TWI278319B

TWI278319B - VGF selective binding agents and methods of treating VGF-related disorders

Info

Publication number: TWI278319B
Application number: TW89114524A
Authority: TW
Inventors: Hai Yan
Original assignee: Amgen Inc
Priority date: 1999-07-21
Filing date: 2000-07-20
Publication date: 2007-04-11

Abstract

The present invention provides novel VGF polypeptides and selective binding agents. The invention also provides host cells and methods for producing VGF polypeptides. The invention further provides VGF pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, and/or prevention of diseases, conditions, and disorders associated with VGF polypeptides.

Description

1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（1) 發明範圍：本發明係關於新穎的VGF多肽和選擇性結合劑。本發明亦關於產製VGF多肽的宿主細胞和方法。本發明更有關於診斷、治療、改善及/或預防與VGF多肽有關之疾病、病況和障礙的VGF醫藥組合物和方法。發明背景： VGF爲在神經元和内分泌細胞中找到的分泌性多肽。 Canu等人，1997，Genomics，45 : 443_46。在下視丘中發現 VGF，並在腦中由電活性、傷害和生理時鐘調節。已經顯示下視丘在攝食和能量輸出的調節上扮演重要的角色，並已經顯示對下視丘的傷害將影響食慾和體重兩者。Schwartz 等人，1995, Am，J. Physiol·，269 : 949-57。亦已經發現 VGF 在代謝上扮演某種角色。已知人類和大鼠VGF的核苷酸和胺基酸序列，並顯示彼此有高度的同種性。Canu等人，在前。VGF基因，最初確認爲2.7kb cDNA片段，在活體外藉著神經營養素 (neurotrophin)，在神經元和内分泌細胞的子群中轉錄。肥胖和食慾不振爲能量體内平衡的瓦解，起因於在能量攝入和支出之間的不平衡。對於有效處理這類病況有需求。對於有效處理惡病質、***、代謝亢進之病況、機能亢進、活動性不足、胰島素產生過多和相關的病況和障礙亦有所需求。發明概述: -4- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) ---------訂— 卷. 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（2 ) 本發明係關於經過分離之多肽，包括在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 0號任一個中陳述的胺基酸序列。較佳的經過分離之多肽，包括在序列識別2號中陳述的胺基酸序列。本發明亦提供經過分離之多肽，包括在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 0號任一個中陳述之胺基酸序列的片段，其中該片段具有如同在序列識別1號、序列識別2號、序列識別 3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之多肽的活性。較佳的經過分離之多肽包括在序列識別2號中陳述之胺基酸序列的片段，其中該片段具有在序列識別2號中陳述之多肽的活性。本發明更提供經過分離之多肽，包括選自包括下列的胺基酸序列： (a)在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之胺基酸序列，具有至少一個保留性的胺基酸置換，且其中該多肽具有在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7 號、序列識別8號、序列識別9號或序列識別1 〇號任一個中 -5 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）一-------------------^--------- AW (請先閱讀背面之注意事項再填寫本頁) _ 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（3 ) 陳述之多肚的活性； (b) 在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之胺基酸序列’具有至少一個胺基酸***，且其中該多肽具有在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之多肽的活性； (c) 在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之胺基酸序列，具有至少一個胺基酸刪除，且其中該多肽具有在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之多肽的活性； (d) 在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之胺基酸序列，具有C -及/或N -終端的截短，且其中該多肽具有在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之 ^紙張尺度適时關家鮮（CNS)A4規格（21〇 X 297公釐） ---------------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 A7 B7 五、發明說明（4 ) 多肽的活性；以及 (e )在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之胺基酸序列，具有至少一個選自包括胺基酸取代、胺基酸 ***、胺基酸刪除、羧基-終端截短和胺基·終端截短的修改，且其中該多肽具有在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 0 號任一個中陳述之多肽的活性。亦提供融合多肽，包括至少一個上述之多肽，與異種胺基酸序列融合。本發明亦提供選擇性結合劑，像是抗體，能夠專一地與至少一個多肽結合，該多肽包括在序列識別1號、序列識別 2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之胺基酸序列。本發明更提供能夠專一地與至少一個多肽之片段結合的選擇性結合劑，該多肽包括在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6 號、序列識別7號、序列識別8號、序列識別9號或序列識別10號任一個中陳述之胺基酸序列，或其天然存在的變體。本發明之選擇性結合劑可以是抗體或其片段，包括但不限於··老鼠的抗體、人類化的抗體、人類的抗體、多株抗本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱） (請先閱讀背面之注意事項再填寫本頁) --------訂---- 卷_ 經濟部智慧財產局員工消費合作社印製 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（5 ) 體、單株抗體、嵌合型抗體、CDR-移植抗體、抗·遺傳性型的抗體和多變區片段（像是Fab或Fab，片段）。本發明的選擇性結合劑包括具有至少一個互補性-決定區，對多肽具有專性的逐擇性結合劑或其片段，該多肽包括在序列識別1 號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之胺基酸序列。本發明之選擇性結合劑可視需要與可檢測標記結合。亦提供能夠拮抗VGF生物活性的選擇性結合劑。較佳的選擇性結合劑或其片段，能夠專一地與包括在序列識別2號中陳述之胺基酸序列或其片段的多肽結合。本發明亦提供醫藥組合物，包括本發明之多肽或選擇性結合劑，以及一或多個在藥學上可接受的調配劑，亦包含在本發明内。碉配劑可以是適當的載劑、佐劑、助溶劑、穩足劑或抗-氧化劑。可利用水溶性的聚合物，像是聚乙二醇和葡聚醣以共價方式修改VGF多肽或選擇性結合劑。可使用本發明之醫藥組合物，提供在治療上有效含量的本發明之VGF多肽或選擇性結合劑。本發明亦提供製造可用來治療VGF相關性疾病、病況或障礙之醫藥的方法。 ^發明更提供治療、預防或改善VGF相關性疾病、病況或障礙的方法，包括對患者投予有效含量的本發明之VGF 多肽或選擇性結合劑。VGF相關性疾病、病況和障礙，包括肥胖、***、惡病質、飲食障礙、aids相關性複合症、代謝尤進之病況、機能宄進、活動性不足和胰島素：生過本紙張尺度適用中國國冢標準(CNS)A4飞格⑽X 297 ‘ΐ·) /—-----------------^--------- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1278319 五、發明說明（6 ) 夕VGF相關〈飲食障礙包括貪食症和神經性厭食症。將、曉亦可提供本發明之方法，投予本發明之vgf多肽或選擇性結合劑的組合。、亦& i、在動物中’ ♦斷VGF相關性疾病、病況和障礙，或對VGF相關性疾病、病況和障礙之感受性的方法，包括決足VGF多肽的存在或含量，並以VGF多肤表現的存在或含量為基礎，診斷VGF相關性疾病、病況和障礙，或對 VGF相關性疾病、病況和障礙的感受性。在診斷ν(^相關性疾病、病況或障礙，或對VGF相關性疾病、病況和障礙的感文性的較佳方法中，該動物為哺乳動物。在更佳的方法中，該動物為人類。本發明亦提供測定受試分子，確認該受試分子與vGF多肽結合的方法。該方法包括使VGF多肽與受試分子接觸，並決定該受試分子與該多肽結合的程度。該方法進一步包括決定這類受試分子是否為VGF多肽的激動劑或拮抗劑。本發明更提供測試分子對VGF多肽之表現，或對VGF多月太之活性的影響的方法。在考量整個說明書時，本發明的這些及其他目標更將_ 得更為明確。圖片簡述：圖1解釋在投予VGF- la(序列識別2號）之後，VGF擊倒老鼠的體重。在第5天停止VGF-1 a的投藥（以箭頭表示）。圖2解釋以VGF-1 (序列識別1號）注射之兔予的抗體力價含量。 --------訂--------- (請先閱讀背面之注意事項再填寫本頁) -9 - 1278319 A7 B7 五、發明說明（7 ) 圖3解釋以VGF-2(序列識別4號）注射之兔子的抗體力價含量。 t明詳述: 在本文中使用的章節開頭僅爲了組織化的目的，並非企圖限制所描述的主題。在本申請案中提及的參考文獻，將其合併於此以作爲參考來表達之。定義_ ，’ VGF多肽” 一詞意指包括在表I中陳述的任何胺基酸序列，以及在本文中描述的相關多肽。相關的多肽包括VGF 多肽片段、正類似物（orthologs)、變體和衍生物，其具有至少一個在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 0號中陳述之任一個VGF多肽的特徵。VGF多肽可以是成熟的多肽，如同在本文中之定義，並可以或可以不具有胺基-終端的甲硫胺酸殘基，依據製備它們的方法而定。1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. INSTRUCTIONS (1) Scope of the Invention: The present invention relates to novel VGF polypeptides and selective binding agents. The invention also relates to host cells and methods for producing VGF polypeptides. The invention further relates to VGF pharmaceutical compositions and methods for diagnosing, treating, ameliorating and/or preventing diseases, conditions and disorders associated with VGF polypeptides. BACKGROUND OF THE INVENTION VGF is a secreted polypeptide found in neurons and endocrine cells. Canu et al., 1997, Genomics, 45: 443_46. VGF is found in the hypothalamus and is regulated in the brain by electrical activity, injury, and physiological clock. It has been shown that the hypothalamus plays an important role in the regulation of feeding and energy output, and it has been shown that damage to the hypothalamus will affect both appetite and body weight. Schwartz et al., 1995, Am, J. Physiol, 269: 949-57. VGF has also been found to play a role in metabolism. The nucleotide and amino acid sequences of human and rat VGF are known and show a high degree of homology to each other. Canu et al. The VGF gene, originally recognized as a 2.7 kb cDNA fragment, is transcribed in a subpopulation of neurons and endocrine cells by a neurotrophin in vitro. Obesity and loss of appetite are the breakdown of energy homeostasis, resulting from an imbalance between energy intake and expenditure. There is a need to effectively handle such conditions. There is also a need for effective treatment of cachexia, infertility, hypermetabolic conditions, hyperactivity, inadequate mobility, hyperinsulin production and related conditions and disorders. SUMMARY OF THE INVENTION: -4- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back and fill out this page) --------- Order - Volume 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. INSTRUCTIONS (2) The present invention relates to isolated polypeptides, including sequence identification No. 1, sequence identification No. 2, sequence identification No. 3, sequence identification 4 No., Sequence Recognition No. 5, Sequence Recognition No. 6, Sequence Identification No. 7, Sequence Identification No. 8, Sequence Identification No. 9, or Sequence Identification amino acid sequence as set forth in any of the No. 10. Preferred isolated polypeptides include the amino acid sequences set forth in Sequence Identification No. 2. The invention also provides isolated polypeptides, including sequence identification No. 1, sequence identification No. 2, sequence identification No. 3, sequence identification No. 4, sequence identification No. 5, sequence identification No. 6, sequence identification No. 7, and sequence identification No. 8. a sequence recognition sequence 9 or a sequence recognition of a fragment of an amino acid sequence as set forth in any of the 10, wherein the fragment has the same sequence identification number 1, sequence recognition number 2, sequence recognition number 3, sequence recognition number 4, sequence The activity of the polypeptide as set forth in any of No. 5, Sequence Recognition No. 6, Sequence Recognition No. 7, Sequence Identification No. 8, Sequence Recognition No. 9, or Sequence Identification No. 1 is identified. A preferred isolated polypeptide comprises a fragment of the amino acid sequence set forth in Sequence Identification No. 2, wherein the fragment has the activity of the polypeptide set forth in Sequence Identification No. 2. The invention further provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) sequence recognition number 1, sequence recognition number 2, sequence recognition number 3, sequence recognition number 4, sequence recognition number 5, sequence Recognizing an amino acid sequence as set forth in any one of No. 6, Sequence Identification No. 7, Sequence Identification No. 8, Sequence Identification No. 9, or Sequence Identification No. 1 having at least one retention amino acid substitution, and wherein the polypeptide has In sequence identification No. 1, sequence identification No. 2, sequence identification No. 3, sequence identification No. 4, sequence identification No. 5, sequence identification No. 6, sequence identification No. 7, sequence identification No. 8, sequence identification No. 9, or sequence identification 1 Any one of the -5 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -------------------^--- ------ AW (please read the notes on the back and fill out this page) _ 1278319 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Print A7 B7 V. Inventions (3) Declared activity; (b ) in sequence identification No. 1, sequence identification No. 2, sequence identification No. 3, sequence identification No. 4, sequence identification No. 5 Sequence recognition No. 6, sequence identification No. 7, sequence recognition No. 8, sequence identification No. 9, or sequence recognition 1 Amino acid sequence as set forth in any of the apostrophes has at least one amino acid insertion, and wherein the polypeptide has a sequence Identification No. 1, Sequence Identification No. 2, Sequence Identification No. 3, Sequence Identification No. 4, Sequence Identification No. 5, Sequence Identification No. 6, Sequence Identification No. 7, Sequence Identification No. 8, Sequence Identification No. 9, or Sequence Identification No. 1 The activity of a polypeptide as stated in (c) Sequence identification No. 1, Sequence identification No. 2, Sequence identification No. 3, Sequence identification No. 4, Sequence identification No. 5, Sequence identification No. 6, Sequence identification No. 7, Sequence identification 8 Amino acid sequence as set forth in any of the SEQ ID NO: No. 9 or Sequence Identification 1 nickname, having at least one amino acid deletion, and wherein the polypeptide has sequence recognition number 1, sequence recognition number 2, sequence recognition number 3 , Sequence Identification No. 4, Sequence Recognition No. 5, Sequence Recognition No. 6, Sequence Recognition No. 7, Sequence Identification No. 8, Sequence Recognition No. 9, or Sequence Identification 1 The activity of the polypeptide stated in any of the nicknames; (d) in the sequence Identification number 1 Sequence identification 2, sequence identification 3, sequence identification 4, sequence identification 5, sequence identification 6, sequence identification 7, sequence identification 8, sequence identification 9 or sequence identification 1 An amino acid sequence having a C- and/or N-terminal truncation, and wherein the polypeptide has sequence recognition number 1, sequence recognition number 2, sequence recognition number 3, sequence recognition number 4, sequence recognition number 5, sequence Identification No. 6, Sequence Identification No. 7, Sequence Identification No. 8, Sequence Identification No. 9, or Sequence Identification 1 No. stated in the paper size, timely (CNS) A4 specification (21〇X 297 mm) ---------------------Book --------- (Please read the notes on the back and fill out this page) 1278319 A7 B7 V. Invention Description (4) Activity of the polypeptide; and (e) Sequence identification No. 1, Sequence identification No. 2, Sequence identification No. 3, Sequence identification No. 4, Sequence identification No. 5, Sequence identification No. 6, Sequence identification No. 7, Sequence identification Amino acid sequence as set forth in any of the No. 8, Sequence Identification No. 9, or Sequence Identification 1 nickname, having at least one selected from the group consisting of amino acid substitutions, a modification of a base acid insertion, an amino acid deletion, a carboxy-terminal truncation, and an amino terminal termination, wherein the polypeptide has sequence identification number 1, sequence recognition number 2, sequence recognition number 3, sequence identification number 4, The activity of the polypeptide as set forth in any one of Sequence Identification No. 5, Sequence Recognition No. 6, Sequence Recognition No. 7, Sequence Identification No. 8, Sequence Identification No. 9, or Sequence Identification No. 10. Fusion polypeptides are also provided, including at least one of the above polypeptides, fused to a heterologous amino acid sequence. The invention also provides a selective binding agent, such as an antibody, capable of specifically binding to at least one polypeptide, which is included in sequence recognition No. 1, sequence identification No. 2, sequence identification No. 3, sequence identification No. 4, sequence identification No. 5 The amino acid sequence set forth in any of Sequence Identification No. 6, Sequence Identification No. 7, Sequence Identification No. 8, Sequence Identification No. 9, or Sequence Identification. The present invention further provides a selective binding agent capable of specifically binding to a fragment of at least one polypeptide, which comprises Sequence Identification No. 1, Sequence Recognition No. 2, Sequence Recognition No. 3, Sequence Recognition No. 4, Sequence Recognition No. 5, Sequence The amino acid sequence set forth in any of No. 6, Sequence Identification No. 7, Sequence Identification No. 8, Sequence Identification No. 9, or Sequence Identification No. 10, or a naturally occurring variant thereof. The selective binding agent of the present invention may be an antibody or a fragment thereof, including but not limited to, an antibody against a mouse, a humanized antibody, a human antibody, and a plurality of anti-paper standards applicable to the Chinese National Standard (CNS) A4 specification (210) X 297 public love) (Please read the note on the back and fill out this page) --------Book---- Volume _ Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1278319 Ministry of Economic Affairs Intellectual Property Bureau staff Consumer Cooperatives Print A7 B7 V. INSTRUCTIONS (5) Body, monoclonal antibody, chimeric antibody, CDR-grafted antibody, anti-hereditary antibody and variable region fragment (like Fab or Fab, fragment) . The selective binding agent of the present invention comprises an excipient binding agent or fragment thereof having at least one complementarity-determining region, which is specific to the polypeptide, and the polypeptide is included in Sequence Identification No. 1, Sequence Identification No. 2, Sequence Identification No. 3 The amino acid sequence set forth in any one of Sequence Identification No. 4, Sequence Recognition No. 5, Sequence Recognition No. 6, Sequence Identification No. 7, Sequence Identification No. 8, Sequence Identification No. 9, or Sequence Identification No. 1 nickname. The selective binding agents of the present invention can be combined with a detectable label as desired. A selective binding agent capable of antagonizing the biological activity of VGF is also provided. Preferred selective binding agents or fragments thereof are capable of specifically binding to a polypeptide comprising the amino acid sequence or a fragment thereof set forth in the Sequence Identification No. 2. The invention also provides pharmaceutical compositions, including polypeptides or selective binding agents of the invention, and one or more pharmaceutically acceptable formulation agents, also included in the invention. The oxime formulation can be a suitable carrier, adjuvant, cosolvent, stabilizing agent or anti-oxidant. Water-soluble polymers, such as polyethylene glycol and dextran, can be used to modify the VGF polypeptide or selective binding agent in a covalent manner. A pharmaceutical composition of the invention may be used to provide a therapeutically effective amount of a VGF polypeptide or a selective binding agent of the invention. The invention also provides methods of making a medicament useful for treating a VGF-associated disease, condition or disorder. The invention further provides a method of treating, preventing or ameliorating a VGF-associated disease, condition or disorder comprising administering to the patient an effective amount of a VGF polypeptide or selective binding agent of the invention. VGF-related diseases, conditions and disorders, including obesity, infertility, cachexia, eating disorders, aids-related complications, metabolic conditions, hyperactivity, lack of activity, and insulin: the size of the paper is applicable to China冢Standard (CNS) A4 Feige (10)X 297 'ΐ·) /—-----------------^--------- (Please read the back of the note first Matters fill out this page) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1278319 V. Invention description (6) 夕 VGF related <Dietary disorders include bulimia and anorexia nervosa. Combinations of the vgf polypeptides of the invention or a selective binding agent can also be administered by the methods of the invention. And also, i. methods for mutating VGF-related diseases, conditions and disorders in animals, or susceptibility to VGF-associated diseases, conditions and disorders, including the presence or amount of a VGF polypeptide, and more VGF Based on the presence or amount of skin manifestations, diagnose VGF-associated diseases, conditions and disorders, or susceptibility to VGF-associated diseases, conditions and disorders. In a preferred method of diagnosing ν (a related disease, condition or disorder, or sensitivity to VGF-associated diseases, conditions, and disorders, the animal is a mammal. In a preferred method, the animal is a human The invention also provides a method of determining a test molecule to confirm binding of the test molecule to a vGF polypeptide, the method comprising contacting a VGF polypeptide with a test molecule and determining the extent to which the test molecule binds to the polypeptide. Included are methods for determining whether such a test molecule is a agonist or antagonist of a VGF polypeptide. The present invention further provides methods for testing the performance of a VGF polypeptide, or the effect of VGF on a multi-monthly activity. These and other goals of the invention will be more clarified. BRIEF DESCRIPTION OF THE DRAWINGS: Figure 1 illustrates VGF knocking down the body weight of mice after administration of VGF-la (sequence recognition No. 2). Stopping VGF-1 on day 5 Administration of a (indicated by an arrow) Figure 2 illustrates the antibody titer content of rabbits injected with VGF-1 (sequence recognition No. 1). -------- Order --------- (Please read the notes on the back and fill out this page) -9 - 1 278319 A7 B7 V. INSTRUCTIONS (7) Figure 3 illustrates the antibody valence content of rabbits injected with VGF-2 (sequence recognition No. 4). t Ming detailed: The chapters used in this article are for organizational purposes only. It is not intended to limit the subject matter described. The references mentioned in the present application are hereby incorporated by reference herein. Any amino acid sequence, and related polypeptides described herein. Related polypeptides include VGF polypeptide fragments, orthologs, variants and derivatives having at least one in sequence recognition number 1, sequence identification number 2 , Sequence Identification No. 3, Sequence Recognition No. 4, Sequence Identification No. 5, Sequence Identification No. 6, Sequence Identification No. 7, Sequence Identification No. 8, Sequence Recognition No. 9, or Sequence Identification No. 10 features of any of the VGF polypeptides stated in No. 10 The VGF polypeptide may be a mature polypeptide, as defined herein, and may or may not have an amino-terminal methionine residue, depending on the method of preparing them.

表I (清先閱讀背面之注音？事項再填寫本頁) ---------訂--- 經濟部智慧財產局員工消費合作社印製 VGF多肽 VGF多肽胺基酸序列(序列識別號碼） VGF-1 AQEEAD AEERRLQEQEELENYIEHVLLHRP(序列識別 1 號） VGF-la AQEEADAEE(序列識別2號） VGF-lb LQEQEELENYIEHVLLHRP(序列識別 3 號） VGF-2 LEGSFLGGSEAGERLLQQGLAQVEA(序列識別 4 號） VGF-3 SQEE APGH(序列識別5號） -10- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1278319 A7 B7 經濟部智慧財產局員工消費合作社印製五、發明說明（9) VGF多肽。例如老鼠和人類的VGF多肽，視為彼此的正類似物。 ’’ VGF多肽變體” 一詞意指包括與在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號中陳述的任何VGF多肽相比較，具有一或多個胺基酸序列的取代（保留性、非-保留性或其混合）、刪除（像是内部的刪除及/或VGF多肽片段），及/或加入（像是内部的加入及/或VGF融合多肽）之胺基酸序列的VGF多肽。變體可以是天然存在的（例如VGF多肽對偶基因變體或VGF多肽正類似物），或人工建構的。 ’’ VGF多肽衍生物’’ 一詞意指已經以化學方式修改的在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別10號中陳述之任何VGF多肽，或如同在本文中定義的VGF多肽片段、正類似物或變體。化學修改可以是，例如，藉著共價附接一或多個聚合物，包括但不限於水溶性聚合物、N -連接或Ο -連接的碳水化合物、糖類和磷酸酯。在與多肽附接之分子的類型或位置上，以與天然存在之VGF多肽不同的方式來修改VGF多肽衍生物。VGF多肽衍生物更包括了刪除一或多個天然附接於 VGF多肽上之化學基團的VGF多肽。 ’’成熟的VGF多肽，，一詞意指缺乏前導序列的VGF多肽。成 -12- (請先閱讀背面之注意事項再填寫本頁) -— 訂--- 卷· 本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） 1278319 A7 B7 經濟部智慧財產局員Η消費合作社印製五、發明說明（熟的VGF多肽亦包括其他的修改，像是胺基·終端（有或無前導序列）及/或羧基-終端的蛋白水解加工，從較大的前驅物中切開較小的多肽，N _連接及/或〇 -連接的糖基化作用及其類似者。 ·· VGF融合多肽”一詞意指一或多個胺基酸（像是異種的肽或多肽）融合在VGF多肽、片段、正類似物、變體或衍生物的胺基或竣基-終端上。 π具有生物活性的VGF多肽一詞意指具有至少一個包括在任一個序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號中陳述之胺基酸序列的多肽之活性特徵的VGF多肽。此外，VGF多肽可像免疫原一樣地活躍；也就是該VGF多肽含有至少一個可使抗體升高的抗原決定位。 •'經過分離的多肽π —詞意指本發明之多肽，其（j )已經從至少大約5 0 %的多核甞酸、脂質、碳水化合物或其他物質中分離出來，這些物質是在從來源細胞中分離出時，與其一起自然地被發現的，（2)該多肽的全部或一部份，並未與 ••經過分離之多肽”自然連接的物質連接（經由共價或非共價之叉互作用），（3 )該多肽與並非自然連接之物質以可操作之方式連接（經由共價或非共價之交互作用），或（4)並非天然存在的。較佳的是，經過分離的多肽實質上不含任何其他的污染多肽，或在其天然環境中找到的其他污染物，其將干擾它的治療、診斷、預防或研究用途。 -------------------^--------- (請先閱讀背面之注音？事項再填寫本頁) 13- 1278319 A7 經濟部智慧財產局員工消費合作社印製五、發明說明（11 ❿叫仕何用來運送密碼資訊至宿主細胞的分子（例如核酸、質體或病毒）。 ”表現載體”一詞意指適合用來轉化宿主細胞，並含有指揮及/或控制***之異種性核酸序列表現之核酸序列的載體。表現包括但不限於諸如轉錄、轉譯，若有***序列存在，還有RNA接合之類的過程。使用"宿主細胞"一詞時，意指已經利用核酸序列轉化，或能夠以核酸序列轉化，然後表現感興趣之選擇基因的細胞。孩名詞包括親代細胞的後代，無論後代是否在形態學上或在遺傳的組成上與原始的4目> 基因即可？ H的視代相同，只要提供選出的 ”同、一性"-詞，如同此項技藝中已知的，意指藉著比較序列定出在二或多個多肽分子，或二或多個核酸分子的序 2間的相關性。在此項技藝中同一性"亦意指在多肤或核魬分子〈間序列相關性的程度，例如，可藉著在長串 ^二或多個核誓酸或二或多個胺基酸序列之間的相配性來 ::二同：性"利靖排列，(若有的話)藉著特定的數予模式或电腦程式（也就是"演算法"）尋址，測量在二或多個序列中較小序列之間的相同配對之百分比。 ^ 詞爲相關的概念，但是與”同-性"顯然有別，巧似性"意指包括相同配對和保留性置換配對之相關性 77。如果兩個多肽序列具有例如1G/2G個相同的胺基 ^百而东下的全部是非保留性置換，此時同—性和類似性的百刀比將都是5 G %。如果在相㈣實财，有上 -------------------訂--------- (請先閱讀背面之注意事項再填寫本頁)Table I (Read the phonetic notes on the back first and then fill out this page) ---------Book --- Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed VGF polypeptide VGF polypeptide amino acid sequence (sequence recognition No.) VGF-1 AQEEAD AEERRLQEQEELENYIEHVLLHRP (Sequence Identification No. 1) VGF-la AQEEADAEE (Sequence Recognition No. 2) VGF-lb LQEQEELENYIEHVLLHRP (Sequence Recognition No. 3) VGF-2 LEGSFLGGSEAGERLLQQGLAQVEA (Sequence Identification No. 4) VGF-3 SQEE APGH (sequence) Identification No. 5) -10- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1278319 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (9) VGF peptide. For example, mouse and human VGF polypeptides are considered to be positive analogs of each other. The term ''VGF polypeptide variant') includes the inclusion and sequence identification number 1, sequence identification number 2, sequence identification number 3, sequence identification number 4, sequence identification number 5, sequence identification number 6, sequence identification number 7, sequence Comparison of any of the VGF polypeptides set forth in recognition number 8, sequence recognition 9 or sequence recognition 1 nickname, having one or more amino acid sequence substitutions (retention, non-retention or mixtures thereof), deletion (like a VGF polypeptide that is an internal deletion and/or VGF polypeptide fragment), and/or an amino acid sequence (such as an internal addition and/or VGF fusion polypeptide). The variant may be naturally occurring (eg, a VGF polypeptide dual) A genetic variant or a positive analog of a VGF polypeptide, or artificially constructed. The term ''VGF polypeptide derivative'' means a sequence that has been chemically modified, Sequence Identification No. 1, Sequence Identification No. 2, Sequence Identification No. 3, Any of the VGF polypeptides set forth in Sequence Recognition No. 4, Sequence Recognition No. 5, Sequence Recognition No. 6, Sequence Recognition No. 7, Sequence Recognition No. 8, Sequence Recognition No. 9, or Sequence Recognition No. 10, or a VGF polypeptide as defined herein. Fragment, positive class Analogous or variant. Chemical modification may be, for example, by covalent attachment of one or more polymers including, but not limited to, water soluble polymers, N-linked or hydrazone-linked carbohydrates, sugars, and phosphates. The VGF polypeptide derivative is modified in a different manner than the naturally occurring VGF polypeptide at the type or position of the molecule to which the polypeptide is attached. The VGF polypeptide derivative further includes deletion of one or more naturally attached to the VGF polypeptide. a chemical group of VGF polypeptide. ''Mature VGF polypeptide,' means the VGF polypeptide lacking the leader sequence. Cheng-12- (please read the back note first and then fill out this page) -- Volume · This paper scale applies to China National Standard (CNS) A4 specification (210 x 297 mm) 1278319 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Η Consumer Cooperative Printed 5, Invention Description (cooked VGF peptide also includes other modifications, like Is an amino-terminal (with or without leader sequence) and/or carboxyl-terminal proteolytic processing, cutting smaller polypeptides from larger precursors, N-linked and/or 〇-linked glycosylation And similar. The term "VGF fusion polypeptide" means that one or more amino acids (such as a heterologous peptide or polypeptide) are fused to an amine or thiol-terminal of a VGF polypeptide, fragment, normal analog, variant or derivative. The term "bioactive VGF polypeptide" means having at least one of sequence identification No. 1, sequence recognition No. 2, sequence recognition No. 3, sequence identification No. 4, sequence identification No. 5, sequence identification No. 6, sequence identification. a VGF polypeptide having a transcriptional characteristic of a polypeptide of the amino acid sequence recited in the nickname No. 7, sequence identification No. 8, sequence identification No. 9, or a VGF polypeptide which is active as an immunogen; that is, the VGF The polypeptide contains at least one epitope that raises the antibody. • 'Isolated polypeptide π - word means a polypeptide of the invention, which (j) has been isolated from at least about 50% of polynuclear decanoic acid, lipids, carbohydrates or other substances, which are from the source cell When isolated, naturally found together, (2) all or a portion of the polypeptide is not linked to a substance that is naturally linked to the isolated polypeptide (via a covalent or non-covalent fork) Interaction (3) the polypeptide is operably linked (via covalent or non-covalent interaction) to a substance that is not naturally linked, or (4) is not naturally occurring. Preferably, it is separated The polypeptide is substantially free of any other contaminating polypeptide, or other contaminants found in its natural environment, which would interfere with its therapeutic, diagnostic, prophylactic or research use. -------^--------- (Please read the phonetic transcription on the back? Please fill out this page again) 13- 1278319 A7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description ( 11 ❿叫仕何 is used to transport password information to the host cell molecule (eg, nucleic acid, plastid or virus). The term "expression vector" means a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and/or controls the expression of the inserted heterologous nucleic acid sequence. Such as transcription, translation, if there is an insertion sequence, there are processes such as RNA conjugation. The use of the term "host cell" means that it has been transformed with a nucleic acid sequence, or can be transformed with a nucleic acid sequence, and then expressed interest The cell of the selected gene. The genus includes the progeny of the parental cell, regardless of whether the progeny is morphologically or genetically composed in the same manner as the original 4 mesh> gene, as long as the selected one is provided. The homologous "-word, as is known in the art, means the correlation between two or more polypeptide molecules, or two or more nucleic acid molecules, by comparison sequences. The identity in the art " also means the degree of sequence correlation in a polypeptide or a nuclear molecule, for example, by long strings of two or more nuclear or two or more amino acids. sequence The match between:: the same: sex " Lijing arrangement, (if any) by a specific number of patterns or computer programs (that is, "algorithm") addressing, measured in two Or the percentage of identical pairs between smaller sequences in multiple sequences. ^ Words are related concepts, but are similar to "same-sex", which is obviously different, and is meant to include the same pairing and retention substitution pairing. Correlation 77. If two polypeptide sequences have, for example, 1G/2G identical amine groups and all of them are non-reserved substitutions, then the homology and similarity of the ratio will be 5 G %. If you are in the phase (four) real money, there are ------------------- order --------- (please read the notes on the back and then fill out this page)

本紙張尺度適財®^i?^NS)A4規格（210 X 297公釐） 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（12 ) 的位置具有保留性置換，此時同一性的百分比仍然是5 0 % ’但類似性的百分比將是7 5 % (〗5 /2 〇 )。因此，在有保留性置換:的案例中，在兩個多肽之間的類似性百分比將比在那兩個多肽之間的同一性百分比更高。 ’’核酸序列”或”核酸分子’，一詞意指DNA或RNA序列。該名到包括由任何已知的DNA和RNA之鹼基類似物形成的分子’像是但不限於4_乙醯基胞嘧啶、8_羥基-Ν6_甲基腺嘌呤、氮丙啶基-胞嘧啶、假異胞嘧啶、5 _ (羧基羥甲基）尿嘧淀、5 -氟尿ρ密淀、5 _溴尿喊淀、5 -羧甲基胺甲基_ 2 -硫代尿喃淀、5 -叛甲基胺甲基尿^密淀、二氫尿喃淀、肌嘗、 Ν 6 -異·戊烯基腺嘌呤、〗-甲基腺嘌呤、丨_甲基假尿嘧啶、 1 -甲基鳥嘌呤、1 -甲基肌苷、2，2 _二甲基鳥嘌呤、2 _甲基腺嘌呤、2 -甲基鳥嘌呤、3 _甲基胞嘧啶、5 _甲基胞嘧啶、N6-甲基腺嘌呤、7_甲基鳥嘌呤、5_甲胺基甲基尿嘧啶、5 -甲氧胺基-甲基硫代尿嘧啶、0 _D—甘露糖基快歐甞（queosine)、5’ -甲氧羰基_甲基尿嘧啶、5 -甲氧基尿嘧啶、2 -甲硫基-N6 -異戊烯基腺嘌呤、尿嘧啶_ 5 _氧基乙酸甲酯、尿嘧啶-5_氧基乙酸、氧基丁氧甞（〇xybut〇x〇sine)、假尿嘧啶、快歐甞、2-硫代胞嘧啶、5_甲基_2_硫代尿嘧啶、 2-硫代尿嘧啶、4-硫代尿嘧啶、5_甲基尿嘧啶、N_尿嘧啶 -5 -氧基乙酸甲基酯、尿嘧啶_ 5 _氧基乙酸、假尿嘧啶、快歐甞、2_硫代胞嘧啶和2,6-二胺基嘌呤。 "天然存在”或”天然的”一詞，當與諸如核酸分子、多肽、宿主細胞及其類似物之類的生物物質連接使用時，意指 -15 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) --1--1 訂--- 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（13) 該物質在自然界中發現，而並非由人類製造的。同樣的，當在本文中使用π非-天然存在，’或”非-天然的"一詞時，意指該物質並非在自然界中發現的，或是在結構上已經由人類修改或合成的。當在本文中使用”以可操作之方式連結” 一詞時，意指侧面序列的排列，其中將這樣描述的側面序列建構或裝配，而得以進行其平常的功能。因此，以可操作之方式與密碼序列連結的側面序列，能夠完成該密碼序列的複製、轉錄及/或轉譯。例如，將密碼序列以可操作之方式與啓動基因連結’此時该啓動基因能夠指揮該密碼序列的轉錄。側面序列不需要與密碼序列相鄰，只要它具有正確的功能即可。因此’例如’可在啓動基因序列和密碼序列之間出現介於其間之未轉譯但會轉綠的序列，而該啓動基因仍被視爲是”以可操作之方式連結’’密碼序列的。有效含量’’和’’在治療上有效的含量”一詞，分別意指所使用之VGF多肽的含量’支持如同在本文中陳述之vgf多肽的一或多個生物活性的可觀察程度。當在本文中使用”在藥學上可接受的載劑"或"在生理學上可接受的載劑”一詞時，意指一或多個適於完成或促進醫藥組合物形式之VGF多肽或VGF選擇性結合劑之遞送的調配物質。 ft選擇性結合劑”一詞意指對VGF多肽有專一性的分子或分子們。當在本文中使用時，，，專一的”或"專一性” 一詞意才曰選擇性結合劑與VGF多肽結合，且不與非-vgf多肽結合 --------鲁--------tTi-------卷 (請先閱讀背面之注音？事項再填寫本頁) -16-The paper scale is suitable for the product ^^i?^NS) A4 specification (210 X 297 mm) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. The description of the invention (12) has a reserved replacement. The percentage of identity is still 50% 'but the percentage of similarity will be 7 5 % (〗〖5 /2). Thus, in the case of a retained substitution: the percent similarity between the two polypeptides will be higher than the percent identity between the two polypeptides. The term 'nucleic acid sequence' or 'nucleic acid molecule' means the DNA or RNA sequence. The name to include molecules formed by any known base analogs of DNA and RNA 'like but not limited to 4_acetylcytosine, 8-hydroxy-Ν6-methyl adenine, aziridine- Cytosine, pseudoisopyrimidine, 5 _ (carboxyhydroxymethyl) uracil, 5-fluorourine ρ, 5 _ bromo uranium, 5-carboxymethylamine methyl -2- thiouran Precipitate, 5-deoxymethylamine methyl urethane, dihydrourethane, muscle taste, Ν6-iso-pentenyl adenine, 〗-methyl adenine, 丨-methyl pseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methyl Pyrimidine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methylthiouracil, 0-D-mannose-based fast oxime Queosine), 5'-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetate, urine Pyrimidine-5-oxyacetic acid, oxybutoxyxanthene (〇xybut〇x〇sine), pseudouracil, fast oxime, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid Methyl ester, uracil_5-oxyacetic acid, pseudouracil, fast oxime, 2_thiocytosine and 2,6-diaminoguanidine. The term "naturally occurring" or "natural" when used in connection with a biological substance such as a nucleic acid molecule, polypeptide, host cell or the like, means -15 - the paper scale applies to the Chinese National Standard (CNS) A4 size (210 X 297 mm) (Please read the note on the back and fill out this page) --1--1 Order--- 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention Description (13) The substance is found in nature and not made by humans. Similarly, when π non-naturally occurring, or 'non-natural' is used herein, it means that the substance is not Found in nature, or structurally modified or synthesized by humans. When the term "operably linked" is used herein, it is meant an arrangement of side sequences in which the side sequences thus described are constructed or assembled to perform their ordinary functions. Thus, the copying, transcription and/or translation of the cryptographic sequence can be accomplished by a side sequence operably linked to the cryptographic sequence. For example, the cryptographic sequence is operably linked to the promoter gene' where the promoter gene is capable of directing transcription of the crypto sequence. The side sequence does not need to be adjacent to the password sequence as long as it has the correct function. Thus, for example, a sequence between the initiation gene sequence and the codon sequence that is untranslated but turns green will be present, and the promoter gene is still considered to be "operably linked" to the cryptographic sequence. The term "therapeutically effective amount" of the effective amounts ''and'', respectively, means that the amount of VGF polypeptide used 'supports an observable degree of one or more biological activities of the vgf polypeptide as set forth herein. When the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" is used herein, it is meant one or more VGFs suitable for completing or promoting the form of a pharmaceutical composition. Formulation of delivery of a polypeptide or VGF selective binding agent. The term "ft selective binder" means a molecule or molecule that is specific for a VGF polypeptide. When used herein, the term "specific" or "specificity" means a selective binder and VGF polypeptide binds and does not bind to non-vgf polypeptide--------Lu--------tTi------- volume (please read the phonetic on the back? Page) -16-

1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（14) 的能力。然而，將知曉該選擇性結合劑亦可能與VGF正類似物結合。所使用的”轉導π —詞，意指從一個細菌中將基因運送至另一個，通常是藉著噬菌體。”轉導”亦意指藉著反轉錄病毒獲得並運送眞核生物細胞的序列。所使用的”轉移感染” 一詞，意指由細胞攝入外來的或外源的DNA，而在已經將外源的DNA導入細胞膜内時，該細胞便已經被”轉移感染''了。許多轉移感染的技術是此項技藝中已熟知的，並在本文中揭示，參見例如，Graham等人，1973，Virology 52 : 456 ; Sambrook 等人，Molecular Cloning, A Laboratory Manual(Cold Spring Harbor Laboratories, 1989) ; Davis 等人，Basic Methods in Molecular Biology (Elsevier，1986);以及Chu等人，1981，Gene 13 ·· 197。可使用這類技術將一或多個外源的DNA部份導入適當的宿主細胞中。當在本文中使用”轉化” 一詞時，意指在細胞遺傳特徵上的改變，且該細胞已經被轉化，此時已經將其修改成含有新的DNA。例如，細胞被轉化，其中從它的自然狀態中以遺傳上的方式修改之。在轉移感染或轉導之後，可將轉化的DNA與細胞重組，藉著以物理方式整合到細胞的染色體内，可暫時以沒有複製的附加體要素之形式維持，或可以質體的形式獨立地複製。當DNA隨著細胞***而複製時，將該細胞視爲已經被穩定地轉化。 VGF多肽的相關性 -17- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------------------訂--------- (請先閱讀背面之注音3事項再填寫本頁) 1278319 A7 B7 經濟部智慧財產局員工消費合作社印製五、發明說明（15 VGF多肤之胺基酸序列的保留性修改，將產生具有類似那些VGF多肽之功能和化學特徵的多肽。相反的，在VGF 多肤之功能及/或化學特徵上的實際修改，可藉著在vgf多肽之胺基酸序列中的選擇取代作用來完成，該胺基酸的選擇取代作用’在其維持（a)取代區中分子主鏈的結構，例如 ’像是片或螺旋的結構，（b)在標靶位置處分子的電荷或忌水性’或（c)巨大的側鏈的作用上，有明顯不同的影響。例如，”保留性的胺基酸置換”可能涉及以非天然的殘基來取代天然胺基酸殘基的作用，使得對該位置處的胺基酸殘基之極性或電荷有較少的或沒有影響。此外，亦可利用丙胺酸取代任何在多肽中的天然殘基，如同先前已經描述之"丙胺酸掃描的突變生成作用”。保留性的胺基酸置換亦包括非天然存在的胺基酸殘基，通常藉著化學肽合成而非藉著在生物系統中的合成作用將其併入。這些包括肽模仿物，以及胺基酸部份的其他相反或倒轉形式。可以常見的側鏈特徵爲基礎，將天然存在的殘基分類： 1 )忌水性的：去甲亮胺酸，Met，Ala，Val，Leu，lie ; 2)中性親水性的：Cys，Ser，Thr ; 3 )酸性的：Asp，Glu ; 4)驗性的：Asn，Gln，His，Lys，Arg ; 5 )影響鏈方位的殘基：Gly, Pro ;以及 6)芳香族的：Trp，Tyr，Phe。例如，非-保留性置換可能涉及一類中的成員與得自另一 18- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -------------------^--------- (請先閱讀背面之注音？事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（16 ) 類 < 成員的交換。可將這類取代的殘基導入VGF多肽與其他VGF多肽正類似物同系的區域中，或導入該分子的非-同系區域中。在進行這類改變時’可考量胺基酸的疏水指數。已經以胺基酸的忌水性和電荷特徵爲基礎，指定每個胺基酸的疏水指數。疏水指數爲：異亮胺酸（+ 4 5);纈胺酸（+ 4 2) ·，免胺酸（ + 3.8);***酸（ + 2·8);半胱胺酸/胱胺酸 ( + 2.5);甲硫胺酸（+1 9);丙胺酸（+ 1 8);甘胺酸（·〇4) ;蘇胺酸（-0·7);絲胺酸（_〇叼；色胺酸（_〇 9);酪胺酸 (-1.3);脯胺酸（_ι6);組胺酸卜3 2广穀胺酸卜3 5厂疲胺醯胺（-3.5);天冬胺酸（-3·5);天冬醯胺卜35);離胺酸（-3。9 );和精胺酸（_ 4 5 )。在此項技藝中’通常知遒疏水之胺基酸指數的重要性，在於將交互作用的生物功能賦與蛋白質（Kyte等人，1982, J· Mol. Biol· 157 : 105-31)。已知可以某些胺基酸取代其他具有類似疏水指數或分數的胺基酸，而仍然維持類似的生物活性。在以疏水指數爲基礎來進行改變時，其疏水指數在士 2内的胺基故取代作用是較佳的，在士 1内的是特佳的，而在± 0 · 5内的是更佳的。在此項技藝中亦瞭解可以親水性爲基礎，有效地進行類似胺基酸的取代，特別是在企圖藉此產生具有生物功能的相等蛋白質或肽，以便在免疫學的具體實施例中使用之處 ’如同在本案例中。蛋白質最大的局部平均親水性，由其相鄰的胺基酸之親水性支配，與其免疫原性和抗原性有關本紙張尺度適用中國國家標準（CNS)A4規格（21〇 x 297公釐） -------------------^--------- (請先閱讀背面之注音？事項再填寫本頁) -19- 1278319 Α7 Β7 五、發明說明（17) ，也就是與蛋白質的生物學特性有關。已經對這些胺基酸殘基指定了下列的親水性值··精胺酸 ( + 3.0)，離胺酸（ + 3.0);天冬胺酸（+ 3〇士丨）；穀胺酸 (+ 3.0 士 1 )，絲胺酸（+ 〇 · 3 );天冬醯胺（+ 〇 . 2 );穀胺醯胺 ( + 0.2);甘胺酸（0);蘇胺酸4);脯胺酸（〇 5±1); 丙胺酸（-0.5);組胺酸（-0·5);半胱胺酸〇);甲硫胺酸（·1.3);織胺酸（_1·5);亮胺酸（U);異亮胺酸 (-1.8);酪胺酸（·2·3);***酸（-2·5);和色胺酸（_ 3.4 )。在以類似的親水性值為基礎來進行改變時，其親水性值在士2内的胺基酸取代作用是較佳的，在士1内的是特佳的，而在土 0 · 5内的是更佳的。亦可以親水性為基礎，從原始的胺基故序列中確認出抗原決定位。這些區域亦稱為，，抗原決定位的核心區"。在想要這類取代時’可由熟諳此藝者決定想要的胺基酸取代（疋否為保邊性或非保留性的）。例如，可使用胺基酸的取代來確認VGF多肽的重要殘基，或增加或減少在本文中描述之VGF多肽的親和力。在表Π中陳述典型的胺基酸取代。 --------------------訂-----— (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 -20- 本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7_ 五、發明說明（18 )1278319 The Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. The invention (14) ability. However, it will be appreciated that the selective binding agent may also bind to a VGF positive analog. The term "transduction π" means the transfer of a gene from one bacterium to another, usually by phage. "Transduction" also means the sequence of obtaining and transporting a nucleus cell by a retrovirus. The term "transfer infection" is used to mean the ingestion of foreign or exogenous DNA by a cell, which has been "transfected" when the foreign DNA has been introduced into the cell membrane. A number of techniques for transferring infection are well known in the art and are disclosed herein, see, for example, Graham et al, 1973, Virology 52: 456; Sambrook et al, Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al., Basic Methods in Molecular Biology (Elsevier, 1986); and Chu et al., 1981, Gene 13 · 197. Such techniques can be used to introduce one or more exogenous DNA portions into appropriate host cells. When the term "transformation" is used herein, it refers to a change in the genetic characteristics of a cell, and the cell has been transformed, at which point it has been modified to contain new DNA. For example, a cell is transformed, which is modified in a genetic manner from its natural state. After transfer of infection or transduction, the transformed DNA can be recombined with the cells, physically integrated into the chromosome of the cell, temporarily maintained as an epigenetic element that is not replicated, or independently in the form of a plastid copy. When DNA replicates as the cell divides, the cell is considered to have been stably transformed. Correlation of VGF Peptide-17- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) -------------------- Order -- ------- (Please read the note on the back of the page and then fill out this page) 1278319 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (15 VGF multi-skin amino acid sequence retention Sexual modifications will result in polypeptides having functional and chemical characteristics similar to those of VGF polypeptides. Conversely, actual modifications in the functional and/or chemical characteristics of VGF polypeptides may be made in the amino acid sequence of the vgf polypeptide. Selective substitution is accomplished by the selective substitution of the amino acid 'where it maintains the structure of the molecular backbone in the (a) substitution region, eg, the structure like a sheet or a helix, (b) the molecule at the target position The effect of charge or water-repellent or (c) large side chains has a distinctly different effect. For example, a "reserved amino acid substitution" may involve the replacement of a natural amino acid residue with a non-natural residue. The effect of having less or no shadow on the polarity or charge of the amino acid residue at that position In addition, alanine can also be substituted for any natural residues in the polypeptide, as previously described for the "mutational production of alanine scanning". Reserved amino acid substitutions also include non-naturally occurring amino acids. Residues, usually by chemical peptide synthesis rather than by synthesis in biological systems. These include peptide mimetics, as well as other opposite or inverted forms of the amino acid moiety. Common side chain features Based on the classification of naturally occurring residues: 1) water-repellent: norleucine, Met, Ala, Val, Leu, lie; 2) neutral hydrophilic: Cys, Ser, Thr; 3) acidic 4: Asn, Gln, His, Lys, Arg; 5) Residues affecting chain orientation: Gly, Pro; and 6) Aromatic: Trp, Tyr, Phe. For example, Non-reserved replacements may involve members of one class and are derived from another 18-paper scale applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------------- ------^--------- (Please read the phonetic on the back? Please fill out this page again) 1278319 Ministry of Economics Intellectual Property Bureau employee consumption cooperatives printed A7 B7 V. Description of invention (16) Class < exchange of members. Such substituted residues can be introduced into the region where the VGF polypeptide is homologous to other VGF polypeptide positive analogs, or introduced into the molecule. In the non-homologous region, the hydrophobicity index of the amino acid can be considered when making such changes. The hydrophobicity index of each amino acid has been specified based on the water-repellent and charge characteristics of the amino acid. Hydrophobic index: isoleucine (+ 4 5); proline (+ 4 2) ·, amino acid ( + 3.8); phenylalanine ( + 2 · 8); cysteine / cystine ( + 2.5); methionine (+1 9); alanine (+ 18); glycine (·〇4); threonine (-0·7); serine (_〇叼; color Aminic acid (_〇9); tyrosine acid (-1.3); valine acid (_ι6); histidine acid 3 2 wide glutamate b 3 5 plant amide (-3.5); aspartic acid (-3·5); aspartame 35); lysine (-3. 9); and arginine (_ 4 5 ). In this art, the importance of the hydrophobic amino acid index is generally known to confer interaction with biological functions (Kyte et al., 1982, J. Mol. Biol. 157: 105-31). It is known that certain amino acids can be substituted for other amino acids having a similar hydrophobic index or fraction while still maintaining similar biological activity. When the change is based on the hydrophobic index, the substitution of the amine group with a hydrophobic index in ±2 is preferred, especially in ±1, and better in ±0.5. of. It is also understood in the art that amino acid-like substitutions can be efficiently performed based on hydrophilicity, particularly in an attempt to produce equivalent proteins or peptides having biological functions for use in specific embodiments of immunology. 'as in this case. The largest local average hydrophilicity of a protein, governed by the hydrophilicity of its adjacent amino acid, is related to its immunogenicity and antigenicity. This paper scale applies to the Chinese National Standard (CNS) A4 specification (21〇x 297 mm) - ------------------^--------- (Please read the phonetic on the back? Please fill out this page again) -19- 1278319 Α7 Β7 V. Description of the invention (17), that is, related to the biological properties of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0), lysine (+3.0); aspartic acid (+3 guansin); glutamic acid (+ 3.0 ± 1), serine (+ 〇 · 3 ); aspartame (+ 〇. 2 ); glutamine (+ 0.2); glycine (0); sulphate 4); guanamine Acid (〇5±1); alanine (-0.5); histidine (-0·5); cysteine citrate); methionine (·1.3); lysine (_1·5); Leucine (U); isoleucine (-1.8); tyrosine (·2·3); phenylalanine (-2·5); and tryptophan (_3.4). When the change is based on a similar hydrophilicity value, the amino acid substitution of the hydrophilicity value in ±2 is preferred, and it is particularly preferable in the case of the soil, and within the soil of 0.5. It is better. The epitope can also be confirmed from the original amine-based sequence based on the hydrophilicity. These areas are also known as, the core area of the anti-origin decision. When such substitutions are desired, the amino acid which is desired by the artist can be substituted (whether it is marginal or non-retentive). For example, substitution of an amino acid can be used to confirm important residues of the VGF polypeptide, or to increase or decrease the affinity of the VGF polypeptides described herein. A typical amino acid substitution is stated in the table. --------------------Book------ (Please read the notes on the back and fill out this page) Printed by the Intellectual Property Office of the Ministry of Economic Affairs -20- The paper size is applicable to China National Standard (CNS) A4 specification (210 x 297 mm). 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7_ V. Invention description (18)

表II 胺基酸的取代作用原始的殘基典型的取代作用較佳的取代作用Table II Substitution of Amino Acids Original Residues Typical Substitutions Preferred Substitutions

Ala Val, Leu, He Val Arg Lys，Gln，Asn Lys Asn Gin Gin Asp Glu Glu Cys Ser, Ala Ser Gin Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn，Gln，Lys，Arg Arg He Leu，Val，Met，Ala，Phe，去甲亮胺酸 Leu Leu 去甲亮胺酸，He，Val，Met，Ala，Phe He Lys Arg，1，4·二胺基-丁酸，Gln，Asn Arg Met Leu, Phe, lie Leu Phe Leu, Val, He, Ala, Tyr Leu Pro Ala Gly Ser Thr，Ala，Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp，Phe，Thr，Ser Phe Val lie，Met, Leu，Phe，Ala,去甲亮胺酸 Leu -21 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ---------------------------- (請先閱讀背面之注意事項再填寫本頁) 1278319 A7 B7 五、發明說明（19 ) 熱讀此藝者將能夠使用已熟知的技術，決定適當的VGF 多肽變體。為了確認該分子中能夠被改變但不破壞生物活性的適田區域，熟請此藝者可瞄準不相信其對活性具有重要丨生的區域。例如，當已知得自相同物種或得自不同物種的類似多肽，具有類似的活性時，熟諳此藝者可將VGF多月太的胺基酸序列與這類類似的多肤做比較。利用這類比較，可確認出在類似多肽中被保留之分子的殘基和部份。將察知在VGF分子中，與這類類似多肽有關之未保留區中進行的改變，將不太可能不利地影響VGF多肽之生物活性及/ 或結構。熟爾此藝者亦將知道，即使在相對上較具保留性的區域，可以在化學上類似的胺基酸來取代天然存在的殘基，同時仍保留活性（保留性的胺基酸殘基之置換）。因此，即使該區域對生物活性或對於結構可能是重要的，仍可使其接受保留性的胺基酸置換，而不破壞生物活性，或不會對該多肽結構有不利的影響。此外’熟清此藝者可回顧結構·功能的研究，確認在類似的多肽中，對活性或結構很重要的殘基。在這類比較的回顧中，可預期在VGF多肽中具有重要性的胺基酸殘基，與在類似多肽中對於活性或結構很重要的胺基酸殘基一致。熟諳此藝者可選擇在化學上類似的胺基酸，來取代在vGF 多肽中，預期它很重要的這些胺基酸殘基。熟諳此藝者亦可分析與類似多肽中之結構有關的三度空間結構和胺基酸序列。回顧這類資訊，熟諳此藝者可預期 VGF多肽的胺基酸排列與其三度空間結構有關。熟請此藝 22- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) ---------訂— 秦· 經濟部智慧財產局員工消費合作社印製 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（2Q) 者可選擇對預期是在蛋白質表面上的胺基酸殘基不會造成基團的改變，因爲這類基團可能涉及與其他分子的重要交互作用。然而，熟諳此藝者可創造在每個胺基酸殘基處含有單一胺基酸取代的受試變體。可使用熟諳此藝者已知的活性測定來篩選變體。可使用這類變體收集關於適當變體的資訊。例如，如果發現特定胺基酸殘基的改變，導致破壞的、不想要地降低或不適當的活性，則將避免帶有這類改變的變體。換句話説，以從這類例行之實驗中收集到的資訊爲基礎，熟諳此藝者將可輕易地決定應該.單獨或與其他突變一起，避免進一步取代何處的胺基酸。許多科學的出版物已經致力於二級結構的預測。參見 Moult，1996，Curr. Opin. Biotechnol. 7 : 422_27 ; Chou等人， 1974，Biochemistry 13 : 222-45 ; Chou 等人，1974,Ala Val, Leu, He Val Arg Lys, Gln, Asn Lys Asn Gin Gin Asp Glu Glu Cys Ser, Ala Ser Gin Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg He Leu, Val, Met, Ala, Phe, Leu Leu Leu Leu Leucine, He, Val, Met, Ala, Phe He Lys Arg, 1,4·Diamino-butyric acid, Gln, Asn Arg Met Leu, Phe, lie Leu Phe Leu, Val, He, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val lie, Met, Leu, Phe , Ala, Norleucine Leu -21 - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) ------------------- --------- (Please read the notes on the back and fill out this page) 1278319 A7 B7 V. INSTRUCTIONS (19) Those who are interested in this program will be able to determine the appropriate VGF peptide using well-known techniques. Variants. In order to identify an adapted field in the molecule that can be altered without destroying the biological activity, the artist is expected to target areas that do not believe that it has an important activity on the activity. For example, when similar polypeptides from the same species or from different species are known to have similar activities, those skilled in the art can compare the amino acid sequence of VGF to a similar polypeptide. Using such comparisons, residues and moieties of molecules that are retained in similar polypeptides can be identified. It will be appreciated that changes in the unretained regions associated with such analogous polypeptides in VGF molecules will be less likely to adversely affect the biological activity and/or structure of the VGF polypeptide. Cooks will also know that even in relatively retentive regions, chemically similar amino acids can be substituted for naturally occurring residues while still retaining activity (retained amino acid residues) Replacement). Thus, even if the region is biologically active or may be important to the structure, it can be replaced by a retentive amino acid without destroying the biological activity or adversely affecting the structure of the polypeptide. In addition, the skilled person can review the structure and function of the residue and identify residues that are important for activity or structure in similar polypeptides. In such retrospective comparisons, amino acid residues of importance in VGF polypeptides are expected to be identical to amino acid residues important for activity or structure in similar polypeptides. Those skilled in the art may choose a chemically similar amino acid to replace these amino acid residues which are expected to be important in the vGF polypeptide. Those skilled in the art can also analyze three-dimensional spatial structures and amino acid sequences associated with structures in similar polypeptides. Looking back at this type of information, those skilled in the art can expect that the amino acid arrangement of the VGF polypeptide is related to its three-dimensional structure. Please familiarize yourself with this art 22- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back and fill in this page) ---------Book - Qin · Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention Description (2Q) The choice of amino acid residues expected to be on the surface of the protein will not cause Changes in groups because such groups may involve important interactions with other molecules. However, those skilled in the art can create test variants containing a single amino acid substitution at each amino acid residue. Variants can be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about appropriate variants. For example, if a change in a particular amino acid residue is found to result in a destructive, undesirably reduced or inappropriate activity, variants with such alterations will be avoided. In other words, based on the information gathered from such routine experiments, those skilled in the art will be able to easily decide which one, alone or in combination with other mutations, to avoid further substitution of the amino acid. Many scientific publications have focused on the prediction of secondary structures. See Moult, 1996, Curr. Opin. Biotechnol. 7: 422_27; Chou et al., 1974, Biochemistry 13: 222-45; Chou et al., 1974,

Biochemistry 113 : 211-22 ; Chou等人，1978, Adv. Enzymol. Relat. Areas Mol· Biol. 47 : 45-48 ; Chou等人，1978，Ann·， Rev. Biochem. 47 : 251-276 ;以及Chou等人，1979，Biophys. J. 26 : 367-84。此外，目前可利用電腦程式來幫助預測二級結構。一種預測二級結構的方法是以同種性模型爲基礎。例如，兩個多肽或蛋白質具有3 0 %以上的序列同一性，或 4 0 %以上的類似性，通常具有類似的結構拓樸學。蛋白質結構之資料庫（PDB)目前的增長，已經提供了二級結構的提高預測性，包括在多肽或蛋白質之結構内可能的折疊數目。參見Holm等人，1999, Nucleic Acids Res·，27 : 244_47。已經暗示在特定的多肽和蛋白質中，有限的折疊數目，且 -23 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1·—^---------------tr--------- (請先閱讀背面之注意事項再填寫本頁) __ 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（21 ) 一旦已經解決結構的決定性數目，結構的預測將變成戲劇化地更精確（Brenner等人，1997，Curr. Opin. Struct· Biol· 7 : 369-76) 〇預測二級結構的其他方法包括”穿線”（Jones, 1997, Cun*· Opin. Struct. Biol· 7 : 377-87 ; Sippl 等人，1996，Structure 4 ·· 15-19)，’’特色分析·’（Bowie等人，1991，Science, 253 : 164-70 ; Gribskov 等人，1990, Methods Enzymol· 183 : 146-59 ; Gribskov等人，1987, Proc. Nat· Acad. Sci. U.S.A. 84 ·· 4355-58 ) ’和"演化連鎖"（參見Holm等人，在前，和Brenner等人，在前）。較佳的VGF多肽變體包括糖基化作用變體，其中與本發明之VGF多肽的胺基酸序列相比較，已經改變糖基化作用位置的數目及/或類型。在一個具體實施例中，VGF多肽變體包括比本發明之V GF多肽的胺基酸序列，更多或更少的 N _键結之糖基化作用位置。以序列：Asn- X - Ser或Asn- X -Thr定出N -键結之糖基化作用位置的特徵，其中以X表示之胺基酸殘基可以是脯胺酸以外的任何胺基酸殘基。產生該序列之胺基酸殘基的取代作用，提供了加入N-键結之碳水化合物鏈可能的新位置。另外，排除該序列的取代作用將移除現存的N-鍵結之碳水化合物鏈。亦提供N-键結之碳水化合物鏈的重新排列，其中除去一或多個N-鍵結之糖基化作用位置（通常是天然存在的那些），並產生一或多個新的 N_键結位置。其他較佳的VGF變體包括半胱胺酸變體，與本發明之VGF多肽的胺基酸序列相比較，其中刪除一或多 -24- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ------------1------^---I----- (請先閱讀背面之注意事項再填寫本頁) 1278319Biochemistry 113: 211-22; Chou et al., 1978, Adv. Enzymol. Relat. Areas Mol· Biol. 47: 45-48; Chou et al., 1978, Ann·, Rev. Biochem. 47: 251-276; Chou et al., 1979, Biophys. J. 26: 367-84. In addition, computer programs are currently available to help predict secondary structure. One method of predicting secondary structure is based on an isogenic model. For example, two polypeptides or proteins have more than 30% sequence identity, or more than 40% similarity, and generally have similar structural topologies. The current growth of the Protein Structure Database (PDB) has provided improved predictability of secondary structure, including possible fold numbers within the structure of the polypeptide or protein. See Holm et al., 1999, Nucleic Acids Res., 27: 244_47. It has been suggested that there are a limited number of folds in a particular polypeptide and protein, and -23 - this paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1·—^-------- -------tr--------- (Please read the note on the back and fill out this page) __ 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (21 Once the decisive number of structures has been resolved, the prediction of the structure will become dramatic and more precise (Brenner et al., 1997, Curr. Opin. Struct Biol. 7: 369-76). Other methods of predicting secondary structure include: Threading" (Jones, 1997, Cun*· Opin. Struct. Biol. 7: 377-87; Sippl et al., 1996, Structure 4 · 15-19), ''Characteristic Analysis·' (Bowie et al., 1991, Science, 253: 164-70; Gribskov et al., 1990, Methods Enzymol 183: 146-59; Gribskov et al., 1987, Proc. Nat. Acad. Sci. USA 84 · 4355-58) 'and" evolution Chain " (see Holm et al., formerly, and Brenner et al., formerly). Preferred VGF polypeptide variants include glycosylation variants in which the number and/or type of glycosylation sites have been altered as compared to the amino acid sequence of the VGF polypeptides of the invention. In a specific embodiment, the VGF polypeptide variant comprises more or less N-linked glycosylation sites than the amino acid sequence of the VGF polypeptide of the invention. The sequence of the glycosylation of the N-bond is determined by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue represented by X may be any amino acid other than valeric acid. Residues. The substitution of the amino acid residue that produces the sequence provides a possible new location for the addition of an N-bonded carbohydrate chain. In addition, excluding the substitution of this sequence will remove the existing N-bonded carbohydrate chain. Also provided is a rearrangement of N-bonded carbohydrate chains, wherein one or more N-bonded glycosylation sites (usually those that occur naturally) are removed and one or more new N-bonds are produced. Knot position. Other preferred VGF variants include cysteine variants which are compared to the amino acid sequence of the VGF polypeptide of the invention wherein one or more of the paper sizes are removed for use in the Chinese National Standard (CNS) A4 specification ( 210 X 297 mm) ------------1------^---I----- (Please read the notes on the back and fill out this page) 1278319

經濟部智慧財產局員工消費合作社印製個半胱胺酸殘基’或以其他的胺基酸(例如絲胺酸⑽”取代。當必須將醫多肽再折疊成具有生物活性之構型時，像是在分離不溶的包涵體之後，半胱胺酸變體是有用的。半胱胺酸變體通常具有比天然蛋白質更少的半胱胺酸殘基 ’且通常具有偶數’使起因於不成對半胱胺酸的交互作用減至最少。此外’可將VGF多肽與同種的多肽融合，形成同種二聚把，或與異種的多肽融合，形成異種二聚體。異種的肽和夕肽包括但不限於··容許刪除及/或***VGF融合多肽的抗原=定位；穿透膜受體蛋白質或其一部份，像是細胞外的功把邯位或穿透膜和細胞内的功能部位；與穿透膜受體蛋白質結合的配體或其一部份；具有催化活性的酵素或其一部份；促進低聚化作用的多肽或肽，像是亮胺酸拉鍊功能部位；增加穩定性的多肽或肽，像是免疫球蛋白恆定區；以及具有與本發明之VGF多肽不同之治療活性的多肽。可在VGF多肽的胺基_終端或羧基-終端進行融合。可直接融泛’不品交聯劑或接合體分子，或可經由交聯劑或接合體分子。交聯劑或接合體分子可以是一或多個胺基酸殘基 ’通常是從大約2 0至大約5 0個胺基酸殘基。亦可將交聯劑或接合體分子設計成帶有可供DNA核酸内切限制酶或蛋白酶切開的位置，以便容許分離融合的部份。將知曉一旦已經建構，便可根據在本文中描述的方法來衍生該融合多肽。在本發明更進一步的具體實施例中，將VGF多肽與人類 IgG之F c片段的一或多個功能部位融合。抗體包括兩個功能 -25- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I I . —tr--------- Aw (請先閱讀背面之注意事項再填寫本頁) 1278319 A7 ___ B7___ 五、發明說明（23 ) 獨立的部份，稱爲’’ F ab ”的可變功能部位，其與抗原結合，以及稱爲π F c"的恆定功能部位，其涉及效應物功能，像是補體的活化作用，以及被巨嗟細胞攻擊。：F c具有長的血清半衰期，而Fab是短命的。Capon等人，1989，Nature 337 :525- 3 1。當與治療性蛋白質一起建構時，f c功能部位可提供較長的半衰期，或是像F c受體結合、蛋白質A結合、補體固定，或許甚至經胎盤傳遞，同上，將這類功能併入。表III概述在此項技藝中已知之某些F c融合的用途。The Ministry of Economic Intelligence's Intellectual Property Office employee consumption cooperative prints a cysteine residue' or is replaced by another amino acid (such as serine (10).) When the medical peptide must be refolded into a biologically active configuration, Cysteine variants are useful, for example, after isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than natural proteins 'and usually have an even number' resulting in failure The interaction with cysteine is minimized. In addition, the VGF polypeptide can be fused to the same polypeptide to form the same dimerization or fused to the heterologous polypeptide to form a heterodimer. The heterologous peptide and the kiln peptide include However, it is not limited to the antigens that allow deletion and/or insertion of VGF fusion polypeptides; localization; transmembrane receptor proteins or a part thereof, such as extracellular functions such as cleavage or penetrating membranes and functional sites within cells a ligand or a part thereof that binds to a membrane-receptor protein; a catalytically active enzyme or a part thereof; a polypeptide or peptide that promotes oligomerization, such as a leucine zipper functional site; Sexual polypeptide a peptide, such as an immunoglobulin constant region; and a polypeptide having a therapeutic activity different from that of the VGF polypeptide of the present invention. The fusion can be carried out at the amine-terminal or carboxyl-terminal of the VGF polypeptide. Or a linker molecule, or may be via a crosslinker or a linker molecule. The crosslinker or linker molecule may be one or more amino acid residues 'typically from about 20 to about 50 amino acids Residues. The cross-linking agent or adaptor molecule can also be designed to have a position for DNA endonuclease restriction enzyme or protease cleavage to allow separation of the fused portion. Once known, it can be The method described in the above is used to derive the fusion polypeptide. In a further embodiment of the invention, the VGF polypeptide is fused to one or more functional portions of the F c fragment of human IgG. The antibody comprises two functions - 25 - paper The scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) II . —tr--------- Aw (Please read the note on the back and fill out this page) 1278319 A7 ___ B7___ V. Description of invention (23) Independent a variable functional part called ''F ab ', which binds to an antigen, and a constant functional part called π F c", which is involved in effector functions, such as activation of complement, and by giant sputum cells. Attack: F c has a long serum half-life, while Fab is short-lived. Capon et al, 1989, Nature 337: 525- 3 1. When constructed with therapeutic proteins, the fc functional site provides a longer half-life, Either such as Fc receptor binding, protein A binding, complement fixation, and perhaps even transplacental delivery, as above, such functions are incorporated. Table III summarizes the use of certain Fc fusions known in the art.

表III F c與治療性蛋白質融合 Fc之形式融合夥伴治療涉及參考文獻 IgGl CD30-L 的 N終端霍奇金氏症；退行性淋巴瘤；T·細胞白血病美國專利第 5,480,981 號老鼠的 Fc- γ 2a IL-10 抗炎症；移植排斥 Zheng等人，1995, J. Immunol. 154 : 5590-600 IgGl TNF受體敗血性休克 Fisher等人，1996， N. Engl. J. Med. 334 ： 1697-1702 ; Van Zee等人， 1996, J· Immunol. 156 ： 2221-30 IgG，IgA，IgMTNF 受體或IgE(除了第一個功能部位之外）炎症，自體免疫障礙美國專利第 5,808,029號 IgGl CD4受體 AIDS Capon等人，1989, Nature337 ： 525-31 IgGl, IgG3 IL-2的N-終端抗-癌症，抗病毒 Harvill 等人，1995, Immunotech. 1 : -26- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) ---------訂— s'. 經濟部智慧財產局員工消費合作社印製 1278319 A7 B7 五、發明說明（24) 95-105 IgGl OPG的C-終端骨關節炎，骨密度 WO 97/23614 IgGl 莱普亭(leptin)的抗-肥胖 PCT/US 97/23183 N-終端，1997年12月11日建檔人類igC , 1 CTLA-4 自體免疫障礙 Linsley, 1991, J. Exp. Med·，174 : 561-69 在一個實例中，可使用熟諳此藝者已知的方法，將人類 IgG绞鏈、CH2和CH3區融合到VGF多肽的胺基·終端或羧基 -終端。可藉著使用蛋白質A親和力管柱，純化所得的VGF 融合多肽。已經發現與F c區融合的肽和蛋白質，在活體内顯示出比未融合的副本，實質上更高的半衰期。再者，F c 區的融合，容許融合多肽的二聚化作用/多聚化作用。該F c 區可以是天然存在的F c區，或可以改變，以便改善某些品質，像是治療品質、循環時間或降低凝集。可藉著已知的方法，輕易地計算相關多肽的同一性和類似性。這類方法包括但不限於在Computational Molecular Biology(A.M. Lesk 編輯，Oxford University Press 1988); Biocomputing ： Informatics and Genome Projects (D.W. Smith 編輯，Academic Press 1993) ; Computer Analysis of Sequence Data (Part 1，A.M. Griffin 和 H.G. Griffin編輯，Humana Press 1994) ; G. von Heinle, Sequence Analysis in Molecular Biology (Academic Press 1987) ; Sequence Analysis Primer (M.Table III Fc and therapeutic protein fusion Fc form fusion partner therapy involving reference IgGl CD30-L N-terminal Hodgkin's disease; degenerative lymphoma; T-cell leukemia US Patent No. 5,480,981 mouse Fc-γ 2a IL-10 anti-inflammatory; transplant rejection Zheng et al, 1995, J. Immunol. 154: 5590-600 IgGl TNF receptor septic shock Fisher et al, 1996, N. Engl. J. Med. 334: 1697-1702 Van Zee et al, 1996, J. Immunol. 156: 2221-30 IgG, IgA, IgMTNF receptor or IgE (except for the first functional site) Inflammation, autoimmune disorders US Patent No. 5,808,029 IgGl CD4 AIDS Capon et al., 1989, Nature 337: 525-31 IgGl, IgG3 IL-2 N-terminal anti-cancer, antiviral Harvill et al, 1995, Immunotech. 1 : -26- This paper scale applies to Chinese national standards ( CNS)A4 specification (210 X 297 mm) (Please read the note on the back and fill out this page) ---------Booking - s'. Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 1278319 A7 B7 V. Description of invention (24) 95-105 C-terminal of IgG1 OPG Osteoarthritis, bone mineral density WO 97/23614 IgGl leptin anti-obesity PCT/US 97/23183 N-terminal, December 11, 1997 filed human igC, 1 CTLA-4 autoimmune disorder Linsley, 1991, J. Exp. Med., 174: 561-69 In one example, human IgG hinge, CH2 and CH3 regions can be fused to the amine group of a VGF polypeptide using methods known to those skilled in the art. Terminal or carboxyl-terminal. The resulting VGF fusion polypeptide can be purified by using a Protein A affinity column. Peptides and proteins fused to the Fc region have been found to exhibit substantially higher half-lives in vivo than unfused copies. Furthermore, fusion of the F c region allows for dimerization/multimerization of the fusion polypeptide. The F c region can be a naturally occurring F c region or can be altered to improve certain qualities such as therapeutic quality, circulation time or reduced agglutination. The identity and similarity of related polypeptides can be easily calculated by known methods. Such methods include, but are not limited to, Computational Molecular Biology (AM Lesk, ed., Oxford University Press 1988); Biocomputing: Informatics and Genome Projects (DW Smith, ed., Academic Press 1993); Computer Analysis of Sequence Data (Part 1, AM Griffin and HG Griffin, ed., Humana Press 1994); G. von Heinle, Sequence Analysis in Molecular Biology (Academic Press 1987); Sequence Analysis Primer (M.

Gribskov和 J. Devereux 編輯，M· Stockton Press 1991);以及 Carillo等人，1988, SIAM J· Applied Math.，48 : 1073 中描述 -27- 尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） (請先閱讀背面之注意事項再填寫本頁) ---------訂— s'. 經濟部智慧財產局員工消費合作社印製 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（25 ) 的那些。設計決定同一性及/或類似性的較佳方法，得到在受試序列之間的最大配對。在可公開獲得的電腦程式中，描述了決定同一性和類似性的方法。決定在兩個序列之間的同一性和類似性之較佳的電腦程式方法，包括但不限於GCG套裝程式，包括 GAP (Devereux等人，1984, Nucleic Acids Res. 12 ： 387 ; Genetics Computer Group, University of Wisconsin, Madison，WI)，BLASTP 和 FASTA (Altschul 等人，1990，J· Mol· Biol· 215 : 403-10)。BLASTX程式可公開獲自 National Center for Biotechnology Information (NCBI)和其他來源 (Altschul 等人，BLAST Manual (NCB NLM NIH，Bethesda， MD) ; Altschul等人，1990，在前）。亦可使用已熟知的 Smith Waterman演算法來決定同一性。某些演算計劃將兩個胺基酸序列排成一直線，可能導致兩個序列中僅有一短區域的相配，而該小的直線排列區可能具有極高的序列同一性，即使在這兩個全長的序列之間並無明顯的關連性。因此，在較佳的具體實施例中，選擇排列的方法（GAP程式）將產生跨越提出申請之多肽至少5 0 個相鄰的胺基酸。例如，使用電腦演算法GAP(Genetics Computer Group， University of Wisconsin，Madison，WI)，爲了使其個別胺基酸最適切地配對，將欲決定其序列同一性百分比的兩個序列排列成一直線（藉著演算法決定”配對的跨越幅度π)。連同演算法一起使用間隙開口罰點（以3 X平均對角線來計算之 -28- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I--------11 --------^--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（26 ) ;”平均對角線”爲所使用之比較矩陣的對角線平均値；”對角線"爲由特定的比較矩陣，對每個完美胺基配對指定的分數或數目）和間隙延伸罰點（其通常爲〇 · 1X間隙開口罰點），，以及比較矩陣，像是PAM 250或BLOSUM 62。亦由演算法使用標準比較矩降（參見Dayhoff等人，5 Altas of Protein Sequence and Structure(附錄 3，1978)(PAM250 比較矩陣）； Henikoff等人，1992, Proc· Natl· Acad· Sci· USA 89 : 10915-19 (BLOSUM 62比較矩陣））。多肽序列比較的較佳參數，包括下列：演算法：Needleman and Wunsch，1970，J. Mol. Biol· 48 ·· 443-53 ; 比較矩陣：BLOSUM62(Henikoff等人，在前）；間隙罰點：1 2 間隙長度罰點：4 類似性之閾値·· 0 利用上述參數可使用GAP程式。上文提及之參數是使用 GAP程式之多肽比較的預設値參數（並未連同末端間隙的罰點一起）。其他典型的演算法，可使用間隙開口罰點、間隙延伸罰點、比較矩陣和類似性之閾値，包括在Program Manual， Wisconsin Package,第9版，1997年9月中陳述的那些。熟諳此藝者將知曉，並將依據待進行的特定比較，像是蛋白質-對-蛋白質、蛋白質-對-DNA ;以及額外地，是否在特定一對的序列之間（在該案例中，GAP或BestFit通常是較佳的）， -29- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ---------------I---訂---I----- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（27) 或是在一個序列與一個大的序列資料庫之間（在該案例中’ FASTA或BLASTA是較佳的）進行比較，來進行特殊的選擇。核酸分子可利用各種方式，輕易地獲得編碼包括VGF多肽之胺基酸序列之多肽的核酸分子，包栝但不限於化學合成、cDNA 或基因組庫篩選，表現庫篩選，及/或cDNA的PCR擴大作用。在本文中使用的重組DNA方法通常是在Sambrook等人， Molecular Cloning ： A Laboratory Manual(Cold Spring Harbor Laboratory Press，1989)，以及Current Protocols in Molecular Biology(Ausubel 等人，編輯，Green Publishers Inc.和 Wiley and Sons 1994)中陳述的那些。可藉著表現選殖，其以表現蛋白質之特性爲基礎，使用陽性純種系的檢測，來確認編碼VGF多肽之胺基酸序列的核酸分子。通常，藉著使抗體或其他結合夥伴（例如受體或配體）與在宿主細胞表面上表現和展示的選殖蛋白質結合，來篩選核酸庫。以可檢測標記修改抗體或結合夥伴，以便確認表現想要純種系的那些細胞。可依據根據下文陳述之説明來經營的重組表現技術，產生這些夕核4酸’並表現編碼的多肤。例如，藉著將編碼 VGF多肽之胺基酸序列的核酸序列***適當的載體中，熟請此藝者可輕易地產生大量想要的核甞酸序列。然後可使用該序列來產製檢測探針或擴大引子。另外，亦可將編碼 VGF多肽之胺基酸序列的多核甞酸，***表現載體内。藉著將表現載體導入適當的宿主内，可大量地產製編碼的 -30- 本紙張尺度適用中國國家標準（CNS)A4規格（21〇 x 297公釐） -------------------^--------- Φ (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（28 ) VGF多肽。另一種獲得適當核酸序列的方法，是聚合酶連鎖反應 (PCR)。在該方法中，使用酵素反轉錄酶，從聚（A) + RNA 或總RNA中製備cDNA。然後將兩個引子，通常是與編碼 V GF多肽之胺基酸序列的cDNA中兩個分開區域互補的，連同諸如Taq聚合酶之類的聚合酶一起，加至cDNA中，而該聚合酶擴大在兩個引子之間的cDNA區域。另一種製備編碼VGF多肽之胺基酸序列的核酸分子的方法，是使用熟諳此藝者已熟知之方法的化學合成，像是由 Engels等人，1989, Angew. Chem. IntL Ed. 28 : 716-3 4 描述的那些。這些方法特別包括核酸合成的磷酸三酯、磷醯胺酸法，和H_膦酸酯法。這類化學合成中的較佳方法，是聚合物·支撑的合成法，使用標準的磷醯胺酸化學。通常，編碼 VGF多肽之胺基酸序列的DNA，長度將是數百個核甞酸。可使用這些方法，以數個片段之方式合成大約100個核甞酸以上的核酸。然後可連接片段，形成VGF基因的全長核苷酸序列。編碼多肽之胺基-終端的DNA片段經常具有ATG，其編碼甲硫胺酸殘基。該甲硫胺酸可以或可以未出現在 VGF多肽的成熟形式中，依據是否將在宿主細胞中產生之多肽設計成從該細胞中分泌出而定。亦可使用熟諳此藝者已知的其他方法。在某些具體實施例中，爲了在特定的宿主細胞中最適切地表現VGF多肽，已經將含有密碼子的核酸變體加以改變。特定的密碼子改變將依據選擇用來表現的VGF多肽和宿 -31 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -------------------^--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（29) 主細胞而定。可藉著各種方法完成這類”密碼子的最佳條件 π，例如藉著選擇在特定宿主細胞中，在高度表現之基因中最喜歡使用的密碼子。可使用合併密碼子頻率表，像是有關於高度表現之細菌基因偏好的密碼子之” Eco_high.Cod" 的電腦演算法，並可由University of Wosconsin Package第 9.0版（Genetics Computer Group，Madison，WI)提供之。其他有用的密碼子頻率表，包括”Celegans_high.codn、 ’’Celegans—low.cod·’、"Drosophila high·codn、"Human high.cod” 、•’Maize—high·cod’’和”Yeast一high.cod’·。在一些案例中，可能想要製備編碼VGF多肽變體的核酸分子。可使用指定位置之突變生成作用、PCR擴大作用或其他適當的方法，來產製編碼變體的核酸分子，其中引子（們）具有想要的點突變（參見Sambrook等人，在前，和 Ausubel等人，在前，關於突變技術的説明）。亦可使用使用由Engels等人，在前，描述之方法的化學合成法來製備這類變體。亦可使用熟諳此藝者已知的其他方法。載體和宿主細胞使用標準的連接技術，將編碼VGF多肽之胺基酸序列的核酸分子***適當的表現載體内。通常選擇在所使用的特定宿主細胞中，具有功能的載體（也就是説，該載體可與宿主細胞機制相容，而得以發生基因的擴大及/或基因的表現 )。可在原核生物、酵母菌、昆蟲（桿狀病毒系統）及/或眞核生物宿主細胞中，擴大/表現編碼VGF多肽之胺基酸序列的核酸分子。宿主細胞的選擇一部份將依據是否將在轉譯 -32- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -------------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 A7 B7 經濟部智慧財產局員工消費合作社印製五、發明說明（3G) 之後修改VGF多肽（例如糖基化及/或磷酸化）。如果是，則酵母菌、昆蟲或哺乳動物宿主細胞是較佳的。關於表j見载體的回顧，參見Meth· Εηζ·，第1 8 5册（D · V· Goeddel編輯， Academic Press 1990)。通常，在任何宿主細胞中使用的表現載體，將含有質體維持和選殖及表現外源性核甞酸序列的序列。這類序列，在某些具體實施例中集體地稱爲”側面序列”，通常將包括一或多個下列的核甞酸序列：啓動基因、一或多個促進子序列、複製起點、轉錄終止序列、含有捐贈者和接受者接合位置的完全***序列、編碼多肤分泌之前導序列的序列、核糖體結合位置、聚腺芬酸化作用序列，***編碼待表現之多肽之核酸的聚交聯劑區，以及可選擇標記要素。在下文中分別討論這些序列。載體可視需要含有編碼”標籤，，的序列，也就是位在Vgf 多月太密碼序列之5 ’或3 ’末端的寡核甞酸分子，該寡核菩酸序列編碼聚His(像是六His)，或其他的標籤，像是FLAG、 ha(血球凝集素流行性感冒病毒）或myc，有對抗它的可蹲 2之抗體存在。通常在表現多肽時，將該標籤與該多肽融 & ’並可做爲從宿主細胞中以親和力純化VGF多肽的工具。可藉著例如管柱層析法，使用對抗該標籤的抗體做爲親和力基質，來完成親和力純化。接著可視需要，藉著各種万法，像是使用某些切開用的肽酶，從經過純化的vgf多肽中移除該標籤。側面序列可以是同種的（也就是得自與宿主細胞相同的物 (請先閱讀背面之注意事項再填寫本頁) ----- 訂--- 卷. -33- 1278319 A7Edited by Gribskov and J. Devereux, M. Stockton Press 1991); and Carillo et al., 1988, SIAM J. Applied Math., 48: 1073. -27- Scales applicable to the Chinese National Standard (CNS) A4 specification (210 x 297) () Please read the notes on the back and fill out this page.) ---------Booking - s'. Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumers Cooperative, Printing 1278319 Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumption Cooperatives A7 B7 V. Those of the invention description (25). The preferred method of designing identity and/or similarity results in a maximum pairing between the tested sequences. Methods for determining identity and similarity are described in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences, including but not limited to GCG suite programs, including GAP (Devereux et al., 1984, Nucleic Acids Res. 12: 387; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-10). The BLASTX program is publicly available from National Center for Biotechnology Information (NCBI) and other sources (Altschul et al, BLAST Manual (NCB NLM NIH, Bethesda, MD); Altschul et al, 1990, supra). The well-known Smith Waterman algorithm can also be used to determine identity. Some calculation schemes align two amino acid sequences in a straight line, which may result in the matching of only one short region in the two sequences, and the small linear alignment region may have extremely high sequence identity even in these two full lengths. There is no significant correlation between the sequences. Thus, in a preferred embodiment, the method of arranging the alignment (GAP program) will result in at least 50 contiguous amino acids spanning the polypeptide of the application. For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, WI), in order to optimally pair individual amino acids, the two sequences whose percentage of sequence identity is to be determined are arranged in a straight line. The algorithm determines the "crossing amplitude of the pairing π." Use the gap opening penalty along with the algorithm (calculated by the 3 X average diagonal -28 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) I--------11 --------^--------- (Please read the notes on the back and fill out this page) 1278319 Ministry of Economics Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (26); "Average diagonal" is the diagonal average of the comparison matrix used; "Diagonal" is a specific comparison matrix, for each A perfect amine-based pairing specifies the fraction or number) and a gap extension penalty (which is typically a 〇·1X gap opening penalty), and a comparison matrix such as PAM 250 or BLOSUM 62. The algorithm also uses the standard to compare the moment drop (see Dayhoff et al., 5 Altas of Protein Sequence and Structure (Appendix 3, 1978) (PAM250 comparison matrix); Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89 : 10915-19 (BLOSUM 62 comparison matrix)). Preferred parameters for peptide sequence comparison include the following: Algorithm: Needleman and Wunsch, 1970, J. Mol. Biol· 48 · 443-53; Comparison matrix: BLOSUM62 (Henikoff et al., former); gap penalty: 1 2 Gap length penalty point: 4 Similarity threshold 値·· 0 Use the above parameters to use the GAP program. The parameters mentioned above are the default 値 parameters compared to the polypeptides of the GAP program (without the penalty for the end gap). Other typical algorithms may use gap opening penalty points, gap extension penalty points, comparison matrices, and similarity thresholds, including those set forth in the Program Manual, Wisconsin Package, 9th Edition, September 1997. Those skilled in the art will know and will rely on specific comparisons to be made, such as protein-to-protein, protein-to-DNA; and additionally, between specific pairs of sequences (in this case, GAP) Or BestFit is usually better), -29- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) ---------------I--- Order---I----- (Please read the notes on the back and fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Print A7 B7 V. Invention Description (27) Or in a sequence and one Large sequence databases (in this case 'FASTA or BLASTA are preferred) are compared for special selection. Nucleic Acid Molecules Nucleic acid molecules encoding polypeptides comprising amino acid sequences of VGF polypeptides can be readily obtained in a variety of ways, including but not limited to chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA. effect. Recombinant DNA methods for use herein are generally found in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989), and Current Protocols in Molecular Biology (Ausubel et al., eds., Green Publishers Inc. and Wiley). And Sons 1994). The nucleic acid molecule encoding the amino acid sequence of the VGF polypeptide can be confirmed by the performance of the protein based on the characteristics of the expressed protein using the detection of the positive pure germline. Typically, the nucleic acid library is screened by binding an antibody or other binding partner (e.g., a receptor or ligand) to a selectin protein that is expressed and displayed on the surface of the host cell. The antibody or binding partner is modified with a detectable label to confirm those cells that express the desired pure line. These polynuclear acids can be produced and expressed in accordance with recombinant expression techniques operated according to the statements set forth below. For example, by inserting a nucleic acid sequence encoding an amino acid sequence of a VGF polypeptide into an appropriate vector, the skilled artisan can readily produce a large number of desired nucleotide sequences. This sequence can then be used to produce a detection probe or to expand the primer. Alternatively, a polynuclear acid encoding an amino acid sequence of a VGF polypeptide can be inserted into an expression vector. By introducing the performance vector into the appropriate host, the large-scale real-time coded -30- paper scale applies to the Chinese National Standard (CNS) A4 specification (21〇x 297 mm) ---------- ---------^--------- Φ (Please read the note on the back and fill in this page) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention Description (28) VGF polypeptide. Another method for obtaining an appropriate nucleic acid sequence is the polymerase chain reaction (PCR). In this method, cDNA is prepared from poly(A) + RNA or total RNA using an enzyme reverse transcriptase. The two primers, usually complementary to two separate regions of the cDNA encoding the amino acid sequence of the V GF polypeptide, are then added to the cDNA along with a polymerase such as Taq polymerase, and the polymerase is expanded The cDNA region between the two primers. Another method of preparing a nucleic acid molecule encoding an amino acid sequence of a VGF polypeptide is by chemical synthesis using methods well known to those skilled in the art, as by Engels et al., 1989, Angew. Chem. IntL Ed. 28: 716 -3 4 Those described. These methods include, inter alia, phosphotriesters for nucleic acid synthesis, phosphoproline methods, and H_phosphonate methods. A preferred method of this type of chemical synthesis is a polymer-supported synthesis using standard phosphonium chemistry. Typically, the DNA encoding the amino acid sequence of the VGF polypeptide will be hundreds of nucleotides in length. These methods can be used to synthesize about 100 nucleic acids of nucleotide or higher in a plurality of fragments. The fragment can then be ligated to form the full length nucleotide sequence of the VGF gene. The amino-terminal DNA fragment encoding the polypeptide often has an ATG encoding a methionine residue. The methionine may or may not be present in the mature form of the VGF polypeptide, depending on whether the polypeptide produced in the host cell is designed to be secreted from the cell. You can also use other methods known to the artist. In certain embodiments, a codon-containing nucleic acid variant has been altered in order to optimally express a VGF polypeptide in a particular host cell. Specific codon changes will be based on the VGF peptide and sink-31 selected for performance. This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------------ -------^--------- (Please read the notes on the back and fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (29) Depending on the main cell. The optimal conditions for such codons can be accomplished by a variety of methods, such as by selecting codons that are most preferred in highly expressed genes in a particular host cell. A combined codon frequency table can be used, such as A computer algorithm for the Eco_high.Cod" codon for highly expressed bacterial gene preferences, and is available from the University of Wosconsin Package version 9.0 (Genetics Computer Group, Madison, WI). Other useful codon frequency tables include "Celegans_high.codn, ''Celegans-low.cod·', "Drosophila high·codn, "Human high.cod", •'Maize-high·cod'' and Yeast-high.cod'. In some cases, it may be desirable to prepare a nucleic acid molecule encoding a variant of a VGF polypeptide. The coding variant can be produced using a mutation-producing action at a specified position, PCR amplification, or other suitable method. Nucleic acid molecules in which the primers have the desired point mutations (see Sambrook et al., supra, and Ausubel et al., supra, for explanation of mutation techniques). Also used by Engels et al. Such variants are prepared by chemical synthesis of the methods described. Other methods known to those skilled in the art can also be used. Vectors and host cells nucleic acid molecules encoding amino acid sequences of VGF polypeptides using standard ligation techniques. Insert into an appropriate expression vector. A vector that has a function in the particular host cell used (ie, the vector is compatible with the host cell mechanism) Enlargement of genes and/or gene expression). Enlargement/expression of nucleic acids encoding amino acid sequences of VGF polypeptides in prokaryotes, yeasts, insects (baculovirus systems) and/or purine host cells Molecules. The selection of host cells will depend on whether the Chinese National Standard (CNS) A4 specification (210 X 297 mm) will be applied at the translation-32-paper scale ------------- ------Book --------- (Please read the note on the back and fill out this page) 1278319 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (3G) Modification of VGF polypeptides (e.g., glycosylation and/or phosphorylation). If so, yeast, insect or mammalian host cells are preferred. For a review of the vectors in Table j, see Meth·Εηζ·, 1st 8 5 (edited by D. V. Goeddel, Academic Press 1990). Typically, expression vectors used in any host cell will contain sequences for plastid maintenance and colonization and for the expression of exogenous nucleotide sequences. Sequences, collectively referred to as "side sequences" in some embodiments, Often will include one or more of the following nucleotide sequences: a promoter gene, one or more promoter sequences, an origin of replication, a transcription termination sequence, a fully inserted sequence containing the junction of the donor and the recipient, prior to encoding the polypeptide secretion The sequence of the leader sequence, the ribosome binding site, the polyadenylation sequence, the poly-crosslinker region encoding the nucleic acid encoding the polypeptide to be expressed, and the selectable marker elements are discussed separately below. The vector may optionally contain a sequence encoding the "tag," which is an oligonucleotide nucleic acid molecule located at the 5' or 3' end of the Vgf multi-month crypto sequence, which encodes a poly-His (like six His ), or other tags, such as FLAG, ha (hemagglutinin influenza virus) or myc, have antibodies against it 2. Usually, when the polypeptide is expressed, the tag is fused with the polypeptide. 'Can be used as a tool for affinity purification of VGF polypeptides from host cells. Affinity purification can be accomplished by, for example, column chromatography using antibodies against the tag as an affinity matrix. The method, such as the use of certain peptidases for excision, removes the tag from the purified vgf polypeptide. The flanking sequence can be homologous (ie, derived from the same host cell (please read the back of the note first) Please fill in this page again) ----- Order--- Volume. -33- 1278319 A7

種及/或品系）、異種的（也就是得自與宿主細胞物不同的物種）、雜種的(也就是得自一種以上來源之側：序列的混合)或合成的，或該侧面序列可以是天然，並正常便具有調節VGF多肽表現的功能。側面序列的了本身可以是任何的眞核生物或原核生物，任何無脊椎動物，或任何植物，其限制條件爲該側面序列在^ 王細胞機制中是具有功能的，並可由其活化。可藉著此項技藝中已熟知的數種方法中任一種用的側面序列。通常，在本文中有用的側面序列-心基因之侧面序列不同·先前已經藉著作圖及/或核酸内切㈣酶4化作用確認，並因此可使用適當的核酸内切限制酶，從通當的組織來源中分離。在一些案例中，側面序列的完整核《序列可能是已知的。在這裏，可使用在本文中描述，有關於核酸合成或選殖之方法，來合成侧面序列。在已知全部或一部份側面序列之處，可使用pcR及/或藉著利用適當的寡核嘗酸及/或得自相同或不同物種之侧面序歹J片#又’篩選基因組庫而獲得。在不知道側面序列之處，可從較大的DNA破片中分離含有側面序列的dna片段，其可能含有例如密碼序列’或甚至是其他的基因或基因們可藉著核酸内切限制酶消化作用，產生適當的dna片段，接著使用瓊脂糖凝膠純化作用、Qiagen®管柱層析法（chats_ worth, CA)，或熟睛此藝者已知的其他方法分離，完成分離作用。熟諳此藝者將輕易地知曉爲了完成該目標而選擇適當的酵素。巧張尺度適财關家標準（Ci^s)A4規格（ χ挪公髮丁 --------β--------- (請先閱讀背面之注意事項再填寫本頁) -34- 1278319 五、發明說明（32 濟部智慧員工消費複製起點通常是那些可購得之眞核生物表現載體的一部份，且孩起點有助於在宿主細胞中擴大該載體。在—些案例中，將載體擴大至確定的副本數目，對於適切地表現 VGF多肽是很重要的。如果選擇的载體不含複製起點位置，可以已知的序列爲基礎’以化學方式合成—個複製起點，並連接到載體内。例如，得自質體pBR322(New E㈣— Biolabs，Bevedy，MA)的複製起點，適合大多數的革蘭氏陰性細菌，且各種起點（例如SV40、多瘤、腺病毒、水疱性口火病毒（VSV)或乳頭狀瘤病毒，像是Hpv或Bpv)，適用於在哺乳動物細胞中選殖載體。一般而言，哺乳動物表現載體不需要複製起點成份（例如，通常僅使用SV40起點，因爲它含有早期啓動基因）。轉錄終止序列通常位在多肽密碼區的3,末端，並用來終士轉錄作用。通常，在原核生物細胞中的轉錄終止序列爲田各G_ C的片段，接著是聚_ τ序列。很容易從庫中選殖該 ^列，或甚至是以載體的一部份購得，亦可輕易地使用核酸泛成的方法來合成之，像是在本文中描述的那些。、可選擇標記基因要素編碼宿主細胞在選擇性培養基中存活$生長所需的蛋白質。典型的可選擇標記基因編碼之蛋白貝其（a)賦與原核生物宿主細胞對抗生素或其他毒素的 =藥性，例如氨卞青黴素、四環素或康黴素；（b)與細胞的營養缺陷互補，·或（C)供應不能獲自複雜培養基的重要養份二較佳的可選擇標記爲康黴素抗藥性基因、氨卞青黴素抗蕖丨生基因和四環素抗藥性基因。關於原核生物和眞核生物 -35- 本紙張尺度適用X 29 τAnd/or strain), xenogeneic (ie from a species different from the host cell), hybrid (ie from the side of more than one source: a mixture of sequences) or synthetic, or the flanking sequence may be Naturally, and normally, it has the function of regulating the expression of VGF polypeptide. The flanking sequence itself may be any deuteron or prokaryote, any invertebrate, or any plant, with the proviso that the flanking sequence is functional in and activated by the king cell mechanism. The side sequences can be used by any of several methods well known in the art. In general, the side sequence of the side sequence-heart gene is different in this article. It has been previously confirmed by mapping and/or endonuclease (4) enzyme 4, and thus the appropriate endonuclease restriction enzyme can be used. Separated from the tissue source. In some cases, the complete kernel of the side sequence "sequences may be known. Here, side sequences can be synthesized using methods described herein for nucleic acid synthesis or colonization. Where all or a portion of the side sequence is known, pcR can be used and/or by using appropriate oligo-acids and/or from the same or different species, the genomic library can be screened. obtain. Where a side sequence is not known, a DNA fragment containing a side sequence may be isolated from a larger DNA fragment, which may contain, for example, a cryptographic sequence' or even other genes or genes that can be digested by restriction endonuclease digestion. The appropriate DNA fragment is generated, followed by separation using agarose gel purification, Qiagen® column chromatography (chats_worth, CA), or other methods known to those skilled in the art to complete the separation. Those skilled in the art will readily know that the right enzyme is chosen to accomplish this goal. Qiao Zhang scale financial standards (Ci ^ s) A4 specifications (χ 公公 - ------------- β--------- (Please read the back of the notes and then fill out this Page) -34- 1278319 V. INSTRUCTIONS (32) The origin of the consumption of the Ministry of Education of the Ministry of Education is usually part of the commercially available DNA display vector, and the starting point of the child helps to expand the vector in the host cell. In some cases, it is important to extend the vector to a defined number of copies for the proper expression of the VGF polypeptide. If the selected vector does not contain an origin of replication, it can be chemically synthesized based on known sequences. The origin of replication is ligated into the vector. For example, the origin of replication from plastid pBR322 (New E(4) - Biolabs, Bevedy, MA) is suitable for most Gram-negative bacteria, and various starting points (eg SV40, multi-tumor) , adenovirus, vesicular fire-fever virus (VSV) or papilloma virus (such as Hpv or Bpv), suitable for selection of vectors in mammalian cells. In general, mammalian expression vectors do not require replication origin components (eg , usually only from SV40 Point, because it contains an early promoter gene. The transcription termination sequence is usually located at the 3' end of the polypeptide cryptodomain and is used for terminal transcription. Typically, the transcription termination sequence in prokaryotic cells is a fragment of G_C in the field, This is followed by a poly_τ sequence. It is easy to clone the column from the library, or even a part of the vector, or it can be easily synthesized using a nucleic acid synthesis method, as described in this article. The selectable marker gene elements encode the host cell to survive in a selective medium for growth of the protein required for growth. A typical selectable marker gene encodes a protein (a) confers a prokaryotic host cell to an antibiotic or other toxin. = Pharmacological, such as ampicillin, tetracycline or ketomycin; (b) complementary to auxotrophy of the cell, or (C) supply of important nutrients that cannot be obtained from complex media. Antibiotic gene, ampicillin anti-twist gene and tetracycline resistance gene. About prokaryote and steroids -35- This paper scale applies X 29 τ

---------------------訂— (請先閱讀背面之注意事項再填寫本頁) s'.--------------------- Order — (Please read the notes on the back and fill out this page) s'.

I 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（33) 宿主細胞的選擇，亦可使用新黴素抗藥性基因。可使用其他的選擇基因擴大將要表現的基因。擴大作用是其中使對於產製對生長具有決定性之蛋白質，需要量較大之基因’反覆地縱排在重組細胞之連續產製的染色體内的過程。哺乳動物細胞之適當可選擇標記的實例，包括二氫葉酸還原酶（DHFR)和胸腺核甞激酶。將哺乳動物細胞轉化物置於％擇壓力下’其中僅有憑藉著出現在載體中的選擇基因，而獨一無二地改編的轉化物能夠存活。藉著將轉化之細胞培養在連續改變在培養基中選擇製劑之濃度的條件下，強加上選擇壓力，藉此導致選擇基因和編碼VGF多肽之DNA的擴大。結果，從經過擴大的DNA*合成增加含量的VGF多肽。核糖體結合位置通常是mRNA開始轉譯所必需的，並藉著 Shine· Dalgarno(原核生物）或Kozak序列（眞核生物）定出其特徵。該要素通常位在啓動基因的3，和待表現之VGF多肽的密碼序列的5 *端。Shine-Dalgarno序列是多變化的，但通常疋I %呤（也就是具有南A - G内含量的）。已經確認出許多 Shine· Dalgarno序列，可輕易地使用在本文中陳述的方法分別合成它們，並可用於原核生物的載體中。可使用前導或信號序列來指揮VGF多肽到宿主細胞之外。通常’編碼信號序列的核苷酸序列係位在VGF核酸分子的密碼區中，或直接位在VGF多肽密碼區的5，末端。已經確認出許多信號序列，可將任何在所選擇之宿主細胞中具有功能的那些與VGF核酸分子一起使用。因此，信號序列 -36- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） )-------------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（34 ) 可以是與VGF核酸分子同種的（天然存在的）或異種的。此外，亦可使用在本文中描述的方法，以化學方式合成信號序列。在大多數的案例中，從宿主細胞中，經由信號肽的存在來分泌VGF多肽，將導致從已經分泌的VGF多肽中移除信號肽。該信號序列可以是载體的成份，或它也可以是 ***载體内之VGF核酸分子的一部份。可將編碼天然VGF多肽信號序列的核甞酸序列與VGF多肽密碼區連接，或可將編碼異種信號序列的核甞酸序列與 VGF多肽密碼區連接。所選擇的異種信號序列應該是可由宿主細胞認出並加工，也就是被信號肽酶切開的信號序列。關於不認得和加工天然VGF多肽之信號序列的原核生物宿主細胞’藉著例如選自包括鹼性磷酸酶、青黴素酶或熱-穩足的腸毒素II前導子的原核生物信號序列，來取代該信號序列。關於酵母菌的分泌，可由酵母菌轉化酶、“因子或酸性磷酸酶前導子，來取代天然VGF多肽的信號序列。在哺乳動物細胞的表現中，天然的信號序列是令人滿意的，雖然其他的哺乳動物信號序列亦可能是適當的。在一些案例中，像是在眞核生物宿主細胞表現系統中想要有糖基化作用之處，可運用各種前序列幻來改軎糖基化作用或產量。例如，可改變特殊信號肤的月太酶切開位置，或加入前序列（Pr〇- sequence)，其亦可影響糖基化作用。最終的蛋白質可在-1位置（相對於成熟蛋白質的第匕個胺基酸），具有一或多個額外胺基酸的附帶表現，其可月匕尚未το王私除。例如，最終的蛋白質產物可具有一或兩 ‘紙張尺度適用中國國家標規格（21〇 X 297公釐） ---------------------------- s, (請先閱讀背面之注音？事項再填寫本頁) -37. 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（35) 個在肽酶切開位置中找到的胺基酸殘基，附接在胺基-終端上。另外，一些酵素切開位置的使用，如果該酵素在成熟多肽内的這類地方切開，亦可產生想要之VGF多肽稍微截短的形式。在許多案例中，藉著一或多個出現在載體内的***序列，增加核酸分子的轉錄；這在眞核生物宿主細胞中產製多肽之處是特別眞實的，尤其是哺乳動物宿主細胞。所使用的***序列可以是在VGF基因内天然存在的，特別是在所使用的基因爲全長的基因組序列或其片段之處。在***序列不是在基因中天然存在的（像是大多數的cDNa)之處，該 ***序列可獲自其他來源。***序列的位置相對於側面序列和VGF基因，通常是很重要的，因爲必須有效地轉錄插入序列。因此，當要轉錄VGF cDNA分子時，***序列的較佳位置是在轉錄起始位置的3，，和聚-A轉錄終止序列的5，。較佳的是，該***序列或***序列們將位ScDNA的一邊或另一邊（也就是5 ’或3 ’），使得它不會妨礙密碼序列。可使用得自任何來源的任何***序列，包括病毒的、原核生物和眞核生物（植物或動物）的，其限制條件爲它與它將插入的宿主細胞是可相容的。在本文中亦包括合成的***序列。在載體中，可視需要使用一個以上的***序列。表現和選殖載體通常將含有可由宿主生物認出的啓動基因，並以可操作之方式與編碼VGF多肽的分子連接。啓^ 基因是位在結構基因之起始密碼子上游（也就是5，）的未轉錄序列（通常在大約100到1000個鹼基對之内），其控制結構 --------------------訂—------ (請先閱讀背面之注意事項再填寫本頁)I 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Description of invention (33) The selection of host cells can also use neomycin resistance genes. Other selection genes can be used to expand the genes to be expressed. The enlarging action is a process in which a gene which is decisive for growth is required to be produced, and a gene which is required to be larger is repeatedly placed in a continuous chromosome of the recombinant cell. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. The mammalian cell transformants are placed under % selection pressure' wherein only the transformant genes that are present in the vector are capable of surviving. By culturing the transformed cells under continuous conditions to change the concentration of the selected preparation in the medium, the selection pressure is imposed, thereby causing the expansion of the selection gene and the DNA encoding the VGF polypeptide. As a result, the VGF polypeptide is increased in content from the expanded DNA* synthesis. The ribosome binding site is usually required for the initial translation of the mRNA and is characterized by Shine Dalgarno (prokaryote) or Kozak sequence (xenon). This element is usually located at the 5* end of the promoter gene and the 5' end of the crypto sequence of the VGF polypeptide to be expressed. The Shine-Dalgarno sequence is highly variable, but is usually 疋I %呤 (that is, having a content in the South A-G). A number of Shine Dalgarno sequences have been identified which can be readily synthesized using the methods set forth herein and can be used in vectors for prokaryotes. A leader or signal sequence can be used to direct the VGF polypeptide out of the host cell. Typically, the nucleotide sequence encoding the signal sequence is in the cryptodomain of the VGF nucleic acid molecule or directly at the 5' end of the cryptodomain of the VGF polypeptide. A number of signal sequences have been identified which can be used with any VGF nucleic acid molecule that is functional in the host cell of choice. Therefore, the signal sequence -36- this paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm))------------------- Order--- ------ (Please read the notes on the back and fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. Description of Invention (34) May be the same species as VGF Nucleic Acid (naturally occurring) Or heterogeneous. In addition, signal sequences can be chemically synthesized using the methods described herein. In most cases, secretion of a VGF polypeptide from a host cell via the presence of a signal peptide will result in the removal of the signal peptide from the already secreted VGF polypeptide. The signal sequence can be a component of a vector, or it can be part of a VGF nucleic acid molecule inserted into a vector. The nucleotide sequence encoding the native VGF polypeptide signal sequence can be ligated to the VGF polypeptide cryptodomain, or the nucleotide sequence encoding the heterologous signal sequence can be ligated to the VGF polypeptide cryptodomain. The heterologous signal sequence selected should be recognizable and processed by the host cell, i.e., the signal sequence cleaved by the signal peptidase. A prokaryotic host cell that does not recognize and process a signal sequence of a native VGF polypeptide is replaced by, for example, a prokaryotic signal sequence selected from the group consisting of alkaline phosphatase, penicillinase or heat-stable enterotoxin II proton. Signal sequence. Regarding the secretion of yeast, the signal sequence of the native VGF polypeptide can be replaced by a yeast invertase, a "factor or an acid phosphatase leader." In the expression of mammalian cells, the natural signal sequence is satisfactory, although other The mammalian signal sequence may also be appropriate. In some cases, such as where glycosylation is desired in the host cell expression system of the scorpion, various pre-sequences can be used to alter glycosylation. Or yield. For example, it can change the position of the special signal peptide, or add the pre-sequence (Pr〇-sequence), which can also affect glycosylation. The final protein can be in the -1 position (relative to maturity) The third amino acid of a protein, with the attendant performance of one or more additional amino acids, which may not be vacated by το王. For example, the final protein product may have one or two 'paper scales for Chinese countries. Standard specifications (21〇X 297 mm) ---------------------------- s, (Please read the phonetic notes on the back first? Fill in this page) -37. 1278319 Ministry of Economic Affairs Intellectual Property Officer Consumer Cooperatives Print A7 B7 V. Description of the Invention (35) Amino acid residues found in the peptidase cleavage site, attached to the amine-terminal. In addition, some enzymes are used in the incision position if the enzyme Incision in such a region within the mature polypeptide may also result in a slightly truncated form of the desired VGF polypeptide. In many cases, transcription of the nucleic acid molecule is increased by one or more insertion sequences present in the vector; The production of a polypeptide in a host cell of a scorpion nucleus is particularly well-established, especially in mammalian host cells. The insertion sequence used may be naturally occurring within the VGF gene, particularly if the gene used is a full-length genomic sequence. Or a fragment thereof. Where the insertion sequence is not naturally found in the gene (such as most cDNa), the insertion sequence can be obtained from other sources. The position of the insertion sequence relative to the side sequence and the VGF gene is usually Very important, because the inserted sequence must be efficiently transcribed. Therefore, when the VGF cDNA molecule is to be transcribed, the preferred position of the inserted sequence is at the transcription start position. 3, and the poly-A transcription termination sequence of 5, preferably, the insertion sequence or the insertion sequence will be located on one side or the other side of the ScDNA (ie 5' or 3') so that it does not interfere A cryptographic sequence. Any insertion sequence from any source, including viral, prokaryotic, and nucleus (plant or animal), may be used, with the proviso that it is compatible with the host cell into which it will be inserted. Also included herein are synthetic insertion sequences. In the vector, more than one insertion sequence may be used as desired. The expression and selection vectors will typically contain a promoter gene recognizable by the host organism and operably associated with the VGF polypeptide. Molecular junction. The gene is an untranslated sequence (usually within about 100 to 1000 base pairs) upstream of the initiation codon of the structural gene (ie, within about 100 to 1000 base pairs), and its control structure ----- --------------- Order —------ (Please read the notes on the back and fill out this page)

1278319 A7 五、發明說明（36 ) 請基因的轉綠。在傳統上可將啓動基因分類成兩種中的一種 •可謗導之啓動基因和組成的啓動基因。可謗導的啓動基因反映在培養條件中的一些改變，像是養份的存在或缺乏或溫度上的改變，開始增加從DNA中轉錄的程度。換句話説，組成的啓動基因發動連續的基因產物產製；也就是在整個基因表現中有較少的或沒有控制。由各種有可能之宿王細胞認得的大量啓動基因，是已爲人所熟知的。藉著經由限制酵素消化，從來源DNA中移出啓動基因，並將想要的啓動基因序列***載體中，將適當的啓動基因以可操作 < 方式與編碼VGF多肽的DNA連接。可使用天然的VGF啓動基因序列，來指揮VGF核酸分子的擴大作用及/或表現。然而，如果與天然的啓動基因相比較，其容許所表現之蛋白質較多的轉錄和較高的產量，且如果它與已經選擇使用之宿主細胞系統是可相容的，則異種的啓動基因是較佳的。通合原核生物宿主使用的啓動基因包括内醯胺酶和乳糖啓動基因系統；鹼性磷酸酶；色胺酸（trp)啓動基因系統 ;以及雜種的啓動基因，像是tae啓動基因。其他已知的細菌啓動基因亦是適當的。已經公告它們的序列，藉此使熟諳此藝者得以將它們與想要的01^八序列連接，按照需要^ 用父聯劑或接合體，以便提供任何有用的限制位置。適合酵母菌宿主使用的啓動基因亦爲此項技藝中已熟知的。可有利地將酵母菌促進子與酵母菌啓動基因一起使用P 。適合哺乳動物宿主細胞使用的啓動基因亦是已熟知的，並包括但不限於獲自病毒之基因組的那些，像是多瘤病毒本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公-_ 39- 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（37 ) 、禽痘病毒、腺病毒（像是腺病毒2)、牛乳頭狀瘤病毒、難肉瘤病毒、細胞巨大病毒、反轉錄病毒、B型肝炎病毒，而最佳的是猿病毒4 0 ( SV40)。其他適當的哺乳動物啓動基因包括異種的哺乳動物啓動基因，例如熱-休克啓動基因和肌動蛋白啓動基因。在控制V G F基因表現上，可能感興趣的其他啓動基因包括，但不限於：SV40早期啓動基因區（Bernoist和Chambon， 1981，Nature 290 : 304-10) ; CMV啓動基因；在勞氏肉瘤病毒之3*長端重複中所含有的啓動基因（Yamamoto等人， 1980，Cell 22 : 787-9 7);疱疹胸腺核甞激酶啓動基因 (Wagner等人，1981，Proc· Natl. Acad· Sci· U.S.A· 78 : 1444-45);金屬硫肤基因的調節序列（Brinster等人，1982，Nature 296 : 3 9 - 4 2 );原核生物表現載體，像是卢·内醯胺酶啓動基因（Villa-Kamaroff等人，1978，Proc· Natl· Acad· Sci· U.S. A. 75 : 3727-31);或 tac啓動基因（DeBoer 等人，1983， Proc. Natl. Acad. Sci. U.S.A·，80: 21-25)。感興趣的還有下列的動物轉錄控制區，其顯示出組織專一性，並已經在基因轉殖的動物中使用：彈性蛋白酶I基因控制區，其在胰臟腺泡細胞中是有活性的（Swift等人，1984, Cell 38 : 639-46 ;Ornitz 等人，1986, Cold Spring Harbor Symp· Quant· Biol. 50 ： 399-409(1986) ; MacDonald, 1987, Hepatology 7 ： 425-515);胰島素基因控制區，其在胰臟卢細胞中是有活性的 (Hanahan，1985，Nature 315 : 115-22 );免疫球蛋白基因控制區，其在淋巴細胞中是有活性的（Grosschedl等人，1984, -40- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------------------^---------^^9. (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 Α7 Β7 五、發明說明（38)1278319 A7 V. INSTRUCTIONS (36) Please turn the gene green. The promoter gene has traditionally been classified into one of two types: a promoter gene that can be used to initiate genes and a promoter. The achievable promoter is reflected in some changes in culture conditions, such as the presence or absence of nutrients or changes in temperature, which begin to increase the degree of transcription from DNA. In other words, the constituent promoter genes initiate a continuous production of the gene product; that is, there is little or no control over the overall gene expression. A large number of promoter genes recognized by various potential scorpion cells are well known. The promoter gene is removed from the source DNA by restriction enzyme digestion, and the desired promoter gene sequence is inserted into the vector, and the appropriate promoter gene is operably linked to the DNA encoding the VGF polypeptide. Natural VGF initiation gene sequences can be used to direct the expansion and/or expression of VGF nucleic acid molecules. However, if compared to the native promoter gene, it allows for more transcription and higher yield of the expressed protein, and if it is compatible with the host cell system that has been selected for use, the heterologous promoter gene is Preferably. Promoter genes for use in prokaryotic host organisms include endoprostase and lactose initiation gene systems; alkaline phosphatase; tryptophan (trp) promoter gene systems; and hybrid promoter genes, such as the tae promoter gene. Other known bacterial promoter genes are also suitable. Their sequences have been published so that those skilled in the art can connect them to the desired sequence and use the parent or adapter as needed to provide any useful restriction. Promoter genes suitable for use in yeast hosts are also well known in the art. It is advantageous to use the yeast promoter together with the yeast promoter gene P. Promoter genes suitable for use in mammalian host cells are also well known and include, but are not limited to, those obtained from the genome of the virus, such as the polyoma virus paper scale applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 public). -_ 39- 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Printed A7 B7 V. Inventions (37), fowlpox virus, adenovirus (like adenovirus 2), bovine papilloma virus, sarcoma virus, cell Huge virus, retrovirus, hepatitis B virus, and the best is prion 4 0 (SV40). Other suitable mammalian promoter genes include heterologous mammalian promoter genes, such as heat-shock promoter genes and actin Promoter genes. Other promoter genes that may be of interest in controlling VGF gene expression include, but are not limited to, the SV40 early promoter gene region (Bernoist and Chambon, 1981, Nature 290: 304-10); the CMV promoter gene; The promoter gene contained in the 3* long-end repeat of sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-9 7); herpes thymidine nuclear kinase kinase promoter gene (Wagner et al. Human, 1981, Proc. Natl. Acad. Sci. USA 78: 1444-45); regulatory sequences of the metal thiophyllin gene (Brinster et al., 1982, Nature 296: 3 9 - 4 2 ); prokaryotic expression vector, Such as the Luinactamase promoter gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-31); or the tac promoter gene (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA·, 80: 21-25). Also interested are the following animal transcriptional control regions, which show tissue specificity and have been used in genetically transgenic animals: elastase I gene control region It is active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409 (1986) MacDonald, 1987, Hepatology 7: 425-515); the insulin gene control region, which is active in pancreatic cells (Hanahan, 1985, Nature 315: 115-22); immunoglobulin gene control region, Active in lymphocytes (Grosschedl et al., 1984, -40- This paper scale applies to Chinese countries) Standard (CNS) A4 specification (210 X 297 mm) --------------------^---------^^9. (Please first Read the precautions on the back and fill out this page.) 1278319 Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, Printed Α7 Β7 V. Inventions (38)

Cell 38 : 647-58 ; Adames 等人，1985, Nature 318 : 533_38 ; Alexander等人，1987, Mol. Cell· Biol·，7 : 1436-44);老鼠乳房腫瘤病毒控制區，其在睪丸、***、淋巴和肥大細胞中是有活性的（Leder等人，1986, Cell 45 : 485-95);白蛋白基因控制區，其在肝臟中是有活性的（Pinkert等人，1987, Genes和Devel. 1 : 268-76) ; α -胎兒·蛋白質基因控制區，其在肝臟中是有活性的（Krumlauf等人，1985, Mol· Cell. Biol·， 5 : 1639-48 ; Hammer等人，1987，Science 235 : 53-58) ; a 1 -抗胰蛋白酵素基因控制區，其在肝臟中是有活.性的（Kelsey 等人，1987，Genes and Devel. 1 : 161-71); 血球蛋白基因控制區，其在體樣細胞中是有活性的（Mogram等人，1985, Nature 315 : 338-40 ; Kollias等人，1986，Cell 46 : 89-94); 骨髓磷脂鹼性蛋白質基因控制區，其在腦中的寡樹突膠質細胞中是有活性的（Readhead等人，1987，Cell 4 8 : 703- 1 2 ) ;肌球蛋白輕鏈-2基因控制區，其在骨骼肌中是有活性的 (Sani，1985, Nature 314 ·· 283- 8 6);以及促性腺釋放荷爾蒙基因控制區，其在下視丘中是有活性的（Mason等人，1986, Science 234 : 1372-78)。可將促進子序列***載體内，增加編碼VGF多肽之DNA 在高等眞核生物中的轉錄。促進子是DNA順向-作用的要素，長度通常爲大約1 0-300個鹼基對，其作用在啓動基因上，增加轉錄。促進子相對上是方位和位置獨立的。已經在轉錄單位的5 ’和3 1發現它們。已知數個獲自哺乳動物基因的促進子序列（例如血球蛋白、彈性蛋白酶、白蛋白、α - -41 - $紙張尺度適用中國國家標準（CNS)A4規格(210 x 297公釐） ---------------------------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（39) 胎兒·蛋白質和胰島素）。然而，通常將使用得自病毒的促進子。SV40促進子、細胞巨大病毒早期啓動基因促進子、多瘤促進子和腺病毒促進子，是眞核生物啓動基因之激活作用中的代表性促進要素。可在VGF核酸分子的5 ’或3 ’處，將促進子接合到載體内，其通常位在啓動基因的5 ’位置。可從起始載體，像是可購得的載體，來製備表現載體。這類載體可以或可以不含有所有想要的側面序列。其中一或多個在本文中描述的側面序列，尚未出現在載體中時，可分別獲得它們，並將其連接到載體内。獲得每個側面序列所使用的方法是熟諳此藝者已熟知的。較佳的載體是與細菌、昆蟲和哺乳動物宿主細胞可相容的那些。這類載體特別包括pCRII、pCR3和pcDNA3.1 (Invitrogen，San Diego, CA)、pBSII (Stratagene，La Jolla，CA) 、pET15(Novagen, Madison，WI)、pGEX(Pharmacia Biotech, Piscataway，NJ)、pEGFP-N2 (Clontech，Palo Alto，CA)、 pETL(BlueBacII，Invitrogen)、pDSR-汉（PCT 出版物編號 WO 90/14363)和pFastBacDual(Gibco_BRL，Grand Island，NY)。其他適當的載體包括，但不限於黏接質體、質體或經過修改的病毒，但將知道該載體系統必須是與選出之宿主細胞可相容的。這類載體包括，但不限於諸如Bluescript®質體衍生物（以高副本數目ColEl·爲基礎的噬菌體質體， Stratagene，Cloning Systems，La Jolla CA)之類的質體，設計用來選殖Taq-擴大PCR產物的PCR選殖質體（例如TOPO™ ΤΑ Cloning® 工具組，PCR2· 1 ® 質體衍生物，Invitrogen， -42- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐1 -----— — — — — — ------11 ^---------^^^1 (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（40)Cell 38: 647-58; Adames et al., 1985, Nature 318: 533_38; Alexander et al., 1987, Mol. Cell Biol, 7: 1436-44); mouse mammary tumor virus control region, in testicular pills, breast Active in lymphoid and mast cells (Leder et al., 1986, Cell 45: 485-95); an albumin gene control region that is active in the liver (Pinkert et al., 1987, Genes and Devel. 1 : 268-76); α-fetal·protein gene control region, which is active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol., 5: 1639-48; Hammer et al., 1987, Science 235: 53-58); a 1 - anti-trypsin gene control region, which is viable in the liver (Kelsey et al., 1987, Genes and Devel. 1 : 161-71); a gene control region that is active in body-like cells (Mogram et al, 1985, Nature 315: 338-40; Kollias et al, 1986, Cell 46: 89-94); myelin basic protein gene control region , which is active in oligodendrocytes in the brain (Readhead et al., 1987, Cell 4 8: 703-12); myosin light -2 gene control region, which is active in skeletal muscle (Sani, 1985, Nature 314 · 283-8); and gonadotropin releasing hormone control region, which is active in the hypothalamus (Mason Et al., 1986, Science 234: 1372-78). Promoter sequences can be inserted into vectors to increase transcription of DNA encoding VGF polypeptides in higher nucleus. Promoters are elements of DNA cis-acting, usually about 10 to 300 base pairs in length, which act on the promoter gene to increase transcription. Promoters are relatively independent in orientation and position. They have been found at 5' and 31 of the transcription unit. Several promoter sequences derived from mammalian genes are known (eg, blood globulin, elastase, albumin, alpha--41 - $ paper scales are applicable to the Chinese National Standard (CNS) A4 specification (210 x 297 mm) - --------------------------- (Please read the notes on the back and fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative System A7 B7 V. Description of invention (39) Fetus, protein and insulin). However, promoters derived from viruses will generally be used. The SV40 promoter, the cell giant virus early promoter gene promoter, the multi-tumor promoter and the adenovirus promoter are representative promoters of the activation of the purine nuclear promoter gene. The promoter may be ligated into the vector at 5' or 3' of the VGF nucleic acid molecule, which is typically located at the 5' position of the promoter gene. Expression vectors can be prepared from starting vectors, such as commercially available vectors. Such vectors may or may not contain all of the desired side sequences. When one or more of the side sequences described herein are not present in the vector, they are separately obtained and ligated into the vector. The method used to obtain each side sequence is well known to those skilled in the art. Preferred carriers are those which are compatible with bacterial, insect and mammalian host cells. Such vectors include, inter alia, pCRII, pCR3 and pcDNA3.1 (Invitrogen, San Diego, CA), pBSII (Stratagene, La Jolla, CA), pET15 (Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacII, Invitrogen), pDSR-Han (PCT Publication No. WO 90/14363) and pFastBacDual (Gibco_BRL, Grand Island, NY). Other suitable carriers include, but are not limited to, plastids, plastids or modified viruses, but it will be appreciated that the vector system must be compatible with the selected host cell. Such vectors include, but are not limited to, plastids such as Bluescript® plastid derivatives (high-copy number ColEl-based phage plastids, Stratagene, Cloning Systems, La Jolla CA) designed to colonize Taq - Expand PCR product selection for PCR products (eg TOPOTM ΤΑ Cloning® tool set, PCR2· 1 ® plastid derivatives, Invitrogen, -42- This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297君1 -----—— — — — — — ------11 ^---------^^^1 (Please read the notes on the back and fill out this page) 1278319 Economy Ministry of Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (40)

Carlsbad，CA)，以及哺乳動物、酵母菌或病毒載體，像是桿狀病毒表現系統（pBacPAK質體衍生物，ciontech，Palo Alto, CA) 〇在已經建構載體，並已經將編碼VGF多肽的核酸分子插入該載體的適當位置之後，可將完成的載體***適合擴大及/或多肽表現的宿主細胞内。可藉著已熟知的方法，包括諸如轉移感染、感染、氯化鈣、電穿透作用、微量注射、脂染體、DEAE-葡聚醣法之類的方法，或其他已知的技術，完成將VGF多肽的表現載體轉化至選出的宿主細胞中。所選擇的方法，一部份將是所使用之宿主細胞類型的函數。這些方法和其他適當的方法是熟諳此藝者已熟知的，並在例如Sambrook等人，在前，中陳述。宿主細胞可以是原核生物的宿主細胞（像是大腸桿菌）或眞核生物的宿主細胞（像是酵母菌、昆蟲或脊椎動物細胞）。當在適當的條件下培養時，該宿主細胞合成VGF多肽，可接著從培養基中收集（如果宿主細胞將其分泌至培養基中 )，或直接從產製它的宿主細胞中收集（如果未分泌該多肽）。將依據各種因素來選擇適當的宿主細胞，像是想要的表現程度、對於活性想要的或必需的多肽修改（像是糖基化作用或磷酸化作用），以及容易折疊成具有生物活性的分子。許多適當的宿主細胞是此項技藝中已知的，並可獲自 American Type Culture Collection(ATCC)，Manassas，VA。實例包括，但不限於哺乳動物細胞，像是中國倉鼠卵巢細胞 (CHO)、CHO DHFR(-)細胞（Urlaub等人，1980，Proc. Natl. -43- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -------------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（41)Carlsbad, CA), and mammalian, yeast or viral vectors, such as the baculovirus expression system (pBacPAK plastid derivatives, ciontech, Palo Alto, CA). The vector has been constructed and the nucleic acid encoding the VGF polypeptide has been constructed. After insertion of the molecule into the appropriate position of the vector, the completed vector can be inserted into a host cell suitable for expansion and/or expression of the polypeptide. This can be accomplished by well-known methods, including methods such as transfer infection, infection, calcium chloride, electroporation, microinjection, lipofect, DEAE-dextran, or other known techniques. The expression vector of the VGF polypeptide is transformed into the selected host cell. Part of the method chosen will be a function of the type of host cell used. These and other suitable methods are well known to those skilled in the art and are described, for example, in Sambrook et al., supra. The host cell may be a host cell of a prokaryote (such as E. coli) or a host cell of a nucleus (such as a yeast, insect or vertebrate cell). When cultured under appropriate conditions, the host cell synthesizes a VGF polypeptide which can then be collected from the culture medium (if the host cell secretes it into the culture medium) or collected directly from the host cell from which it was produced (if not secreted) Peptide). Appropriate host cells will be selected based on various factors, such as the degree of expression desired, the desired or necessary polypeptide modification (such as glycosylation or phosphorylation) for the activity, and the ease of folding into biologically active molecule. Many suitable host cells are known in the art and are available from the American Type Culture Collection (ATCC), Manassas, VA. Examples include, but are not limited to, mammalian cells such as Chinese hamster ovary cells (CHO), CHO DHFR (-) cells (Urlaub et al., 1980, Proc. Natl. -43- This paper scale applies to Chinese National Standards (CNS) A4 size (210 X 297 mm) ------------------- Order --------- (Please read the notes on the back and fill out this page. 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (41)

Acad· Sci. U.S.A· 97 : 4216_20)、人類胚胎腎臟（HEK) 293 或 293 T細胞，或3 T 3細胞。選擇適當的哺乳動物宿主細胞，以及轉化、培養、擴大、篩選、產物產製和純化的方法，爲此項技藝中已知的。其他適當的哺乳動物細胞株，是猴子COS-1和COS- 7細胞株，以及CV-1細胞株。更多代表性的哺乳動物宿主細胞，包括靈長類細胞株和嚅齒類細胞株，包括轉化的細胞株。正常的二倍體細胞，衍生自在活體外原始組織培養物的細胞品系，以及原始的移植物，亦是適合的。候選者細胞可能在基因型上，在選擇基因中是有缺陷的，或可含有優勢作用的選擇基因。其他適合的哺乳動物細胞株包括但不限於老鼠神經胚細胞瘤N2 A細胞、 HeLa、老鼠L - 9 29細胞、衍生自Swiss、Balb_ c或NIH老鼠的 3 T 3細胞株、BHK或HaK倉鼠細胞株。這些細胞株中的每一個都是熟諳蛋白質表現技藝者已知的，並可由其取得。像適當之宿主細胞一樣有用的是細菌的細胞。例如，在生物技術學的範圍内，已熟知各種品系的大腸样菌（例如 HB101、DH5汉、DH10和MC1061)可做爲宿主細胞。在該方法中，亦可使用各種品系的枯草桿菌（B . subtilis)、假單胞菌屬（Pseudomonas spp·)、其他的桿菌屬（Bacillus spp·)、鏈黴菌屬（Streptomyces spp.)，及其類似物。熟諳此藝者已知許多品系的酵母菌細胞，亦可用來做爲表現VGF多肽的宿主細胞。較佳的酵母菌細胞包括例如酒酵母菌（Saccharomyces cerivisae)和巴斯德畢赤酵母菌（Pichia pastoris)。 -44- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -------------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（42) 另外，亦可在想要之處使用昆蟲細胞系統，來表現VGF 多肽。在例如Kitts等人，1993，Biotechniques，14 : 810-17 ; Lucklow，1993，Curr. Opin. Biotechnol. 4 : 564-72 ;以及 Lucklow等人，1993, J. Virol., 67 : 4566-79 中描述了這類系統。較佳的昆蟲細胞爲S f - 9和Hi5( Invitrogen)。亦可使用基因轉殖的動物來表現糖基化的VGF多肽。例如，可使用基因轉殖的產乳動物（例如，牛或山羊），並在動物的乳汁中獲得該糖基化的多肽。亦可使用植物來產製 VGF多肽，然而，一般在植物中發生的糖基化作用與在哺乳動物細胞中產生的不同，並可能導致糖基化的產物不適合人類的治療用途。多肽的產製可使用熟諳此藝者已熟知的標準培養基，來培養包括 VGF多肽表現載體的宿主細胞。培養基通常將含有所有細胞生長和存活所需的養份。適合用來培養大腸桿菌的培養基，包括例如Luria肉湯（LB)及/或Terrific肉湯（TB)。適合用來培養眞核生物細胞的培養基包括Roswell Park Memorial Institute 培養基 1640(RPMI 1640)、Minimal Essential 培養基 (MEM)，及 / 或 Dulbecco’s Modified Eagle培養基（DMEM)，它們全都要按照所培養之特殊細胞株的需要，補充血、清及/ 或生長因子。適合昆蟲培養的培養基，是按照需要補充酵母菌溶胞產物（yeastolate)、乳清蛋白水解產物，及/或胎牛血清的Grace’s培養基。通常，加入可用來選擇經過轉移感染或轉化之細胞生長 -45- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -------------------^--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（43 ) 的抗生素或其他化合物，做爲培養基的添加物。所使用的化合物，將由出現在用來轉化該宿主細胞之質體上的可選擇標記要素指定。例如，可選擇標記要素是康黴素抗藥性 ’則加至培養基中的化合物將是康黴素。其他用於選擇性生長的化合物包括氨卞青黴素、四環素和新黴素。可使用此項技藝中已知的標準方法，評估由宿主細胞產製之VGF多肽的含量。這類方法包括，但不限於西方墨點分析、SDS-聚丙烯醯胺凝膠電泳、非·變性之凝膠電泳、高效液體層析（HPLC)分離、免疫沉澱法及/或諸如〇ΝΑ結合凝膠移轉測定之類的活性測定。如果已經將VGF多肽設計成由宿主細胞分泌，則可在細胞培養基中發現大部份的多肽。然而，如果該VGF多肽並未從宿主細胞中分泌，則它將出現在細胞質及/或核中（就眞核生物宿主細胞而言），或是在細胞溶質中（就革蘭氏陰性細菌的宿主細胞而言）。關於位在宿主細胞之細胞質及/或核中（就眞核生物宿主細胞而言），或是在細胞溶質中（就細菌的宿主細胞而言）的 VGF多肽，可使用熟諳此藝者已知的任何標準技術，從宿主細胞中萃取細胞内的物質（包括革蘭氏陰性細菌的包涵體 )。例如，可藉著法式壓榨（Frence Press)、均質化作用及/ 或超聲波振盪，接著離心，使宿主細胞溶解，釋、放出胞漿周圍/細胞質的内容物。如果VGF多肽已經在細胞溶質中形成包涵體，通常包涵體可與内及/或外側的細胞膜結合，並因此將在離心之後， -46- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319Acad. Sci. U.S.A. 97: 4216_20), human embryonic kidney (HEK) 293 or 293 T cells, or 3 T 3 cells. Selection of appropriate mammalian host cells, as well as methods of transformation, culture, expansion, screening, product production, and purification, are known in the art. Other suitable mammalian cell lines are the monkey COS-1 and COS-7 cell lines, and the CV-1 cell line. More representative mammalian host cells, including primate cell lines and carious cell lines, include transformed cell lines. Normal diploid cells, derived from cell lines of the original tissue culture in vitro, as well as the original graft, are also suitable. Candidate cells may be genotyped, defective in the selection gene, or may contain a dominant gene for selection. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2 A cells, HeLa, mouse L-929 cells, 3 T3 cell lines derived from Swiss, Balb_c or NIH mice, BHK or HaK hamster cells. Strain. Each of these cell lines is known to and is available to those skilled in the art of protein expression. As useful as a suitable host cell is a bacterial cell. For example, in the context of biotechnology, it is well known that various strains of large intestine-like bacteria (e.g., HB101, DH5 Han, DH10, and MC1061) can be used as host cells. In this method, various strains of B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp., and Its analogues. Yeast cells of many strains known to the art are also useful as host cells for expressing VGF polypeptides. Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris. -44- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) ------------------- Order -------- - (Please read the notes on the back and fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Print A7 B7 V. Instructions for Invention (42) Alternatively, you can use the insect cell system to perform where you want. VGF polypeptide. In, for example, Kitts et al, 1993, Biotechniques, 14: 810-17; Lucklow, 1993, Curr. Opin. Biotechnol. 4: 564-72; and Lucklow et al, 1993, J. Virol., 67: 4566-79 This type of system is described. Preferred insect cells are Sf-9 and Hi5 (Invitrogen). Gene-transformed animals can also be used to express glycosylated VGF polypeptides. For example, a gene-transferred lactating animal (e.g., cow or goat) can be used and the glycosylated polypeptide can be obtained in the milk of an animal. Plants can also be used to produce VGF polypeptides. However, glycosylation typically occurring in plants differs from that produced in mammalian cells and may result in glycosylated products that are unsuitable for human therapeutic use. Production of Polypeptides Host cells, including VGF polypeptide expression vectors, can be cultured using standard media well known to those skilled in the art. The medium will usually contain all the nutrients needed for cell growth and survival. Suitable mediums for cultivating E. coli include, for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitable media for culturing scorpion nucleated cells include Roswell Park Memorial Institute medium 1640 (RPMI 1640), Minimal Essential Medium (MEM), and/or Dulbecco's Modified Eagle Medium (DMEM), all according to the particular cell line being cultured. Need to supplement blood, clear and / or growth factors. A medium suitable for insect culture is Grace's medium supplemented with yeast yeast lysate, whey protein hydrolysate, and/or fetal bovine serum as needed. Usually, the addition can be used to select cells that have undergone transfer infection or transformation -45- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -------------- -----^--------- (Please read the notes on the back and fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Print A7 B7 V. Inventions (43) Antibiotics Or other compounds, as an additive to the medium. The compound used will be designated by an optional marker element that appears on the plastid used to transform the host cell. For example, if the selectable marker element is oxytocin resistance' then the compound added to the medium will be oxytetracycline. Other compounds for selective growth include ampicillin, tetracycline, and neomycin. The amount of VGF polypeptide produced by the host cell can be assessed using standard methods known in the art. Such methods include, but are not limited to, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, high performance liquid chromatography (HPLC) separation, immunoprecipitation, and/or hydrazine binding. Activity assays such as gel transfer assays. If the VGF polypeptide has been designed to be secreted by the host cell, most of the polypeptide can be found in the cell culture medium. However, if the VGF polypeptide is not secreted from the host cell, it will appear in the cytoplasm and/or nucleus (for the nucleus host cell) or in the cytosol (as in the case of Gram-negative bacteria) For host cells). For VGF polypeptides located in the cytoplasm and/or nucleus of the host cell (in the case of a sputum host cell), or in the cytosol (in terms of the host cell of the bacterium), known to those skilled in the art Any standard technique for extracting intracellular material (including inclusion bodies of Gram-negative bacteria) from host cells. For example, the host cell can be lysed by the French press, homogenization and/or ultrasonic vibration, followed by centrifugation to release and release the cytoplasmic/cytoplasmic contents. If the VGF polypeptide has formed inclusion bodies in the cytosol, usually the inclusion bodies can bind to the inner and/or outer cell membranes, and therefore will be applied to the Chinese National Standard (CNS) A4 specification after centrifugation. X 297 mm) -------------------- Order --------- (Please read the notes on the back and fill out this page) 1278319

五、發明說明（44) 經濟部智慧財產局員工消費合作社印製發現主要在小球狀的物質中。然後在pIi値兩端，劑的存在下，像是在驗性pH値下爲二疏蘇糖醇，或在酸性 PH値下爲如8羧乙基膦，以諸如清潔劑 '胍、胍衍生物、尿素或尿素衍生物之類的促溶劑處理該小球狀的物質，以便釋放、打破分開，並使該包涵體溶解。然後可使用凝膠电永、免疫沉澱法或其類似者，分析溶解的vgf多肽。如果想要分離VGF多肽，可使用標準方法，像是在本文中和在Mam〇n等人，！ 990, Meth Enz，〗82 · 264_75中描述的那些，來完成分離作用。在一些案例中，VGF多肽在分離時可能不具有生物活性。可使用各種’’再折疊”或將多肽轉變爲其三級結構，並產生二硫鍵的方法，使生物活性復原。這類方法包括使溶解的多肽暴露在大约7的？11値下，並在特定濃度之促溶劑的 2在下。促溶劑的選擇與用來溶解包涵體之方法的選擇非常類似，但通常使用較低濃度的促溶劑，且不一定要與溶解作用所使用的促溶劑一樣。在大部份的案例中，再折疊/ 氧化作用的溶液亦將含有還原劑，或按特定比例之還原劑加=氧化形式，產生特殊的氧化還原電位，容許在形成蛋白質的半胱胺酸橋時，發生二硫鍵的洗牌。一些常用的氧化還原對偶，包括半胱胺酸/胱胺、穀胱甘肽（GSH)/二硫雙 GSH、氯化銅、二硫蘇糖醇（DTT)/二嘍烷dTt，以及2,2-巯基乙醇（bME)/二硫-b(ME)。在許多例子中，可以使用或可把而要共溶劑，以便增加再折疊的效率，而爲了此一目的，較g用的試劑包括甘油、各種分子量的聚乙二醇、精 -47- 本紙張尺度適财_家標準（CNS)A4規格⑽x 297公髮） -------訂--------- (請先閱讀背面之注咅？事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（45) 胺酸及其類似物。如果在表現VGF多肽時，未形成明顯程度的包涵體，此時將主要在離心細胞勻漿之後的上清液中發現多肽。可從上清液中，使用諸如在本文中描述的那些之類的方法，進一步地分離多肽。可使用各種技術，完成從溶液中純化VGF多肽。如果已經合成多肤，使其在它的瘦基或胺基·終端含有諸如7^組胺酸（VGF多肽/六His)之類的標籤，或其他小的肽，像是 FLAG(Eastman Kodak Co.，New Haven，CT)或 myc(Invitrogen， Carlsbad，CA)，便可在一-步法中，藉著使該溶液通過其中管柱基質對該標籤具有高度親和力的親和力管柱來純化之。例如，聚組胺酸以高親和力和專一性與鎳結合。因此，可使用鎳的親和力管柱（像是Qiagen®鎳管柱）來純化VGF多肽/ 聚 His。參見，例如Current Protocols in Molecular Biology §10.11.8(Ausubel等人，編輯，Green Publishers Inc.和 Wiley and Sons 1993)。此外，可經由單株抗體的使用來純化VGF多肽，該單株抗體能夠專一地認出並與VGF多肽結合。在較好是部份或完全純化VGF多肽，使得它部份或實質上不含污染物之處，可使用熟諳此藝者已知的標準方法。這類方法包括’但不限於藉著電泳分離，接著電洗脱、各種類型的層析（親和力、免疫親和力、分子篩和離子交換）、HPLC和製備等電焦聚（n Is〇prime"機械/技術，H〇efer Scientific，San Francisco，CA)。在一些案例中。可混合二或 -48 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公复） ----1---------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 A7 B7 五、發明說明（46) 多個純化技術，達到增加的純度。 (請先閱讀背面之注意事項再填寫本頁) 亦可藉著化學合成方法（像是固相肽合成），使用此項技藝中已知的技術，像是由Merrifield等人，1963，】.八111· Chem. Soc· 85 : 2149 ; Houghten等人，1985，Proc. Natl. Acad· Sci. USA 82 : 5132，以及 Stewart 和 Young, Solid Phase Peptide Synthesis(Pierce Chemical Co. 1984)陳述的那些，來製備VGF多肽。可合成在其胺基·終端有或無甲硫胺酸的這類多肽。可使用在這些參考文獻中陳述的方法，將化學合成的VGF多肽氧化，形成二硫橋。預期化學合成的VGF多肽具有與以重組方式產生，或從天然來源中純化之相對應 VGF多肽可相比擬的生物活性，並因此可與重組或天然的 VGF多肽交替使用。其他獲得VGF多肽的方法是經由從生物試樣中純化，像是其中天然發現VGF多肽的起源組織及/或液體。可使用如同在本文中描述之蛋白質純化的方法，來經營這類純化作用。在純化期間，可監視VGF多肽的出現，例如，使用對抗重組產製之VGF多肽或其肽片段而製備出的抗體。經濟部智慧財產局員工消費合作社印製許多產製多肽的其他方法，是此項技藝中已知的，並可使用這些方法來產製對VGF多肽具有專一性的多肽。參見，例如Roberts等人，1997, Proc. Natl· Acad. Sci· U.S.A. 94 : 12297-303，其描述在mRNA與其編碼之肽之間，融合蛋白質的產製。亦參見Roberts，1999, Curr· Opin· Chem· Biol. 3 : 268-73 。美國專利第5,763，192號；第5,814,476號；第5,723,323號和 -49- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（47) 第5,817,483號，描述了產製肽或多肽的方法。這藉著產製機會基因或其片段來進行，然後將這些基因導入宿主細胞中，其產生一或多個由該機會基因編碼的蛋白質。然後筛選宿主細胞，確認產生具有想要活性之肽或多肽的那些Z 種系。其他產製肽或多肽的方法，描述在Athersys，lnc.提出的 PCT/US98/20094(W099/15650)中。已知爲”爲了基因發現的基因表現之隨機激活作用（Random Activation of Gene Expression for Gene Discovery)"(RAGE-GD)，該方法藉著就地重組方法，涉及内源性基因表現的激活，或基因的過度_ 表現。例如，藉著將調節序列整合到標靶細胞中，激活或增加内源性基因的表現，該標靶細胞能夠藉著非-同種的或達法的重組作用，激活基因的表現。首先使標靶DNA接受照射’並***遺傳的啓動基因。啓動基因最後設置在基因前面的破損之處，發動基因的轉錄。結果導致想要之肽或多月太的表現。將知曉亦可使用這些方法來產生總括性的IL -1 7類似蛋白質表現庫，隨後可在各種測定中，像是生化測定、細胞測定和整個生物體的測定（例如植物、老鼠等等），用來進行高輸出入總量的表現型篩選。合成熟諳此藝者將知曉在本文中描述的VGF多肽分子，可藉著重組及其他方法產製。選擇性結厶劑 -50 - 本紙張尺度適用中國國家標準（CNS)A4規格（21G x 297公髮） —----------------1--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 A7 B7V. INSTRUCTIONS INSTRUCTIONS (44) Printed by the Consumers' Cooperatives of the Intellectual Property Office of the Ministry of Economic Affairs. It was found mainly in small spherical matter. Then, at both ends of the pIi値, in the presence of a reagent, such as di-threitol at an illustrative pH, or at a acidic pH, such as 8 carboxyethylphosphine, derived from a detergent such as 胍, 胍The spheroidal substance is treated with a solubilizing agent such as a urea or a urea derivative to release, break apart, and dissolve the inclusion body. The dissolved vgf polypeptide can then be analyzed using gel electrophoresis, immunoprecipitation or the like. If you want to isolate VGF peptides, use standard methods, as in this article and in Mam〇n et al. 990, Meth Enz, 〗 82 82 264_75 to complete the separation. In some cases, the VGF polypeptide may not be biologically active upon isolation. Various methods of 'refolding' or converting a polypeptide into its tertiary structure and producing a disulfide bond can be used to restore biological activity. Such methods include exposing the solubilized polypeptide to about 7 値 11 ,, and At a specific concentration of the solvent 2, the choice of the solubilizing agent is very similar to the method used to dissolve the inclusion body, but usually a lower concentration of the solubilizing agent is used, and does not have to be the same as the solubilizing agent used for the dissolution. In most cases, the refolding/oxidizing solution will also contain a reducing agent, or a specific proportion of reducing agent plus oxidized form, to produce a specific redox potential that allows for the formation of protein-containing cysteine. When bridged, the shuffle of disulfide bonds occurs. Some common redox couples include cysteine/cystamine, glutathione (GSH)/disulfide GSH, copper chloride, dithiothreitol ( DTT) / dioxane dTt, and 2,2-mercaptoethanol (bME) / disulfide-b (ME). In many cases, a co-solvent may be used or may be added to increase the efficiency of refolding, and For this purpose, the reagent kit for g Glycerin, polyethylene glycol of various molecular weights, fine-47- This paper scale is suitable for wealth _ family standard (CNS) A4 specifications (10) x 297 metric) ------- order --------- ( Please read the note on the back? Please fill out this page. 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. Description of Invention (45) Aminic acid and its analogues. If the VGF polypeptide is not formed, it is not formed. A significant degree of inclusion bodies, at which point the polypeptide will be found primarily in the supernatant after centrifugation of the cell homogenate. The polypeptide may be further isolated from the supernatant using methods such as those described herein. Purification of the VGF polypeptide from solution using a variety of techniques. If the polypeptide has been synthesized, it contains a label such as 7^ histidine (VGF polypeptide/six His) at its lean or amine terminal. Other small peptides, such as FLAG (Eastman Kodak Co., New Haven, CT) or myc (Invitrogen, Carlsbad, CA), can be used in a one-step process by passing the solution through a column matrix The tag has a high affinity affinity column to purify it. For example, poly group The acid binds to nickel with high affinity and specificity. Therefore, a nickel affinity column (such as a Qiagen® nickel column) can be used to purify the VGF polypeptide/polyHis. See, for example, Current Protocols in Molecular Biology §10.11.8 ( Ausubel et al., ed., Green Publishers Inc. and Wiley and Sons 1993). In addition, VGF polypeptides can be purified via the use of monoclonal antibodies that are specifically recognized and bind to VGF polypeptides. Where it is preferred to partially or completely purify the VGF polypeptide such that it is partially or substantially free of contaminants, standard methods known to those skilled in the art can be used. Such methods include, but are not limited to, separation by electrophoresis followed by electroelution, various types of chromatography (affinity, immunoaffinity, molecular sieves, and ion exchange), HPLC, and preparative isoelectric focusing (n Is〇prime" Technology, H〇efer Scientific, San Francisco, CA). In some cases. Can be mixed two or -48 - This paper scale is applicable to China National Standard (CNS) A4 specification (210 X 297 public) ----1--------------- order--- ------ (Please read the notes on the back and fill out this page) 1278319 A7 B7 V. INSTRUCTIONS (46) Multiple purification techniques to achieve increased purity. (Please read the notes on the back and fill out this page.) Chemical synthesis methods (such as solid phase peptide synthesis) can also be used, using techniques known in the art, such as by Merrifield et al., 1963, ed. VIII 111· Chem. Soc·85: 2149; Houghten et al., 1985, Proc. Natl. Acad. Sci. USA 82: 5132, and those stated by Stewart and Young, Solid Phase Peptide Synthesis (Pierce Chemical Co. 1984), To prepare a VGF polypeptide. It is possible to synthesize such polypeptides with or without methionine at their amine groups. The chemically synthesized VGF polypeptide can be oxidized using the methods set forth in these references to form a disulfide bridge. Chemically synthesized VGF polypeptides are expected to have comparable biological activities as corresponding VGF polypeptides produced recombinantly or purified from natural sources, and thus can be used interchangeably with recombinant or native VGF polypeptides. Other methods of obtaining VGF polypeptides are via purification from biological samples, such as tissues and/or liquids from which the VGF polypeptide is naturally found. Such purification can be carried out using methods such as protein purification as described herein. During purification, the appearance of a VGF polypeptide can be monitored, for example, using an antibody prepared against a recombinantly produced VGF polypeptide or a peptide fragment thereof. Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperatives. Many other methods of producing polypeptides are known in the art and can be used to produce polypeptides that are specific for VGF polypeptides. See, for example, Roberts et al, 1997, Proc. Natl. Acad. Sci. U.S.A. 94: 12297-303, which describes the production of fusion proteins between mRNA and its encoded peptide. See also Roberts, 1999, Curr· Opin·Chem. Biol. 3: 268-73. US Patent Nos. 5,763,192; 5,814,476; 5,723,323 and -49- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. INSTRUCTIONS (47) No. 5,817,483, describes a method of producing a peptide or polypeptide. This is done by the production opportunity gene or a fragment thereof, which is then introduced into a host cell which produces one or more proteins encoded by the opportunistic gene. The host cells are then screened to confirm those Z lines that produce the peptide or polypeptide with the desired activity. Other methods of producing peptides or polypeptides are described in PCT/US98/20094 (W099/15650) by Athersys, lnc. Known as "Random Activation of Gene Expression for Gene Discovery" (RAGE-GD), which involves the activation of endogenous gene expression by in situ recombination methods, Or overexpression of a gene. For example, by integrating a regulatory sequence into a target cell, activating or increasing the expression of an endogenous gene, the target cell can be activated by a non-homologous or Dafa recombination action. The expression of the gene. First, the target DNA is irradiated and inserted into the genetic promoter. The promoter is finally placed at the break in front of the gene, and the transcription of the gene is initiated. The result is that the desired peptide or multi-month too. It is known that these methods can also be used to generate a library of generic IL-7 similar protein expressions, which can then be used in various assays, such as biochemical assays, cell assays, and assays for whole organisms (eg, plants, mice, etc.). To perform phenotypic screening of high total input and output. Those skilled in the art will be aware of the VGF polypeptide molecules described herein, which can be recombined and He produced the method. Selective Conjunctive Agent-50 - This paper scale applies to China National Standard (CNS) A4 specification (21G x 297 metric) —----------------1 --------- (Please read the notes on the back and fill out this page) 1278319 A7 B7

經濟部智慧財產局員工消費合作社印製五、發明說明（48) 選擇性結合劑••一詞意指對一或多個VGF多肽具有專一性的分子。適當的選擇性結合劑包括，但不限於抗體及= 衍生物、多肽和小分子。使用此項技藝中已知的方法來製備適當的選擇性結合劑。本發明代表性的VGF多肽選擇性結合劑，能夠與VGF多肽的特定部份結合，藉此抑制該多肽與VGF多肽受體結合。 ~ 杬原’’ 一詞意指能夠被選擇性結合劑結合的分子或分子的一部份，並額外地能夠使用於動物中，產生能夠與^抗原之抗原決定位結合的抗體。抗原可具有一或多個抗原決定位。 ”抗原決定位，，一詞意指在任何分子中，能夠被選擇性結合劑認出，並在該結合劑的一或多個抗原-結合區與其結合的部份。抗原決定位通常包括分子之具有化學活性的表面基團，像是胺基酸或糖側鏈，其具有特定的三度空間結構特徵，以及特定的電荷特徵。”抑制抗原決定位，，和，，中^抗原決定位” 一詞，意指當抗原決定位與選擇性結合劑結合時 ’導致含有抗原決定位之分子在活體内、在活體外或就地喪失其生物活性，較佳的是在活體内，包括vgf多肽與 VGF多肽受體的結合。、田在本文中使用抗原_結合區” 一詞時，意指選擇性結合劑的邵份，其含有與抗原產生交互作用的胺基酸殘基，並賦與該結合劑對抗原的專-性和親和力。較佳的是，抗原· 結合區將是老鼠來源的。在其他的具體實施例中，抗原_結合區可衍生自其他的動物物種，特別是諸如兔子、大鼠或 -51 - ^紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐)'--- -------------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperatives. V. INSTRUCTIONS (48) Selective binders •• The term means a molecule that is specific for one or more VGF polypeptides. Suitable selective binding agents include, but are not limited to, antibodies and = derivatives, polypeptides and small molecules. Suitable selective binders are prepared using methods known in the art. A representative VGF polypeptide selective binding agent of the invention is capable of binding to a particular portion of a VGF polypeptide, thereby inhibiting binding of the polypeptide to a VGF polypeptide receptor. The term "杬原" refers to a molecule or molecule that can be bound by a selective binding agent and can additionally be used in an animal to produce an antibody that binds to the epitope of the antigen. The antigen may have one or more antigenic positions. The term "antigenic epitope" means a moiety which, in any molecule, is recognized by a selective binding agent and binds to one or more antigen-binding regions of the binding agent. The epitope is usually comprised of a molecule. Chemically active surface groups, such as amino acids or sugar side chains, which have specific tertiary structural characteristics and specific charge characteristics." Inhibition of epitopes, and, The term, meaning that when an epitope binds to a selective binding agent, the molecule that contains the epitope is lost in vivo, in vitro or in situ, preferably in vivo, including vgf The binding of a polypeptide to a VGF polypeptide receptor. The term "antigen-binding region" as used herein, refers to a portion of a selective binding agent that contains an amino acid residue that interacts with an antigen and is assigned Specificity and affinity for the antigen with the binding agent. Preferably, the antigen binding region will be of mouse origin. In other embodiments, the antigen-binding region may be derived from other animal species, particularly such as rabbit, rat or -51 -^ paper scales applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). '--- -------------------Book --------- (Please read the notes on the back and fill out this page) 1278319

經濟部智慧財產局員工消費合作社印製五、發明說明（49) 倉鼠之類的«類。例如，本發明之選擇性結合劑的抗原_ 結合區’最好是衍生自對人類VGF多肽有專一性的#人類抗體。編碼這類非人_體之DNA的較佳來源，包括產製抗體的細胞株’像是-般稱爲雜種瘤的雜種細胞株。諸如抗體和抗體片段之類，與VGF多肽結合的選擇性結合劑，均在本發明的範園内。抗體可以是多株的，包括單專一性的多株的；單株的；重組的，·嵌合的·人類化的，像是CDR-移植的；人類的；單鏈的；及/或雙重專一性的；以及其片段、變體或衍生物。抗體片段包括抗體與在 VGF夕肽上之抗原決定位結合的那些部份。這類片段的實例包括藉著酵素切開全長之抗體而產生的Fat^aF(ab广片段。其他的結合片段包括藉著重組DNA技術產生的那些，像疋表現含有編碼抗體可變區之核酸序列的重組質體。多株抗體是衍生自以抗原免疫之動物的血清之抗體分子的異種族群。抗原爲能夠與抗體結合的分子或分子的一部份，其另外還能夠謗使動物產生能夠與該抗原之抗原決定位結合的抗體。抗原可具有一或多個抗原決定位。上文提到的專一性反應，意圖表示抗原將以高度選擇之方式，與其相對應的抗體反應’而不與可能由其他抗原唤起之大批的其他抗體反應。針對VGF多肤的多株抗體’通常在動物（例如兔子或老氣）中，藉著多次的皮下或腹腔内注射VGF多肽和佐劑來產製。將VGF多肽與在待免疫物種中爲免疫原性的载劑蛋白質偶聯可能是有用的，像是鎖孔緘血藍蛋白、血清、白蛋白 -52- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ------------------^訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 A7 B7 經濟部智慧財產局員工消費合作社印製五、發明說明（50) 、牛甲狀腺球蛋白或黃豆胰蛋白酶抑制劑。再者，亦可使用諸如明礬之類的凝集劑來促進免疫反應。在免疫之後，將動物採血，並測定血清之抗-VGF抗體力價。單株抗體含有實質上均質的，對抗原專一的抗體族群，該族群含有實質上類似的抗原決定位結合位置。這類抗體可以是任何類型的免疫球蛋白，包括〗gG、IgM、IgE、IgA 、GILD及其亞類型。可在活體外、就地或在活體内，培養產生本發明之單株抗體的雜種瘤。在活體内或就地產製高力價，是較佳的產製方法。可使用任何方法產製針對VGF多肽的單株抗體，其提供由在培養中之連續細胞株產製的抗體分子。適當之製備單株抗體的方法，包括Kohler等人，1975, Nature 256 ·· 495 - 9 7 的雜種瘤方法，以及人類Β·細胞雜種瘤方法（Kozbor，1984, J. Immunol. 133 : 3001 ; Brodeur等人，Monoclonal Antibody Production Techniques and Applications 51-63(Marcel Dekker， Inc.，1987)。本發明亦提供產製與VGF多肽起反應之單株抗體的雜種瘤細胞株。較佳的抗-VGF選擇性結合劑包括在活體内，以競爭方式抑制與人類VGF多肽結合的單株抗體，或實質上具有相同的專一性結合特徵之抗體，以及其片段或區域。可在 Harlow和 Lane，Antibodies : A Laboratory Manual(Cold Spring Harbor Laboratories, 1989) ; Current Protocols inMinistry of Economic Affairs, Intellectual Property Bureau, employee consumption cooperatives, printing, five, invention description (49) such as hamsters. For example, the antigen-binding region' of the selective binding agent of the present invention is preferably a human antibody derived from a human VGF polypeptide. A preferred source of DNA encoding such a non-human body, including a cell line producing antibodies, is a hybrid cell line that is generally referred to as a hybridoma. Selective binding agents, such as antibodies and antibody fragments, which bind to VGF polypeptides, are within the scope of the invention. Antibodies may be multi-strained, including single-species multiple plants; single-plant; recombinant, chimeric, humanized, like CDR-grafted; human; single-stranded; and/or double Specific; and its fragments, variants or derivatives. Antibody fragments include those that bind to the epitope on the VGF peptide. Examples of such fragments include Fat^aF (ab broad fragments) produced by excision of full length antibodies by enzymes. Other binding fragments include those produced by recombinant DNA techniques, such as sputum containing nucleic acid sequences encoding variable regions of antibodies. Recombinant plastids. Multiple antibodies are heterogeneous populations of antibody molecules derived from the serum of an animal immunized with an antigen. The antigen is part of a molecule or molecule that binds to the antibody and additionally enables the animal to An antigen-binding antibody of the antigen. The antigen may have one or more epitopes. The specific reaction mentioned above is intended to indicate that the antigen will react with its corresponding antibody in a highly selective manner' A large number of other antibody reactions that may be aroused by other antigens. Multiple antibodies against VGF polypeptides are typically produced by multiple subcutaneous or intraperitoneal injections of VGF polypeptides and adjuvants in animals such as rabbits or old air. It may be useful to couple a VGF polypeptide to a carrier protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum. Albumin-52- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) ------------------^ --- (Please read the note on the back and fill out this page) 1278319 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed five, invention instructions (50), bovine thyroglobulin or soybean trypsin inhibitor. An agglutinating agent such as alum can also be used to promote the immune response. After immunization, the animal is bled and the serum anti-VGF antibody titer is determined. The monoclonal antibody contains a substantially homogeneous, antigen-specific antibody population. , the population contains substantially similar epitope binding sites. Such antibodies can be any type of immunoglobulin, including gG, IgM, IgE, IgA, GILD, and subtypes thereof. Can be in vitro, in situ or In vivo, a hybridoma producing the monoclonal antibody of the present invention is cultured. It is a preferred method for producing a high-yield valence in vivo or in a real estate. Any method can be used to produce a monoclonal antibody against a VGF polypeptide. Provided by a continuous cell line in culture Body molecule. Suitable methods for preparing monoclonal antibodies, including the hybridoma method of Kohler et al., 1975, Nature 256 · 495 - 9 7 , and the human Β·cell hybridoma method (Kozbor, 1984, J. Immunol. 133 : 3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (Marcel Dekker, Inc., 1987). The present invention also provides hybridoma cell lines which produce monoclonal antibodies reactive with VGF polypeptides. Preferred anti-VGF selective binding agents include monoclonal antibodies that compete in a competitive manner with human VGF polypeptides, or antibodies that have substantially the same specific binding characteristics, as well as fragments or regions thereof. Available in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Current Protocols in

Immunology(Colligan等人，編輯，Greene Publishing Assoc, and Wiley Interscience，1993);以及 Muller，1988，Meth. -53- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -------------------^--------- (請先閱讀背面之注音？事項再填寫本頁) 1278319 Α7 Β7 五、發明說明（51)Immunology (Colligan et al., ed., Greene Publishing Assoc, and Wiley Interscience, 1993); and Muller, 1988, Meth. -53- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -- -----------------^--------- (Please read the phonetic on the back? Please fill in this page again) 1278319 Α7 Β7 5, invention description (51 )

Enzymol. 92 : 589-601中找到藉著競爭抑制作用來決定單株抗體專一性和親和力的較佳方法。 (請先閱讀背面之注意事項再填寫本頁) 為了用來做為治療劑，可修改本發明之單株抗體。一個具體實施例為”嵌合型抗體’’，其中重（H)及/或輕（L)鏈的一部份與在衍生自特定物種之抗體中的相對應序列是相同或同種的，或屬於特定的抗體類型或亞類型，而剩下的鏈（們）則是與在衍生自其他物種之抗體中的相對應序列是相同或同種的，或屬於其他的抗體類型或亞類型。亦包括這類抗體的片段，只要其顯示想要的生物活性即可。參見美國專利第 4,816,567號；Morrison等人，1985, Proc· Natl· Acad· Sci· 81 : 6851-55 ° 嵌合型抗體及其產製方法為此項技藝中已知的。參見 Cabilly等人，1984, Proc. Natl· Acad· Sci· U.S.A. 81 : 3273-77 ;Morrison 等人，1984，Proc. Natl. Acad. Sci. U.S.A. 81 : 6851_55 ; Boulianne 等人，1984，Nature 312 : 643-46 ; Neuberger 等人，1985，Nature 314 : 268-70 ; Liu等人，1987, Proc. Natl. Acad· Sci. U.S.A. 84 : 3439-43 ;以及 Harlow和 Lane j在前。經濟部智慧財產局員工消費合作社印製當在本文中使用”嵌合型抗體” 一詞時，包括單價的、二價的或多價的免疫球蛋白。單價的嵌合型抗體是二聚體 (HL)，藉著嵌合的Η鏈經由二硫橋與嵌合的L鏈結合而形成。二價的嵌合型抗體是四聚體（H2L2)，藉著兩個HL二聚體經由至少一個二硫橋結合而形成。亦可藉著例如使用聚集 CH區（例如從IgM Η鏈或//鏈），來產製多價的嵌合型抗體。 -54- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1278319 A7 五、發明說明（52 ) 本發明之嵌合型抗體亦可包括個別的Η及/或l免疫球蛋白鏈。較佳的嵌合型Η鏈包括衍生自對Vgf多肽有專一性之非-人類抗體之Η鏈的抗原·結合區，其至少連接人類η鏈C 區（CH)的一部份，像是CHi*ch2。較佳的嵌合型L鏈包括折生自對VGF多肽有專一性之非-人類抗體之l鏈的抗原·結合區，其至少連接人類L鏈C區（C L)的一部份。具有相同或不同可變區結合專一性之嵌合型Η鏈和L鏈的選擇性結合劑，亦可根據此項技藝中已知的方法，藉著適當地結合個別的多肽鏈來製備。參見，例如CurrentA preferred method for determining the specificity and affinity of a single antibody by competitive inhibition is found in Enzymol. 92: 589-601. (Please read the precautions on the back and fill out this page.) In order to be used as a therapeutic agent, the monoclonal antibodies of the present invention can be modified. A specific embodiment is a "chimeric antibody" wherein a portion of the heavy (H) and/or light (L) chain is the same or the same as the corresponding sequence in an antibody derived from a particular species, or It belongs to a specific antibody type or subtype, and the remaining strands are the same or the same as the corresponding sequences in antibodies derived from other species, or belong to other antibody types or subtypes. Fragments of such antibodies, as long as they exhibit the desired biological activity. See U.S. Patent No. 4,816,567; Morrison et al., 1985, Proc. Natl. Acad. Sci. 81: 6851-55 ° chimeric antibodies and Production methods are known in the art. See Cabilly et al., 1984, Proc. Natl. Acad. Sci. USA 81: 3273-77; Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 6851_55; Boulianne et al, 1984, Nature 312: 643-46; Neuberger et al, 1985, Nature 314: 268-70; Liu et al, 1987, Proc. Natl. Acad. Sci. USA 84: 3439-43; And Harlow and Lane j are in the front. Co-operative printing When the term "chimeric antibody" is used herein, it includes monovalent, bivalent or multivalent immunoglobulins. Monovalent chimeric antibodies are dimers (HL), by incorporation The conjugated guanidine chain is formed by binding to a chimeric L chain via a disulfide bridge. The bivalent chimeric antibody is a tetramer (H2L2) formed by the combination of two HL dimers via at least one disulfide bridge. Multivalent chimeric antibodies can also be produced by, for example, using an aggregated CH region (eg, from an IgM Η chain or//chain) -54- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210) X 297 mm) 1278319 A7 V. INSTRUCTIONS (52) The chimeric antibodies of the invention may also comprise individual purines and/or l immunoglobulin chains. Preferred chimeric chains include those derived from Vgf polypeptides. An antigenic binding region of a specific non-human antibody Η chain that binds at least a portion of the human η chain C region (CH), such as CHi*ch2. The preferred chimeric L chain includes a degenerate An antigen-binding region of a l-chain of a non-human antibody specific for a VGF polypeptide, which is linked at least to the human L chain C region (CL) Partially selective binding agents for chimeric Η and L chains having the same or different variable regions in combination with specificity may also be suitably combined with individual polypeptide chains according to methods known in the art. preparation. See, for example, Current

Protocols in Molecular Biology(Ausubel等人，編輯，Green Publishers Inc· and Wiley and Sons 1994)和 Harlow等人，在前。使用該方法，可將表現嵌合型H鏈（或其衍生物）之宿主細胞’與表現嵌合型L鏈（或其衍生物）的宿主細胞分開培養 ’並分別回收免疫球蛋白鏈，然後再連結。另外，亦可共同培養宿主細胞，並同時容許鏈在培養基中結合，接著回收已經組合好的免疫球蛋白。本發明亦提供選擇性結合劑的”衍生物”，像是其抗體、片段、區域或衍生物，該名詞包括由截短或經過修改之基因編碼的那些蛋白質，產生在功能上類似免疫球蛋白片段的分子物種。該修改包括，但不限於加入細胞毒性蛋白質之过傳序列密碼’像是植物和細菌的毒素。可從本發明任何的宿主細胞中產製片段和衍生物。另外，亦可使抗_vGF 選擇性結合劑，像是抗體、其片段或區域，在活體外與細胞毒性蛋白質或化合物結合，提供細胞毒性之抗-Vgf抗體 -55- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) ----- 訂---- 經濟部智慧財產局員工消費合作社印製 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（53) ，其將選擇性地殺死具有VGF多肽受體的細胞。適當的片段包括，例如Fab、Fab’、F ( ab，）2和F v。這些片段缺乏完整抗體的Fc片段，從循環中較迅速地清除，並可具有比完整抗體更少的非-專一性組織結合。參見Wahl等人，1983, J. Nucl· Med· 24 : 316-25。使用此項技藝中已熟知的方法，從完整的抗體中產製這些片段，例如藉著利用諸如木瓜蛋白酶（產生Fab片段）或胃蛋白酶（產生F ( abf) 2片段）之類酵素的蛋白水解切開作用。由本發明之單株抗體認出這些抗原-結合區及/或抗原決定位的身份，提供了產製額外之單株抗體所必需的資訊，其具有類似的結合特徵和治療或診斷之用途，與本申請案之具體實施例並行。在另一個具體實施例中，本發明之單株抗體爲”人類化’f 的抗體。將非-人類化抗體人類化的方法，爲此項技藝中已熟知的。參見美國專利第5,585,089號和第5,693,762號。一般而言，人類化之抗體具有一或多個從非人類之來源導入的胺基酸殘基。例如，可使用在此項技藝中描述的方法 (Jones等人，1986，Nature 321 : 522-25 ; Riechmann 等人， 1998，Nature 332 : 323-27 ; Verhoeyen等人，1988，Science 239 ·· 1534G6)，藉著以人類抗體的相對應區域取代至少一部份嚆齒類的互補性-決定區（CDR)，來進行人類化作用。創造抗體分子之抗原-結合區（也就是Fab或可變區片段）的重組DNA版本的技術，其繞過單株抗體的產製，亦包括在本發明的範圍内。在該技術中，可從得自免疫動物的免疫系統之細胞中，萃取抗體-專一性信使RNA分子，並轉錄 -56- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I---r---------------訂--------- ΑΨ (請先閱讀背面之注音？事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7___ 五、發明說明（54 ) 成cDNA。然後將該cDNA選殖到細菌表現系統中。適合實行本發明之這類技術的一個實例，使用具有前導序列的噬菌體λ載體系統，其引起已經表現的Fab蛋白質移動至近膜間隙（在細菌細胞膜和細胞壁之間），或待分泌。可迅速地產製，並針對與抗原結合的那些來篩選大量具有功能的Fab 片段。特別將這類VGF-結合分子（具有對vgf多肽之專一性的Fab片段），包括在在本文中使用的”抗體”一詞内。藉著將传自具有適當之抗原-專一性的老鼠抗體分子之基因’與得自具有適當之生物活性（像是能夠激活人類補體並調解ADCC)的人類抗體分子之基因接合在一起，發展出產製嵌合型抗體的技術，亦在本發明的範園内。M〇rris〇n等人，1984, Proc· Natl· Acad. Sci. U.S.A. 81 : 6851-55 ·，Neuberger 等人，1984, Nature，312 : 604-08。藉著該技術產製的選擇性結合劑’像疋抗體’亦在本發明的範圍内。將知曉本發明並不限於老鼠或大鼠的單株抗體；事實上，可使用人類抗體。可藉著使用人類的雜種瘤獲得這類抗體。參見 Cote 等人 ’ MOnoclonal Antibodies and Cancer Therapy，77(1985)。因此，與VGF多肽結合的完整人類抗體亦包括在本發明中。藉著以VGF抗原（可視需要與載劑結合 )免疫能夠在缺乏内源的免疫球蛋白產生之下，產製人類抗體之節目的基因轉殖動物（例如老鼠），來產製這類抗體。參見，例如 Jakobovits 等人，1993, Proc. Natl. Acad· Sci. U.S.A. 90 : 2551-55 ; Jakobovits 等人，1993，Nature 362 : 255_ 58 ; Bruggemann 等人，1993, Year inlmmuno. 7 : 33-40。 -57- 本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） 1·---^---------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 A7 B7 五、發明說明（55) (請先閱讀背面之注意事項再填寫本頁) 抗-遺傳性型（抗-Id)的抗體是認得獨特決定位的抗體， 4決足位通常與抗體之抗原-結合位置有關。可藉著以欲對其製備抗-1 d的單株抗體，免疫與該單株抗體之來源相同物種和基因型（例如老鼠品系）的動物，來製備抗d抗體。經過免疫的動物將認得，並藉著對這些遺傳性型決定位產生抗體（抗-Id抗體），對經過免疫之抗體的遣傳性型決定位產生反應。參見，例如美國專利第4,699,880號。抗-Id抗體亦可用來做為’’免疫原"，在另一個動物中謗發免疫反應，產生所謂的抗-抗-1 d抗體。抗-抗-1 d抗體在抗原決定位方面，可能與謗發抗-Id之原始的單株抗體相同。因此，藉著使用對抗單株抗體之遺傳性型決定位的抗體，可能確認出其他表現具有相同專一性之抗體的純種系。本發明亦包括與V G F多肽結合的人類抗體。使用能夠在缺乏内源的免疫球蛋白產生之下，產製人類抗體之節目的基因轉殖動物（例如老鼠），藉著以VGF抗原免疫（也就是具有至少六個相鄰的胺基酸），其可視需要與載劑結合，來產製這類抗體。參見，例如Jakobovits等人，1993，Proc. Natl.Protocols in Molecular Biology (Ausubel et al., ed., Green Publishers Inc. and Wiley and Sons 1994) and Harlow et al., supra. Using this method, a host cell expressing a chimeric H chain (or a derivative thereof) can be cultured separately from a host cell expressing a chimeric L chain (or a derivative thereof) and the immunoglobulin chain can be separately recovered, and then Connect again. Alternatively, the host cells can be cultured together, while allowing the chains to bind in the medium, followed by recovery of the already assembled immunoglobulins. The invention also provides "derivatives" of selective binding agents, such as antibodies, fragments, regions or derivatives thereof, which include those proteins encoded by truncated or modified genes that produce functionally similar immunoglobulins. The molecular species of the fragment. Such modifications include, but are not limited to, the addition of a cytotoxic protein to an over-sequence code' such as a toxin of plants and bacteria. Fragments and derivatives can be produced from any of the host cells of the invention. In addition, anti-vGF selective binding agents, such as antibodies, fragments or regions thereof, can be combined with cytotoxic proteins or compounds in vitro to provide cytotoxic anti-Vgf antibodies-55- This paper size is applicable to Chinese countries. Standard (CNS) A4 specification (210 X 297 mm) (Please read the note on the back and fill out this page) ----- Order---- Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative print 1278319 Ministry of Economics wisdom The Property Bureau Staff Consumer Cooperatives prints A7 B7 V. Inventive Note (53), which will selectively kill cells with VGF polypeptide receptors. Suitable fragments include, for example, Fab, Fab', F(ab,)2, and Fv. These fragments lack the Fc fragment of the intact antibody, are cleared more rapidly from the circulation, and may have less non-specific tissue binding than intact antibodies. See Wahl et al., 1983, J. Nucl. Med. 24: 316-25. These fragments are produced from intact antibodies using methods well known in the art, for example by proteolytic cleavage using enzymes such as papain (which produces Fab fragments) or pepsin (which produces F(abf)2 fragments). effect. Recognizing the identity of these antigen-binding regions and/or epitopes by the monoclonal antibodies of the invention provides information necessary for the production of additional monoclonal antibodies, which have similar binding characteristics and therapeutic or diagnostic uses, Specific embodiments of the present application are in parallel. In another embodiment, the monoclonal antibodies of the invention are "humanized" antibodies. Methods for humanizing non-humanized antibodies are well known in the art. See U.S. Patent No. 5,585,089 and No. 5,693,762. In general, humanized antibodies have one or more amino acid residues introduced from a non-human source. For example, the methods described in this art can be used (Jones et al., 1986, Nature). 321 : 522-25 ; Riechmann et al, 1998, Nature 332: 323-27; Verhoeyen et al, 1988, Science 239 · 1534G6), by replacing at least a portion of the caries with corresponding regions of human antibodies Complementarity-determination region (CDR) for humanization. Techniques for creating recombinant DNA versions of antigen-binding regions (ie, Fab or variable region fragments) of antibody molecules that bypass the production of monoclonal antibodies, Also included within the scope of the invention is the ability to extract antibody-specific messenger RNA molecules from cells of the immune system derived from immunized animals, and to transcribe -56- the paper scale applies to Chinese national standards (CNS) )A4 Grid (210 X 297 mm) I---r---------------Book--------- ΑΨ (Please read the phonetic on the back? This page) 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7___ V. Invention Description (54) cDNA formation. The cDNA was then cloned into a bacterial expression system. An example of such a technique suitable for practicing the present invention, A phage lambda vector system with a leader sequence is used which causes the already expressed Fab protein to move to the membrane proximal space (between the bacterial cell membrane and the cell wall), or to be secreted. It can be rapidly produced and screened for a large number of those bound to the antigen. Functional Fab fragments. In particular, such VGF-binding molecules (Fab fragments having specificity for vgf polypeptides) are included within the term "antibody" as used herein. By being passed from the appropriate antigen - The gene for the specific mouse antibody molecule' is combined with the gene derived from a human antibody molecule with appropriate biological activity (such as the ability to activate human complement and mediate ADCC) to develop a technique for producing chimeric antibodies. In this Ming Fan's Garden. M〇rris〇n et al., 1984, Proc·Natl·Acad. Sci. USA 81: 6851-55 ·, Neuberger et al., 1984, Nature, 312: 604-08. The selective binding agent 'like sputum antibody' is also within the scope of the invention. It will be appreciated that the invention is not limited to monoclonal antibodies to mice or rats; in fact, human antibodies can be used. Such antibodies can be obtained by using human hybridomas. See Cote et al. 'MOnoclonal Antibodies and Cancer Therapy, 77 (1985). Thus, intact human antibodies that bind to VGF polypeptides are also included in the present invention. Immunization with a VGF antigen (which can be combined with a carrier as needed) enables the production of such antibodies by gene-transforming animals (e.g., mice) that produce human antibody programs in the absence of endogenous immunoglobulin production. See, for example, Jakobovits et al, 1993, Proc. Natl. Acad. Sci. USA 90: 2551-55; Jakobovits et al, 1993, Nature 362: 255_58; Bruggemann et al, 1993, Year inlmmuno. 7: 33-40 . -57- This paper size is applicable to China National Standard (CNS) A4 specification (210 x 297 mm) 1·---^--------------- --- (Please read the notes on the back and fill out this page) 1278319 A7 B7 V. Inventions (55) (Please read the notes on the back and fill out this page) Anti-hereditary type (anti-Id) An antibody is an antibody that recognizes a unique determinant, and a 4 position is usually associated with the antigen-binding position of the antibody. An anti-d antibody can be prepared by immunizing an animal having the same species and genotype (e.g., a mouse strain) as the source of the monoclonal antibody by preparing a monoclonal antibody against -1 d. The immunized animal will recognize and produce an antibody (anti-Id antibody) to these hereditary determinants, which will respond to the deferred determinant of the immunized antibody. See, for example, U.S. Patent No. 4,699,880. Anti-Id antibodies can also be used as ''immunogens" to elicit an immune response in another animal to produce a so-called anti-anti-1 d antibody. The anti-anti-1 d antibody may be identical in epitope to the original monoclonal antibody against the anti-Id. Therefore, by using an antibody against the hereditary type of the monoclonal antibody, it is possible to identify other pure lines of antibodies which exhibit the same specificity. The invention also encompasses human antibodies that bind to a V G F polypeptide. A genetically modified animal (eg, a mouse) capable of producing a human antibody program in the absence of endogenous immunoglobulin production, immunized with a VGF antigen (ie, having at least six adjacent amino acids) It can be combined with a carrier as needed to produce such antibodies. See, for example, Jakobovits et al., 1993, Proc. Natl.

Acad. Sci. U.S.A· 90 : 2551-55 ; Jakobovits等人，1993, Nature 362 : 255-58 ; Bruggemann 等人，1993, Year in Immuno. 7 : 33 經濟部智慧財產局員工消費合作社印製。在一種方法中，藉著使内源的部位不能編碼其中的重和輕免疫球蛋白鏈，並將編碼人類重和輕鏈蛋白質的部位插入其基因組内，來產製這類基因轉殖的動物。然後使部份修改的動物，也就是具有比完全互補之修改更少的那動物些雜交，獲得具有所有想要之免疫系統修改的動物。 -58- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公董） 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7___ 五、發明說明（56 ) 在投予免疫原時，這些基因轉殖的動物產生帶有人類（而非例如老鼠的）胺基酸序列的抗體，包括可變區，其對這些抗原是免疫專一性的。參見pct申請案第PCT/US96/05928號和 PCT/US93/06926號。其他的方法描述在美國專利第 5,545,807 號、PCT 申請案第 PCT/US91/245 號和 PCT/GB89/01207號，以及歐洲專利出版物第546〇73Β1號和 546073 A1號中。亦可藉著在宿主細胞中表現重組的DNA，或藉著在如同本文描述之雜種瘤細胞中表現，來產製人類抗體。在另一個具體實施例中，亦可從噬菌體-展示庫中產製人類抗體（Hoogenboom等人，1991，J· Mol. Biol· 227 ·· 381 ; Marks 等人，1991，J. Mol· Biol· 222 : 581)。這些方法經由在絲狀嗟菌體的表面上展示抗體節目，模仿免疫選擇，並藉著噬菌體與選擇之抗原的結合來完成後續的選擇。在PCT 申請案第PCT/US98/17364號中描述了一種這類的技術，其描述使用這類方法，分離MPL-和msk-受體的高親和力與功说枯彳/1»的抗體。嵌合的、CDR移植和人類化的抗體，通常是藉著重組方法產製。將編碼該抗體的核酸導入宿主細胞中，並使用在本文中描述的，以及此項技藝中已知的材料和程序來表現之。在較佳的具體實施例中，在哺乳動物宿主細胞中產製抗體，像是CHO細胞。可藉著在宿主細胞中表現重組的 DNA，或藉著在如同本文描述之雜種瘤細胞中表現，來產製單株（例如人類）抗體。 -59- 本紙張尺度適用中國國家標準(CNS)A4規格（210 X 297公釐1 '-------------------訂--------- (請先閱讀背面之注音？事項再填寫本頁) 1278319 A7 B7 五、發明說明（57 ) (請先閱讀背面之注意事項再填寫本頁) 可在任何已知的測定方法中使用本發明之抗-VGF抗體，像是競爭性結合測定、直接和間接的三明治測定，以及免疫沉澱測定（Sola, Monoclonal Antibodies : A Manual of Techniques 147-158(CRC Press，Inc·，1987))，來檢測和定量 VGF多肽。抗體將以適合欲使用之測定方法的親和力，與 VGF多肽結合。爲了診斷的應用，在某些具體實施例中，可利用可檢測部份來標示抗-VGF抗體。可檢測部份可以是任一個能夠直接或間接產生可檢測信號的部份。例如，可檢測部份可以是放射性同位素，像是 3H、14C、32P、35S、125I、99Tc、ulIn 或67Ga ;螢光或化學發光化合物，像是螢光素異硫代氰酸酯、若丹明（rhodamine)或蟲螢光素；或酵素，像是驗性磷酸酶、卢-半乳糖甞酶或辣根過氧化酶（Bayer等人，1990, Meth. Enz., 184 : 138-63) 〇經濟部智慧財產局員工消費合作社印製競爭性結合測定依賴已標示之標準物（例如VGF多肽，或其在免疫學上具有反應性的部份）和受試試樣分析物（VGF 多肽）競爭與限制含量之抗-VGF抗體結合的能力。在受試試樣中VGF多肽的含量，與已經與抗體結合之標準物的含量成反比。欲協助決定已經結合之標準物的含量，通常抗體在競爭作用之前或之後是不溶的，使得與該抗體結合之標準物和分析物，可便利地從仍維持未結合之標準物和分析物中分離出來。三明治測定通常涉及兩個抗體的使用，其分別能夠與待檢測及/或定量之蛋白質不同的免疫原部份或抗原決定位結 -60- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）經濟部智慧財產局員工消費合作社印製 1278319 A7 B7 五、發明說明（58 ) 合。在二明治測定中’受試試樣的分析物通常與固定在固體支撑物上的第一個抗體結合，隨後第二個抗體與該分析物結合，因此形成不溶的三-份複合體。參見，例如美國專利第4,376,110號。第二個抗體本身可利用可檢測部份來標不（直接的三明治測定），或可使用以可檢測部份標示的抗_ 免疫球蛋白抗體來測量（間接的三明治測定）。例如，一種類型的三明治測定是酵素·連接的免疫吸附測定（ELISA)，在這種案例中，可檢測部份爲酵素。 %擇性結合劑，包括抗-VGF抗體，亦可用於在活體内的成像。可將以可檢測部份標示的抗體投予動物，較佳的是進入血流中，並測定在宿主中已標示之抗體的存在和位置。可利用在動物中，無論是藉著核磁共振、放射線學或其他此項技藝中已知的檢測方法可檢測到的任何部份來標示抗體。本發明之選擇性結合劑，包括抗體，均可用來做爲治療劑。這些治療劑通常是激動劑或拮抗劑，它們分別促進或降低至少一種VGF多肽的生物活性。在一個具體實施例中，本發明之拮抗劑抗體是能夠專一地與V(JF多肽結合，並能夠抑制或排除VGF多肽在活體内或在活體外之功能活性的抗體或其結合片段。在較佳的具體實施例中，選擇性結合劑，例如拮抗劑抗體，將抑制VGF多肽之功能活性至少大約5〇%，更佳的是至少大約8〇%。在另—個具體實施例中，選擇性結合劑可以是抗_VGF多肽抗體，其能夠與vgf 多肽的結合夥伴（配體或受體）進行交互作用，藉此在活體本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐 ----------11—.-------4^^--------^--------- C請先間讀背面Μ涑意事頊再填寫本貢) \ , -61 - 經濟部智慧財產局員工消費合作社印製 1278319 、發明說明（59 ) 蔽ΐί ir二制或㈣VGF多肤的活性。可藉著此項技 :、"師選測定，確認選擇性結合劑，包括激動劑和私杬劑抗-VGF多肽抗體。本發明亦關於包括VGF選擇性結合劑（像是抗體）和其他用 ’在生物試樣中檢測VGF多肽含量之試劑的工具組。這類試劑可包括可檢測標記、阻斷血清、陽性和陰性對照組試樣，以及檢測試劑。 j匕學衍生物可由热爾此藝者製備以化學方式修改之VGF多肽的衍生物，待到在本文中描述的揭示内容。以與_天然附接於該多月太上之为子的型式和位置不同的方式，修改Vgf多肽衍生物。衍生物可包括藉著刪除一或多個天然-附接之化學基團而形成的分子。可藉著共價附接一或多個聚合物，來修改 VGF多肽。例如，通常選擇水溶性的聚合物，使得其所附接的蛋白質不會在含水的環境中沉澱，像是生理學的環境。聚合物的混合物亦包括在適當聚合物的範圍内。較佳的是，爲了終產物製品的治療用途，該聚合物將是在藥學上可接受的。聚合物可以分別具有任何的分子量，並可以是分支或未分支的。聚合物通常分別具有在大約2kDa到大約lOOkDa之間的平均分子量（"大約” 一詞代表在水溶性聚合物的製品中，有些分子將比所陳述的分子量更重，有些則更輕）。每個聚合物的平均分子量最好是在大約5kDa到大約50kDa之間，更佳的是在大約12kDa到大約40kDa之間，而最佳的是在大 62 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） :I*---^---------------訂--------- Φ (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（60 ) 約20kDa到大約35kDa之間。適當的水溶性聚合物或其混合物，包匕括仁不限於N _键|士或0 _鍵結的碳水化合物、糖、蹲酸酷 … , ^ 酉曰聚乙二醇（PEG)(包括已經用來衍生蛋白質之PEG的形式，包括單 _ 坑氧基或芳氧基-聚乙二醇），單甲每；早甲虱基_聚乙二醇、葡聚醣（像是低分子量的葡聚醣，例如大外』久約6kD)、纖維素，或其他以碳水化合物爲基礎的聚合物，窄、甘 . 來_(N•乙晞基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚環氧丙烷/環氧乙烷丘聚物、聚氧乙晞化的多元醇（例如甘油）和聚乙缔醇。本發明亦包括雙重功能的交聯分子，其可用方』用來製備共價附接的 VGF多肽多聚體。一般而言，可在任何用來使蛋白質與已活化之聚合物分子反應的適當條件下，進行化學衍生作用。製備多肽之化學衍生物的方法，通常將包括下列步驟：（a)使多肽與已活化（聚合物分子（像是聚合物分子的反應性酯或醛衍生物），在藉此使VGF多肽附接到一或多個聚合物分子上的條件下反應；（b)獲得反應產物。將以已知的參數和想要的結果爲基礎’決定最適切的反應條件。例如，聚合物分子對蛋白質之比例越高，則附接之聚合物分子的百分比就越大。在一個具體實施例中，VGF多肽衍生物可在胺基-終端具有單一的聚合物分子邵份。參見，例如美國專利第5,234,784 號。可使用此項技藝中已知的任何聚乙二醇化反應，專一地完成多肽的聚乙二醇化作用。例如在下列的文獻中描述了 -63 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------------------訂--------- (請先閱讀背面之注咅？事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（61 ) 這類反應：Francis等人 ’ 1992，Focus on Growth Factors 3 : 4-10 ;歐洲專利出版物第154316號和第4〇1384號；以及美國專利第4，179,337號。例如，可經由與反應性聚乙二醇分子（或類似的反應性水溶性聚合物）的醯化反應或烷基化反應，如同在本文中的描述，完成聚乙二醇化作用。關於醯化作用’選出的聚合物應該具有單一的反應性酯基團。關於還原性坑基化作用，選出的聚合物應該具有單一的反應性酸基團。反應性的醛，例如是聚乙二醇丙醛，其爲對水穩定的’或其單_ C i 氧基或芳氧基衍生物（參見美國專利第 5,252,714號）。在另一個具體實施例中，可以化學方式將Vgf多肽與生物素偶聯。然後容許生物素/VGF多肽分子與抗生物素蛋白結合’導致四價的抗生物素蛋白/生物素/VGF多肽分子。 VGF多肽亦可與二硝基苯酚（DNp)或三硝基苯酚（TNp)共價結合，並使所得的共軛物與抗-DNP或抗-TNP_IgM—起沉殿，形成1 〇價的十體共軛物。通常’可藉著投予本發明之VGF多肽衍生物，包括在本文中描述有關於VGF多肽的那些，來減輕或調節病況。然而，在本文中揭示之VGF多肽衍生物與非-衍生的分子相比較’可能具有額外的活性，增加或減少的生物活性，或其他的特徵’像是增加或減少的半衰期。性之其他抽揚詷節劑的測定在一些場合，可能想要確認爲VGF多肽活性之抑揚調節劑的分子’也就是激動劑或拮抗劑。可使用一或多種篩選 -64- 本紙張尺錢财國國家標準（CNS)A4規格（210 X 297公爱） '----- 1·---^---------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 A7 B7 經濟部智慧財產局員工消費合作社印製五、發明說明（62 ) 測足，像疋在本文中描述的 ^ ^ P二’確$忍调卽VGF多肤之天然或合成的分子0可以在活w ^ 古外 < 万式或在活體内之方式，藉著注射，或藉著口服遞送、适、植入裝置或其類似物，來投予這類分子。受試分子”意指在評估調節丨^ Μ即（也洗疋增加或減少）VGF多肽活性之能力下的分子。最當 > 瑕$見的是，受試分子將直接與 VGF多肽產生交互作用。炊Acad. Sci. U.S.A. 90: 2551-55; Jakobovits et al., 1993, Nature 362: 255-58; Bruggemann et al., 1993, Year in Immuno. 7 : 33 Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative. In one method, a gene-transferred animal is produced by making the endogenous site unable to encode the heavy and light immunoglobulin chains therein and inserting portions encoding human heavy and light chain proteins into its genome. . The partially modified animal, i.e., the animal with fewer modifications than the fully complementary, is then hybridized to obtain an animal with all desired immune system modifications. -58- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 dongdong) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7___ V. Invention description (56) When investing in immunogens, these Gene-transformed animals produce antibodies with amino acid sequences of humans (rather than, for example, mice), including variable regions, which are immunospecific to these antigens. See PCT Application No. PCT/US96/05928 and PCT/US93/06926. Other methods are described in U.S. Patent No. 5,545,807, PCT Application Nos. PCT/US91/245, and PCT/GB89/01207, and European Patent Publication Nos. 546,73, and 546,073. Human antibodies can also be produced by expressing recombinant DNA in a host cell, or by expression in a hybridoma cell as described herein. In another embodiment, human antibodies can also be produced from phage-display libraries (Hoogenboom et al., 1991, J. Mol. Biol. 227 381; Marks et al., 1991, J. Mol. Biol. 222 : 581). These methods mimic the immune selection by displaying antibody programs on the surface of the filamentous fungus, and by subsequent combination of the phage and the selected antigen. One such technique is described in PCT Application No. PCT/US98/17364, which describes the use of such methods to isolate antibodies of the high affinity and function of the MPL- and msk-receptors. Chimeric, CDR-grafted and humanized antibodies are usually produced by recombinant methods. The nucleic acid encoding the antibody is introduced into a host cell and expressed using materials and procedures as described herein, as well as known in the art. In a preferred embodiment, antibodies, such as CHO cells, are produced in a mammalian host cell. A single (e.g., human) antibody can be produced by expressing recombinant DNA in a host cell, or by expressing it in a hybridoma cell as described herein. -59- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm 1 '------------------- order------- -- (Please read the phonetic on the back? Please fill out this page again) 1278319 A7 B7 V. Inventions (57) (Please read the notes on the back and fill out this page) You can use this in any known measurement method. Inventive anti-VGF antibodies, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Sola, Monoclonal Antibodies: A Manual of Techniques 147-158 (CRC Press, Inc., 1987)) The VGF polypeptide is detected and quantified. The antibody will bind to the VGF polypeptide with an affinity appropriate for the assay to be used. For diagnostic applications, in certain embodiments, a detectable moiety can be utilized to label the anti-VGF antibody. The detecting portion may be any portion capable of directly or indirectly generating a detectable signal. For example, the detectable portion may be a radioisotope such as 3H, 14C, 32P, 35S, 125I, 99Tc, ulIn or 67Ga; Or a chemiluminescent compound such as luciferin isothiocyanate, if Rhodamine or luciferin; or an enzyme such as an assay phosphatase, lu-galactosidase or horseradish peroxidase (Bayer et al., 1990, Meth. Enz., 184: 138-63) 〇 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperatives Print competitive binding assays rely on labeled standards (eg, VGF polypeptides, or immunologically reactive portions thereof) and test-like analytes (VGF peptides) The ability to compete with a restricted amount of anti-VGF antibody. The amount of VGF polypeptide in the test sample is inversely proportional to the amount of the standard that has been bound to the antibody. To assist in determining the amount of standard that has been bound, usually the antibody It is insoluble before or after the competition, so that the standards and analytes bound to the antibody can be conveniently separated from the standards and analytes that still remain unbound. Sandwich assays typically involve the use of two antibodies, It can be different from the immunogenic part or antigen-determining node of the protein to be detected and/or quantified -60- This paper scale is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) Ministry of Intellectual Property, Intellectual Property Bureau, Staff Consumer Cooperative, Printed 1278319 A7 B7 V. Inventive Note (58) In the second Meiji determination, the analyte to be tested is usually combined with the first antibody immobilized on a solid support. The second antibody then binds to the analyte, thus forming an insoluble three-part complex. See, for example, U.S. Patent No. 4,376,110. The second antibody itself can be labeled with a detectable moiety (direct sandwich assay) or can be measured using an anti-IG immunoglobulin antibody labeled as a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in which case the detectable moiety is an enzyme. % Selective binding agents, including anti-VGF antibodies, can also be used for imaging in vivo. The antibody labeled with the detectable moiety can be administered to the animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host can be determined. Any part that can be detected in an animal, whether by nuclear magnetic resonance, radiology or other detection methods known in the art, can be used to label the antibody. The selective binding agents of the invention, including antibodies, can be used as therapeutic agents. These therapeutic agents are typically agonists or antagonists which promote or reduce the biological activity of at least one VGF polypeptide, respectively. In a specific embodiment, the antagonist antibody of the present invention is an antibody or a binding fragment thereof which is capable of specifically binding to V (JF polypeptide) and capable of inhibiting or excluding the functional activity of the VGF polypeptide in vivo or in vitro. In a preferred embodiment, a selective binding agent, such as an antagonist antibody, will inhibit the functional activity of the VGF polypeptide by at least about 5%, more preferably at least about 8%. In another embodiment, The sex binding agent may be an anti-VGF polypeptide antibody that is capable of interacting with a binding partner (ligand or receptor) of the vgf polypeptide, thereby applying the Chinese National Standard (CNS) A4 specification (210 X 297) on a living paper scale.青----------11-.-------4^^--------^--------- C Please read the back Μ涑顼顼顼顼 ) ) \ \ \ \ \ \ \ \ \ \ \ \ 12 12 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 783 , " teacher-selected assays, confirming selective binding agents, including agonists and private anti-VGF polypeptide antibodies. The invention also relates to including VG F selective binding agents (such as antibodies) and other tools that use 'reagents for detecting VGF polypeptide content in biological samples. Such agents may include detectable labels, blocking serum, positive and negative control samples, And a detection reagent. The derivative of the chemically modified VGF polypeptide can be prepared by the artist, and the disclosure described herein is to be attached to the natural one over the moon. The Vgf polypeptide derivative is modified in a manner different for the type and position of the sub. Derivatives may include molecules formed by deleting one or more naturally-attached chemical groups. One or more may be attached by covalent attachment. a polymer to modify the VGF polypeptide. For example, a water-soluble polymer is usually selected such that the attached protein does not precipitate in an aqueous environment, such as a physiological environment. A mixture of polymers is also included. Within the scope of the polymer. Preferably, the polymer will be pharmaceutically acceptable for the therapeutic use of the final product article. The polymer may have any molecular weight, respectively, and may be Branched or unbranched. The polymers generally have an average molecular weight of between about 2 kDa and about 100 kDa, respectively. (The term "about" means that in a product of a water soluble polymer, some molecules will be heavier than the stated molecular weight, Some are lighter.) The average molecular weight of each polymer is preferably between about 5 kDa and about 50 kDa, more preferably between about 12 kDa and about 40 kDa, and most preferably at a size of 62 paper. China National Standard (CNS) A4 specification (210 X 297 mm): I*---^--------------- order --------- Φ (please Read the notes on the back and fill out this page. 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Print A7 B7 V. Invention Description (60) Between 20kDa and about 35kDa. Suitable water-soluble polymers or mixtures thereof, not limited to N _ bond | 士 or 0 _ bonded carbohydrates, sugar, citric acid cool ..., ^ 酉曰 polyethylene glycol (PEG) (including already The form of PEG used to derivatize proteins, including mono-peptidyloxy or aryloxy-polyethylene glycol), monomethyl-peri-methionyl-polyethylene glycol, dextran (like low molecular weight Portuguese) Glycans, such as large outside, about 6kD), cellulose, or other carbohydrate-based polymers, narrow, sweet, _ (N• acetylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, Polypropylene oxide/ethylene oxide latent polymer, polyoxyethylated polyol (such as glycerin) and polyethylene alcohol. The invention also encompasses dual function cross-linking molecules which can be used to prepare covalently attached VGF polypeptide multimers. In general, chemical derivatization can be carried out under any suitable conditions for reacting the protein with the activated polymer molecule. A method of preparing a chemical derivative of a polypeptide will generally comprise the steps of: (a) rendering the polypeptide with an activated molecule (such as a reactive ester or aldehyde derivative of a polymer molecule), thereby attaching the VGF polypeptide The reaction is carried out under conditions of one or more polymer molecules; (b) the reaction product is obtained. The optimum reaction conditions will be determined based on known parameters and desired results. For example, polymer molecules versus proteins The higher the ratio, the greater the percentage of polymer molecules attached. In one embodiment, the VGF polypeptide derivative may have a single polymer molecule at the amine-terminus. See, for example, U.S. Patent No. No. 5,234,784. The pegylation of the polypeptide can be specifically performed using any PEGylation reaction known in the art. For example, the following documents describe -63. The paper scale applies to the Chinese National Standard (CNS). A4 size (210 X 297 mm) -------------------- Order --------- (Please read the note on the back? Fill in this page) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperation Printed A7 B7 V. INSTRUCTIONS (61) Such reactions: Francis et al. '1992, Focus on Growth Factors 3: 4-10; European Patent Publications Nos. 154,316 and 4, 1384; and US Patent 4 No. 179,337. For example, PEGylation can be accomplished via a deuteration or alkylation reaction with a reactive polyethylene glycol molecule (or a similar reactive water soluble polymer) as described herein. The selected polymer should have a single reactive ester group for deuteration. For reductive pit-based, the selected polymer should have a single reactive acid group. Reactive aldehydes, for example, are poly Ethylene glycol propionaldehyde, which is water-stable or its mono-C i oxy or aryloxy derivative (see U.S. Patent No. 5,252,714). In another embodiment, the Vgf polypeptide can be chemically conjugated. Coupling with biotin. The biotin/VGF polypeptide molecule is then allowed to bind to avidin' resulting in a tetravalent avidin/biotin/VGF polypeptide molecule. The VGF polypeptide can also be combined with dinitrophenol (DNp) or Trinitrogen The phenol (TNp) is covalently bound, and the resulting conjugate is incubated with anti-DNP or anti-TNP_IgM to form a ten-valent conjugate of the ten-valent conjugate. VGF polypeptide derivatives, including those described herein with respect to VGF polypeptides, are used to alleviate or modulate conditions. However, the VGF polypeptide derivatives disclosed herein may have additional activity compared to non-derived molecules. Or reduced biological activity, or other characteristics 'like increased or decreased half-life. Determination of other sputum sputum agents in some cases, may want to be identified as a molecule of the inhibitor of VGF polypeptide activity' An agonist or antagonist. Can use one or more screens -64- This paper ruler money national standard (CNS) A4 specifications (210 X 297 public) '----- 1·---^--------- ------Set--------- (Please read the notes on the back and fill out this page) 1278319 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (62) Foot, like 疋二二 ' ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Such molecules are administered by oral delivery, suitable, implanted devices or the like. "Test molecule" means a molecule that is capable of assessing the ability to modulate (or also increase or decrease) the activity of a VGF polypeptide. Most is > 瑕$ see that the test molecule will interact directly with the VGF polypeptide. Function.炊

…、而，吓企圖包括間接調節VGF 多肽活性的受試分子，像是_荽 ^ 疋褙耆衫響VGF基因表現，或藉者與VGF多肽結合夥伴（例如受體或配體）結合。在一個且體實施例中，受試分子將以至少大約1〇、之親和力常數與VGF多肽結合，較佳的是大_.8μ，更佳的是大約1〇^ ，而最佳的是大約i(r1GM。 VGF多肽激動劑或拮抗劑可以是蛋白質、肽、碳水化合物、脂質或小分子量的分子，其與VGF多肽產生交互作用而調節其㈣。調節VGF多肽表現的分子包括與編碼vgf 多肽 < 核酸互補的核酸，或與指揮或控制VGF多肽表現之核酸序列互補的核酸，而其係擔任表現的反義調節者。一旦藉著與VGF多肽之交互作用確認出受試分子，便可進一步評估該分子增加或減少VGF多肽之活性的能力。可以數種格式完成受試分子與VGF多肽之交互作用的測量，包括以細胞爲基礎的結合測定、膜結合測定、溶液_相測定和免疫測定。一般而言，將受試分子與VGf多肽一起培養一段特定的時間，並藉著一或多種測量生物活性的測定，定出VGF多肽的活性。 65- 本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） 1·1 —— ·_---------------訂--------- $ (請先閱讀背面之注咅？事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（63) 亦可在免疫測定中使用多株或單株抗體，直接測定受試分子與VGF多肽的交互作用。另外，亦可在溶液和免疫測定中，使用含有如同在本文中描述之抗原決定位標籤的 VGF多肽之修改形式。在VGF多肽經由與結合夥伴（例如受體或配體）之交互作用，來展現生物活性的事件中，可使用各種在活體外的測定，測量VGF多肽對相對應之結合夥伴（像是選擇性結合劑、受體或配體）的結合作用。可使用這些測定，就其增加或減少VGF多肽與其結合夥伴結合的比例和/或程度的能力，來篩選受試分子。在一個測定中，將VGF多肽固定在微量滴定盤的孔中。然後可依序（按任一種次序）或同時將放射性標示的VGF多肽結合夥伴（例如碘化的VGF多肽結合夥伴）和受試分子加至孔中。在培養之後，沖洗該孔並進行放射性計數，使用閃爍計數器，定出結合夥伴與VGF多肽結合的程度。通常，將測試在一段濃度範圍内的分子，並爲了在結果評估中的精確性，可使用一系列缺少一或多個受試測定中之要素的對照組孔。該方法的另一種選擇涉及倒轉蛋白質的”位置"，也就是將VGF多肽結合夥伴固定在微量滴定盤的孔中，與受試分子和放射性標示之VGF多肽一起培養，並決定VGF多肽結合的程度。參見，例如Current Protocols in Molecular Biology,第 1 8 章（Ausubel等人，編輯，Green Publishers Inc.，and Wiley and Sons 1995) 〇除了放射性標示之外，亦可使VGF多肽或其結合夥伴與生物素結合，然後可使用連接酵素，像是辣根過氧化酶 -66 _ 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I.—_---------------^--------- (請先閱讀背面之注咅？事項再填寫本頁) 1278319 A7 B7 經濟部智慧財產局員工消費合作社印製五、發明說明（64 (HRP)或鹼性磷酸酶（AP)的鏈黴菌抗生物素蛋白，檢測生物素基化之蛋白質的存在，其可以比色的方式，或藉著瞄準鏈黴菌抗生物素的螢光來檢測。在將複合體與連接 hrp的酵素連接之鏈黴菌抗生物素蛋白一起培養之後，爲了檢測的目的，亦可使用針對VGF多肽或VGF多肽結合= 伴的抗體，並使其與生物素結合。口夕亦可藉著附接於瓊脂糖小珠、丙烯酸小珠，或其他類型的這類惰性固相受酶質上，來固定VGF多肽或VGF多肽結合夥伴。可將受酶質-蛋白質複合體放在含有互補蛋白質Z 受試化合物的溶液中。在培養之後，可藉著離心使小珠沉澱，並使用在本文中描述的方法，評估在VGF多肽及其結合夥伴之間的結合量。另外，亦可將受酶質-蛋白質複合體固定在管柱中，並使受試分子和互補的蛋白質通過該管柱。然後可使用在本文中描述的任何技術（例如放射性標示或抗體結合），評估在VGF多肽及其結合夥伴之間形成的複合體。用來確認在VGF多肽結合蛋白質與VGF多肽結合夥伴之間，增加或減少複合體形成之受試分子的其他在活體外的測定’是表面質粒基因組共振檢測器系統，像是BIAc〇re測定系統（Pharmacia，Piscataway，NJ)。按照製造者的指示使用 BIAc〇re系統。該測定基本上涉及VGF多肽或VGF多肽結合夥伴與位在檢測器中之塗覆葡聚醣的感應器晶片的共價結合。然後可將受試化合物與其他互補的蛋白質，同時或連續地注射到含有感應器晶片的小室内。可以在分子質量上 -67- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I---— 丨！！ -----—訂·! ----- (請先閱讀背面之注意事項再填寫本頁) 1278319..., and, in an attempt, include a test molecule that indirectly regulates the activity of a VGF polypeptide, such as a VGF gene, or a VGF polypeptide binding partner (e.g., a receptor or a ligand). In one embodiment, the test molecule will bind to the VGF polypeptide with an affinity constant of at least about 1 Torr, preferably greater than 8. 8 μ, more preferably about 1 μM, and most preferably about i (r1GM. VGF polypeptide agonist or antagonist may be a protein, peptide, carbohydrate, lipid or small molecular weight molecule that interacts with the VGF polypeptide to modulate it. (4) The molecules that regulate the expression of the VGF polypeptide include and encode a vgf polypeptide. < A nucleic acid complementary to a nucleic acid, or a nucleic acid complementary to a nucleic acid sequence that directs or controls the expression of a VGF polypeptide, which acts as an antisense regulator of expression. Once the test molecule is identified by interaction with the VGF polypeptide, Further assessing the ability of the molecule to increase or decrease the activity of the VGF polypeptide. Measurements of the interaction of the test molecule with the VGF polypeptide can be performed in several formats, including cell-based binding assays, membrane binding assays, solution-phase assays, and immunizations. In general, the test molecule is incubated with the VGf polypeptide for a specific period of time and the VGF is determined by one or more measurements of biological activity. Peptide activity 65- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 x 297 mm) 1·1 ——·_--------------- Order-- ------- $ (Please read the note on the back? Please fill out this page again) 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. Instructions for Invention (63) Can also be used in immunoassay Multi-strain or monoclonal antibodies directly measure the interaction of the test molecule with the VGF polypeptide. Alternatively, modifications of the VGF polypeptide containing the epitope tag as described herein may also be used in solution and immunoassays. In the event that a VGF polypeptide exhibits biological activity via interaction with a binding partner (eg, a receptor or a ligand), various in vitro assays can be used to measure the binding partner of the VGF polypeptide to the corresponding binding partner (eg, selective binding) Binding of agents, receptors or ligands. These assays can be used to screen for test molecules in terms of their ability to increase or decrease the ratio and/or extent of binding of a VGF polypeptide to its binding partner. In one assay, VGF is used. Polypeptide immobilized in a microtiter plate The radiolabeled VGF polypeptide binding partner (eg, an iodinated VGF polypeptide binding partner) and the test molecule can then be added to the well sequentially (in either order) or simultaneously. After incubation, the well is rinsed and processed. Radioactivity counting, using a scintillation counter, to determine the extent to which the binding partner binds to the VGF polypeptide. Typically, molecules will be tested over a range of concentrations, and for accuracy in the assessment of results, a series of missing one or more The control wells of the elements in the assay are determined. Another option for this method involves the "position" of the inverted protein, ie, the VGF polypeptide binding partner is immobilized in the well of the microtiter plate, with the test molecule and the radiolabeled VGF. The polypeptide is cultured together and determines the extent of VGF polypeptide binding. See, for example, Current Protocols in Molecular Biology, Chapter 18 (Ausubel et al., ed., Green Publishers Inc., and Wiley and Sons 1995). In addition to radiolabeling, VGF polypeptides or their binding partners and biotin can also be used. Combination, then connect enzymes, such as horseradish peroxidase-66 _ This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) I.—_--------- ------^--------- (Please read the note on the back? Please fill out this page again) 1278319 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (64 (HRP) or alkaline phosphatase (AP) streptavidin, detecting the presence of biotinylated proteins, which can be detected in a colorimetric manner or by fluorescence directed against streptavidin After culturing the complex with streptavidin linked to the hrp-binding enzyme, an antibody against VGF polypeptide or VGF polypeptide binding = can also be used for detection purposes and allowed to bind to biotin. Mouth can also be attached to agarose beads, The oleic acid beads, or other types of such inert solid phases, are enzymatically bound to immobilize the VGF polypeptide or VGF polypeptide binding partner. The enzyme-protein complex can be placed in a solution containing the complementary protein Z test compound. After incubation, the beads can be pelleted by centrifugation and the amount of binding between the VGF polypeptide and its binding partner can be assessed using the methods described herein. Alternatively, the enzyme-protein complex can be immobilized. In the column, the test molecule and the complementary protein are passed through the column. The technique formed between the VGF polypeptide and its binding partner can then be assessed using any of the techniques described herein (eg, radiolabeling or antibody binding). Complex. Other in vitro assays used to identify a test molecule that increases or decreases complex formation between a VGF polypeptide-binding protein and a VGF polypeptide binding partner' is a surface plasmid genomic resonance detector system, such as BIAc〇 Re assay system (Pharmacia, Piscataway, NJ). The BIAc〇re system was used according to the manufacturer's instructions. The assay essentially involves a VGF polypeptide or a VGF polypeptide. Covalent bonding of the partner to the dextran-coated sensor wafer located in the detector. The test compound and other complementary proteins can then be injected simultaneously or continuously into the chamber containing the sensor wafer. In terms of molecular mass -67- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) I---- 丨! ! ------订·! ----- (Please Read the notes on the back and fill out this page. 1278319

發明說明（65) 經濟部智慧財產局員工消費合作社印製的改變爲基礎，評估結合之互補蛋白質的含量，而該分予質量上的改變在物理學上與感應器晶片之塗覆葡聚醣的邊有關，並可藉著檢測器系統來測量分子質量上的改變。在一些案例中，可能想要一起評估二或多個受試化合物 ’其增加或減少在VGF多肽和VGF多肽結合夥伴之間形成複合體的能力。在這些案例中，可藉著同時或連續地將這類額外的受試化合物（們）加至第一個受試化合物中，輕易地修改在本文中陳述的測定。在該測定中剩下的步驟，仍如同在本文中的陳述。可有利地使用在活體外的測定，像是在本文中描述的那些，就對在VGF多肽和VGF多肽結合夥伴之間複合體形成的影響，來篩選大量的化合物。該測定可自動篩選在噬菌體展示、合成肽和化學合成庫中產製的化合物。亦可在細胞培養中，使用表現VGF多肽或VGF多肽結合夥伴的細胞和細胞株，來篩選增加或減少在VGF多肽和VGF 多肽結合夥伴之間形成複合體的化合物。可從任何哺乳動物中獲得細胞和細胞株，但較佳的將是得自人類和其他靈長類、犬或嚆齒類來源的。在受試分子的存在或缺乏之下，評估VGF多肽與在表面表現VGF多肽結合夥伴之細胞的結合’並藉著例如流動血球計數法（cytometry)，使用對 VGF多肽結合夥伴之生物素基化的抗體，決定結合的程度。可有利地使用細胞培養測定，進一步評估在本文描述的蛋白質結合測定中，得到正分數的化合物。亦可使用細胞培養來篩選藥物候選者的影響力。例如， -68- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）」1·---：----------------訂--------- f請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1278319 A7 ---------— B7 _^ 五、發明說明（66 ) 藥物候選者可減少或增加VGF基因的表現。在某些具體實施例中，可在使細胞培物養暴露在藥物候選者之下後，測量所產生之VGF多肽或VGF多肽片段的含量。在某些具體實施例中，可檢測藥物候選者對細胞培養物的實際影響。例如’特定基因的過度-表現，可能對細胞培養物具有特定的影響。在這類案例中，可測試藥物候選者增加或減少基因表現的能力，或其預防或抑制對細胞培養物之特定影響的能力。在其他的實例中，特殊代謝產物的產生，像是多肽的片段，可能導致或與疾病或病理狀況有關。在這類案例中’可測試藥物候選者在細胞培養物中減少這類代謝產物之產製的能力。内化的蛋白質可使用tat蛋白質序列（得自HIV)將蛋白質内化至細胞内。參見，例如Falwell等人，1994, Proc· Natl· Acad· Sci. U.S.A. 91 : 664-68。例如已經描述了 HIV tat蛋白質之1 1個胺基酸序列（Y-G_R_K-K-R_R-Q_R-R-R ;序列識別1 1號）（稱爲”蛋白質轉導功能部位”，或TATPDT)越過細胞的細胞質膜和核膜調節遞送。參見 Schwarze等人，1999, Science 285 : 1569-72 ;以及Nagahara等人，1998, Nat· Med· 4 : 1449-52。在這些程序中，製備可在腹腔内投藥之後貫穿組織的FITC-構築體 (FITC-標示之 G-G-G-G_ Y-G_R-K-K-R-R-Q-R-R-R ;序列識別 1 2號），並藉著螢光·激活的細胞分類（FACS)分析，檢測這類構築體與細胞的結合。以tat_ _ gal融合蛋白質處理細胞 ’將證實- gal的活性。在注射之後，可在許多組織中檢 -69- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐) I*---：---------------訂--------- Φ (請先閱讀背面之注咅？事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（67 ) 測這類構築體的表現，包括肝臟、腎臟、肺臟、心臟和腦組織。咸相信這類構築體經歷一些程度的伸展，以便進入細胞，並在進入細胞之後可能需要再折疊。因此，將知曉可使用tat蛋白質序列，内化想要的多肽至細胞内。例如，使用tat蛋白質序列，可以細胞内之方式投予VGF拮抗劑（像是抗-VGF選擇性結合劑、小分子、可溶性受體或反義寡核苷酸），而抑制VGF分子的活性。當在本文中使用"VGF分子” 一詞時，意指如同在本文中定義之VGF 核酸分子和VGF多肽兩者。在想要之處，亦可使用這類程序，將VGF多肽本身從内部投予細胞。亦參見Straus，1999, Science 285 : 1466-67 〇治療用途可使用本發明之VGF多肽和選擇性結合劑來治療、診斷、改善或預防許多急性和慢性的疾病或病況，包括在本文中提及的那些。 VGF相關性疾病、病況和障礙，包括肥胖、***、惡病質、飲食障礙、AIDS相關性複合症、代謝宄進之病況、機能尤進、活動性不足和胰島素產生過多。由不想要之VGF多肽含量引起或調節的其他疾病，亦包括在本發明的範圍内。不想要的含量包括過量的VGF多肽和在正常含量之下的VGF多肽。 VGF多肽組合物和投藥治療性組合物亦在本發明的範圍内。這類VGF多肽的醫藥組合物可包括在治療上有效含量的VGF多肽，與針對適 -70- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I.—^----------------訂-------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（68) 當之投藥形式而選擇的在藥學上或在生理學上可接受之調配劑混合。醫藥組合物可包括在治療上有效含量的一或多個VGF多肽選擇性結合劑，與針對適當之投藥形式而選擇的在藥學上或在生理學上可接受之調配劑混合。可接受的調配物質最好是在其所使用的劑量和濃度上，對接受者是無毒性的。醫藥組合物可含有修改、維持或保存例如該組合物之p Η 値、滲透性、黏性、澄清度、顏色、等張性、味道、無菌性、穩定性、解離或釋放速率、吸收或貫穿的調配物質。適當的調配物質包括，但不限於胺基酸（像是甘胺酸、穀胺醯胺、天冬醯胺、精胺酸或離胺酸）、抗微生物製劑、抗氧化劑（像疋抗壞血敗、亞硫酸鈉或亞硫酸氫鈉）、緩衝溶液（像是硼酸鹽、碳酸氫鹽、Tris- HC1、擰檬酸鹽、嶙酸鹽或其他的有機酸）、填充劑（像是甘露糖醇或甘胺酸）、螯合劑（像疋乙一胺四乙fe( EDT A))、錯合劑（像是咖啡驗、聚乙晞吡咯烷酮、/? _環糊精或羥丙基_卢-環糊精）、填料、單醣類、雙醣類和其他的碳水化合物（像是葡萄糖、甘露糖或糊精）、蛋白質（像是血清白蛋白、明膠或免疫球蛋白）、著色劑、調味劑和稀釋劑、乳化劑、親水性聚合物（像是聚乙晞吡咯烷酮）、低分子量的多肽、形成鹽的抗衡離子（像是鈉）、防腐劑（像是卞二甲羥銨、苯甲酸、水楊酸、乙基求硫代水楊酸鈉、苯乙醇、對羥苯甲酸甲酯、對羥苯甲酸丙醋、氯己定（chlorhexidine)、山梨酸或過氧化氫）、溶劑（像是甘油、丙二醇或聚乙二醇）、糖醇類（像是甘露糖醇或山梨糖 -71 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱） -I.---^---------------訂--------- ΜΨ (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（69) 醇）、懸浮劑、表面活性劑或濕潤劑（像是普魯卡因 (pluronics ; PEG ;脱水山梨糖醇酯；多乙氧基醚，像是多乙氧基醚20或多乙氧基醚80 ;三通；三甲醇氨基甲烷 (tromethamine);卵璘脂；膽固醇或泰洛沙泊（tyloxapal)、穩定性促進劑（像是蔗糖或山梨糖醇）、張力促進劑（像是鹼金屬鹵化物-較佳的是氯化鈉或钟-或甘露糖醇或山梨糖醇）、遞送媒劑、稀釋劑、賦形劑及/或藥學佐劑。參見 Remington’s Pharmaceutical Sciences(第 1 8 版，A.R. Gennaro 編輯，Mack Publishing Company 1990。將由熟諳此藝者依據，例如打算使用的投藥途徑、遞送格式和想要的劑量，來決定最適切的醫藥組合物。參見，例如 Remington’s Pharmaceutical Sciences，在前0 這類組合物可能影響VGF分子的物理狀態、穩定性、在活體内釋放的速率，以及在活體内清除的速率。在醫藥組合物中，主要媒劑或載劑的性質.，可以是含水的或不-含水的。例如，適合注射的媒劑或載劑可以是水、生理鹽水溶液或人造的腦脊髓液，可能補充在非經腸投藥之組合物中常見的其他物質。中性的緩衝生理鹽水或與血清白蛋白混合的生理鹽水，是較具代表性的媒劑。其他代表性的醫藥組合物包括大約pH 7. 0-8· 5的Tris緩衝溶液，或大約pH 4. 0 - 5 . 5的醋酸緩衝溶液，其更可包括山梨糖醇或適當的取代。在本發明的一個具體實施例中，可藉著將具有想要純度的選出之組合物與任意的調配劑（Remington’s Pharmaceutical Sciences，在前）混合成冷)東乾燥餅的形式， -72- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I.---.---------------訂--------- ΜΨ (請先閱讀背面之注音？事項再填寫本頁) 1278319Description of the invention (65) Based on the changes printed by the Intellectual Property Office of the Intellectual Property Office of the Ministry of Economic Affairs, the content of the complementary protein is evaluated, and the quality change is physically coated with the dextran of the sensor wafer. The edge is related and the detector system can be used to measure changes in molecular mass. In some cases, it may be desirable to assess together two or more test compounds' which increase or decrease the ability to form a complex between a VGF polypeptide and a VGF polypeptide binding partner. In these cases, the assays set forth herein can be readily modified by adding such additional test compounds (s) to the first test compound simultaneously or continuously. The remaining steps in this assay are still as set forth herein. It is advantageous to use in vitro assays, such as those described herein, to screen for a large number of compounds in terms of the effect of complex formation between the VGF polypeptide and the VGF polypeptide binding partner. This assay automatically screens for compounds produced in phage display, synthetic peptides, and chemical synthesis libraries. Cells and cell lines that exhibit VGF polypeptide or VGF polypeptide binding partners can also be used in cell culture to screen for compounds that increase or decrease the formation of a complex between the VGF polypeptide and the VGF polypeptide binding partner. Cells and cell lines can be obtained from any mammal, but preferably will be derived from humans and other primate, canine or carious sources. In the presence or absence of a test molecule, assessing the binding of a VGF polypeptide to a cell that exhibits a VGF polypeptide binding partner on the surface and using biotinylation of a VGF polypeptide binding partner by, for example, cytometry The antibody determines the extent of binding. Cell culture assays can be advantageously used to further evaluate compounds that give a positive fraction in the protein binding assays described herein. Cell culture can also be used to screen the influence of drug candidates. For example, -68- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)" 1·---:---------------- Order-- ------- f Please read the notes on the back and fill out this page.) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1278319 A7 ---------- B7 _^ V. Description of the Invention ( 66) Drug candidates can reduce or increase the performance of the VGF gene. In certain embodiments, the amount of VGF polypeptide or VGF polypeptide fragment produced can be measured after exposure of the cell culture to a drug candidate. In certain embodiments, the actual effect of the drug candidate on the cell culture can be detected. For example, 'excessive-expression of a particular gene may have a specific effect on cell culture. In such cases, the ability of a drug candidate to increase or decrease gene expression, or its ability to prevent or inhibit a particular effect on a cell culture, can be tested. In other instances, the production of specific metabolites, such as fragments of a polypeptide, may result in or be associated with a disease or pathological condition. In such cases, drug candidates can be tested for their ability to reduce the production of such metabolites in cell culture. Internalized Proteins Proteins can be internalized into cells using the tat protein sequence (derived from HIV). See, for example, Falwell et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91: 664-68. For example, the 11 amino acid sequence of the HIV tat protein (Y-G_R_K-K-R_R-Q_R-RR; sequence recognition No. 1) (referred to as "protein transduction functional site", or TATPDT) has been described as crossing the cell. The cytoplasmic membrane and nuclear membrane regulate delivery. See Schwarze et al, 1999, Science 285: 1569-72; and Nagahara et al., 1998, Nat. Med. 4: 1449-52. In these procedures, FITC-constructs (FITC-labeled GGG-G_Y-G_R-KKRRQRRR; sequence recognition No. 12) that can be inserted through the tissue after intraperitoneal administration are prepared and classified by fluorescence-activated cells. (FACS) analysis to detect the binding of such constructs to cells. Treatment of cells with the tat__gal fusion protein will confirm the activity of gal. After injection, it can be inspected in many tissues. -69- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) I*---:------------ ---Book--------- Φ (Please read the note on the back? Please fill out this page again) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (67) The performance of such structures includes liver, kidney, lung, heart and brain tissue. It is believed that such constructs undergo some degree of stretching in order to enter the cell and may need to be refolded after entering the cell. Thus, it will be appreciated that the desired polypeptide can be internalized into the cell using the tat protein sequence. For example, using a tat protein sequence, a VGF antagonist (such as an anti-VGF selective binding agent, a small molecule, a soluble receptor or an antisense oligonucleotide) can be administered intracellularly while inhibiting the activity of the VGF molecule. When the term "VGF molecule" is used herein, it is meant both VGF nucleic acid molecules and VGF polypeptides as defined herein. Where desired, such procedures can also be used to internalize the VGF polypeptide itself. Administration of cells. See also Straus, 1999, Science 285: 1466-67. Therapeutic uses The VGF polypeptides and selective binding agents of the invention can be used to treat, diagnose, ameliorate or prevent many acute and chronic diseases or conditions, including Those mentioned in this article. VGF-related diseases, conditions and disorders, including obesity, infertility, cachexia, eating disorders, AIDS-related complexes, hypermetabolic conditions, functional dysfunction, lack of mobility, and excessive insulin production Other diseases caused or modulated by unwanted VGF polypeptide content are also included within the scope of the invention. Unwanted amounts include excess VGF polypeptide and VGF polypeptide below normal levels. VGF polypeptide composition and administration Compositions are also within the scope of the invention. Pharmaceutical compositions of such VGF polypeptides may comprise a therapeutically effective amount of a VGF polypeptide, and This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) I.—^---------------- Order-------- (Please Read the following notes on the back and fill out this page.) 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Print A7 B7 V. Description of Invention (68) Pharmacologically or physiologically acceptable formulation selected as the form of administration The pharmaceutical composition may comprise a therapeutically effective amount of one or more VGF polypeptide selective binding agents, in admixture with a pharmaceutically or physiologically acceptable formulation selected for the appropriate pharmaceutical form. Preferably, the formulated formulation is non-toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions may contain modifications, maintenance or storage, for example, p Η , permeability, viscosity, Formulations for clarity, color, isotonicity, taste, sterility, stability, dissociation or release rate, absorption or penetration. Suitable formulations include, but are not limited to, amino acids (eg glycine, glutamine) Guanamine, aspartame, arginine or lysine) Antimicrobial agents, antioxidants (like sputum ascorbic acid, sodium sulfite or sodium bisulfite), buffer solutions (such as borate, bicarbonate, Tris-HC1, citrate, citrate or other organic Acid), filler (such as mannitol or glycine), chelating agent (such as 一乙-amine tetraethylene fe (EDT A)), complexing agent (like coffee test, polyethylpyrrolidone, /? _ ring Dextrin or hydroxypropyl _lu-cyclodextrin), fillers, monosaccharides, disaccharides and other carbohydrates (like glucose, mannose or dextrin), proteins (like serum albumin, gelatin or Immunoglobulins), colorants, flavorings and diluents, emulsifiers, hydrophilic polymers (such as polypyridyl pyrrolidone), low molecular weight peptides, salt-forming counterions (like sodium), preservatives (like Is dimethyl hydroxyammonium, benzoic acid, salicylic acid, ethyl thiosalicylate, phenylethyl alcohol, methyl paraben, propyl propyl chlorate, chlorhexidine, sorbic acid or Hydrogen peroxide), solvent (like glycerin, propylene glycol or polyethylene) Alcohol), sugar alcohols (like mannitol or sorbose-71 - this paper scale applies to China National Standard (CNS) A4 specifications (210 X 297 public) -I.---^------ ---------Book--------- ΜΨ (Please read the note on the back and fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention Description (69) alcohols, suspending agents, surfactants or humectants (like procaine (pluronics; PEG; sorbitan ester; polysorbate, like polysorbate 20 or more Oxyether 80; tee; tromethamine; egg yolk; cholesterol or tyloxapal, stability enhancer (like sucrose or sorbitol), tonicity enhancer (like Alkali metal halides - preferably sodium chloride or bell- or mannitol or sorbitol, delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants. See Remington's Pharmaceutical Sciences (18th edition, edited by AR Gennaro, Mack Publishing Company 1990. The most appropriate pharmaceutical composition will be determined by the skilled artisan, such as the route of administration, delivery format and intended dosage. See, for example, Remington's Pharmaceutical Sciences, compositions such as top 0 may affect the physical state of VGF molecules, stability, rate of release in vivo, and rate of clearance in vivo. In pharmaceutical compositions, the primary vehicle or The nature of the carrier may be aqueous or non-aqueous. For example, a vehicle or carrier suitable for injection may be water, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with a composition for parenteral administration. Other substances commonly found. Neutral buffered saline or physiological saline mixed with serum albumin is a more representative vehicle. Other representative pharmaceutical compositions include Tris at pH 7. 0-8·5. A buffer solution, or an acetic acid buffer solution of about pH 4.0-5.5, which may further comprise sorbitol or a suitable substitution. In one embodiment of the invention, the selected composition of the desired purity can be combined with any formulation (Remington's Pharmaceutical Sciences, prior) in the form of a cold dry east cake, -72-ben The paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). I.---.--------------- Order--------- ΜΨ (Please read the phonetic on the back first? Then fill in this page) 1278319

經濟部智慧財產局員工消費合作社印製戍含水的落液，來製備可儲存的VGF多肽組 p 亦可使用諸如蔗糖之類的適當賦形劑，以冷。此外，式調配VGF多肽產物。 7，粟乾燥物的形爲了非經腸遞送，可選擇VGF多肽醫藥組合物亦可爲了吸入或爲了經由消化道遞送，像是口服^握组合物。這類在藥學上可接受之組合物的製：：：藝的範圍内。 /、技凋配成份出現的濃度，爲投藥處可接受的。例如，用將組合物維持在生理學的pH値下，4在稍低之_値下，通常在從大約5到大約8之間PH値範圍内的緩衝溶液。當企圖非經腸投藥時，在本發明中使用之治療性組合物可以是不含熱原的、非經腸可接受的含水溶液形式，其包括在藥學上可接受之載劑中想要的VGF分子。特別適合非經肪注射的媒劑爲無菌的蒸餘水，在其中將VGf分子調配成無菌、等張的溶液，並適當地防腐。其他的製備可能涉及將想要的分子與諸如可注射之中心體、生物可腐蚀的顆粒、聚合的化合物（像是聚乳酸或聚甘醇酸）、小珠或微脂粒之類的製劑一起調配，提供經控制或持續釋放的產物，然後經由儲存處注射來遞送。亦可使用透明質酸，且其可能具有在循環中增進持續期間的作用。其他導入想要分子的適當方法’包括可植入的藥物遞送裝置。在一個具體實施例中，可調配吸入用的醫藥組合物。例如，可將VGF多肽調配成吸入用的乾粉。亦可爲了以氣溶膠遞送，將VGF多肽或核酸分子吸入溶液與推進劑一起調 -73- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ·---------------訂--------- (請先閲讀背面之注音？事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（7Ί ) 配。在另一個具體實施例中，可將溶液霧化。在PCT出版物第W0 94/20069號中進一步描述了肺臟投藥，其描述以化學方式修改之蛋白質的肺臟遞送。亦止圖口服投予特定的調配物。在本發明的一個具體實施例中，可將以此形式投藥的VGF多肽與或不與在傳統上用來調和諸如錠劑和膠囊之類固體劑量形式的那些載劑一起調配。例如，可將膠囊設計成在胃腸道中的某處釋放該凋配物之活性部份，此時發揮最大的生物利用性，並將前_ 全身性降解減至最少。可包含其他的製劑，以促進v多肽的吸收。亦可使用稀釋劑、調味劑、低熔點的蠟、植物油、潤滑劑、懸浮劑、錠劑崩解劑和黏合劑。其他的醫藥組合物可能涉及在帶有適合製造錠劑之無毒性賦形劑的混合物中，VGF多肽的有效量。藉著將錠劑$ 解於無菌的水中，或其他適當的媒劑中，可製備單位·劑量形式的溶液。適當的賦形劑包括，但不限於惰性稀釋劑，像是碳酸鈣、碳酸鈉或碳酸氫鈉，乳糖或磷酸鈣，或黏合劑，像是澱粉、明膠或***樹膠；或潤滑劑，像是1 酸鎂、硬脂酸或滑石。曰其他的VGF多肽醫藥組合物，對熟請此藝者將是明顯的，包括涉及在持續-或經控制-遞送之調配物中之VGF 的調配物。調配各種其他的持續或經控制.之遞送方二技術，像是微脂粒載劑、生物可腐蚀的微顆粒，的小珠和儲存處注射，亦爲熟諳此藝者已知的^ 如™嶋829，其描述用來遞送醫藥組合物之 ‘紙張尺度適用中關家標準（CNS)A4規格（210 X 297公髮 I.---.---------------訂--------- (請先閱讀背面之注音？事項再填寫本頁) -74 1278319 A7 B7 五、發明說明（72 ) 釋放的多孔隙聚合微顆粒。持續釋放之製品的額外實例包括以成形物品之形式的半透性聚合物材料，例如薄膜或微膠囊。持續釋放的材料可包括聚酯類、水膠體、聚交酯（美國專利第3,773,919號和歐洲專利出版物第〇58481號）、L·穀胺酸和r乙基-L_穀胺酸酯的共聚物（Sidman等人，1983, Biopolymers 22 : 547-56)、聚（2-羥乙基異丁烯酸酯）（Langer等人，1981, J· Biomed.The Ministry of Economic Affairs' Intellectual Property Office staff consumption cooperative prints 戍 aqueous drops to prepare a storable VGF polypeptide group p can also be cooled using appropriate excipients such as sucrose. In addition, the VGF polypeptide product is formulated. 7. Shape of millet dry for Veterinary delivery The VGF polypeptide pharmaceutical composition may also be selected for inhalation or for delivery via the digestive tract, such as an oral composition. Such formulations are within the scope of the pharmaceutically acceptable compositions. /, the concentration of the compound with the ingredients, is acceptable for the drug administration. For example, a buffer solution in the range of pH 之间 from about 5 to about 8 is maintained at a physiological pH of the composition, 4 at a slightly lower level. When attempted parenteral administration, the therapeutic composition for use in the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution, which is desired in a pharmaceutically acceptable carrier. VGF molecule. A vehicle which is particularly suitable for non-fat injection is sterile distilled water in which the VGf molecule is formulated into a sterile, isotonic solution and suitably preserved. Other preparations may involve the preparation of the desired molecule with a formulation such as an injectable centrosome, bioerodible particles, a polymeric compound (such as polylactic acid or polyglycolic acid), beads or vesicles. The formulation provides a controlled or sustained release product which is then delivered via injection at the reservoir. Hyaluronic acid can also be used, and it may have an effect of enhancing the duration during circulation. Other suitable methods for introducing the desired molecule' include implantable drug delivery devices. In a specific embodiment, a pharmaceutical composition for inhalation can be formulated. For example, a VGF polypeptide can be formulated into a dry powder for inhalation. Alternatively, in order to deliver by aerosol, the VGF polypeptide or nucleic acid molecule inhalation solution is adjusted with the propellant -73- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ·------ ---------Book--------- (Please read the phonetic on the back? Please fill out this page again) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention Description (7Ί) Match. In another embodiment, the solution can be atomized. Lung administration, which describes the lung delivery of a chemically modified protein, is further described in PCT Publication No. WO 94/20069. Also, the administration of specific formulations is also orally administered. In a particular embodiment of the invention, the VGF polypeptide administered in this form may or may not be formulated with those carriers conventionally used to modulate solid dosage forms such as lozenges and capsules. For example, the capsule can be designed to release the active portion of the ingestion somewhere in the gastrointestinal tract, where maximum bioavailability is achieved and pre-systemic degradation is minimized. Other formulations may be included to promote absorption of the v-polypeptide. Diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders can also be used. Other pharmaceutical compositions may involve an effective amount of a VGF polypeptide in a mixture with a non-toxic excipient suitable for the manufacture of a tablet. A solution in unit dose form can be prepared by dissolving the tablet in sterile water or other suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate, or binders such as starch, gelatin or gum arabic; or lubricants, such as 1 Magnesium, stearic acid or talc.曰 Other VGF polypeptide pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving VGF in a sustained- or controlled-delivered formulation. Formulating various other continuous or controlled delivery techniques, such as microlipid carriers, bioerodible microparticles, beads, and storage injections, are also known to those skilled in the art.嶋 829, which describes the 'paper scale applicable to the National Standards (CNS) A4 specification for the delivery of pharmaceutical compositions (210 X 297 issued I.---.------------- --Book --------- (Please read the phonetic on the back? Please fill out this page again) -74 1278319 A7 B7 V. INSTRUCTIONS (72) Release of porous polymeric microparticles. Additional examples include semipermeable polymeric materials in the form of shaped articles, such as films or microcapsules. Sustained release materials may include polyesters, hydrocolloids, polylactides (U.S. Patent No. 3,773,919 and European Patent Publications) Co., Ltd. No. 58481), a copolymer of L-glutamic acid and r-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22: 547-56), poly(2-hydroxyethyl methacrylate) ) (Langer et al., 1981, J. Biomed.

Mater. Res. 15 : 167-277 和 Langer，1982，Chem. Tech. 12 : 9 8 - 105)、乙晞乙酸乙烯酯（Langer等人，在前），或聚-D(- )-3 -羥基丁酸（歐洲專利出版物第號）。持續釋放的組合物亦可包括微脂粒，其可藉著此項技藝中已知的數種方法中的任一種來製備。參見，例如EppStein等人，1985Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12: 9 8 - 105), ethyl acetate (Langer et al., former), or poly-D (-)-3 Hydroxybutyric acid (European Patent Publication No. 1). Sustained release compositions may also include vesicles, which may be prepared by any of several methods known in the art. See, for example, EppStein et al., 1985

Proc· Natl. Acad· Sci. USA 82 : 3688-92 ;以及歐洲專利出版物第〇36676號、第〇88〇46號和第丨43949號。在活體内投藥所使用的VGF醫藥組合物，通常必須是益菌的。這可藉著通過無菌過濾膜過濾而完成。在組合物爲冷凍乾燥物之處，可在冷凍乾燥和重建之前或之後，經營使用該方法的滅菌作用。可以冷凍乾燥之形式或在溶液中，儲存非經腸投藥的組合物。此外，通常將非經腸2 =人經濟部智慧財產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) 物放在具有滅菌通氣孔的容器中，例如且古7 4" 巧π共有可被皮下注針頭刺穿之塞子的靜脈内溶液袋或小瓶。一旦已經調配好醫藥組合物，便可以溶液、懸浮液、^ 膠、乳劑、固體或脱水或冷凍乾燥散劑的形式#，、二履j凝在無菌的小瓶中。這類調配物可以現成可田、、：儲存机J用 < 形式，或以本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -75 - 1278319 經濟部智慧財產局員工消費合作社印製 A7 五、發明說明（73 ) 在技藥〈則需要重建（例如冷滚乾燥）的形式儲存。 =定：具體實施例中，本發明針對產生單…劑量投藥二組。工具組可分別含有具有脱水蛋白質的第- :备：和具有含水調配物的第二個容器。在本發明的範 ’吓包括含有隔成一和多個隔間之預先，裝的注射筒 (例如欲體注射筒和溶解注射筒（ly〇syringes))的工具組。在治療上使用之VGF醫藥組合物的有效含量，將依據治療的前後關係和目標而定。熟諳此藝者將知曉治療的適當剑里3里，並因此一部份將依據所衍生之分子，代表將使用何= VGF分子，投藥的途徑，以及患者的大小（體重、體表面％或器g的大小）和狀況（年齡和一般健康狀況）。因此，臨床醫師將滴定劑量，並修改投藥途徑，以便獲得最佳的治療效果。典型的劑量範圍可以從大約〇丨微克/公斤到最高大約100毫克/公斤或更多，係依據上文提及的因素。在其他的具體實施例中，劑量範圍可從0 J微克/公斤到最高大約100毫克/公斤；或1微克/公斤到高達大約100毫克/ 公斤；或5微克/公斤到高達大約ι〇〇毫克/公斤。投藥的頻率將依據在待使用之調配物中Vgf分手的藥物動力學參數。通常，臨床醫師將投予組合物，直到達到獲知想要之效果的劑量爲止。因此，可在一段時間内，以單一劑量、二或多個劑量投予組合物（可以或可以不含相同含量的想要分子），或經由植入之裝置或套管連續輸液。由熟諳此藝者例行地進行更精細地區別適當的劑量，並在例行由他們完成之職務的範圍内。可經由使用適當的劑量_反應 -76· 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I.---.---------------^--------- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1278319 A7 -*---^_E___________ __ 五、發明說明（74 ) 數據，確定適當的劑量。又丁醫椠、、且合物的途徑，係根據已知的方法，例如口服，·經由靜脈内、腹腔内、大腦内（實質内）、腦室内、肌肉内眼内、動脈内、門脈内（intraportal)或病灶内的路徑注射藉著持續釋放的系統；或藉著植入裝置。在想要之處可藉著團塊注射，或藉著輸液或植入裝置連續投予該組合物。另外或額外地，亦可經由膜、海绵，或其他已經在其中吸附或包封想要分子之適當物質的植入，局部投予該組合物。在使用植入裝置之處，可將該裝置植入任何適當的組織或器官中，並可經由擴散、按時-釋放之團塊或連續投藥來遞送想要的分子。在一些案例中，可能想要以在活體外之方式來使用VGF 夕肽醫樂組合物。在這類例子中，使已經從患者中移出之細胞、組織或器官，在後續將該細胞、組織或器官移植回該患者内之前，先暴露在VGF多肽醫藥組合物之下。在其他的案例中，可藉著植入某些已經使用像是在本文中描述的那些方法，以遺傳方式設計，表現和分泌VGF多肽的細胞，來遞送VGF多肽。這類細胞可以是動物或人類的細胞，並可以是自體的、異種的或異種的。該細胞可視需要爲永存不死的。爲了降低免疫學反應的改變，可將細胞包膠，以避免周圍組織的浸潤。包膠的材料通常是生物可相容的、半-通透的聚合封入物或膜，其容許蛋白質產物 (們）的釋放，但防止該細胞被患者的免疫系統，或被其他 -77- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1·1——·---------------訂---------^9, (請先閱讀背面之注音？事項再填寫本頁) A7 1278319 B7__ 五、發明說明（75 ) 來自周圍組織的有害因素破壞。如同在本文中討論的，可能想要以一或多種VGF多肽處理已經分離的細胞族群（像是幹細胞、淋巴球、紅血球、軟骨細胞、神經元及其類似物）。這可藉著使經過分離的細胞直接暴露在多肽之下來完成，在該處其係以可通過細胞膜的形式。、本發明的其他具體實施例，係關於在活體外產製治療性多肽，並藉著基因治療或細胞治療來產製和遞送治療性多肽的細胞和方法（例如同種重組作用及/或其他的重組產製方法）兩者。可使用同種或其他的重組方法來修改含有正常無聲轉錄的VGF基因，或表現-低下之基因的細胞，並藉此產生表現出在治療上有效含量之VGF多肽的細胞。同種重組作用最初是為了瞒準基因，以便在具有轉錄活性的基因中謗發或修正突變而發展出的技術。Kucherlapati， 1989, Prog, in Nucl. Acid Res· & Mol· Biol. 36 : 301。發展基本的技術，成為將特定突變導入哺乳動物基因組之特定區内（Thomas等人，1986，Cell 44 : 419-28 ; Thomas和 Capecchi， 1987，Cell 51 : 503-12 ; Doetschman等人，1988，Proc. Natl· Acad. Sci. U.S.A· 85 : 8583-87)，或在有缺陷的基因内修正特定突變的方法（Doetschman等人，1987, Nature 330 : 576-78)。在美國專利第5,272,071號；歐洲專利出版物第 9193051 號和 505500 號；PCT/US90/07642 和 PCT 出版物第 WO 91/09955號中描述了代表性的同種重組技術。經由同種重組作用，可藉著將其與瞄準DNA附接，指揮 -78- >紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱1 I.---^---------------^--------- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1278319 A7 B7 五、發明說明（76 ) 待***基因組内的DNA序列至感興趣基因的特定區域。瞄準DNA是與基因組DNA之該區互補的（同種的）核菩酸序= 。在DNA進行複製的期間内，使與基因組特定區互補的小片瞄準DNA與親代股接觸。已經***細胞内的dna雜交是一般的特性，並因此經由共享的同種區，與其他片的内源 DNA重組。如果該互補股與含有突變或不同序列，或額外核苷酸的寡核謀酸附接，則由於重組的結果，也將它併入新合成的股中。由於校對的功能，可能以DNA的新序列做爲模板。因此，將轉移的DNA併入基因組中。與那些瞒準DNA片附接的是可與VGF多肽之表現產生交互作用或控制它的DNA區域’例如側面序列。例如，在足以影響編碼想要之VGF多肽的DNA轉錄的附近和方位，將啓動基因/促進子要素、抑制者基因，或外源的轉錄調節要素***想要宿主細胞的基因組中。控制要素控制了在宿主細胞基因組中出現的DNA邵份。因此，可不藉編碼vgf基因本身之DNA的轉錄，而寧可藉著與dna調節斷片偶聯之瞒準DNA(含有與感興趣之内源基因具有同種性的區域）的使用’來完成想要之VGF多肽的表現，其提供内源的基因序列，具有可認出VGF基因轉錄的信號。在典型的方法中，經由在細胞基因組内，藉著導入包括至少一個凋郎序列、表現序列和接合捐贈者位置的dna，在預先選擇位置處的同種重組作用，改變了想要之標革巴基因在細胞中的表現（也就是想要的内源細胞基因）。事實上以這類方式將這些成份導入染色體（基因組的）DNA内，結 -79- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) --------訂---- s'. 經濟部智慧財產局員工消費合作社印製 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（77 ) 果產生了新的轉錄單位（其中出現在該DNA構築體中的調節序列、表現序列和接合捐贈者位置，以可操作之方式與内源的基因連接）。由於將這些成份導入染色體之DNA内的結果，改變了想要之内源基因的表現。改變的基因表現，如同在本文中的描述，包括活化（或謗發表現）正常在獲得它的細胞中是無聲（未表現）的基因，以及增加在獲得它的細胞中並未以生理學上顯著程度表現之基因的表現。具體實施例進一步包括改變調節或謗導的模式，使其與在獲得它的細胞中發生之調節或謗導的模式不同，並降低（包括排除）在獲得它的細胞中表現之基因的表現。一種可藉著使用同種重、组作用，來增加或引起從細胞的内源VGF基因中產製VGF多肽的方法，涉及首先使用同種重組作用，將得自位置-專一的重組系統之重組序列（例如 Cre/loxP，FLP/FRT)(Sauer，1994，Curr. Opin. Biotechnol. 5 : 521-27 ; Sauer, 1993，Methods Enzymol·，225 : 890-900)，放在細胞的内源基因組VGF多肽密碼區的上游（也就是5 f)。將含有與剛好放在基因組VGF多肽密碼區上游之位置同種的重組位置的質體，連同適當的重組酶（recombinase)酵素一起導入經過修改的細胞株中。該重組酶引起質體經由質體的重組位置，整合到在該細胞株中，剛好位在基因組 VGF多肽密碼區之上游的重組位置内（Baubonis和Sauer， 1993, Nucleic Acids Res. 21 : 2025.29 ; O’Gorman等人，1991， Science 251 ·· 1351-55)。已知可增加轉錄的任何側面序列（ -80- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱Ί~ 1·---“---------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 五、發明說明（78 ) 例如促進子/啓動基因、***序二轉#促進子），如果在孩質體中位在適當之處，將以這類 L一、、靖万式整合，產生新的或經過修改的轉錄單位，導致從細晌干丨守双攸、、，田胞的内源VGF基因中，重新或增加VGF多肽的產製。更多的方法使用以已經將位置專—的重組序列，正好放在細胞的内源基因組VGF多肽密碼區之上游的細胞株，使用同種重組作用在細胞株之基因組的別處，導入第二個重組位置。然後將適當的重组酶酵素導入兩個-重組_位置的細胞株内，謗發重組的事件（刪除、倒位和移位）（Sauer, 1994, Curr. Opin. Biotechnol. 5 ： 521-27 ; Sauer ? 1993,Proc. Natl. Acad. Sci. USA 82: 3688-92; and European Patent Publication Nos. 36676, 〇88〇46 and 丨43949. VGF pharmaceutical compositions for administration in vivo must generally be probiotic. This can be done by filtration through a sterile filtration membrane. Where the composition is a lyophilizate, the sterilization of the method can be carried out before or after lyophilization and reconstitution. The parenterally administered composition can be stored in a lyophilized form or in solution. In addition, usually the parenteral 2 = Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing (please read the note on the back and then fill out this page) in a container with sterile vents, for example, and ancient 7 4" A π-intravenous solution bag or vial that can be pierced by a hypodermic needle. Once the pharmaceutical composition has been formulated, it can be in the form of a solution, suspension, gel, emulsion, solid or dehydrated or lyophilized powder, in a sterile vial. Such formulations may be ready for use in the field, or in the form of a storage machine J, or in accordance with the Chinese National Standard (CNS) A4 specification (210 X 297 mm) - 75 - 1278319 Consumer Cooperatives Print A7 V. Invention Notes (73) In the form of technical drugs, they need to be rebuilt (for example, cold-rolled dry). = Determination: In a specific embodiment, the present invention is directed to the production of a single ... dose administration of two groups. The tool set may each contain a first-prepared product having a dehydrated protein and a second container having an aqueous formulation. In the scope of the present invention, a kit comprising a pre-filled syringe (e.g., a syringe and a lysing syringe) is provided that is divided into one and a plurality of compartments. The effective amount of the VGF pharmaceutical composition to be used therapeutically will depend on the context and objectives of the treatment. Those who are familiar with this artist will know the appropriate sword for treatment, and therefore part of it will be based on the molecule derived, which will represent the VGF molecule, the route of administration, and the size of the patient (weight, body surface% or device) The size and condition of g (age and general health). Therefore, the clinician will titrate the dose and modify the route of administration in order to obtain the best therapeutic effect. Typical dosages can range from about 〇丨 micrograms/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may range from 0 J micrograms/kg up to about 100 mg/kg; or 1 microgram/kg to up to about 100 mg/kg; or 5 micrograms/kg up to about ι mg. /kg. The frequency of administration will depend on the pharmacokinetic parameters of the Vgf breakup in the formulation to be used. Typically, the clinician will administer the composition until the dose at which the desired effect is achieved is reached. Thus, the composition can be administered in a single dose, in two or more doses over a period of time (may or may not contain the same amount of the desired molecule), or continuously infused via an implanted device or cannula. Routinely, the artist is routinely fine-tune the appropriate doses and is within the scope of routine duties performed by them. Can be used by using the appropriate dose_reaction-76· This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) I.---.-------------- -^--------- (Please read the notes on the back and fill out this page.) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1278319 A7 -*---^_E___________ __ V. Invention Description (74 ) Data to determine the appropriate dose. The route of the medicinal preparation and the compound is based on known methods, such as oral administration, via intravenous, intraperitoneal, intracerebral (intra-substantial), intraventricular, intramuscular, intra-arterial, intra-articular, portal vein. The path within the intraportal or lesion is injected by a system of sustained release; or by an implanted device. Where desired, the composition can be administered by bolus injection or by infusion or an implant device. Additionally or additionally, the composition may be administered topically via implantation of a film, sponge, or other suitable substance in which the desired molecule has been adsorbed or encapsulated. Where an implant device is used, the device can be implanted into any suitable tissue or organ and the desired molecule can be delivered via diffusion, time-release agglomerates or continuous administration. In some cases, it may be desirable to use the VGF oxime peptide therapeutic composition in an in vitro manner. In such instances, the cells, tissues or organs that have been removed from the patient are exposed to the VGF polypeptide pharmaceutical composition prior to subsequent transplantation of the cells, tissues or organs into the patient. In other cases, VGF polypeptides can be delivered by implanting certain cells that have been genetically engineered, expressed and secreted with VGF polypeptides, using methods such as those described herein. Such cells may be cells of an animal or human and may be autologous, xenogeneic or xenogeneic. The cells can be immortalized as needed. To reduce the change in the immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissue. The encapsulated material is typically a biocompatible, semi-permeable polymeric encapsulant or membrane that permits release of the protein product, but prevents the cell from being affected by the patient's immune system, or by other The paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). 1·1——·--------------- Order---------^ 9, (Please read the phonetic transcription on the back? Please fill out this page again) A7 1278319 B7__ V. INSTRUCTIONS (75) Destruction of harmful factors from surrounding tissues. As discussed herein, it may be desirable to treat a population of cells that have been isolated (such as stem cells, lymphocytes, red blood cells, soft bone cells, neurons, and the like) with one or more VGF polypeptides. This can be accomplished by exposing the isolated cells directly to the polypeptide where it is in the form of a cell membrane. Other embodiments of the invention relate to cells and methods for producing and delivering therapeutic polypeptides by gene therapy or cell therapy in vitro (eg, homologous recombination and/or others) Reorganized production methods) both. The same or other recombinant methods can be used to modify a VGF gene containing normal silent transcription, or a cell exhibiting a low-lower gene, and thereby producing a cell exhibiting a therapeutically effective amount of a VGF polypeptide. The same type of recombination was originally developed to modulate genes to develop or modify mutations in genes with transcriptional activity. Kucherlapati, 1989, Prog, in Nucl. Acid Res· & Mol· Biol. 36: 301. The development of basic techniques has become the introduction of specific mutations into specific regions of the mammalian genome (Thomas et al., 1986, Cell 44: 419-28; Thomas and Capecchi, 1987, Cell 51: 503-12; Doetschman et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8583-87), or a method for modifying specific mutations within a defective gene (Doetschman et al., 1987, Nature 330: 576-78). Representative homologous recombination techniques are described in U.S. Patent No. 5,272,071; European Patent Publication Nos. 9193051 and 505500; PCT/US90/07642 and PCT Publication No. WO 91/09955. Through the same kind of recombination, it can be attached to the aiming DNA, and the -78- > paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 public 1 I.---^---- -----------^--------- (Please read the notes on the back and fill out this page) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1278319 A7 B7 V. Invention Description (76) The DNA sequence to be inserted into the genome to a specific region of the gene of interest. The targeting DNA is a nuclear homologous sequence that is complementary to the region of the genomic DNA = during the replication of the DNA, A small piece of complementary genomic specific region is targeted to the DNA and is in contact with the parental strand. DNA hybridization that has been inserted into the cell is a general property and thus recombines with the endogenous DNA of other fragments via the shared homologous region. If the complementary strand contains mutations Or different sequences, or oligonucleotide attachment of additional nucleotides, is also incorporated into the newly synthesized strand due to the result of recombination. Due to the function of proofreading, it is possible to use a new sequence of DNA as a template. , the transferred DNA is incorporated into the genome. Attached to those DNA fragments A DNA region that interacts with or controls the expression of a VGF polypeptide, such as a flanking sequence. For example, in a vicinity and orientation sufficient to affect transcription of a DNA encoding a desired VGF polypeptide, the promoter/promotor element, suppressor gene will be activated. , or an exogenous transcriptional regulatory element inserted into the genome of the desired host cell. The control element controls the DNA fragment present in the host cell genome. Therefore, instead of encoding the DNA encoding the vgf gene itself, it is preferred to The dna regulates the use of fragment-coupled cDNA (containing a region homologous to the endogenous gene of interest) to perform the desired VGF polypeptide expression, providing an endogenous gene sequence with recognizable VGF Signals for gene transcription. In a typical method, by introducing a DNA that includes at least one of the lang sequences, the expression sequences, and the DNA of the donor's position within the genome of the cell, the same recombination at the preselected position changes the mind. The performance of the target gene in the cell (that is, the desired endogenous cell gene). In fact, these are in this way. Imported into the chromosome (genome) DNA, knot-79- This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back and fill out this page) ---- ----订---- s'. Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing A7 B7 V. Invention Description (77) The result is a new transcription unit ( The regulatory sequences, expression sequences, and joining donor positions present in the DNA construct are operably linked to endogenous genes). The result of introducing these components into the DNA of the chromosome changes the performance of the desired endogenous gene. Altered gene expression, as described herein, includes activation (or bursting) of a gene that is normally silent (not expressed) in the cells from which it is obtained, and increased in the cells from which it is obtained is not physiologically A significant degree of performance of the gene. Particular embodiments further include altering the mode of regulation or stimuli to distinguish it from the pattern of modulation or stimuli that occurs in the cell from which it is obtained, and to reduce (including excluding) the expression of genes that are expressed in the cells from which it is obtained. A method for producing or producing a VGF polypeptide from an endogenous VGF gene of a cell by using the same seed weight, group action, involving first using a homologous recombination action, a recombination sequence derived from a position-specific recombination system (eg, Cre/loxP, FLP/FRT) (Sauer, 1994, Curr. Opin. Biotechnol. 5: 521-27; Sauer, 1993, Methods Enzymol·, 225: 890-900), the endogenous genomic VGF polypeptide code placed in the cell Upstream of the zone (ie 5 f). A plastid containing a recombination site homologous to the position immediately upstream of the genomic region of the genomic VGF polypeptide is introduced into the modified cell strain along with an appropriate recombinase enzyme. The recombinase causes the plastid to pass through the plastid recombination site and integrate into the recombination site in the cell line just upstream of the genomic region of the genomic VGF polypeptide (Baubonis and Sauer, 1993, Nucleic Acids Res. 21 : 2025.29; O'Gorman et al., 1991, Science 251 · 1351-55). Any side sequence known to increase transcription (-80- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 public Ί~1·---"----------- ----Book--------- (Please read the notes on the back and fill in this page) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 V. Invention Description (78) For example, Promoter / The promoter gene, the insertion sequence, the second promoter, and the promoter, if they are in the proper position in the plastid, will be integrated with this type of L, and jing, to produce new or modified transcription units, resulting in内丨丨、、、、、、、、、、、、、、重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新重新The cell line upstream of the cryptodomain of the VGF polypeptide is introduced into the second recombination site using the same recombination in the genome of the cell strain, and then the appropriate recombinase enzyme is introduced into the cell line of the two-recombinant _ position, and the hair is generated. Recombination events (deletion, inversion, and shifting) (Sauer, 1994, Curr. Opin. B Iotechnol. 5 : 521-27 ; Sauer ? 1993,

Methods Enzymol·，225 : 890-900)，其將產生新的或經過修改的轉錄單位，導致從細胞的内源VGF基因中，重新或增加VGF多肽的產製。從細胞的内源VGF基因中，增加或謗發VGF多肽表現的其他方法，涉及增加或謗發基因或基因們（例如轉錄因子）的表現，及/或降低基因或基因們（例如轉錄阻遏物）的表現，其多少導致從細胞的内源VGF基因中，重新或增加VGF多肽的產製。該方法包括將非-天然存在的多肽（例如包括與轉錄因子功能部位融合之位置專一的DNA結合功能部位的多肽）導入細胞内，得到從細胞的内源VGF基因中，重新或增加VGF多肽產製的結果。亦企圖包括VGF多肽的細胞治療’例如植入產生VGF多肽的細胞。該具體實施例涉及植入能夠合成並分泌VGF多肽之生物活性形式的細胞。這類產製VGF多肽的細胞’可以 -81 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公Μ ) .,1、---^---------------訂--------- ί請先閱讀背面之注意事項再填寫本頁} 1278319Methods Enzymol, 225: 890-900), which will generate new or modified transcription units, result in the re- or increased production of VGF polypeptides from the endogenous VGF gene of the cell. Other methods of increasing or eliciting the expression of a VGF polypeptide from the endogenous VGF gene of a cell, involving increasing or expressing the expression of genes or genes (eg, transcription factors), and/or reducing genes or genes (eg, transcriptional repressors) The performance of the VGF polypeptide is re- or increased from the endogenous VGF gene of the cell. The method comprises introducing a non-naturally occurring polypeptide (for example, a polypeptide comprising a site-specific functional site that is fused to a functional site of a transcription factor) into a cell, thereby obtaining or regenerating the VGF polypeptide from the endogenous VGF gene of the cell. The result of the system. Cellular therapies including VGF polypeptides, such as implantation of cells that produce VGF polypeptides, are also contemplated. This particular embodiment relates to the implantation of cells capable of synthesizing and secreting a biologically active form of a VGF polypeptide. Such VGF-producing cells can be used in the Chinese National Standard (CNS) A4 specification (210 X 297 cm). 1,---^----------- ----Book --------- ί Please read the notes on the back and then fill out this page} 1278319

五、發明說明（79) (請先閱讀背面之注意事項再填寫本頁) 是屬於VGF多肽之天然生產者的細胞，或可以是重組的細胞，已經藉著以編碼想要之VGF多肽的基因，或以增加 VGF多肽表現之基因轉化，增加其產生vgf多肽的能力。可藉著適合用來遞送基因，並促進其表現和分泌的载體，來完成這類修改。爲了使在待投予VGF多肽之患者中可能的免疫學反應減至最少，像是可能在投予外來物種之多肤時發生的’最好產製VGF多肽的天然細胞，是屬於人類來源的，並產生人類的VGF多肽。同樣的，產製VGF多肽的重組細胞，最好是利用含有編碼人類VGF多肽之基因的表現載體轉化的。可將被植入的細胞包膠，以避免周圍組織的浸潤。可植入患者中的人類或非-人類的動物細胞，在生物可相容、半 -通透的聚合封入物或膜中，其容許VGF多肽的釋放，但防止该細胞被患者的免疫系統，或被其他來自周圍組織的有害因素破壞。另外，亦可將患者自己的細胞，在活體外轉化成可產製VGF多肽，直接植入患者中，而不需這類的包膠作用。經濟部智慧財產局員工消費合作社印製活細胞的包膠技術，爲此項技藝中已知的，並可例行地完成包膠細胞的製備，和將其植入患者。例如，Baetge等人（PCT出版物第W0 95/05452號和PCT/US94/09299)描述了含有遺傳工程設計之細胞的膜膠囊，以便有效地遞送生物活性的分子。該膠囊爲生物可相容的，並容易取回。該膠囊包膠以重組DNA分子轉移感染的細胞，該重組DNA分子包括以可操作之方式與啓動基因連接之具有生物活性之分子 -82 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1278319 A7 B7 經濟部智慧財產局員工消費合作社印製五、發明說明（80) 的DNA序列密碼，其在植入哺乳動物宿主中時，並未經歷在活體内的向下調節。該裝置提供了將得自活細胞的分子遞送至接受者内的特定位置。此外，參見美國專利第 4,892,538號；5,011，472號和5,106,627號。包膠活細胞的系統描述在？(^出版物第〜0 91/1〇425號（八€1^〇1^1*等人）。亦參見？(^出版物第賈0 91/10470號（八61^8〇1^1*等人）；界11111等人，1991，Exper. Neurol. 113 : 322-29 ; Aebischer等人，1991， Exper. Neurol· 111 : 269-75 ;以及Tresco等人 ’ 1992，ASAIO 3 8 : 1 7-23 〇亦想像VGF多肽之在活體内和在活體外的基因治療遞送。基因治療技術的一個實例，是使用編碼V GF多肽的核酸（基因組DNA、cDNA及/或合成的DNA)，其以可操作之方式與組成的或可謗導的啓動基因連接，形成”基因治療之DNA 構築體”。啓動基因可以是與内源的VGF基因同種或異種的，其限制條件爲它在將***該構築體的細胞或組織類型中，是具有活性的。基因治療之DNA構築體的其他成份，可視需要包括爲了位置-專一的整合作用而設計的DNA分子（例如可用於同種重組作用的内源序列）、組織-專一的啓動基因、促進子或無聲序列（silencer)、能夠提供勝過親代細胞之選擇性優勢的DNA分子、可用來做爲標記以便確認轉化細胞的DNA分子、陰性選擇系統、細胞專一性結合劑（像是例如爲了瞄準細胞）、細胞-專一的内化因子、促進從載體中表現的轉錄因子，以及使載體能夠產製的因子。然後可使用病毒或非_病毒的載體，將基因治療之DNA構 -83- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I-—”---------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（81 ) 築體導入細胞内（在活體外或在活體内）。一種導入基因治療之DNA構築體的方法，是藉著如同在本文中描述之病毒載體。某些載體，像是反轉錄病毒載體，會將DNA構築體遞送至細胞的染色體DNA内，並將基因整合到染色體的 DNA内。其他的載體將具有附加體之功能，並將基因治療之DNA構築體維持在細胞質内。在另外的具體實施例中，爲了在標靶細胞中控制VGF基因的表現，可包含調節要素。這類要素可反映適當的效應物而打開。以此方式便可在想要之時表現治療性的多肽。一種傳統的控制方法涉及使用小分子的二聚體或雷帕洛體 (rapalog)，二聚含有小分子-結合功能部位的嵌合型蛋白質和能夠發動生物加工的功能部位，像是DNA-結合蛋白質或轉錄激活蛋白質（參見PCT出版物第W0 96/41865、W0 97/31898和W0 97/3 1899號）。可使用蛋白質的二聚作用來發動轉殖基因的轉錄。另一種調節技術，使用將從感興趣之基因中製備的蛋白質，以凝集物或集群之形式保存在細胞内的方法。以融合蛋白質之形式來表現感興趣的基因，其包括有條件的凝集功能部位，導致凝集的蛋白質保留在内質網中。被儲存的蛋白質在細胞中是穩定和無活性的。然而，可藉著投予藥物（例如小分子的配體）釋放該蛋白質，其移除有條件的凝集功能邵位’並藉此專一地打破分開凝集物或集群，而得以從細胞中分泌該蛋白質。參見Aridor等人，2000，Science 287 : 816- 1 7 和 Rivera等人，2000, Science 287 : 826-30 〇 -84- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I.—.------------------------ (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（82) 其他適當的控制方法或基因開關包括，但不限於在本文中描述的系統。使用米非司酮（Mifepristone) (RU486)做爲黃體酮的拮抗劑。經過修改之黃體酮受體配體·結合功能部位與黃體酮拮抗劑的結合，藉著形成兩個轉錄因子的二聚體，然後越過進入細胞核與DNA結合，而激活轉錄作用。修改該配體-結合功能部位，排除了受體與天然配體結合的能力。經過修改的類固醇荷爾蒙受體系統，進一步描述在美國專利第5,364,791號和PCT出版物第W0 96/40911號和W0 97/10337號中。另一種控制系統使用蜕皮激素（果蠅類固醇荷爾蒙），其結合並激活蜕皮激素受體（細胞質之受體）。然後該受體移位至核，與專一的DNA反應要素（得自蜕皮激素-反應性基因的啓動基因）結合。蛻皮激素受體包括轉活化功能部位、 DNA-結合功能部位和發動轉錄的配體-結合功能部位。在美國專利第5,514,578號和PCT出版物第W0 97/38117、W0 96/37609和WO 93/03162號中進一步描述了虫兑皮激素系統。其他的控制方法使用正的四環素-可控制轉活化劑 (transactivator)。該系統涉及與激活轉錄作用之多肽連接的突變tet阻遏物蛋白質DNA-結合功能部位（突變的tet R-4胺基酸改變，其導致相反的四環素-調節之轉活化劑蛋白質，也就是説，其在四環素的存在下與tet操縱基因結合）。在美國專利第5,464,758號、5,650,298號和5,654,168號中描述了這類系統。在 Innovir Laboratories Inc·的美國專利第 5,741，679 號和 -85- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1·---.---------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 Α7 Β7 五、發明說明（83) 5,834，186號中描述了其他的表現控制系統和核酸構築體。可經由局部注射或藉著其他適當之病毒或非-病毒的遞送載體，藉著將編碼VGF多肽的核酸分子導入細胞内，完成在活體外的基因治療。Hefti, 1994, Neurobiology 25 : 1418· 3 5。例如，爲了遞送至標靶細胞，可使編碼VGF多肽的核酸分子包含在與腺有關的病毒（A AV)載體内（參見，例如 Johnson，PCT出版物第W0 95/34670號；PCT申請案第 PCT/US95/07178號）。重組的AAV基因組通常含有位在編碼 VGF多肽之DNA序列侧面的逆向終端重覆段，以可操作之方式與具有功能的啓動基因和聚腺甞酸化作用序列連接。另一種適當的病毒載體包括，但不限於反轉錄病毒、腺病毒、單純癌療病毒、慢病毒、肝炎病毒、微小病毒、乳頭狀瘤多瘤空泡化病毒、痘病毒、π病毒、冠狀病毒、桿形病毒、副黏液病毒和乳頭狀瘤病毒載體。美國專利第 5,672,344號描述了在活體内病毒-調節之基因運送系統，其涉及重組的趨神經之HSV-1載體。美國專利第5,399,346號提供了加工處理，藉著遞送在活體外處理，***編碼治療性蛋白質之DNA斷片的人類細胞，提供患者治療性蛋白質的實例。其他實行基因治療技術的方法和材料，描述在美國專利第5,631，236號（涉及腺病毒載體）、5,672,510號（涉及反轉錄病毒載體）、5,635,399號（涉及表現細胞***素之反轉錄病毒載體）。V. INSTRUCTIONS (79) (Please read the note on the back and then fill out this page) is a cell belonging to the natural producer of VGF polypeptide, or may be a recombinant cell, which has been used to encode the desired VGF polypeptide. , or by increasing the expression of a VGF polypeptide, increasing its ability to produce a vgf polypeptide. Such modifications can be made by a vector suitable for delivering the gene and promoting its expression and secretion. In order to minimize the possible immunological response in patients to be administered VGF polypeptide, such as the natural cells that may produce VGF polypeptides that may occur when the skin of a foreign species is administered, it is of human origin. And produce a human VGF polypeptide. Similarly, recombinant cells producing a VGF polypeptide are preferably transformed using a expression vector containing a gene encoding a human VGF polypeptide. The implanted cells can be encapsulated to avoid infiltration of surrounding tissue. Human or non-human animal cells that can be implanted in a patient, in a biocompatible, semi-permeable polymeric enclosure or membrane that permits release of the VGF polypeptide, but prevents the cell from being affected by the patient's immune system, Or destroyed by other harmful factors from surrounding tissues. In addition, the patient's own cells can be converted into a VGF polypeptide that can be produced in vitro and implanted directly into the patient without the need for such encapsulation. The Department of Economic Intelligence's Intellectual Property Office, the Staff Consumer Cooperative, prints the encapsulation technology for living cells, which is known in the art and routinely completes the preparation of encapsulated cells and implants them into patients. For example, Baetge et al. (PCT Publication No. WO 95/05452 and PCT/US94/09299) describe membrane capsules containing genetically engineered cells for efficient delivery of biologically active molecules. The capsule is biocompatible and easy to retrieve. The capsule encapsulates the infected cell with a recombinant DNA molecule comprising a biologically active molecule operably linked to the promoter gene - 82 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1278319 A7 B7 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed V. Inventive Note (80) DNA sequence code, which does not undergo downward regulation in vivo when implanted in a mammalian host . The device provides for delivery of molecules derived from living cells to specific locations within the recipient. In addition, see U.S. Patent Nos. 4,892,538; 5,011,472 and 5,106,627. What is the system for encapsulating living cells? (^ Publication No. ~0 91/1〇425 (eight €1^〇1^1*, etc.). See also? (^ Publication No. 0 0 91/10470 (8 61^8〇1^1) * et al.); 11111 et al., 1991, Exper. Neurol. 113: 322-29; Aebischer et al., 1991, Exper. Neurol 111: 269-75; and Tresco et al. '1992, ASAIO 3 8: 1 7-23 〇 also envisions gene therapy delivery of VGF polypeptides in vivo and in vitro. One example of gene therapy technology is the use of nucleic acids encoding VP polypeptides (genomic DNA, cDNA and/or synthetic DNA), An operably linked to a constitutive or achievable promoter gene to form a "DNA construct for gene therapy." The promoter gene may be homologous or heterologous to the endogenous VGF gene, with the restriction that it will be inserted The cell or tissue type of the construct is active. Other components of the DNA construct of the gene therapy may optionally include DNA molecules designed for position-specific integration (eg, endogenously available for recombination) Sequence), tissue-specific promoter gene, promoter or silent sequence ( Silencer), a DNA molecule capable of providing a superior advantage over the parental cell, a DNA molecule that can be used as a marker to confirm transformed cells, a negative selection system, a cell-specific binding agent (such as, for example, to target cells), cells - a specific internalization factor, a transcription factor that promotes expression from the vector, and a factor that enables the vector to be produced. The DNA of the gene therapy can then be applied to China using a viral or non-viral vector. National Standard (CNS) A4 Specification (210 X 297 mm) I-—”---------------Book--------- (Please read the back of the note first) Matters fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. Invention Description (81) The body is introduced into the cell (in vitro or in vivo). A DNA construct introduced into gene therapy The method is by a viral vector as described herein. Certain vectors, such as retroviral vectors, deliver the DNA construct into the chromosomal DNA of the cell and integrate the gene into the DNA of the chromosome. The carrier will have The function of the body, and maintain the DNA construct of the gene therapy in the cytoplasm. In another specific embodiment, in order to control the expression of the VGF gene in the target cell, regulatory elements may be included. Such elements may reflect appropriate effects. Open in this way. In this way, a therapeutic polypeptide can be expressed when desired. A traditional control method involves the use of small molecule dimers or rapalog, which contain small molecule-binding functions. A chimeric protein at a site and a functional site capable of eliciting bioprocessing, such as a DNA-binding protein or a transcriptional activator protein (see PCT Publication No. WO 96/41865, WO 97/31898 and WO 97/3 1899). The dimerization of proteins can be used to initiate transcription of the transgenic genes. Another modulating technique uses a method in which the protein prepared from the gene of interest is stored in the cell in the form of agglomerates or clusters. The gene of interest is expressed in the form of a fusion protein that includes a conditional agglutination function site, resulting in retention of the agglutinated protein in the endoplasmic reticulum. The stored protein is stable and inactive in the cell. However, the protein can be released by administering a drug (e.g., a ligand for a small molecule) which removes the conditional agglutination function 'and thereby specifically breaks down the agglutination or cluster to secrete the cell from the cell. protein. See Aridor et al., 2000, Science 287: 816- 1 7 and Rivera et al., 2000, Science 287: 826-30 〇-84- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) I.—.------------------------ (Please read the notes on the back and fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumption Cooperative Printed A7 B7 V. INSTRUCTIONS (82) Other suitable control methods or gene switches include, but are not limited to, the systems described herein. Mifepristone (RU486) was used as an antagonist of progesterone. The modified progesterone receptor ligand-binding functional site binds to the progesterone antagonist and activates transcription by forming a dimer of two transcription factors and then passing into the nucleus to bind to the DNA. Modification of the ligand-binding functional site excludes the ability of the receptor to bind to the natural ligand. The modified steroid hormone receptor system is further described in U.S. Patent No. 5,364,791 and PCT Publication Nos. WO 96/40911 and WO 97/10337. Another control system uses ecdysone (Drosophila steroid hormone), which binds to and activates the ecdysone receptor (the cytoplasmic receptor). The receptor is then transferred to the nucleus and bound to a specific DNA response element (a promoter derived from the ecdysone-reactive gene). The ecdysone receptor includes a transactivation functional site, a DNA-binding functional site, and a ligand-binding functional site that initiates transcription. The insect dermatological hormone system is further described in U.S. Patent No. 5,514,578 and PCT Publication Nos. WO 97/38117, WO 96/37609, and WO 93/03162. Other control methods use a positive tetracycline-controlled transactivator. The system involves a mutant tet repressor protein DNA-binding functional site linked to a polypeptide that activates transcription (a mutant tet R-4 amino acid change that results in an opposite tetracycline-regulated transactivator protein, that is, It binds to the tet operator in the presence of tetracycline). Such systems are described in U.S. Patent Nos. 5,464,758, 5,650,298 and 5,654,168. US Patent Nos. 5,741,679 and -85- of Innovir Laboratories Inc. apply to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1·---.-------- -------Book--------- (Please read the note on the back and fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed Β7 Β7 5, invention description (83) Other performance control systems and nucleic acid constructs are described in 5,834,186. Gene therapy in vitro can be accomplished by local injection or by other suitable viral or non-viral delivery vectors by introducing a nucleic acid molecule encoding a VGF polypeptide into a cell. Hefti, 1994, Neurobiology 25: 1418· 3 5. For example, a nucleic acid molecule encoding a VGF polypeptide can be included in a gland-associated virus (A AV) vector for delivery to a target cell (see, for example, Johnson, PCT Publication No. WO 95/34670; PCT Application No. PCT/US95/07178). The recombinant AAV genome typically contains a reverse terminal repetitive segment flanking the DNA sequence encoding the VGF polypeptide, operably linked to a functional promoter gene and a polyadenylation sequence. Another suitable viral vector includes, but is not limited to, retroviruses, adenoviruses, cancer-only viruses, lentiviruses, hepatitis viruses, parvoviruses, papilloma polyfollicular vacuoles, poxviruses, piviruses, coronaviruses , rod-shaped virus, paramyxovirus and papilloma virus vectors. U.S. Patent No. 5,672,344 describes a viral-regulated gene delivery system in vivo involving a recombinant neurotropic HSV-1 vector. U.S. Patent No. 5,399,346 provides a process for providing a therapeutic protein for a patient by delivering a human cell that is processed in vitro and inserted into a DNA fragment encoding a therapeutic protein. Other methods and materials for performing gene therapy techniques are described in U.S. Patent No. 5,631,236 (involving adenoviral vectors), 5,672,510 (involving retroviral vectors), and 5,635,399 (involving retroviral vectors expressing cytokinins) .

非病毒的遞送方法包括，但不限於微脂粒-調節之運送、裸露的DNA遞送（直接注射）、受體-調節的運送（配體-DNA -86- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I.—.---------------^--------- (請先閱讀背面之注意事項再填寫本頁) 1278319Non-viral delivery methods include, but are not limited to, liposome-mediated delivery, naked DNA delivery (direct injection), and receptor-mediated delivery (ligand-DNA-86- this paper size applies to Chinese national standards (CNS) A4 size (210 X 297 mm) I.—.---------------^--------- (Please read the notes on the back and fill in the form) Page) 1278319

經濟部智慧財產局員工消費合作社印製 :合體）、電穿透作用、磷酸鈣沉澱法和微顆粒撞擊基因槍）。基因治療之材料和方法，亦可包 7 1如 ^ 匕括可謗導的啓動基因、組織-專一的促進子_啓動基因、爲 A ., I义直-寻一之整奋作用而設計的DNA序列、能夠提供勝過賴你硯代細胞心選擇性優勢的DNA序列、確認轉化細胞之標記、陰性的選擇系統以及表現控制系統（安全性測量）、細胞_專一的=合劑 (爲了瞄準細胞）、細胞-專一的内化因子，和由載體促進表現的轉錄因子，以及載體製造的方法。這類實行基因治療技術的額外方法和材料，描述在美國專利第4,97〇，154號（涉及電穿透技術）、5,679,559號（描述基因遞送之含有脂蛋白的系統）、5,676,954號（涉及微脂粒載劑）、5,593,875號（描述嶙酸鈣轉移感染的方法）和4,945,050號（描述在細胞中以藉此使顆粒貫穿細胞表面並併入細胞内部之速度，來推動生物活性之顆粒的方法），以及PCT出版物第w〇 9 6 / 40958 號（涉及核配體）。亦企圖使VGF基因治療或細胞治療，更包括在相同或不同的細胞（們）中，遞送一或多個額外的多肽（們）。可將這類細胞分別導入患者中，或可在單一的可植入裝置中含有這些細胞’像是上述的包膠膜，或可藉著病毒載體分別修改這些細胞。在細胞中經由基因治療來增加内源的VGF多肽表現的方法，是將一或多個促進子要素***VGF多肽的啓動基因中，促進子要素可在那裏擔任增加VGF基因之轉錄活性的職務。將以想要在其中激活基因的組織爲基礎，來選擇所使 87 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） Γ·---:----------------訂--------- C請先閱讀背面之注意事項再填寫本頁}Printed by the Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative: fit), electroporation, calcium phosphate precipitation and microparticle impact gene gun). Gene therapy materials and methods can also be designed to include the promoter gene, the tissue-specific promoter, the promoter gene, and the A., I-straight-seeking effect. DNA sequence, DNA sequence that provides superiority over the selectivity of your cell, confirmation of transformed cell markers, negative selection system, and expression control system (safety measurement), cell-specific = mixture (for targeting cells) ), cell-specific internalization factors, and transcription factors that promote expression by vectors, and methods of vector production. Additional methods and materials for performing such gene therapy techniques are described in U.S. Patent No. 4,97,154 (incorporating Electroporation Technology), No. 5,679,559 (Liquid-containing System for Gene Delivery), No. 5,676,954 (involving Microlipid carrier), No. 5,593,875 (method describing calcium transfer infection of citrate) and No. 4,945,050 (described in the cell to promote the flow of particles through the cell surface and into the interior of the cell) Method), and PCT Publication No. WO 9 9 40958 (involving nuclear ligands). It is also attempted to deliver VGF gene therapy or cell therapy, including one or more additional polypeptides, in the same or different cells. Such cells may be introduced into the patient separately, or may be contained in a single implantable device' such as the above-described encapsulated membrane, or these cells may be modified by viral vectors, respectively. A method of increasing the expression of an endogenous VGF polypeptide via gene therapy in a cell is to insert one or more promoter elements into the promoter gene of the VGF polypeptide, where the promoter element can serve to increase the transcriptional activity of the VGF gene. Based on the organization in which the gene is to be activated, the 87 paper scales will be selected for the Chinese National Standard (CNS) A4 specification (210 X 297 mm) Γ·---:------- ---------Book --------- C Please read the notes on the back and then fill out this page}

經濟部智慧財產局員工消費合作社印製 1278319 五、發明說明（85) 用的促進子要素-已知促進子要素在將選擇的组織中賦與啓動基因活化。例如，若要在τ細胞中”打開"編碼VGF多肽的基因，則可使用lck啓動基因促進子要素。在此，可使用標準選殖技術，將欲加入之轉錄要素有功能的部份***含有 VGF多肽啓動基因的DNA片段中（並可視需要***載體及/ 或側面序列的5·及/或3·内）。然後可在活體外或在活體内，將該構築體，稱爲"同種重組作用的構築體”導入想要的細胞内。亦可使用基因治療，藉著修改内源啓動基因的核甞酸序列來降低VGF多肽的表現。通常經由同種的重組方法來完成這類修改。例如，可設計含有全部或一部份就失活作用選出之VGF基因之啓動基因的〇ΝΑ分子，以便移除並/或置換調節轉錄之啓動基因的碎片。例如，可使用標準的分子生物學技術’刪除啓動基因之轉錄活化劑的TATA盒子及/ 或結合位置；這類刪除可抑制啓動基因的活性，藉此抑制相對應之VGF基因的轉錄。在啓動基因中刪除TAT a盒子或轉錄活化劑結合位置，可藉著產生包括全部或相關部份之 VGF多肽啓動基因（得自與待調節之vgf基因相同或相關的物種）的DNA構采體來完成，其中經由一或多個核嘗酸的取代、刪除及/或***，使一或多個TATA盒子及/或轉錄活化劑結合位置之核：y：酸突變。結果，TATA盒子及/或活化劑結合位置具有降低之活性，或變成完全失活的。該構築體，通常亦將含有至少大約50 0個鹼基的DNA，相當於與已經修改之啓動基因斷片相鄰，天然的（内源的）5 ’和3，DNA序列 -88- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） L---,----------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 A7 B7 五、發明說明（86 ) ，可直接或按照在本文中的描述，經由病毒載體將其導入適當的細胞内（在活體外或在活體内）。通常，構築體整人到細胞的基因組DNA内，將經由同種的重組作用，其中在啓動基因構築體中的5’和3，DNA序列，可經由與内源的染色體DNA雜交，來協助整合經過修改的啓動基因區。 VGF多肽的用涂可在適合待治療之病況處，將VGF多肽與一或多種細胞 ***素、生長因子、抗生素、消炎劑及/或化學治療劑混合使用（同時或連續）。亦可在想要抑制一或多個VGF多肽的活性之處，使用其他的方法。可藉著與表現控制序列（形成三股螺旋）或VgF mRNA互補並與其雜交的核酸分子，來完成這類抑制作用。例如，可將反義的DNA或RNA分子導入細胞内，其具有與 VGF基因中至少一部份互補的序列。在本文中揭示使用 VGF基因之序列，藉著可利用的技術，來設計的反義探針。通常’每個這類的反義分子，將與每個選出之VgF基因的起始位置（5 ’末端）互補。當反義的分子與相對應的vgf mRNA雜X時，便阻礙或減少了該mRNA的轉譯。反義的抑制劑提供了在細胞或器官中，與降低或缺乏Vgf多肽有關的資訊。另外’可使用基因治療來創造一或多個VGF多肽的負面優勢之抑制劑。在該場合中，可製備編碼每個選出之多肽的突變多肽的DNA，並使用如同在本文中描述之病毒或非-病毒的方法，將其導入患者的細胞中。通常將這類突 (請先閱讀背面之注意事項再填寫本頁) > I I Μ·· a·· β ΗΒΗ 經濟部智慧財產局員工消費合作社印製 -89- 經濟部智慧財產局員工消費合作社印製 1278319 A7 ---------B7_______ 五、發明說明（87 ) 變體分別設計成在其生物學角色中，與内源的多肽競爭。此外，亦可使用VGF多肽作爲免疫原，無論其是否具有生物活性’也就是説該多肽含有至少一個可使抗體升高的抗原決定位。爲了在活體内或在活體外的診斷目的，可使用與VGF多肽結合的選擇性結合劑（如同在本文中的描述），包括但不限於使用已標示之形式，來檢測在體液或細胞 4樣中VGF多肽的存在。亦可使用抗體來預防、治療或診所弄多疾病和障礙，包括在本文中提及的那些。抗體可與 VGF多肽結合，而得以減少或阻斷vgf多肽的至少一個活性特徵，或可與多肽結合，以便增加VgF多肽的至少一個活性特徵（包括藉著增加VGF多肽的藥物動力學）。利用表現選殖策略’可使用本發明之Vgf多肽來選殖VGF 少月太文體。可在結合測定中使用以放射性標示（〗25-破）的 vgf多肽，或附上親和力/活性·標籤的vgf多肽（像是F c融合或驗性磷酸酶融合），來確認表現VGI7多肽受體的細胞類型或細胞株或組織。可將從這類細胞或組織中分離出的 RNA轉變爲cDNA，選殖到哺乳動物表現載體中，並轉移感 ¥•到哺乳動物細胞内（像是cos或293細胞），而產生表現庫。然後可使用以放射性標示或附加標籤的VGf多肽做爲親和力配體，從該庫中確認並分離在其表面上表現VGF多肽文體的細胞亞組。然後可從這些細胞中分離DNA，並轉移感染到哺乳動物細胞内，產生第二個表現庫，其中表現 VGF多肽受體之細胞的溶離份，比在原始庫中的更高許多倍。可反覆地重覆該增強作用的過程，直到分離出含有本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐 1·—-----------------訂--------- (請先閱讀背面之注音？事項再填寫本頁) -90- 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（88 ) VGF多肽受體的單一重組純種系爲止。vgf多肽受體的分離，可用來確認或發展VGF多肽發送信號之路徑的新穎激動劑和拮抗劑。這類激動劑和拮抗劑包括可溶的vgf多肽艾m、抗-VGF多肤受體抗體、小分子或反義的寡核菩酸，然後可使用b們來治療、預防或診斷一或多種在本文中描述的疾病或障礙。下列的實例僅企圖做爲解釋之用，不應解釋爲以任何方式限制本發明之範圍。實例1 :抗_VGF多肽抗體的產製藉著利用由 Amgen Boulder Peptide Technology 團體，使用標準肽合成草案合成的VGF多肽注射兔子，來產製對抗 VGF多肽的抗體。在肽合成之後，集合得自不同批的vgf 多肽，並冷凍乾燥之。藉著利用HC1的水解作用，定出每個 VGF多肽的肽内含量。欲從缺乏半胱胺酸殘基的VGF多肽（VGF-1;序列識別1 號或VGF-2 ;序列識別4號；表I)來製備多株的抗-VGF多肽抗體’將5¾克VGF多肽和〇·5毫升Imject® EDC共輛緩衝溶液（Pierce，Rockford，IL)轉移感染到2毫升的小瓶中，並旋轉該混合物。以2毫升無菌的水稀釋2 0毫克小瓶的 Imject®鎖孔緘血藍蛋白（pierce)，並將〇 5毫升的該溶液加至VGF多肽溶液中。在加入鎖孔緘血藍蛋白之後，將〇 j 毫升的EDC( 1 0毫克E DC (Pierce)在1毫升無菌的水中）加至 VGF多肽中，並在室溫下培養該溶液至少2小時。在培養之後，將共軛的VGF多肽移至Spectra/Pro 6透析 -91 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ;--------------------訂------ (請先閱讀背面之注咅？事項再填寫本頁) s'. 1278319 A7 B7 五、發明說明（89) (請先閱讀背面之注意事項再填寫本頁) 管（Spectrum Laboratories，Rancho Dominguez，CA)中，具有 1000之分子量截止値，並在4°C下以2公升PBS透析過夜，以便從試樣中移除EDTA，已知其爲抗-凝固劑。將經過透析、共輛》的VGF多肤溶液移至1 5毫升的錐形管中，並加入 PBS，使該溶液之體積達到4毫升。將各1毫升的等份移至2 毫升有螺旋蓋的試管中，將每個等份吸至2毫升玻璃注射筒中，然後再吸1毫升Titermax⑧研究佐劑（CytRx，Norcross， Georgia) 〇將注射筒與1 8號混合針頭連接，並將該溶液混合成乳劑狀。然後將該混合物移至兩個1毫升的注射筒中，以肌肉内注射到兔子體内。以〇。1毫升VGF多肽/佐劑溶液，在兩個注射位置注射兔子，並在之後4週和6週補強。在第二次補強之後，對兔子進行試驗採血（移出大約5毫升），然後在兩週之後再度進行試驗採血。如果認爲採血的結果是可接受的，便再度補強兔子，然後在這次補強之後兩週，每週採血一次持續六週（移出大約4 0毫升）。圖1和2解釋了以VGF-1或VGF- 2注射兔子的抗體力價含量。經濟部智慧財產局員工消費合作社印製欲從具有半胱胺酸殘基的VGF多肽來製備多株的抗-VGF 多肽抗體，將5毫克VGF多肽和0.5毫升Imject®順丁晞二醯亞胺共軏緩衝溶液（Pierce)轉移感染到2毫升的小瓶中，並旋轉該混合物。以2毫升無菌的水稀釋2 0毫克小瓶的 Imject®鎖孔緘血藍蛋白（Pierce)，並將0 · 5毫升的該溶液加至VGF多肽溶液中。在加入鎖孔緘血藍蛋白之後，在室溫下培養VGF多肽溶液至少2小時。 -92- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（9〇 ) 在培養之後，將共軛的VGF多肽移至Specti*a/Pro 6透析管中，並在4 X：下以2公升P BS透析過夜，以便從試樣中移除 EDTA。將經過透析、共軛的VGF多肽溶液移至1 5毫升的錐形管中，並加入PBS，使該溶液之體積達到4毫升。將各1 毫升的等份移至2毫升有螺旋蓋的試管中，將每個等份吸至 2毫升玻璃注射筒中，然後再吸1毫升Titermax®研究佐劑 (CytRx) 〇將注射筒與1 8號混合針頭連接，並將該溶液混合成乳劑狀。然後將該混合物移至兩個1毫升的注射筒中，以肌肉内注射到兔子體内。以〇 . 1毫升VGF多肽/佐劑溶液，在兩個注射位置注射兔子，並在之後4週和6週補強。在第二次補強之後，對兔子進行試驗採血（移出大約5毫升），然後在兩週之後再度進行試驗採血。如果認爲採血的結果是可接受的，便再度補強兔子，然後在這次補強之後兩週，每週採血一次持續六週（移出大約40毫升）。實例2 : VGF多肽在擊倒老鼠中的生物活性使用由 Amgen Boulder Peptide Technology 圑體，使用標準肽合成草案合成的VGF多肽，來分析VGF多肽在擊倒老鼠中的生物活性。在肽合成之後，集合得自不同批的VGF多肽，並冷凍乾燥之。藉著利用HC1的水解作用，定出每個 VGF多肽的肽内含量。從 Steve Salton of Mt. Sinai School of Medicine, New York, NY獲得VGF基因擊倒老鼠。在投予VGF多肽之前，在LD 1 2 : 1 2的條件（也就是1 2小時明亮和1 2小時黑暗）下飼養 -93 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I.--------------------^--------- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1278319 A7 ----------_B7 五、發明說明（91 ) 擊倒老鼠。纟注射VGF多肽之前24小時，將擊倒老鼠分別關在籠子中，並移至暫時的飼養室，在實驗剩下的期間，在LD 12 : 12的條件下將老鼠飼養於此。藉著腹腔内注射2毫克VGF_la(序列識別2號；表j )，將 VGF多肽每天兩次投予擊倒老鼠（早上9 :⑽和下午6 :⑽）。在注射之前，以aCSF溶液緩衝溶液（126.6 mMNaC1，2.6Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumers Co., Ltd. 1278319 V. INSTRUCTIONS (85) Promoter elements used - known promoter elements are activated in the selected tissues. For example, to "open" a gene encoding a VGF polypeptide in a tau cell, the lck can be used to initiate a gene promoter element. Here, a standard selection technique can be used to insert a functional portion of the transcription element to be added. The DNA fragment containing the VGF polypeptide promoter gene (and optionally inserted into the vector and/or the side sequence of 5· and/or 3·). The construct can then be referred to as " in vitro or in vivo. The construct of the same recombination action is introduced into the desired cell. Gene therapy can also be used to reduce the expression of VGF polypeptides by modifying the nucleotide sequence of the endogenous promoter. Such modifications are usually done via the same recombination method. For example, a purine molecule containing all or a portion of the promoter gene of the VGF gene selected for inactivation can be designed to remove and/or replace fragments of the promoter that regulate transcription. For example, standard molecular biology techniques can be used to delete the TATA box and/or binding site of the transcriptional activator of the promoter; such deletions can inhibit the activity of the promoter gene, thereby inhibiting transcription of the corresponding VGF gene. Deletion of the TAT a cassette or transcriptional activator binding site in the promoter gene by generating a DNA construct that includes all or a relevant portion of the VGF polypeptide promoter gene (derived from the same or related species as the vgf gene to be regulated) This is accomplished by nucleation of one or more TATA boxes and/or transcription activator binding sites via one or more nucleotide substitutions, deletions, and/or insertions: y: acid mutation. As a result, the TATA box and/or activator binding site has reduced activity or becomes completely inactive. The construct, which will typically also contain at least about 50 bases of DNA, is equivalent to adjacent to the modified promoter gene fragment, native (endogenous) 5' and 3, DNA sequence-88-this paper size Applicable to China National Standard (CNS) A4 specification (210 X 297 mm) L---,---------------- order--------- (please first Read the precautions on the back page and fill out this page.) 1278319 A7 B7 5. Inventive Note (86), which can be introduced into appropriate cells (in vitro or in vivo) via a viral vector, either directly or as described herein. . Usually, constructing the human body into the genomic DNA of the cell will be through the same kind of recombination, wherein the 5' and 3, DNA sequences in the promoter gene construct can be integrated with the endogenous chromosomal DNA to assist in the integration. Modified promoter gene region. The VGF polypeptide can be used in combination with one or more cytokinins, growth factors, antibiotics, anti-inflammatory agents and/or chemotherapeutic agents (simultaneously or continuously) at a condition suitable for the treatment to be treated. Other methods may also be used where it is desired to inhibit the activity of one or more VGF polypeptides. Such inhibition can be accomplished by nucleic acid molecules that are complementary to and hybridize to a expression control sequence (forming a triple helix) or VgF mRNA. For example, an antisense DNA or RNA molecule can be introduced into a cell having a sequence that is complementary to at least a portion of the VGF gene. Antisense probes designed using the VGF gene sequence and by available techniques are disclosed herein. Typically, 'each of these antisense molecules will be complementary to the starting position (5' end) of each of the selected VgF genes. When the antisense molecule is heterozygous to the corresponding vgf mRNA, the translation of the mRNA is blocked or reduced. Antisense inhibitors provide information about the reduction or lack of Vgf polypeptides in cells or organs. In addition, gene therapy can be used to create inhibitors of the negative advantages of one or more VGF polypeptides. In this case, DNA encoding the mutant polypeptide of each of the selected polypeptides can be prepared and introduced into the cells of the patient using a virus or non-viral method as described herein. Usually such a sudden (please read the note on the back and fill out this page) > II Μ·· a·· β ΗΒΗ Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing -89- Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative Printing 1278319 A7 ---------B7_______ V. INSTRUCTION DESCRIPTION (87) Variants are designed to compete with endogenous polypeptides in their biological roles, respectively. In addition, a VGF polypeptide can also be used as an immunogen, whether or not it is biologically active' that is, the polypeptide contains at least one epitope that raises the antibody. For diagnostic purposes in vivo or in vitro, a selective binding agent (as described herein), including but not limited to the use of the indicated form, for detection in body fluids or cells can be used. The presence of a VGF polypeptide. Antibodies can also be used to prevent, treat or diagnose a variety of diseases and disorders, including those mentioned herein. The antibody can bind to the VGF polypeptide to reduce or block at least one of the active characteristics of the vgf polypeptide, or can bind to the polypeptide to increase at least one activity characteristic of the VgF polypeptide (including by increasing the pharmacokinetics of the VGF polypeptide). The VGF f polypeptide of the present invention can be used to select VGF sylvestris using the performance selection strategy. A vgf polypeptide that is radiolabeled ("25-broken") or a vgf polypeptide with an affinity/activity tag (such as an F c fusion or a phosphatase fusion) can be used in the binding assay to confirm the expression of the VGI7 polypeptide. The cell type or cell line or tissue of the body. RNA isolated from such cells or tissues can be converted to cDNA, cloned into a mammalian expression vector, and transferred to mammalian cells (such as cos or 293 cells) to produce a library of expression. A radiolabeled or tagged VGf polypeptide can then be used as a affinity ligand from which a subset of cells expressing VGF polypeptide motifs on their surface is identified and isolated. DNA can then be isolated from these cells and transferred to mammalian cells to produce a second library of expression in which the fraction of cells expressing the VGF polypeptide receptor is many times higher than in the original pool. This process of enhancement can be repeated over and over until the separation of the Chinese National Standard (CNS) A4 specification (210 X 297 mm 1·--------------- ---Order--------- (Please read the phonetic on the back? Please fill out this page again) -90- 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (88) A single recombinant pure germline of a VGF polypeptide receptor. The isolation of a vgf polypeptide receptor can be used to identify or develop novel agonists and antagonists of the pathway by which a VGF polypeptide transmits signals. Such agonists and antagonists include soluble vgf Polypeptides, anti-VGF polypeptide receptor antibodies, small molecules or antisense oligonucleotides can then be used to treat, prevent or diagnose one or more of the diseases or disorders described herein. The use of the invention is not intended to be construed as limiting the scope of the invention in any way. Example 1: Production of anti-VGF polypeptide antibodies by using the VGF synthesized by the Amgen Boulder Peptide Technology group using a standard peptide synthesis draft Peptide injection into rabbits to produce anti-VGF polypeptide After the peptide synthesis, the vgf polypeptides from different batches are pooled and lyophilized. By using the hydrolysis of HC1, the intra-peptide content of each VGF polypeptide is determined. Desire from the lack of cysteine residues VGF polypeptide (VGF-1; SEQ ID NO: 1 or VGF-2; SEQ ID NO: 4; Table I) to prepare multiple strains of anti-VGF polypeptide antibody' will share 53⁄4 g of VGF polypeptide with 毫升·5 ml of Imject® EDC Transfer the buffer solution (Pierce, Rockford, IL) to a 2 ml vial and rotate the mixture. Dilute the 20 mg vial of Imject® keyhole limpet hemocyanin (pierce) with 2 ml of sterile water and 5 ml of this solution was added to the VGF polypeptide solution. After adding the keyhole limpet hemocyanin, 〇j ml of EDC (10 mg E DC (Pierce) in 1 ml of sterile water) was added to the VGF polypeptide. And cultivate the solution for at least 2 hours at room temperature. After culturing, transfer the conjugated VGF polypeptide to Spectra/Pro 6 dialysis-91 - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 PCT) ;-------------------- Order ------ (Please read the note on the back first? Fill in this page) s'. 1278319 A7 B7 V. Inventions (89) (Please read the note on the back and fill out this page) Tube (Spectrum Laboratories, Rancho Dominguez, CA) has a molecular weight cutoff of 1000, It was dialyzed against 2 liters of PBS overnight at 4 ° C to remove EDTA from the sample, which is known as an anti-coagulant. The lysed, co-cultured VGF skin solution was transferred to a 15 ml conical tube and PBS was added to bring the volume of the solution to 4 ml. Transfer each 1 ml aliquot to a 2 ml screw-capped test tube, pipet each aliquot into a 2 ml glass syringe, and then inhale 1 ml of Titermax8 study adjuvant (CytRx, Norcross, Georgia). The barrel was connected to a No. 18 mixing needle and the solution was mixed into an emulsion. The mixture was then transferred to two 1 ml syringes for intramuscular injection into rabbits. Take 〇. One milliliter of VGF polypeptide/adjuvant solution was injected into the rabbit at two injection sites and reinforced at 4 and 6 weeks later. After the second boost, the rabbits were tested for blood collection (about 5 ml removed) and then tested again two weeks later. If the result of the blood collection is considered acceptable, the rabbit will be reinforced again, and then two weeks after the reinforcement, blood is collected once a week for six weeks (about 40 ml removed). Figures 1 and 2 illustrate the antibody titer content of rabbits injected with VGF-1 or VGF-2. The Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, prints a variety of anti-VGF polypeptide antibodies from VGF polypeptides with cysteine residues, 5 mg of VGF polypeptide and 0.5 ml of Imject® cis-butane diimine. The conjugated buffer solution (Pierce) was transferred to a 2 ml vial and the mixture was spun. A 20 mg vial of Imject® hemocyanin (Pierce) was diluted in 2 ml of sterile water and 0.5 ml of this solution was added to the VGF polypeptide solution. After addition of the keyhole limpet hemocyanin, the VGF polypeptide solution was incubated for at least 2 hours at room temperature. -92- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (9〇) After cultivation, conjugate The VGF polypeptide was transferred to a Specti*a/Pro 6 dialysis tube and dialyzed against 2 liters of P BS overnight at 4 X: to remove EDTA from the sample. The dialyzed, conjugated VGF polypeptide solution was transferred to a 15 ml conical tube and PBS was added to bring the volume of the solution to 4 ml. Transfer each 1 ml aliquot to a 2 ml screw-capped test tube, pipet each aliquot into a 2 ml glass syringe, and then inhale 1 ml of Titermax® Research Adjuvant (CytRx). A mixing needle No. 8 was attached and the solution was mixed into an emulsion. The mixture was then transferred to two 1 ml syringes for intramuscular injection into rabbits.兔子. 1 ml of VGF polypeptide/adjuvant solution, rabbits were injected at two injection sites and reinforced at 4 and 6 weeks later. After the second boost, the rabbits were tested for blood collection (about 5 ml removed) and then tested again two weeks later. If the result of the blood collection is considered acceptable, the rabbit will be re-enhanced, and then two weeks after the reinforcement, blood is collected once a week for six weeks (about 40 ml removed). Example 2: Biological activity of VGF polypeptides in knockdown mice The biological activity of VGF polypeptides in knockdown mice was analyzed using a VGF polypeptide synthesized by the Amgen Boulder Peptide Technology steroid using standard peptide synthesis. After peptide synthesis, the VGF polypeptides from different batches were pooled and lyophilized. The intra-peptide content of each VGF polypeptide was determined by utilizing the hydrolysis of HC1. VGF gene knockdown mice were obtained from Steve Salton of Mt. Sinai School of Medicine, New York, NY. Before the VGF polypeptide is administered, it is raised under the conditions of LD 1 2 : 1 2 (that is, 12 hours of light and 12 hours of darkness) -93 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297) I)-.------------------^--------- (Please read the notes on the back and fill out this page) Intellectual Property Bureau employee consumption cooperative printed 1278319 A7 ----------_B7 V. Invention description (91) Knock down the mouse. Twenty-four hours prior to the injection of the VGF polypeptide, the knockdown mice were individually housed in cages and transferred to a temporary breeding room where the mice were housed under LD 12:12 for the remainder of the experiment. VGF polypeptide was administered twice daily to knock down mice by intraperitoneal injection of 2 mg VGF_la (SEQ ID NO: 2; Table j) (9:10 am and 6:10 pm). ACSF solution buffer solution (126.6 mM NaC1, 2.6 before injection)

mMKCl, 2.0 mM MgCl 2^1.4 mM CaCl2? 10 mM NaP〇4? pH 7. 4)將VGF-1 a稀釋成1 6 %。擊倒老鼠注射VGF_ u多肽五天，並每天測量每隻老鼠的體重。在停用VGF_la多肽之後，再每天測量處理老鼠的體重三天。圖3解釋在投予VGF_ 1 a之後，VGF擊倒老鼠的體重。五隻經過處理的老鼠中，有三隻體重增加㈧—丨5%。從該實驗中，不可能決定體重的增加是否是增加食物或水攝取的結果。實例3 : VGF多肽_Fc融合蛋白皙以在其瘦基-或胺基_終端帶有人類免疫球蛋白IgG重鏈ρ c 區的融合蛋白質來表現VGF多肽。使用適當的pcr引子和標準PCR擴大程序，藉著在骨架中融合編碼人類IgG1之F c區和編碼VGF-1 (序列識別1號）、VGF-1 b (序列識別3號）或 VGF· 2 (序列識別4號）多肽的DNA序列，來製備VGF多肽-Fc融合構築體。使用標準技術，以核酸限制内切酶Ndel和BamHI消化藉著 PCR產生之VGF多肽-Fc融合產物，連接到載體pAMG21内，然後將其轉化至合格的大腸捍菌品系2596細胞中。就產 -94- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） L—-------------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319mMKCl, 2.0 mM MgCl 2^1.4 mM CaCl2? 10 mM NaP〇4? pH 7. 4) VGF-1 a was diluted to 16%. Knockdown mice were injected with VGF_u peptide for five days and the body weight of each mouse was measured daily. After the VGF_la polypeptide was stopped, the body weight of the treated mice was measured daily for three days. Figure 3 illustrates that VGF knocked down the body weight of mice after administration of VGF_1a. Three of the five treated mice gained weight (eight) - 5%. From this experiment, it is impossible to determine whether the increase in body weight is the result of increased food or water intake. Example 3: VGF polypeptide Fc fusion protein V A VGF polypeptide is expressed as a fusion protein with a human immunoglobulin IgG heavy chain ρ c region at its lean- or amino-terminal terminus. The appropriate PCR primer and standard PCR amplification program are used to fuse the F c region encoding human IgG1 and encode VGF-1 (sequence recognition No. 1), VGF-1 b (sequence recognition No. 3) or VGF·2 in the backbone. The DNA sequence of the (sequence recognition No. 4) polypeptide was used to prepare a VGF polypeptide-Fc fusion construct. The VGF polypeptide-Fc fusion product produced by PCR was digested with the nucleic acid restriction endonucleases Ndel and BamHI using standard techniques, ligated into the vector pAMG21, and then transformed into qualified E. coli strain 2596 cells. On the production -94- This paper scale applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) L-------------------- Order---- ----- (Please read the notes on the back and fill in this page) 1278319

經濟部智慧財產局員工消費合作社印製五、發明說明（92 ) 生重組蛋白質產物，和擁有具有正確核嘗酸序列之基因融合的能力，來篩選純種系。在3 7 C下使在大腸桿菌品系QM221中含有f c融合構築體的細菌培養物，在含有5 〇毫克/毫升康黴素的Luria肉湯中生長。在培養基中加入合成的自動謗導物N-(3_氧代己醯基）-DL-高絲胺酸内酯至終濃度2〇毫微克/毫升之後，完成從luxPR啓動基因中謗發基因產物的表現。在3 7 °c下培養該培養物額外的3小時。在該次培養之後，藉著顯微鏡檢查細菌培養物中包涵體的存在，然後藉著離心收集。藉著再懸浮於含有1 0 % /?-鏡基乙醇的Laemmli試樣缓衝溶液中，直接溶解細胞小球，並藉著SDS_PAGE分析之。藉著首先利用高壓均質化作用（也就是經過14,000磅/平方英吋2次）使細胞在水中破裂，來純化vgf多肽-F c融合蛋白質。藉著在J_6B中以4200RPM離心1小時，收獲包涵體，並以1 /1 0之比例，將包涵體溶解於6 μ胍、50 mM Tris、8 mM DTT，pH 8.7中 1 小時。然後在 2 Μ尿素、50 mM Tris、 160 mM精胺酸、3 mM半胱胺酸，pH 8. 5中，將已溶解的混合物稀釋2 0倍。在冰冷的環境中攪拌該混合物過夜，藉著超過濾濃縮成大約1 0倍，然後以10 mM Tris，1 · 5 Μ尿素， pH 9稀釋成3倍。然後以醋酸將該混合物的pH値調整到pH 5。藉著離心移除沉澱物，並將上清液裝入以2〇 mM NaAc， 100 mM NaCl，pH 5平衡過的SP -瓊脂糖速流管柱中（1 〇毫克/毫升蛋白質裝載；室溫）。使用2 0 -倍管柱體積，在相同緩衝溶液中，範圍在從約lOOmMNaCl到5〇〇mMNaCl的梯度 -95- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） :ΙΓΙ1ί---------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（93 ) ，從管柱中洗脱出蛋白質。將得自管柱的集合物稀釋3倍，並裝入在 20 mM NaAc, 150 mM NaCl, pH 5 的 SP-瓊脂糖HP 管柱（1 〇毫克/毫升蛋白質裝載；室溫）中。使用2 0 -倍管柱體積，在相同緩衝溶液中，範圍在從約150 mM NaCl到400 mM NaCl的梯度，洗脱出蛋白質。收集峰値並過濾之。使用在本文中描述的方法，或熟諳此藝者已知的方法，評估VGF多肽-F c融合蛋白質的活性。實例4 :在基因轉殖的老鼠中表現老鼠的VGF多肽欲評估VGF多肽的生物活性，使用標準草案來製備在賽納普辛（synapsin)啓動基因控制之下編碼老鼠VGF多肽的構築體。預期該構築體的遞送會引起病理學上的改變，這提供了 VGF多肽功能的情報。同樣的，使用標準草案，製備含有在肌動蛋白啓動基因控制之下的全長VGF多肽的構築體。預期該構築體的遞送將導致無所不在的表現。欲產生這些構築體，使用PCR擴大編碼老鼠VGF多肽的模板DNA序歹ij，使用相當於想要序列之5 ,和；31末端的引子，且其併入限制酵素位置，容許將經過擴大的產物***表現載體内。在擴大作用之後，使用標準重組的DNA技術，以凝膠純化PCR產物，以適當的限制酵素消化，並連接到表現載體内。參見 Graham 等人，1997，Nature Genetics, 17 : 272-74和 Ray等人，1991, Genes Dev. 5 : 2265-73 〇在連接之後，藉著電穿透作用使用反應混合物轉化大腸桿菌宿主品系，並就藥物的抗藥性來選擇轉化物。從所選出的菌落中分離質體DNA，並使其接受DNA定序，證實適 -96 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） L--------------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（94 ) 當之***序列的存在，並沒有突變。經由兩輪的CsCl密度梯度離心來純化VGF多肽表現載體，利用適當的限制酵素切開，並藉著凝膠電泳法純化出含有老鼠VGF多肽轉殖基因之已經弄直的片段。以2微克/毫升之濃度，將已經純化的片段再懸浮於5mMTris，pH7.4和0.2mMEDTA中。按照描述（？<^出版物第界0 97/23614號），注射得自 BDFlxBDFl交配的單-細胞胚胎。在（302恆溫箱中培養該胚胎過夜，並將1 5 - 2 0個兩個-細胞的胚胎移至假懷孕之CD1 雌鼠的輸卵管中。如下藉著整合轉殖基因至基因組DNA試樣中的PCR擴大作用，篩選獲自植入微量注射之胚胎的後代。在5 5 QC下，以2 0微升耳朵緩衝溶液（20 mM Tris, pH 8·0, 10 mM EDTA，0.5%SDS和500毫克/毫升蛋白酶K)消化耳朵的碎片過夜。然後以200微升ΤΕ稀釋試樣，並在使用適當引子的PCR反應中使用2微升的耳朵試樣。Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives. V. INSTRUCTIONS (92) The ability to combine recombinant protein products with the ability to fuse genes with the correct nucleotide sequence to screen for pure lines. A bacterial culture containing the f c fusion construct in E. coli strain QM221 was grown at 3 7 C in Luria broth containing 5 mg/ml of oxytetracycline. The synthetic autoinducer N-(3-oxohexyl)-DL-homuryl lactone was added to the medium to a final concentration of 2 ng/g, and the gene product from the luxPR promoter was completed. Performance. The culture was incubated for an additional 3 hours at 3 7 °C. After this incubation, the presence of inclusion bodies in the bacterial culture was examined by microscopy and then collected by centrifugation. The cell pellet was directly lysed by resuspending in a Laemmli sample buffer solution containing 10% /?-mirrylethanol and analyzed by SDS_PAGE. The vgf polypeptide-F c fusion protein was purified by first disrupting the cells in water using high pressure homogenization (i.e., 14,000 psi). The inclusion bodies were harvested by centrifugation at 4200 RPM for 1 hour in J_6B, and the inclusion bodies were dissolved in 6 μ胍, 50 mM Tris, 8 mM DTT, pH 8.7 for 1 hour at a ratio of 1 / 10 . The dissolved mixture was then diluted 20-fold in 2 Μ urea, 50 mM Tris, 160 mM arginine, 3 mM cysteine, pH 8.5. The mixture was stirred overnight in an ice-cold environment, concentrated to about 10 times by ultrafiltration, and then diluted to 3 times with 10 mM Tris, 1.5 Μ urea, pH 9. The pH of the mixture was then adjusted to pH 5 with acetic acid. The precipitate was removed by centrifugation, and the supernatant was loaded into a SP-Sepharose column (1 〇 mg/ml protein loaded at 2 mM NaAc, 100 mM NaCl, pH 5; room temperature; ). Using a 20-fold column volume, in the same buffer solution, ranging from about 100 mM NaCl to 5 mM NaCl-95-this paper scale applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm): ΙΓΙ1ί---------------Book--------- (Please read the note on the back and fill out this page) 1278319 Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative A7 B7 V. Description of the invention (93), eluting proteins from the column. The pool-derived pool was diluted 3 fold and loaded into a SP-Sepharose HP column (1 〇 mg/ml protein loading; room temperature) at 20 mM NaAc, 150 mM NaCl, pH 5. Protein was eluted using a 20-fold column volume in a gradient from about 150 mM NaCl to 400 mM NaCl in the same buffer solution. Collect peaks and filter them. The activity of the VGF polypeptide-F c fusion protein is assessed using the methods described herein, or by methods known to those skilled in the art. Example 4: Expression of VGF polypeptides in mice in genetically transformed mice To assess the biological activity of VGF polypeptides, standard drafts were used to prepare constructs encoding mouse VGF polypeptides under the control of the synapsin promoter. Delivery of the construct is expected to cause pathological changes that provide insight into the function of the VGF polypeptide. Similarly, a construct containing a full-length VGF polypeptide under the control of an actin-inducing gene was prepared using the standard draft. Delivery of the construct is expected to result in ubiquitous performance. To create these constructs, PCR is used to amplify the template DNA sequence 歹 ij encoding the mouse VGF polypeptide, using primers corresponding to the 5, and 31 ends of the desired sequence, and incorporating them to limit the position of the enzyme, allowing for the expanded product. Insert into the expression vector. After expansion, the PCR product is gel purified using standard recombinant DNA techniques, digested with appropriate restriction enzymes, and ligated into the expression vector. See Graham et al, 1997, Nature Genetics, 17: 272-74 and Ray et al, 1991, Genes Dev. 5: 2265-73. After ligation, the reaction mixture is used to transform E. coli host lines by electroporation. Transformants are selected for drug resistance. The plastid DNA was isolated from the selected colonies and subjected to DNA sequencing, confirming that the paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) L------ --------------Book --------- (Please read the notes on the back and fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. INSTRUCTIONS (94) There is no mutation in the presence of the inserted sequence. The VGF polypeptide expression vector was purified by two rounds of CsCl density gradient centrifugation, cut with appropriate restriction enzymes, and the fragment that had been straightened containing the mouse VGF polypeptide transgene was purified by gel electrophoresis. The purified fragment was resuspended in 5 mM Tris, pH 7.4 and 0.2 mM EDTA at a concentration of 2 μg/ml. Single-cell embryos mated from BDFlxBDF1 were injected as described (? <^ Publication No. 0 97/23614). The embryos were cultured overnight in a 302 incubator and 1 5 - 20 two-cell embryos were transferred to the oviduct of a pseudopregnant CD1 female. The recombinant gene was ligated into the genomic DNA sample as follows. PCR amplification to screen for progeny from embryos implanted with microinjection. At 5 5 QC, with 20 μl of ear buffer solution (20 mM Tris, pH 8·0, 10 mM EDTA, 0.5% SDS and 500) Mice/ml protease K) Digest the debris of the ear overnight. The sample was then diluted with 200 μl of sputum and 2 μl of the ear sample was used in a PCR reaction using the appropriate primer.

確認出基因轉殖的創始動物，並用來產生F 1老鼠。欲這樣進行，從F 1老鼠中移出一部份的脾臟、肝臟和全部的腦，並使用總RNA萃取工具組（Qiagen)從脾臟中分離總細胞 RNA，並藉著RT-PCR定出轉殖基因的表現。使用 Superscript™前擴大系統（Gibco· BRL)，如下將從脾臟回收的RN A轉變爲cDNA。使用適當的引子，位在表現載體序列中，並在VGF多肽轉殖基因之3 ’，從轉殖基因的轉錄本中發動cDNA合成。在70 °C下，將1 0毫克從創始者和對照組之肝臟、腦和脾臟製備的總RNA與ImM引子一起培養10分鐘，並放在冰上。然後以10 mM Tris_HCl, pH 8.3，50 mM -97- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） I-—-----------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1278319 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（95) KC1，2.5 mM MgCl2, dNTP各 10 mM，0.1 mM DTT和200單位的 Superscript II反轉錄酶補充該反應。在42°C下培養50分鐘之後，藉著加熱至7 2 °C 1 5分鐘使該反應中止，並在3 7。(3下以2單位RNA酶Η消化2 0分鐘。然後使用對老鼠VGF多肽專一的引子，藉著PCR擴大試樣。使得自轉殖基因陽性之F 1老鼠的小孩雜交，產生ρ 2同種接合子的老鼠。按照在本文中的描述，定出VGF mRNA在 F 2老鼠中的表現程度。宜i列5 考鼠VGF多肽在F2基因轉殖老鼠中的决舲法押在安樂死之前，先將獲自實例4的F2基因轉殖之老鼠稱重’藉著異氟規麻醉’並藉著心臟穿刺抽血。使試樣接受血液學和血清化學分析。在最後的放血之後完成x射線攝影。在巨觀的解剖時，使主要的内臟器官接受重量分析。在巨觀的解剖之後，移出組織（也就是肝臟、脾臟、胰臟、胃、整個胃腸道、腎臟、呼吸器官、皮膚和乳腺、骨骼、腦、心臟、肺臟、胸腺、氣管、食道、甲狀腺、腎上腺、膀胱、淋巴結和骨骼肌），並在10%經過缓衝的Zn_福馬林中固足，以供組織學的檢查。在固定之後，在石蠟塊中加工組織，並獲得3毫米的切片。所有的切片均以蘇木素和曙紅染色，然後進行組織學分析。如下使基因轉殖和對照組老鼠的脾臟、淋巴結和派亞氏腺（Peyer，S patch)接受免疫組織學分析，利用B細胞和丁細胞專-性抗體。將福馬林固定石蠟包埋的切片脫蠟，並在去離子水中水合。以3%過氧化氫使該切片中止，以蛋白質 ---------------------訂--------- (請先閱讀背面之注意事項再填寫本頁) -98- 1278319 A7 B7 五、發明說明（96) (請先閱讀背面之注意事項再填寫本頁) 阻斷劑（Lipshaw，Pittsburgh，P A )阻斷之，並在大鼠單株抗· 老鼠 B 2 20 和 CD3 (Harlan，Indianapolis，IN)中培養。藉著生物素基化的兔抗-大鼠免疫球蛋白和帶有DAB做爲色原 (BioTek，Santa Barbara，CA)的過氧化酶共軏之鏈黴菌抗生物素蛋白（BioGenex，San Ramon，CA)，來檢測抗體的結合。以蘇木素對切片進行對比染色。在驗屍之後，移出得自基因轉殖動物和對照組孩子的脾臟和胸腺之MLN和切片。藉著以注射筒的平端對1 〇〇毫米尼龍細胞遽網（Becton Dickinson，Franklin Lakes，NJ)的底部，溫和地磨碎組織，來製備單一細胞的懸浮液。將細胞沖洗兩次，計數，然後以每種組織大約1X10- 6個細胞，以2 0微升之體積，將其與0.5微克〇〇16/3 2(卩〇7^111/11)卩(：阻斷劑一起培養1 〇分鐘。然後在2 - 8 °C下，在帶有0.5微克對抗 CD90.2 (Thy-1.2)、CD45R(B220)、CDllb(Mac-l)、Gr-1、CD4 或CD8之FITC或 PE-共軏之單株抗體（PharMingen, San Diego, CA)的100微升體積之PBS(缺乏Ca+和Mg + )、0.1%牛血清白蛋白和0.01 %疊氮化鈉中，染色試樣3 0分鐘。在抗體結合之後，沖洗細胞，然後藉著流體血球計數法，在 FACScan(Becton Dickinson)上分析之。經濟部智慧財產局員工消費合作社印製已經從較佳具體實施例的觀點描述了本發明，熟諳此藝者了解將發生改變和修改。因此，企圖使附綠的申請專利範園涵蓋所有在本發明提出申請之範圍内的這類相等的改 0 -99- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1278319 A7 B7 五、發明說明（97 ) 序列一覽表 <il〇> Yan, Hai <120> VGF選擇性結合劑及治療VGF相關性障礙的方法 <130> 00-423-A i j<l40> j kl50> 50/144^43 jci51> 1999-07-21 j <cl60> 10 i |l70> Patentln Ver. 2.0<|210> 1 <2ll> 30 5： :212>蛋白贺 |213>挪威大鼠 <Ι400> 1 ^ Gin Glu Glu Ala Asp Al« Glu Glu Arg Arg Leu Gin G：LU Gin Gill I 5 10 15 GLu Len Glu Ash 20The founder of the gene transfer was identified and used to produce F1 mice. To do this, remove a portion of the spleen, liver, and whole brain from F 1 mice and isolate total cellular RNA from the spleen using the total RNA extraction tool set (Qiagen) and determine by RT-PCR. Gene expression. The RN A recovered from the spleen was converted to cDNA using the SuperscriptTM pre-expansion system (Gibco·BRL) as follows. Using appropriate primers, the expression vector sequence is located in the 3' of the VGF polypeptide transgene, and cDNA synthesis is initiated from the transcript of the transgene. 10 mg of total RNA prepared from the liver, brain and spleen of the founder and control group was incubated with ImM primer for 10 minutes at 70 ° C and placed on ice. Then use 10 mM Tris_HCl, pH 8.3, 50 mM -97- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) I---------------- ---Order--------- (Please read the note on the back and fill out this page) 1278319 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. Invention Description (95) KC1, 2.5 mM The reaction was supplemented with MgCl2, dNTP 10 mM each, 0.1 mM DTT and 200 units of Superscript II reverse transcriptase. After incubation at 42 ° C for 50 minutes, the reaction was stopped by heating to 72 ° C for 15 minutes and at 37. (3 times, digested with 2 units of RNase for 20 minutes. Then use the primer specific to the mouse VGF polypeptide to expand the sample by PCR. The children of the F 1 mouse that are positive for the transgenic gene are crossed to produce the ρ 2 homozygous. According to the description in this article, the degree of expression of VGF mRNA in F 2 mice was determined. It is recommended that the VGF polypeptide in the F2 gene transfer mouse be euthanized before euthanasia. The F2 gene-transferred mice from Example 4 were weighed 'anesthetized with isoflurane' and were bled by cardiac puncture. The samples were subjected to hematology and serum chemistry analysis. X-ray photography was completed after the final bleeding. When the anatomy of the giant anatomy, the main internal organs are subjected to weight analysis. After the anatomy of the giant nectar, the tissues are removed (ie, the liver, spleen, pancreas, stomach, entire gastrointestinal tract, kidneys, respiratory organs, skin and breasts, Bone, brain, heart, lung, thymus, trachea, esophagus, thyroid, adrenal gland, bladder, lymph nodes, and skeletal muscle) and fixed in 10% buffered Zn_formalin for histological examination. After the determination, the tissue was processed in paraffin blocks and 3 mm sections were obtained. All sections were stained with hematoxylin and eosin and then histologically analyzed. The genes were transfected and the spleens, lymph nodes and mice of the control mice were as follows. Peyer's gland (Peyer, S patch) was subjected to immunohistological analysis using B-cell and butyl-specific antibodies. The formalin-fixed paraffin-embedded sections were dewaxed and hydrated in deionized water. Hydrogen peroxide stops the slice, and the protein ------------------------------ (please read the notes on the back and fill in This page) -98- 1278319 A7 B7 V. Description of Invention (96) (Please read the notes on the back and fill out this page) Blockers (Lipshaw, Pittsburgh, PA) block and resist in rats · Cultured in mice B 2 20 and CD3 (Harlan, Indianapolis, IN) by biotinylated rabbit anti-rat immunoglobulin and with DAB as a chromogen (BioTek, Santa Barbara, CA) Oxidase conjugated streptavidin (BioGenex, San Ramon, CA) to detect antibody binding. Sections were subjected to contrast staining. After autopsy, MLN and sections of the spleen and thymus from the gene-transforming animals and control children were removed. By means of a flat end of the syringe, 1 〇〇 mm nylon cell 遽 net (Becton Dickinson, Franklin At the bottom of Lakes, NJ, gently grind the tissue to prepare a single cell suspension. Rinse the cells twice, count, and then approximately 1×10-6 cells per tissue, in a volume of 20 μl, It was incubated with 0.5 μg of 〇〇16/3 2 (卩〇7^111/11) 卩 (: blocker for 1 〇 minutes. Then at 2-8 ° C, with 0.5 μg of anti-CD90.2 (Thy-1.2), CD45R (B220), CDllb (Mac-l), Gr-1, CD4 or CD8 FITC or PE-co-軏Samples were stained for 30 minutes in a single microbody (PharMingen, San Diego, CA) in 100 microliter volumes of PBS (lacking Ca+ and Mg+), 0.1% bovine serum albumin, and 0.01% sodium azide. After antibody binding, the cells were washed and analyzed by FACScan (Becton Dickinson) by fluid blood cell counting. Printed by the Ministry of Economic Affairs, Intellectual Property Office, Employees' Consumer Cooperatives The present invention has been described in terms of preferred embodiments, and those skilled in the art will appreciate that changes and modifications will occur. Therefore, it is attempted to make the Green Application Patent Park cover all such equivalents in the scope of the application of the present invention. 0-99- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). 1278319 A7 B7 V. INSTRUCTIONS (97) Sequence Listing <il〇> Yan, Hai <120> VGF selective binding agent and method for treating VGF-related disorders <130> 00-423-A i j<L40> j kl50> 50/144^43 jci51> 1999-07-21 j <cl60> 10 i |l70> Patentln Ver. 2.0<|210> 1 <2ll> 30 5: :212>213>NordicRat<Ι400> 1 ^ Gin Glu Glu Ala Asp Al« Glu Glu Arg Arg Leu Gin G: LU Gin Gill I 5 10 15 GLu Len Glu Ash 20

Glu His Val l»6ii 2j^u ttia Airg Piro 30 <210> 2 中1> 9 < + 12：>蛋白質挪威大鼠 <4 A3 00> 2 a Gin Glu Glu Ala A3p Ala Glu Glu 1 5 ___!/----------------訂---------(請先閱讀背面之注意事項再填寫本頁) _ 經濟部智慧財產局員工消費合作社印製 <2 <2 <2 <2 <4 X〇> 3 11> 19 12>蛋白質挪烕大鼠 ^^p〇> 3 L〇l Gin Glu Gin Gl-u Glu Leu Glu A6n Tyr He Glu His Val Leu Leu ^ 5 10 15Glu His Val l»6ii 2j^u ttia Airg Piro 30 <210> 2 in 1> 9 < + 12:> Protein Norwegian Rat <4 A3 00> 2 a Gin Glu Glu Ala A3p Ala Glu Glu 1 5 ___!/----------------Book---------(Please read the notes on the back and fill out this page) _ Ministry of Economic Affairs Intellectual Property Office staff Consumer Cooperative Print <2 <2 <2 <2 <4 X〇> 3 11> 19 12>Protein Rats ^^p〇> 3 L〇l Gin Glu Gin Gl-u Glu Leu Glu A6n Tyr He Glu His Val Leu Leu ^ 5 10 15

HiiHii

Pro <2l〇> 4 <2il> 25 <2：^2>蛋白質挪威大鼠Pro <2l〇> 4 <2il> 25 <2:^2> protein Norwegian rat

<4C 0> 4 100 本紙張尺度，用中國國家標準（CNS)A4規格（210 X 297公釐0 1278319 A7B7 五、發明說明（98 )<4C 0> 4 100 paper size, using Chinese National Standard (CNS) A4 specification (210 X 297 mm 0 1278319 A7B7 V. Invention description (98)

Lep Glu Gly Ser Phe Leu Gly Gly Ser Glu Ala Gly Glu Arg Leu Leu 15 X0 15 GIq Gin Gly Leu Ala Gin Val Glu Ala 20 25 <2 <2 <2 <2 10> 5 ll> 8 12>蛋白質 13>挪威大鼠Lep Glu Gly Ser Phe Leu Gly Gly Ser Glu Ala Gly Glu Arg Leu Leu 15 X0 15 GIq Gin Gly Leu Ala Gin Val Glu Ala 20 25 <2 <2 <2 <2 10> 5 ll> 8 12> Protein 13> Norwegian Rat

<m〇> 5 Se!r Gin <31u Glu Ala Pro Gly 1 5 I <2jl0> 6 <211> 3-5 <2jl2>蛋白質 βΐ3>挪威大鼠 <40Ό> 6 Hiis Phe His His Ala Leu Pro Pro Ala Arg His His Pro Asp Leu Glu 15 10 15<m〇> 5 Se!r Gin <31u Glu Ala Pro Gly 1 5 I <2jl0> 6 <211> 3-5 <2jl2> Protein βΐ3>Norwegian Rat <40Ό> 6 Hiis Phe His His Ala Leu Pro Pro Ala Arg His His Pro Asp Leu Glu 15 10 15

Gin AXa Λ Λ Λ Λ 0 12 3 1111 ---2131212-- V V vv 7 60 蛋白質挪威大鼠 LI--*----------------訂--- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 <4〇0> 7 G：(n Ala Glu Ala Thr Axg Gin Ala Ala Ala Gin Glu Glu Arg Leu Ala jl 5 10 15 A^p Leu Ala Ser Asp Leu Leu Leu Gin Tyr Leu Leu Gin Gly Gly Ala I 20 25 30 I AI*g Gin Arg Asp Leu Gly Gly Arg Qly Leu Gin Glu Thr Gin Gin Glu j 3S 40 45 I Arg Glu Αεπ Glu Arg Glu Glu Glu Ala Glu Gin Glu ! 50 55 60 I <210> 8 <211> 46 <#12：>蛋白質 <p13>挪威大鼠 400> δ op. .y Gly Gly Glu Asp Glu Val Gly Glu Glu Asp Glu Glu Ala Ala Ql " 5 10 15 A[La Glu Ala Glu Ala Glu Glu Ala Glu Arg Ala Arg Gin Asn Ala Leu j 20 25 30 丨 -101 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） s'. 1278319 A7 B7 五、發明說明（99) jLeu Phe Ala Glu Glu dlu Asp Gly Glu Ala Gly Ala Gin Asp I 35 40 45Gin AXa Λ Λ Λ Λ 0 12 3 1111 ---2131212-- VV vv 7 60 Protein Norwegian Rat LI--*---------------- Order--- (Please Read the notes on the back and fill out this page. Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumers Co., Ltd. <4〇0> 7 G:(n Ala Glu Ala Thr Axg Gin Ala Ala Ala Gin Glu Glu Arg Leu Ala jl 5 10 15 A^p Leu Ala Ser Asp Leu Leu Leu Gin Tyr Leu Leu Gin Gly Gly Ala I 20 25 30 I AI*g Gin Arg Asp Leu Gly Gly Arg Qly Leu Gin Glu Gin Gin Glu j 3S 40 45 I Arg Glu Αεπ Glu Arg Glu Glu Glu Ala Glu Gin Glu ! 50 55 60 I <210> 8 <211> 46 <#12:>Protein<p13>Nordic Rat 400> δ op. .y Gly Gly Glu Asp Glu Val Gly Glu Glu Asp Glu Glu Ala Ala Ql " 5 10 15 A[La Glu Ala Glu Ala Glu Glu Ala Glu Arg Ala Arg Gin Asn Ala Leu j 20 25 30 丨-101 - This paper scale applies to Chinese national standards ( CNS) A4 size (210 x 297 mm) s'. 1278319 A7 B7 V. Description of invention (99) jLeu Phe Ala Glu Glu dlu Asp Gly Glu Ala Gly Ala Gin Asp I 35 40 45

I j<210> 9 |<211> 49 I <212>蛋白質卜213>挪威大鼠 ί <400> 9I j<210> 9 |<211> 49 I <212>protein 213>Norwegian rat ί <400>

Asp Ala Glu Gly Thr GZu Glu Gly Gly Glu Glu Asp Asp Asp Asp Olu 15 10 15Asp Ala Glu Gly Thr GZu Glu Gly Gly Glu Glu Asp Asp Asp Asp Olu 15 10 15

Glu M隹t ·Ά»ρ Pro Gin Thx lie Asp Ser Leu lie Glu Leu Ser Thir ILy色 20 25 30Glu M隹t ·Ά»ρ Pro Gin Thx lie Asp Ser Leu lie Glu Leu Ser Thir ILy 20 25 30

Leu His Leu Pro Ala Asp Asp Val val ser lie He Glu C3lu val Glu 35 40 45 ! Glu j <210> 10 i <211> 75 j 蛋白質 j <213〉挪威大鼠 I · j <400> 10 J Asn Ala Pro Pro Glu Pro Val Pro Pro Pro Arg Ala Ala Pro Ala Pro ；1 5 10 15 j Thr His val Arg Ser Pro Gin Pro Pro Pro Pro Ala Pro Ala Arg Asp I 20 2S 30Leu His Leu Pro Ala Asp Asp Val val ser lie He Glu C3lu val Glu 35 40 45 ! Glu j <210> 10 i <211> 75 j Protein j < 213 > Norwegian rat I · j <400> 10 J Asn Ala Pro Pro Glu Pro Val Pro Pro Pro Arg Ala Ala Pro Ala Pro ;1 5 10 15 j Thr His val Arg Ser Pro Gin Pro Pro Pro Pro Ala Pro Ala Arg Asp I 20 2S 30

II

[Glu Leu Pro Asp Trp Asn Glu Val Le\i Pro Pro Trp Asp Airg Glu Glu ! 3S 40 45[Glu Leu Pro Asp Trp Asn Glu Val Le\i Pro Pro Trp Asp Airg Glu Glu ! 3S 40 45

II

J j Acp Glu Val Phe Pro Pro Gly Pro Tyr His Pro Phe Pro Asn Tyr lie ! 50 55 60J j Acp Glu Val Phe Pro Pro Gly Pro Tyr His Pro Phe Pro Asn Tyr lie ! 50 55 60

I j Arg Pro Arg Thv Leu Gin Pro pro Ala Ser Ser ί 65 70 75 I.---.---------------訂---------^^^1 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 -102- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）I j Arg Pro Arg Thv Leu Gin Pro pro Ala Ser Ser ί 65 70 75 I.---.---------------Book---------^^ ^1 (Please read the note on the back and fill out this page) Printed by the Intellectual Property Office of the Intellectual Property Office of the Ministry of Economic Affairs -102- This paper scale applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

Claims

1278i 4524號專利申請案中文申請專利範圍替換本(93年10月）六、申請專利範圍 A8 B8 C8 D81278i Patent Application No. 4524 Replacement of Chinese Patent Application (October 1993) VI. Patent Application A8 B8 C8 D8

1. 一種單株抗體或其片段，其專一地與包括選自包括下列之胺基酸序列的多肽結合： (a) 在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之胺基酸序列；以及 (b) 在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 0 號任一個中陳述之胺基酸序列的片段；或其天然存在的變體。 2 · —種單株抗體或其片段，其專一地與包括在序列識別2 號中陳述之胺基酸序列的多肽，或其片段結合。 3 ·根據申請專利範圍第1或2項之單株抗體，其為抗體或其片段。根據申請專利範圍第丨或2項之單株抗體，其為人類化的抗體。根據申請專利·第…項之單株抗體，其為人類抗體或其片段。 6. 根據申請專利範圍第1或2項之體或其片段。單株抗體，其為嵌合型抗A monoclonal antibody or fragment thereof, which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) in sequence recognition No. 1, sequence recognition No. 2, sequence recognition No. 3, sequence recognition 4 No., Sequence Recognition No. 5, Sequence Recognition No. 6, Sequence Recognition No. 7, Sequence Identification No. 8, Sequence Identification No. 9, or Sequence Identification 1 Amino acid sequence as stated in any of the nicknames; and (b) in sequence recognition 1 No., sequence identification No. 2, sequence identification No. 3, sequence identification No. 4, sequence identification No. 5, sequence identification No. 6, sequence identification No. 7, sequence identification No. 8, sequence identification No. 9, or sequence identification No. 1 A fragment of the amino acid sequence stated; or a naturally occurring variant thereof. 2 - A monoclonal antibody or fragment thereof, which specifically binds to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof. 3. A monoclonal antibody according to claim 1 or 2 of the patent application, which is an antibody or a fragment thereof. A monoclonal antibody according to the second or second aspect of the patent application, which is a humanized antibody. According to the monoclonal antibody of the patent application, it is a human antibody or a fragment thereof. 6. According to the scope of claim 1 or 2 of the patent application or its fragments. Chimeric antibody

根據申請專利範圍第1或2項植之抗體或其片段。 <單株抗體，其為CDR-移 8· 根據申清專利範圍第1或2項之單株抗體，其為抗遺傳性An antibody or fragment thereof, according to claim 1 or 2 of the patent application. <Monobody antibody, which is CDR-shifted. 8. According to the patent of the patent of the scope of claim 1 or 2, it is anti-hereditary.

1278319 A8 B8 C8 _____D8 六、申請專利範圍 ~^ 型的抗體或其片段。 9 ·根據申請專利範圍第1或2項之單株抗體，其為可變區片段。 1 0 ·根據申請專利範圍第8項之可變區片段，其為F ab或F ab，片段。 1 1 . 一種單株抗體或其片段，包括至少一個互補性-決定區，其對具有在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別丨〇號任一個中陳述之胺基酸序列的多肽具有專一性。 1 2 ·根據申請專利範圍第丨或2項之單株抗體，其與可檢測標記結合。 1 3 ·根據申睛專利範圍第1或2項之單株抗體，其拮抗多肽的生物活性。 14· 一種用來治療、預防或改善VGF多肽_相關性疾病、障礙或病況的醫藥組合物，包括有效含量的根據申請專利範圍第1或2項之單株抗體。 15·根據申請專利範圍第14項之醫藥組合物，其中該多肽相關性疾病、障礙或病況為肥胖。 16·根據申請專利範圍第14項之醫藥組合物，其中該多肽相關性疾病、障礙或病況，係選自包括***、惡病質、飲食障礙、AIDS相關性複合症、代謝亢進之病況、機能尤進、活動性不足和胰島素產生過多。 17.根據申請專利範圍第16項之醫藥組合物，其中該飲食障 -2-1278319 A8 B8 C8 _____D8 VI. Application for patents ~^ Type of antibody or fragment thereof. 9. A monoclonal antibody according to claim 1 or 2 of the patent application, which is a variable region fragment. 1 0. A variable region fragment according to item 8 of the patent application, which is a F ab or F ab, a fragment. 1 1. A monoclonal antibody or fragment thereof, comprising at least one complementarity-determining region, the pair having sequence recognition number 1, sequence recognition number 2, sequence recognition number 3, sequence recognition number 4, sequence recognition number 5, sequence Polypeptides that recognize the amino acid sequence set forth in any of Sequence No. 6, Sequence Recognition No. 7, Sequence Identification No. 8, Sequence Identification No. 9, or Sequence Identification Nucleus are specific. 1 2 · A monoclonal antibody according to item 丨 or item 2 of the patent application, which is combined with a detectable label. 1 3 · A monoclonal antibody according to item 1 or 2 of the scope of the patent application, which antagonizes the biological activity of the polypeptide. A pharmaceutical composition for treating, preventing or ameliorating a VGF polypeptide-associated disease, disorder or condition, comprising an effective amount of a monoclonal antibody according to claim 1 or 2 of the patent application. The pharmaceutical composition according to claim 14, wherein the polypeptide-related disease, disorder or condition is obesity. The pharmaceutical composition according to claim 14, wherein the polypeptide-related disease, disorder or condition is selected from the group consisting of infertility, cachexia, eating disorder, AIDS-related complex disease, hypermetabolic condition, and function. Advance, lack of activity and excessive insulin production. 17. The pharmaceutical composition according to claim 16 of the patent application, wherein the eating disorder -2-

1278319 A8 B8 C8 _____D8_ __ 六、申請專利範圍礙為貪食症和神經性厭食症。 1 8 · —種藉著以多肽免疫動物而產生的單株抗體，該多肽包括在序列識別1號、序列識別2號、序列識別3號、序列識別4號、序列識別5號、序列識別6號、序列識別7號、序列識別8號、序列識別9號或序列識別1 〇號任一個中陳述之胺基酸序列。 I9· 一種雜種瘤，其產製能夠與根據申請專利範圍第丨或2項之多肽結合的單株抗體。1278319 A8 B8 C8 _____D8_ __ VI. Scope of application for patents Impeding bulimia and anorexia nervosa. 1 8 · A monoclonal antibody produced by immunizing an animal with a polypeptide, which is included in sequence recognition No. 1, sequence identification No. 2, sequence identification No. 3, sequence identification No. 4, sequence identification No. 5, sequence identification 6 No., Sequence Identification No. 7, Sequence Identification No. 8, Sequence Identification No. 9, or Sequence Identification 1 Amino acid sequence as stated in any of the nicknames. I9· A hybridoma which produces a monoclonal antibody capable of binding to a polypeptide according to the formula or item 2 of the patent application.