-胺基酸連接子(如序列辨識號碼：11 所申示，即 Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser)連接至人類 igG4 Fc 之鏈區域。IpK-a_Ala_ Fc構柒體含有連接至在2區域間具一個胺基酸(Aia)的人類 IgGIFc 之絞鏈區域之 iFN-α。編碼 IFN-oi (16) Fc 及 IFN_a-Ala- Fc之DNA片段係分別***pcDNA3表現質體之多選殖位置。經純化質體DNA係接著藉電穿孔用於轉染NS〇細胞。安定一轉形細胞株係在G418存在下篩選。細胞株表現此等連接子變体接著在旋轉培養瓶中生長。收集培養上清液並使用蛋白質A 親和管柱製備純化的蛋白質。經純化蛋白質係用於相同病毒細胞病變效應抑制分析(如實例丨所述）。IFN_〜Ala_Fc及iFN々 (16) Fc二者顯示具有相當之活性(圖& 2· IFNp-Fc連接子變體之構築連接至Fc (卿也）之干擾素_β之大量不同構築體係經製備以測疋連接子長度在IFNj3-Fc融合體活性之效果。此等構築體之胺基酸序列係列於下表i : U:\TYPE\LLTSYC-2.DOC 1 -2- 577896 表1 :各種INFp-Fc之轉譯胺基酸序列連接子變體J INFP及IgG4(Fc)絞鏈間之連接子序列 INFp-⑵ Fc GlySer (SEQ ID NO:3) INFP-(8)Fc GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:4) ENFp-(12)Fc Gly S erGly Gly Gly Gly S erGly Gly Gly Gly S er (SEQIDNO:5) INFp-(18)Fc GlyGlyGlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGly GlySer (SEQ ID NO:6) INFp-(23)Fc GlyGlyGlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGly GlySerGlyGlyGlyGlySer (SEQ ID NO:7) INFp-(30)Fc Gly Gly Gly S erGly Gly Gly S erGly Gly Gly Gly Gly S erGly Gly Gly Gly Gly S erGly Gly Gly Gly S er Gly Gly Gly Gly S er (SEQ ID NO:8) INFp-(40)Fc GlyGlyGlySerGlyGlyGlySerGlyGlyGlyGlyGlySerGlyGly GlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGly GlyGlySerGlyGlyGlyGlySer (SEQ ID NO:9)Amendment 1. Example I: The half-life of the INFa_Fc fusion showed a significant increase over natural Naα. The disclosure (incorporated by reference) of US Patent No. 5,723,25 describes a linker with the following sequence: Ser Gly Gly Ser (Sequence Identification Number: 10) Production of _a_Fc (ri) fusion. The specific activity of this fusion was analyzed in vitro by Daudi cells to be 77 x 10 s units / micromolar. In a later analysis of the inhibition of cytopathic effects, the fusion showed a specific antiviral activity of 22 × 10 8 IU / pmole, which was lower than that of unmodified interferon μ 3 8 × 109 IU / fimole. In an attempt to increase the specific activity of the fusion, the linker was extended to increase flexibility and reduce steric hindrance. Gly Gly ser (Jly (5ly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser (sequence identification number: linker is used. Another difference of the new fusion is that its Fc component is r4Fc, and It is not rlFc. In vitro analysis of viral cytopathic effect inhibition in vitro shows that the new fusion has a 1K 2.2X10 IU / jimole specific antiviral activity that is doubled compared to the old one. However, it is still less than unmodified interferon-α 2- 3 times, which has a specific activity of 3 8 > < 10 9 IU / pmole in the same analysis. However, in the in vivo pharmacokinetic study of primates, the serum half-life of the novel fusions requested by the present invention is Unmodified interferon is about 40 times longer. The clearance half-life after subcutaneous administration of the fusion is approximately 120 times longer. When subcutaneously administered, the fusion is also well absorbed. The new fusion has a significant increase in AUC (area under the curve) It indicates that it is more effective than natural interferon-α and has nothing to do with its lower specific activity. U: \ TYPE \ LUI \ SYC-2.DOC 1 577896 The following experiments were performed to determine the use of linkers of different lengths for cytopathic activity Example Π: Various linkers in IF Study on the effect of N-Fc on cytopathic activity 1. Comparison of EFN-α (16) Fc and IFN-a-Ala-Fc The effect of linker skin is by comparing IFN-a (16) Fc and IFNa-Ala-Fc And tested. IFN-a (16) Fc contains a 16-amino acid linker (as indicated by the sequence identification number: 11, namely Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser) Human igG4 Fc chain region. The IpK-a_Ala_ Fc construct contains iFN-α linked to the hinge region of human IgGIFc with an amino acid (Aia) between 2 regions. It encodes IFN-oi (16) Fc and IFN_a-Ala- Fc DNA fragments were inserted into pcDNA3 to express the multiple selection sites of plastids. Purified plastid DNA lines were then used to transfect NS0 cells by electroporation. A stable cell line in the presence of G418 Screening. Cell strains showing these linker variants were then grown in spinner flasks. Culture supernatants were collected and purified proteins were prepared using Protein A affinity columns. The purified proteins were used for the same viral cytopathic effect inhibition analysis ( As described in Example 丨). IFN_ ~ Ala_Fc and iFN々 (16) Fc both show comparable Activity (Figure & 2. Construction of IFNp-Fc linker variants A large number of different construction systems of interferon_β linked to Fc (Qing Ye) were prepared to measure the effect of the linker length on IFNj3-Fc fusion activity . The amino acid sequence of these constructs is shown in the following table: U: \ TYPE \ LLTSYC-2.DOC 1 -2- 577896 Table 1: Various amino acid sequence linker variants J INFP and INFp-Fc Linker sequence between IgG4 (Fc) hinges INFp-⑵ Fc GlySer (SEQ ID NO: 3) INFP- (8) Fc GlyGlyGlySerGlyGlyGlySer (SEQ ID NO: 4) ENFp- (12) Fc Gly S erGly Gly Gly Gly Gly S erGly Gly Gly Gly S er (SEQIDNO: 5) INFp- (18) Fc GlyGlyGlyGlySerGlyGlyGlyGerGlyGlyGlyGlySerGlyGlyGlyGlySly (SEQ ID NO: 6) INFp- (23) Fc GlyGlyGlySerGlyGlyGlyGlyGerGerGlyGerGlySerGlyGer S erGly Gly Gly S erGly Gly Gly Gly Gly S erGly Gly Gly Gly Gly S erGly Gly Gly Gly S er Gly Gly Gly Gly S er (SEQ ID NO: 8) INFp- (40) Fc GlyGlyGlySerGlyGlyGlySerGlyGlyGlyGlyGlySerGlyGly GlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGly GlyGlySerGlyGlyGlyGlySer (SEQ ID NO: 9)

3. IFNp-Fc連接子變體之表現分別***含有不同IFNp-Fc連接子變體之DNA序列於pcDNA3 表現質體之多轉殖位置。經純化DNA係接著用於放電穿孔轉染NSO細胞。安定一轉形細胞株在G418存在下篩選。表現此等連接子變體之細胞株接著在缺乏G418之情況下生長。收集培養上清液並經由〇.22μπι濾膜過濾。使用純化ΐΡΝβ-Fc蛋白質作為標準品，藉PCFIA估計IFNp-Fc之濃度。培養上清液之濃度估計含有連接子肽2.8, 12, 18, 23, 30及40個胺基酸之 Π^ίβ-Fc 變體分別為 5.4, 22.5, 15.9, 5.7, 10.2, 5.5 及 4.5pg/ml。此等上清液係用於下列體外細胞病變效果實驗。 4. 使用IFNp-Fc變體之體外細胞病變效應分析在96孔平盤中，人類肺癌A549細胞以含5% FBS之DMEM 調成含5X104細胞之ι〇0μΐ/孔。平盤在5 % c〇2培養箱中在 37 C下培養24小時。稀釋含有IFN-Fc連接子變體之培養上清 U:\TYPE\LUI\SYC-2.DOC 1 577896 液。此等溶液接著使用含5% FBS之DMEM在96孔平盤中製成2倍連續稀釋。添加100微升之經稀釋樣品至每一孔中且平盤在培養箱中在37°C下另培養24小時。去除上清液且添加 encephalomyocarditis (EMC)至ΙΟΟμΙ/孔(病毒自病毒貯存液以含5% FBS之D15稀釋1 : 2000)。平盤接著在5 % C02培養箱中在37°C下培養48小時。去除培養上清液且以PBS清洗2 次。接奢以對甲酸固定細胞；並以金沙(giemsa)染劑染色並接著在室溫下放置5分鐘。此後，平盤以自來水溫和沖洗數次。0 添加甲醇至每一孔中並使用Dynatech MR5000 ELISA讀取器在 63〇 nm讀取孔。具IFNp-Fc融合體之數種實驗結果，如圖2所示，及示於下列貫例II之標題1之2種不同IFNa-Fc融合體之結果，顯示細胞病變效應，不論使用何種連接子，均不會顯著改變。進一步之對IFNa- (16) Fc融合體之一之體内實驗係如下述進行。貪例羾：動物腫瘤模型 1·小鼠之腫瘤起始化 φ 雕 CB17/scid 小鼠(Charles River Laboratories ; 7 週半大)係以位於下右側Daudi Burkitt淋巴細胞，總體積ι〇〇μ1，經皮下接種。每組5隻動物測試4種不同的細胞密度(表2)。注射位置在接種後監測1天並接著在接種後3週内每天監測。藉測徑器測量可觸及之腫瘤。測定腫瘤體積並使用公式，ν=4 xyz / 3 ’計算1其中2Χ，2y及2ζ為腫瘤之3個垂直直徑及2 個測量之平均。时 YPE\LUr'SYc.2D〇Cl -4- 577896 對接種而言，在體外在含10%胎牛血清之D15培養基之100毫升旋轉瓶中，細胞生長至具94%存活率之0.6X106/毫升之密度。在300g下離心1〇分鐘以收取細胞，在冷pBS中清洗2 次，並在PBS再懸浮至所需密度。計數細胞及Tryptan Bhe染色以確認細胞密度及存活率。表2 :細密度及接種途徑細胞密度 1 物編號 555# PBS —5--^~— 0.5X106/100ul PBS 5 SV 2.5X106/100ul PBS 5 s c* 1.25X107/100u1 PBS 5 ； * 人類腫瘤異種移殖在接種4週後之注射位置之在125xl〇7群組中，係組成為可檢測的。一週後，腫瘤收取率達到8〇%且經過完整測驗研究期間可維持此量。可觸及腫瘤成長至動物體重之 10-15% 約需 3 週(2.5-3.5 週）。在2.5X106及0.5X106群組中，收取率在第9週半後可到達 60%。腫瘤並不會殺死小鼠且沒有轉移跡象。因此，結論為1.25Xl〇7DaudiBurkitt淋巴細胞之皮下接種在約 4週可產生約80%腫瘤。 2.體内抗增殖研究 ⑴每日劑量實驗以12.5X106 Daudi Burkitt淋巴細胞接種之23隻小鼠係隨機分配到四處理群之一(如表3所示）。Rofern a (IFN_…2a， Hoffmann La Roche，Nutley，NJ)及 IFN-α (16)-2a-Fc (具有如序列辨識號碼11之連接子)之處理開始於腫瘤接種後之日。所 U:\TYPE\LUrSYC-2.DOC 1 577896 有動物每日皮下投劑於頸背及治療持續8週。在治療期間，每3-4天監測動物之腫瘤發展，並如上述測量腫瘤大小。治療期後，另持續每週觀察動物6個月，該動物於治療停止後並沒有腫瘤。分別在IFN-a_2a-Fc治療終結後及R0feron A治療終結1，2及 3週後，在最後投劑日，1，2及4週後逆眼窩收集血液24小時。藉ELISA測定血清干擾素濃度。表3 :劑量，途徑及時間表3. Performance of IFNp-Fc linker variants DNA sequences containing different IFNp-Fc linker variants were inserted at multiple transgenic positions of pcDNA3 expression plastids, respectively. The purified DNA lines were then used to transfect NSO cells by spark perforation. A stable-transformed cell line was screened in the presence of G418. Cell lines expressing these linker variants then grow in the absence of G418. The culture supernatant was collected and filtered through a 0.22 μm filter. Using purified HPNβ-Fc protein as a standard, PCFIA was used to estimate the concentration of IFNp-Fc. The concentration of the culture supernatant was estimated to contain linker peptides 2.8, 12, 18, 23, 30 and 40 amino acid β-Fc variants of 5.4, 22.5, 15.9, 5.7, 10.2, 5.5 and 4.5pg, respectively. / ml. These supernatants were used in the following in vitro cytopathic effect experiments. 4. Analysis of in vitro cytopathic effects using IFNp-Fc variants In a 96-well plate, human lung cancer A549 cells were adjusted to 5 × 104 cells at ΙΟΟμΐ / well with DMEM containing 5% FBS. Plates were incubated in a 5% CO2 incubator at 37 C for 24 hours. Dilute the culture supernatant U: \ TYPE \ LUI \ SYC-2.DOC 1 577896 solution containing the IFN-Fc linker variant. These solutions were then made to 2-fold serial dilutions in 96-well flat plates using DMEM containing 5% FBS. Add 100 microliters of the diluted sample to each well and incubate the plate in an incubator at 37 ° C for another 24 hours. The supernatant was removed and encephalomyocarditis (EMC) was added to 100 μl / well (the virus was diluted from the virus stock solution with D15 containing 5% FBS 1: 2000). The plate was then incubated in a 5% CO2 incubator at 37 ° C for 48 hours. The culture supernatant was removed and washed twice with PBS. Cells were then fixed with formic acid; stained with gimesa stain and then left at room temperature for 5 minutes. Thereafter, the pan was gently rinsed several times with tap water. 0 Methanol was added to each well and the wells were read at 630 nm using a Dynatech MR5000 ELISA reader. The results of several experiments with IFNp-Fc fusions are shown in Figure 2, and the results of two different IFNa-Fc fusions shown in Title 1 of Example II below show cytopathic effects, regardless of the connection No significant change will occur. Further, in vivo experiments on one of the IFNa- (16) Fc fusions were performed as follows. Examples: Animal tumor model 1. Tumor initiation of mice φ CB17 / scid mice (Charles River Laboratories; 7 and a half weeks old) are Daudi Burkitt lymphocytes located on the lower right side, with a total volume of 〇00μ1, Subcutaneous inoculation. Four animals of each group were tested for 4 different cell densities (Table 2). The injection site was monitored for 1 day after vaccination and then daily for 3 weeks after vaccination. Touch the tumor with a caliper. Measure the tumor volume and use the formula, ν = 4 xyz / 3 ′ to calculate 1 where 2 ×, 2y and 2ζ are the average of 3 vertical diameters and 2 measurements of the tumor. YPE \ LUr'SYc. 2D〇Cl -4- 577896 For inoculation, the cells grew to 0.6X106 / with 94% survival rate in vitro in a 100 ml spinner bottle containing 10% fetal bovine serum in D15 medium. Density in milliliters. Centrifuge at 300 g for 10 minutes to collect cells, wash twice in cold pBS, and resuspend in PBS to the desired density. Cells were counted and stained with Tryptan Bhe to confirm cell density and viability. Table 2: Fine density and cell density of inoculation route 1 Object No. 555 # PBS —5-^ ~ — 0.5X106 / 100ul PBS 5 SV 2.5X106 / 100ul PBS 5 sc * 1.25X107 / 100u1 PBS 5; * Human tumor xenograft Groups that were colonized at the injection site 4 weeks after inoculation were detectable in the 125x107 group. After one week, the tumor collection rate reached 80% and it was maintained during the full test study period. It can take about 3 weeks (2.5-3.5 weeks) for palpable tumors to grow to 10-15% of the animal's weight. In the 2.5X106 and 0.5X106 groups, the collection rate can reach 60% after 9 and a half weeks. The tumor did not kill the mouse and showed no signs of metastasis. Therefore, it was concluded that subcutaneous inoculation of 1.25 × 107 DaudiBurkitt lymphocytes produced about 80% of tumors in about 4 weeks. 2. In vivo anti-proliferation study ⑴ Daily dose experiment 23 mice lines inoculated with 12.5 × 106 Daudi Burkitt lymphocytes were randomly assigned to one of the four treatment groups (as shown in Table 3). The treatment of Rofern a (IFN _... 2a, Hoffmann La Roche, Nutley, NJ) and IFN-α (16) -2a-Fc (with a linker such as sequence identification number 11) started on the day after tumor inoculation. All U: \ TYPE \ LUrSYC-2.DOC 1 577896 animals are administered subcutaneously daily on the nape of the neck and the treatment lasts for 8 weeks. During the treatment period, the animals were monitored for tumor development every 3-4 days, and tumor size was measured as described above. After the treatment period, the animals were observed for another 6 months each week. The animals did not have tumors after the treatment was stopped. Blood was collected in the eye socket for 24 hours after the end of IFN-a_2a-Fc treatment and 1, 2 and 3 weeks after the completion of Roferon A treatment. The serum interferon concentration was determined by ELISA. Table 3: Dose, route and schedule

群組劑量投藥途徑時間表控制組稀釋 s.c. 每曰 Roferon A 1Χ106ΐυ/100μ1 s.c. 每曰 IFN-a-Fc 1Χ106ΐυ/100μ1 s.c. 每曰 IFN-a-Fc 1Χ105ΐυ/100μ1 s.c. 每曰 ⑵IFN-α對腫瘤收取率及腫瘤進展之效應不同治療群組之腫瘤發展示於表4。在控制組動物，第1個腫瘤檢測於接種後24天及此後6天内，動物7/8 (87.5 %)發展腫瘤。腫瘤檢測之平均時間為25.1 ± 2.3天(不包括在第75 天發展腫瘤之老鼠）。在Roferon A治療之動物，第1個腫瘤在接種後32天成為可測得的。另兩週後，87.5%發展腫瘤。平均腫瘤檢測時間為39.6 ± 4.7天(t > t0.05 (12)，P < 〇.〇5)。 Roferon A延遲腫瘤發展約2週。在2種劑量之IFN-a-2a-Fc 治療，經過完整投劑期後可完全避免Daudi淋巴發展腫瘤。在較低劑量，在治療程序後第2及19天，2隻小鼠發展腫瘤。1 X 106 IU/天群組之小鼠及在1 X 105 IU/每日之剩餘6 U:\TYPE\LLTSYC-2.DOC 1 577896 隻小鼠在治療後仍維持6週無腫瘤(表句。該實驗重覆i次仍具相似結果(如表4所示）。表4 :在CB17/scid小鼠之腫瘤發展(實驗^Group dose administration schedule schedule control group dilution sc per Roferon A 1 × 106ΐυ / 100μ1 sc per IFN-a-Fc 1 × 106ΐυ / 100μ1 sc per IFN-a-Fc 1 × 105ΐυ / 100μ1 sc per IFN-α tumor recovery rate And the effect of tumor progression. Tumor development in different treatment groups is shown in Table 4. In the control group, the first tumor was detected within 24 days and 6 days after inoculation, and 7/8 (87.5%) of the animals developed tumors. The average time for tumor detection was 25.1 ± 2.3 days (excluding mice that developed tumors on day 75). In animals treated with Roferon A, the first tumor became measurable 32 days after vaccination. After another two weeks, 87.5% developed tumors. The average tumor detection time was 39.6 ± 4.7 days (t > t0.05 (12), P < 0.05). Roferon A delayed tumor development for about 2 weeks. After two doses of IFN-a-2a-Fc treatment, Daudi lymphoma can be completely prevented from developing tumors after the complete administration period. At lower doses, 2 mice developed tumors on days 2 and 19 after the treatment procedure. Mice in the 1 X 106 IU / day group and the remaining 6 U at 1 X 105 IU / day: TYPE LLTSYC-2.DOC 1 577 896 mice remained tumor-free for 6 weeks after treatment (table sentence The experiment was repeated i times with similar results (as shown in Table 4). Table 4: Tumor development in CB17 / scid mice (Experimental ^

R*133 104 103 115 110 113 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 6/20/98 6/20/98 6/20/98 6/22/98 6/26/98 6/28/98 7/1/98 7/6/98 7/6/98 7/7/98 7/9/98 24 24 24 24 26 30 32 35 40 40 41 /to 25.1±2.3R * 133 104 103 115 110 113 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 5/27/98 5 / 27/98 5/27/98 5/27/98 5/27/98 6/20/98 6/20/98 6/20/98 6/22/98 6/26/98 6/28/98 7 / 1/98 7/6/98 7/6/98 7/7/98 7/9/98 24 24 24 24 26 30 32 35 40 40 41 / to 25.1 ± 2.3

1X106IU/天投藥 ⑶INF-α對腫瘤生長率之效應 1°/〇，腫瘤生長率在控制組及當腫瘤生長至約老鼠體重之 Roferon A處理動物係非常接近。在控制組動物，平均腫瘤体和在2週增加10倍，然而R〇fer〇n a處理之小鼠顯示9倍之增加。表5 :在不同治療之腫瘤收取率群組治療腫瘤收取率(％) (N=8) 控制組 Roferon A IFN a-2a-Fc IFN a-2a-Fc 稀釋 1X106 IU/lOOul lX106IU/100ul 1X105 IU/lOOul 100 (8/8) 87.5(7/8) 0 25.0(2/8) U:\TYPE\LUI\SYC-2.DOC 1 ⑷血清IFN-α濃度之定量 IFN-α及IFN-a-2a-Fc之血清濃度係藉ELISA程序測定。在 Roferon A處理之小鼠’在最後投劑後24小時無法檢測脈_ a-2a。在IFN-a-2a-Fc處理小鼠，治療終結後22天，血清 IFN_a-2a-Fc 濃度為 3、g/ml (1 X 106 似天群組）及〇 7|Lig/m〇1 (1 X 105 IU/天群組)(表6)。在治療結束後，其在1至22天間之血清濃度減少。數據指出IFN-a-2a-Fc在老鼠具有約1週之半衰期(皮下投藥1 X 1〇6 IU/天或1 X 1〇5 ιυ/天8週後）。表 6 ·血清 iFN-a-2a 濃度(pg/ml) 治療 ___治療終結後天數__ _ 一 1 8 22 1X10; IU 25.370±6.885 12.080土3.477 3.477土0.525 N-a-2a-Fc 1X10 IU 2.766士 1·138 1.549士0.536 0.691 士0.141 0 eron A Undetectable Undetectable Undetectable ⑸具增加投劑區間之實驗在此實驗，每3天提供R〇feron A 1 X 1 〇6 IU及每3天及每週投劑 1 X 1〇6 IU IFN-a_2a-Fc。結果示於表 7。3 天之 Roferon A 1 X 106 IU，與控制組比較，並未顯示對抗腫瘤形成之任何保硬’然而每3天及每週投藥之1 X 1〇6 IU IFN-a-Fc在8週治療期可有效抑劑腫瘤形成。此抑制在治療期後可延伸7 週。表7 ’具增加投劑週期之動物之腫瘤發展腫瘤收取率(％) (N=8) ~~100 (8/8)"""" 100(8/8) N/A N/A 治療 - R〇fer〇n a 1〇6 ιυ/3 天 IFN-Fc 106 IU/3 天每週腫瘤發展之平均時間 (天） 21.1 士 1.1 22.0 土 1.9 N/A N/A 577896 ⑺已成型Daudi Burkitt淋巴瘤之初步研究具已成型5週大Daudi Burkitt淋巴瘤之2隻小鼠係經IFN-a-Fc (106 IU/每日）治療。1〇天後，在動物中之二者觀察·到完全退化(表8)。具已成型6·5週Daudi淋巴瘤之其它2隻小鼠係以106 IU Roferon A每3天處理8週。在後者之小鼠，腫瘤體積迅減少，自2.7cm3及4.6cm3衰退至〇.3cm3，在前2週減少83%至94%。完全退化並未達成。表8 :控制組小鼠之腫瘤退化老鼠I D. 曰期腫瘤體積(cm3) 416 11/20/98 0.195 (7mmX7.6 mm) 11/24/98 0.161 (6.4mmX7.6 mm) 11/25/98 可觸及 11/26/98 可觸及 11/27/98 完全退化 453 11/20/98 0.858 (10 mm X 16 mm) 11/24/98 0.393 (6.4 mm X 7.6 mm, 7.6mm X7.6mm) 11/25/98 可觸及 11/26/98 可觸及 11/27/98 可觸及 11/28/98 勉強可觸及 11/29/98 完全退化摄要及結掄 1·具1個胺基酸或16個胺基酸之連接子之iFN-a-2a-Fc融合體說明在病毒細胞病變分析之相等活性。 2·具大差異連接子長度之IFN-p-Fc融合體說明在病毒細胞病變分析具類似效果。 3· Roferon A 1X106 IU/天治療延遲Daudi B細胞淋巴瘤發展2週(t > U:\TYPE\LLTSYC-2D0C1 577896 t〇.〇5〇2)，P > 0·05)。經由整個投劑期間，IFN-a_2a_Fc丨χ 1〇6犯/天完全抑制腫瘤形成及該抑制在治療終結後可延伸6個月。完全抑制亦部ς顯示於 1 X 105 IU/天 IFN_a-2a-Fc 處理小鼠。 IRofen A 1X106 IU/3天治療對腫瘤發展並未呈現任何 Burkkt淋巴瘤可藉IFN_a-Fc (1 X i〇6 IU/3天)或謂*2叫1 χ ι〇6师每週）而冗全抑制’且抑制在治療程序後可持續至少7週。 5·初步數據說明當以IFN-oc-2a-Fc 1〇6 IU/每日治瘆, 不口你川天，已成型5週大 Daud! Burk泔淋巴瘤可完全退化。！FN_a_2a_Fc治療開始前以ι〇6⑴ Roferon A/每3天治療7週’ Daudi Bu她淋巴瘤在2週内可達到9〇%之腫瘤體積減少。 ° 應瞭解用於此中之期間及表現僅為例示，並非限制，且本發明係定義於申請專利範圍，且包括彼等申請專利範圍之所有相等物。 μ1X106IU / day administration ⑶ The effect of INF-α on tumor growth rate is 1 ° / 〇. The tumor growth rate is very close in the control group and the Roferon A-treated animal line when the tumor grows to about the weight of the mouse. In the control group animals, the average tumor volume increased 10-fold at 2 weeks, whereas Roferona-treated mice showed a 9-fold increase. Table 5: Tumor collection rate in different treatment groups Tumor collection rate (%) (N = 8) Control group Roferon A IFN a-2a-Fc IFN a-2a-Fc dilution 1X106 IU / 100ul lX106IU / 100ul 1X105 IU / lOOul 100 (8/8) 87.5 (7/8) 0 25.0 (2/8) U: \ TYPE \ LUI \ SYC-2.DOC 1 定量 Quantitative serum IFN-α concentration IFN-α and IFN-a- The serum concentration of 2a-Fc was determined by ELISA procedure. Roferon A-treated mice ' failed to detect Vein_a-2a 24 hours after the last dose. In IFN-a-2a-Fc-treated mice, the serum IFN_a-2a-Fc concentration was 3, g / ml (1 X 106 sky group) and 〇7 | Lig / m〇1 ( 1 X 105 IU / day group) (Table 6). After the end of treatment, his serum concentration decreased between 1 and 22 days. The data indicate that IFN-a-2a-Fc has a half-life in mice of about 1 week (subcutaneously administered 1 X 106 IU / day or 1 X 105 μv / day after 8 weeks). Table 6 · Serum iFN-a-2a concentration (pg / ml) Treatment ___ Days after the end of treatment __ _ 1 8 22 1X10; IU 25.370 ± 6.885 12.080 ± 3.477 3.477 ± 0.525 Na-2a-Fc 1X10 IU 2.766 11.138 1.549 0.50.536 0.691 0.10.141 0 eron A Undetectable Undetectable Undetectable 实验 Experiment to increase the dosage interval In this experiment, Roferon A 1 X 1 〇6 IU is provided every 3 days and every 3 days and weekly Agent 1 X 106 IU IFN-a_2a-Fc. The results are shown in Table 7. Compared with the control group, Roferon A 1 X 106 IU for 3 days did not show any hardening against tumor formation. However, 1 X 106 IU of IFN-a administered every 3 days and every week -Fc is effective in suppressing tumor formation during the 8-week treatment period. This inhibition can be extended for 7 weeks after the treatment period. Table 7 'Tumor development rate of tumors in animals with increased dosing cycles (%) (N = 8) ~~ 100 (8/8) " " " " 100 (8/8) N / AN / A Treatment-R〇fer〇na 1〇6 ιυ / 3 days IFN-Fc 106 IU / 3 days Average weekly tumor development time (days) 21.1 ± 1.1 22.0 ± 1.9 N / AN / A 577896 ⑺ Daudi Burkitt has been formed Preliminary studies of lymphoma Two mice with 5-week-old Daudi Burkitt lymphoma were treated with IFN-a-Fc (106 IU / day). After 10 days, complete degradation was observed in both of the animals (Table 8). The other 2 mice with Daudi lymphoma that had been forming for 6.5 weeks were treated with 106 IU Roferon A every 3 days for 8 weeks. In the latter mice, the tumor volume decreased rapidly, from 2.7 cm3 and 4.6 cm3 to 0.3 cm3, which decreased by 83% to 94% in the first 2 weeks. Complete degradation was not achieved. Table 8: Tumor degenerating mice in control group I D. Tumor volume in the first stage (cm3) 416 11/20/98 0.195 (7mmX7.6 mm) 11/24/98 0.161 (6.4mmX7.6 mm) 11/25 / 98 accessible 11/26/98 accessible 11/27/98 fully degraded 453 11/20/98 0.858 (10 mm X 16 mm) 11/24/98 0.393 (6.4 mm X 7.6 mm, 7.6mm X7.6mm ) 11/25/98 can be touched 11/26/98 can be touched 11/27/98 can be touched 11/28/98 barely can be touched 11/29/98 completely degraded and crusted 1. with 1 amino acid Or iFN-a-2a-Fc fusions of 16 amino acid linkers demonstrate equivalent activity in viral cytopathic analysis. 2. IFN-p-Fc fusions with large differences in linker length indicate similar effects in viral cytopathic analysis. 3. Roferon A 1X106 IU / day treatment delays the development of Daudi B-cell lymphoma for 2 weeks (t > U: \ TYPE \ LLTSYC-2D0C1 577896 t0.055), P > 0.05). Throughout the dosing period, IFN-a_2a_Fc | χ 106 deaths / day completely inhibited tumor formation and the inhibition was extended for 6 months after the end of treatment. Complete inhibition was also shown in 1 X 105 IU / day IFN_a-2a-Fc treated mice. IRofen A 1X106 IU / 3-day treatment does not show any Burkkt lymphoma for tumor development. It can be redundant with IFN_a-Fc (1 X i〇6 IU / 3 days) or so-called * 2 called 1 x 〇〇〇06 division per week) and redundant 'Total inhibition' and the inhibition can continue for at least 7 weeks after the treatment procedure. 5. Preliminary data show that when IFN-oc-2a-Fc 106 IU / day is used for treatment of dysentery, Daud! Burk's lymphoma that has been formed for 5 weeks can completely degenerate. !! FN_a_2a_Fc treatment was started with ι〇6⑴ Roferon A / 7 weeks every 3 days. Daudi Bu her lymphoma achieved a 90% reduction in tumor volume within 2 weeks. ° It should be understood that the periods and performances used herein are merely examples and are not limiting, and the invention is defined in the scope of patent applications and includes all equivalents of their scope of patent applications. μ

U:\TYPE\LLTSVC-2.DOC 1 -10- 577896 SDS-PAGE及LAIFN之西方圖 108 — 80 — ! 丨：. . ; · ; 50.9 — : I 34 — I 27.3 *— : I 16.9 — | ·:, kD .........·-· ... —一— * ····*·--- - · ** M A B C Μ A 1微克LAIFN載於10% SDS膠之每一道中 A : Poncean S染色之總蛋白質 B :抗-hlgG Fc MAb之免疫染色 C :抗hIFNA MAb之免疫染色 R:減少狀沉 N:非減少狀況 Μ:如指出之分子量標記 U:\TYPE\LUI\SYC-2.DOC 1 -11 -U: \ TYPE \ LLTSVC-2.DOC 1 -10- 577896 Western picture of SDS-PAGE and LAIFN 108 — 80 —! 丨:.;; 50.9 —: I 34 — I 27.3 * —: I 16.9 — | · :, kD .........--...---* · · · * *----** MABC Μ A 1 μg LAIFN is contained in 10% SDS gel per A: Total protein stained by Poncean S B: Immunostaining of anti-hlgG Fc MAb C: Immunostaining of anti-hIFNA MAb R: Decrease N: Non-reduced condition M: As indicated by molecular weight marker U: \ TYPE \ LUI \ SYC-2.DOC 1 -11-