TWI264467B - Human anti-CD40 antibodies and methods of making and using same - Google Patents

Human anti-CD40 antibodies and methods of making and using same Download PDF

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TWI264467B
TWI264467B TW090110333A TW90110333A TWI264467B TW I264467 B TWI264467 B TW I264467B TW 090110333 A TW090110333 A TW 090110333A TW 90110333 A TW90110333 A TW 90110333A TW I264467 B TWI264467 B TW I264467B
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antibody
human
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activity
cells
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Toshifumi Mikayama
Nobuaki Takahashi
Xingjie Chen
Stephen P Schoenberger
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Kirin Brewery
Jolla Inst Allergy Immunolog
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The invention is directed to human antibodies that bind CD40 (e.g., human CD40), methods of producing the antibodies and methods of use. Invention human CD40 antibodies include antibodies that can modulate one or more activities of CD40, such as increasing or decreasing cell proliferation. Invention human CD40 antibodies are therefore useful for increasing or decreasing a CD40 activity in order to alter CD40 activity in vivo.

Description

12644671264467

經濟部智慧財產局員工消費合作社印製 先前資料 本案申請專利優先於U.s. Pat系列號6〇/2〇〇,6〇1，公告於 2000年4月2 8日。 技術領域 ,本木疋有關結合至抗原之人類抗體，較特別是結合至人 類CD40之人類抗一 CD4〇抗體，其可調控cD4〇活性。 背景 免疫系統是保護身體拮抗感染作用物的重要角色。然而 已雀知泎夕疾病階段及/或失調症是由於免疫反應不正常 或非欲求之活化作用所致。常見的實例包括移植物的宿主 疾病(GVHD) ’器官或移植物排斥，發炎，及與自體免疫 有關 < 疾病，如多發性硬化（MS)，全身性紅斑狼瘡 (SLE)，及類風濕性關節炎（RA)。 一般而ΊΓ ’免疫反應由於組織傷害或感染之結果而活 化。此二例子均涉及許多免疫系統效應物細胞之補給及活 化作用（即B -及T -淋巴細胞，巨噬細胞，嗜伊紅白血球， 嗜中性白血球等）於經由一系列複雜的細胞'細胞交互作用 之撝同過私中。免疫反應拮抗外來蛋白質之典型劇本如 下·爲抗原呈現細胞（APC’s)如巨噬細胞或樹突細胞的補 獲炙外來蛋白質，係在APC細胞表面上處理及呈現。循環 的T-輔助細胞（Τη細胞），其可表現免疫球蛋白，並確認 (即結合）所呈現之抗原，係在ApC之活化作用下。經活化 的丁 η細胞再活化適合的β _細胞純系，以增殖及分化成漿 細胞，再產生及分泌可對準拮抗外來抗原之體液抗體。所 -4- > ^----「訂---------線 (請先閱讀背面之注意事項再填寫本頁} 本纸張尺度適用中國國家標準（CNS)A4規格（2】0 297公釐） 1264467 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（2 ) 分泌之抗體與可表現外來蛋白質之任何細胞結合，對準此 細胞以爲其他免疫效應物細胞所破壞。 當Τ η細胞與B細胞接觸，可刺激B細胞增殖及免疫球蛋 白（Ig)類由IgM轉換成IgG，IgA或IgE類。各種受體-配體交 互作用涉及於對T -依賴性抗原反應中，Τ η細胞及B細胞間 調介之接觸。特言之，CD40-CD40配體（CD40L)成對，是 達成此細胞-細胞交互作用具關键性的。CD40L並不在休 眠的Τ Η細胞上表現，但在細胞與Τ -依賴性抗原接觸後被 謗生。Β細胞經由Β -細胞表面上之CD40抗原而爲CD40L所 刺激，並在IL-4配合下，可調介IgE之分泌。 CD40L及CD40均爲穿膜糖蛋白，分別屬於腫瘤壞死因子 (TNF)及TNF受體（TNF-R)—族。人類CD40是一種有2 7 7個 胺基酸之肽，是有分子量3 0,600，19個胺基酸之分泌訊號 肽，大部份含有疏水性胺基酸及在細胞外區域中有富含半 胱胺酸的特色。蛋白質含有一段推想的領導子序列，穿膜 區域及許多其他與膜結合之受體蛋白質共有的特色。 人類CD40L是一種II型膜-結合之 Exp. Med. 176:1543 (1992))。鼠類 CD40L 也已被選殖 (Arm it age et al·，Nature 357:80 (1992))。CD40L 在其 C -末端 有一個細胞外區域，穿膜區域，及在N -末端有細胞内區 域。可溶性CD40L包括CD40L的細胞外區域（胺基酸4 7至 2 6 1 )。在無任何共刺激物下，CD40L可謗導B細胞之增殖 作用，且在細胞動素下也可謗使免疫球蛋白之產製。 CD40L活性由CD40L細胞夕卜區域與CD40之結合而調介。 -5- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ----i--------^^衣---^----:丨訂---------線 (請先閱讀背面之注意事項再填寫本頁) 1264467 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 ) C D 4 0表現在B淋巴細胞上’並除了誘導I g類-轉換作用 外也參與於許多的B細胞功能，如可作用如特異抗原之輔 因子’及B細胞有絲***中的某些淋巴激素，避免細胞之 預期叱亡’及啓動B細胞黏附至其他細胞。c d 4 0也可表現 在B -細胞以外的細胞型式中，包括巨噬細胞，樹突細胞， 胸腺上皮細胞，蘭氏細胞及内皮細胞。這些發現導致目前 之想法，即CD40經由調介T _細胞與B -細胞以及其他細胞 型式間之交互作用，在免疫調控中扮演廣泛角色。在此觀 點之支持下，巨嗤細胞及樹突細胞中CD40之刺激作用， 是抗原呈現中T -細胞活化作用所必要（Gruss et al.，Leuk. Lymphoma，24:393 (1997)) 〇 證據顯示，CD40也參與於組織之發炎。已示出，巨噬細 胞對發炎介質IL-12及氧化氮之產製是和CD40有關的 (Buhlmann and Noelle，J. .Clin. Immunol. 16:83 (1996))。在 内皮細胞，頃發現CD40L對CD40之刺激作用可謗使E-選 擇素，ICAM-1及VCAM-1之表面表現，促進白血球與發炎 邵位之黏附作用（Buhlmann and Noelle，J. Clin· Immunol· 16:83 (1996) ; Gruss et al.，Leuk，Lymphoma，24:393 (1997))。最後，在上皮及造血系腫瘤以及腫瘤浸潤之内皮 細胞上，CD40過度表現冬研究顯示CD40。在腫瘤生長及/ 或血管生成中扮演重要角色（Gruss，et al·，Leuk，Lymphoma， 24:393 (1997) ; Kluth et al·，Cancer Res·，57:891 (1997))。 由於CD40在體液免疫學中之角色，因此針對調控CD40 之治療策略可用於治療許多與免疫有關之失調症。例如， -6- 本纸張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ί —^--------^--------^---------. (請先閱讀背面之注意事項再填寫本頁) 1264467Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives. Previous information The patent application in this case takes precedence over the U.s. Pat serial number 6〇/2〇〇, 6〇1, announced on April 28, 2000. In the technical field, the human cockroach is associated with a human antibody that binds to an antigen, and more particularly to a human anti-CD4 〇 antibody that binds to human CD40, which modulates cD4 〇 activity. Background The immune system is an important role in protecting the body against antagonism. However, it has been known that the stage of the disease and/or the disorder is caused by an abnormal immune response or an undesired activation. Common examples include graft host disease (GVHD) 'organ or graft rejection, inflammation, and autoimmune associations< diseases such as multiple sclerosis (MS), systemic lupus erythematosus (SLE), and rheumatoid Arthritis (RA). In general, the immune response is activated as a result of tissue damage or infection. Both of these examples involve the replenishment and activation of many immune system effector cells (ie, B- and T-lymphocytes, macrophages, eosinophils, neutrophils, etc.) via a series of complex cell 'cells The interaction is the same as private. A typical script for antagonizing foreign proteins by the immune response is as follows: antigen-presenting cells (APC's) such as macrophage or dendritic cells, which are processed and presented on the surface of APC cells. Circulating T-helper cells (Τη cells), which express immunoglobulins, and confirm (ie bind) the antigens present, under the activation of ApC. The activated Dn cells reactivate the appropriate β-cell pure line to proliferate and differentiate into plasma cells, and then produce and secrete humoral antibodies that align against the foreign antigen. -4- > ^---- "Order --------- line (please read the note on the back and then fill out this page) This paper size applies to the Chinese National Standard (CNS) A4 specification ( 2] 0 297 mm) 1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperative Printed 5, Invention Description (2) Secreted antibodies bind to any cell that can express foreign proteins, and align this cell with other immune effector cells Destruction. When Τ η cells are in contact with B cells, they can stimulate B cell proliferation and convert immunoglobulin (Ig) from IgM to IgG, IgA or IgE. Various receptor-ligand interactions involve T-dependence In the antigenic reaction, the contact between Τη cells and B cells is regulated. In particular, the CD40-CD40 ligand (CD40L) is paired, which is critical for achieving this cell-cell interaction. CD40L is not dormant. It is expressed on Η cells, but it is produced after contact with Τ-dependent antigen. Β cells are stimulated by CD40L via CD40 antigen on the surface of Β-cell, and can be adjusted with IL-4. Secretion of IgE. CD40L and CD40 are transmembrane glycoproteins, which are tumor necrosis Factor (TNF) and TNF receptor (TNF-R)-family. Human CD40 is a peptide with 277 amino acids, which is a secretory signal peptide with a molecular weight of 3,600,19 amino acids. Contains a hydrophobic amino acid and is rich in cysteine in the extracellular region. The protein contains a conceivable leader sequence, transmembrane region and many other features shared with membrane-bound receptor proteins. Human CD40L Is a type II membrane-binding Exp. Med. 176:1543 (1992)). Mouse CD40L has also been selected (Arm it age et al., Nature 357:80 (1992)). CD40L in its C- There is an extracellular region at the end, a transmembrane region, and an intracellular region at the N-terminus. Soluble CD40L includes the extracellular region of CD40L (amino acid 4 7 to 261). Without any costimulatory substance, CD40L It can induce the proliferation of B cells, and can also produce immunoglobulin under cytokines. CD40L activity is regulated by the combination of CD40L cells and CD40. -5- This paper scale applies to China National Standard (CNS) A4 Specification (210 X 297 mm) ----i--------^^衣---^----:丨定----- ----Line (please read the note on the back and fill in this page) 1264467 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (3) CD 4 0 is expressed on B lymphocytes Induction of I g-switching is also involved in many B-cell functions, such as cofactors such as specific antigens and certain lymphoid hormones in B cell mitosis, avoiding the expected collapse of cells and initiate B cell adhesion. To other cells. c d 4 0 can also be expressed in cell types other than B-cells, including macrophages, dendritic cells, thymic epithelial cells, Langerhans cells and endothelial cells. These findings led to the current idea that CD40 plays a broad role in immune regulation via the interaction between T-cells and B-cells and other cell types. With the support of this view, the stimulatory effect of CD40 in giant sputum cells and dendritic cells is necessary for T-cell activation in antigen presentation (Gruss et al., Leuk. Lymphoma, 24:393 (1997)). It is shown that CD40 is also involved in the inflammation of the tissue. It has been shown that macrophage production of IL-12 and nitric oxide in the inflammatory mediators is associated with CD40 (Buhlmann and Noelle, J. Clin. Immunol. 16:83 (1996)). In endothelial cells, it was found that the stimulatory effect of CD40L on CD40 can promote the surface expression of E-selectin, ICAM-1 and VCAM-1, and promote the adhesion of leukocytes to inflamed spinal sites (Buhlmann and Noelle, J. Clin. Immunol · 16:83 (1996); Gruss et al., Leuk, Lymphoma, 24:393 (1997)). Finally, on epithelial and hematopoietic tumors and tumor-infiltrating endothelial cells, CD40 overexpression winter studies showed CD40. It plays an important role in tumor growth and/or angiogenesis (Gruss, et al., Leuk, Lymphoma, 24: 393 (1997); Kluth et al., Cancer Res, 57: 891 (1997)). Because of the role of CD40 in humoral immunology, therapeutic strategies for regulating CD40 can be used to treat many immune-related disorders. For example, -6- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ί —^--------^--------^---- -----. (Please read the notes on the back and fill out this page) 1264467

經濟部智慧財產局員工消費合作社印製 五、發明說明（4 ) CD40活性之抑制可減低移植物對宿主疾病（GVHD)，移植 物排斥，及自體免疫疾病如多發性硬化（MS)，全身性紅斑 狼瘡（SLE)，及某些型式之關節炎。制劑也可充作 抗發炎化合物，且因此可用於治療各種炎及過敏狀況，如 氣。而，類風濕性關郎炎，自體移植排斥，發炎性腸疾，各 皮膚狀況’及牛皮癣。CD40之抑制劑也可充作抗腫瘤 劑，及其他過度增殖狀況之抑制劑。 CD40活性之促進劑或刺激劑也可用於增加體液免疫力以 抗阻感染劑’如病毒或細菌病原體。促進劑也可用於刺激 或加強體液免疫力以拮抗腫瘤。此外，CD40活性之促進 劑或刺激劑也可用於促進記憶力，由是可改善免疫反應之 速度或強健。 在動物模式中直接拮抗CD40或CD40L之單株抗體顯示， CD40刺激作用之抑制對於GVHD，自體移植物排斥，類風 濕性關節炎，SLE，MS及B-細胞淋巴瘤具有療效益處 (Buhlmann and Noelle，J· Clin. Immunol，16:83 (1996))。因 此C D 4 0之抗體具有成爲南度有效治療劑之潛力…具拮抗性 之抗-CD40抗體可充作免疫遏止劑/抗炎劑，而具激動性之 抗-CD40抗體可充作免疫刺激劑以追加免疫反應，如於免 疫系統受損之個體中。許多抗-CD40抗體目前正發展中， 其可能是鼠類的，彼合的或人類化的。然而，非人類或人 類化抗體因爲對非人類邵份發展出免疫反應，因此效力上 受限。本發明針對此問題並提出相關之優點。 發明要點 本纸張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱） 卜！2-----------Μ---:丨訂--------- (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 1264467 Α7 五、發明說明（5 ) 本發明提出可與CD40(如人類CD40)特異地結合，且可 調控一種以上CD40活性之經分離的人類抗- CD40抗體及其 片段（如scFv，Fab，Fab，或F(ab，）2)。抗體可以是多株或單 株。特殊具體實例包括示爲no. 11及7 2之抗體，及示爲卩1-!〇2，F5-152，F2-103，F5-77，F5-157 及 F4-465 之融合瘤。 其特殊之片段包括示爲no. 11及7 2之抗體片段，或由示爲 FM02，F5-152，F2-103，F5-77，F5-157及F4-465之融合瘤 所產生之抗體。人類CD40抗體特異的輕及重鏈或其片 段，示於SEQ ID NOs:10，11，12，13，14及15。 另外的實施例包括具有CD40結合特異性標示爲no. 11或· 7 2 之抗體，或由標示爲 Fi-i〇2，F5-152，F2-103，F5-77， F5-157及F4-465之融合瘤所產生之抗體。又另外的實施例 包括具有C D 4 0调郎活性標tf爲η 〇. 11或7 2之抗體’或由標 示爲 Fl-102，F5_152，F2-103，F5-77，F5-157及F4-465之融 合瘤所產生之抗體。 可爲人類CD40抗體調控之CD40活性（即增加或減低）包 括如CD40傳訊活性。特殊活性包括細胞增殖之調控（如B -細胞）或蛋白質表現（如CD95，CD80或CD86)。人類CD40抗 體包括可減低或增加CD40配體（如CD40L)至CD40結合之抗 體。人類CD40抗體在有或無CD40配體，如CD40L下可調 控CD40活性。 · 人類C D 4 0抗體包括含有λ或K輕键序列或重键序列之抗 體。 人類CD40抗體包括經修飾型式，如胺基酸置換，加入或 刪除，以及含有異質的功能性區域及不同的分子實體，而 -8- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） —✓--------裝——；—訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 A7 B7 經濟部智慧財產局員工消費合作社印制衣 五、發明說明（6 ) 可在人類CD40抗體上提供互補的不同的功能性。因此也 提出被可偵測標記之CD40抗體。 也提出包括有人類CD40抗體之藥學調和物。也提出可表 現人類CD40抗體之宿主細胞（如融合瘤）。也提出編碼人類 CD40抗體之核酸，以及含有編碼人類CD40抗體之核酸之 宿主細胞。 提出可調控CD40活性之人類CD40抗體之製法。方法包 括如對可表現人類免疫球蛋白之動物（如老鼠）投予CD40或 其免疫原片段；篩選所投予之動物是否有人類CD40抗體 之表現；選出可產生人類CD40抗體之動物，自可產生人 類CD40抗體之動物中分離抗體；並決定人類CD40抗體是 否可調控CD40活性，由是產製可調控CD40活性之人類 CD40抗體。 也提出可調控CD40活性之人類單株CD40抗體之製法。 方法包括如，對可表現人類免疫球蛋白之動物（如老鼠）投 予人類CD40或其免疫原片段；自可產製人類CD40抗體之 動物中分離脾細胞；將脾細胞與骨髓瘤細胞融合以產生融 合瘤；並針對可調控CD40活性之人類CD40抗體進行融合 瘤之表現篩選，由是產製出可調控CD40活性之人類單株 CD40抗體。 由本方法產生之抗體，包括自本發明方法產生之融合瘤 中分離之單株抗體，也提出。 提出調控CD40傳訊活性之方法，包括將可表現CD40之 細胞與調控量之人類CD40抗體接觸。CD40傳訊活性調控 -9 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） • Γ - - 1^ —— — — — — ϋ I ϋ I I I I I ϋ — — — — — — — (請先閱讀背面之注意事項再填寫本頁) 1264467 經濟部智慧財產局員工消費合作社印？衣 A7 B7 五、發明說明（7 ) 的包括增加或減少細胞之增殖（如B -細胞）或蛋白質的表現 (如0095,0080或〇086)。方法視所需包括0040配體之加 入，如CD40L。具體實例包括其中CD40在細胞中是屬人類 白勺。 在另外的具體實例中，方法的實行是利用具有結合特異 性之抗體，具有CD40調控活性，或具有與示爲no. 11或72 之抗體具CD40結合親和力，a*Fl-102，F5-152，F2-103，F5-77，F5-157或F4-465之融合瘤所產生之抗體° 方法可在個體中（如人類）實行，以調控個體中一種以上 的CD40活性。因此也提出增加個體中CD40傳訊活性之方 法及偵測法。在一個具體實例中，方法包括對個體投予藥 量之人類抗-CD40抗體，其係可增加CD40傳訊活性。在另 一具體實例中，方法包括對個體投予可減低CD40傳訊活 性之劑量之人類抗-CD40抗體。 方法可在個體中實行（如人類）以改善失調症或治療與 CD40活性有關之狀況。即，似乎可反應增加或減低的 cd4〇活^生t失調#或μ況（如#失調#或另大況有關m矣 群被減低，個體改善等），可利用人類CD40抗體予以治 療。因此，也提出治療個體失調症或狀況之方法。在一個 具體實例中，狀況包括僻體免疫失調症或非欲求的免疫反 應，方法包括對個體投予可降低CD40活性劑量之人類抗-CD40抗體，由是可舒緩免疫失調症或抑制非欲求之免疫 反應。在另一具體實例中，狀況包括個體之免疫失調症， 且方法包括對個體投予可降低CD40活性劑量之人類抗- -10- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ,·—f-------裝--------訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（8 ) CD40抗體，由是舒緩免疫失調症。 以本發明人類CD40抗體可治療之特異的免疫失調症及非 欲求之免疫反應包括如：宿主對移植細胞，組織或器官之 排斥，發炎，自體免疫力，.淋巴瘤，白血病或骨髓瘤。另 外的免疫失調症包括如免疫缺失，細胞增殖失調症（如良 性增生或癌症/腫瘤）。 另外的方法包括在個體中謗生或刺激免疫反應。在一個 具體實例中，纟法包括對個體投予可增加。_活性劑量 之人類抗-CD40抗體，由是謗生或刺激免疫反應。以人類 圓抗體刺激之特殊免疫反應，包括如拮抗細胞増殖失 調症之免疫反應（如良性增生或癌症/腫瘤）或拮抗病原體 感染之免疫反應。 治療方法包括以人類CD40抗體預防性治療。如預防治療 方法之標的個體包括有失調症或個體危險性之個體，其中 若有任何與特殊失調症有關之症狀也是很少呈現，但是易 感受此失碉症的。 也提出在樣品或細胞中偵測CD4〇存在之方法。方法包括 如，將有或疑似有CD40之樣品，或可表現或疑似可^現 CD40之細胞，與人類〇：1；)4〇抗體接觸，並偵測樣品或細胞 中有否CD40之存在。可·分析之樣品包括如：組織，流體 或來自個體之其他檢品。 也提出偵測與個體中增加的或減低的CD40表現有關之失 調症有否存在之方法。方法包括如，將具有或疑似具有 CD40之樣品或可表現或疑似可表現CD4〇之細胞，其中樣 -11 - 本纸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) 1264467 A7 B7 經濟部智慧財產局員工消費合作社印創衣 五、發明說明（9 ) 品或細胞來自或存在於個體中（如人類），與人類CD40接 觸，並在對照比較下偵測樣品或細胞中增加的或減低之 CD40表現之存在，由是可偵測個體中與增加的或減低之 CD40表現有關之失調症之存在。 本發明一個以上具體實例之詳述示於附圖及以下説明 中。本發明其他的特色，目標及優點，由説明及附圖及申 請專利範圍中顯而易見。 附圖說明 圖1示出下列之Ramos細胞染色活性：（A)抗-人類CD40-FITC陽性對照組及抗-人類CD40抗體，nos· (B) 11 ; (C) 3 66 ; (D) 72 ; (E) 30。由有限稀釋分離出之抗體，其染色 活性之決定如實例1所述。 圖2示出抗-人類CD40抗體之作用：（A) no. 30，72，366 及（B) no· 11，5C3及G28，在Ramos B細胞CD95之表現上， 並與CD40L比較。加入抗體或可溶性CD40配體，濃度依橫 軸所示。以實例1之FACS方法偵測CD95之表現。垂直軸代 表CD95表現之高峰數値。 圖3示出人類抗-人類CD40抗體nos. 30，72及366在Ramos B細胞中阻斷CD95表現之能力。抗體依所示之之濃度與可 溶性CD40配體一起加入。由實例1之FACS法偵測CD95之 表現。水平橫軸顯示可溶性CD40配體之濃度，縱軸爲 CD95表現之高峰値。 圖4示出人類抗-人類CD40抗體在人類周邊B細胞增殖上 之作用，於（A) CD40L存在或（B)不存在（1微克/毫升）下歷 -12- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ϋ ϋ I I n ϋ ϋ I ϋ I > I I n n ϋ ϋ ϋ 訂---------線 (請先閱讀背面之注意事項再填寫本頁) 1264467 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（10 ) 3天。水平軸表示蛋白質抗體或可溶性CD40L濃度。縱軸 表示由人類B細胞所納入之[3h]-胸嘗，如實例1所述。 圖5示出Ramos細胞CD95染色活性，是在（A)人類IgG(實 線）或Fl-l〇2(虛線）抗體；（B) CD40L及人類IgG (實線）或 F 1 -10 2 (虛線）抗體培育一夜之後，f 1 _ 1 〇 2純化自腹水液。 圖6示出Ramos細胞CD95染色活性，將在（A)人類I gG(實 線）或F4-465或F5-152(虛線）抗體··及（b) CD40L，及人類 IgG(實線）或與F4-465或F5-152(虛線）抗體培育一夜之 後。F4-465及F5-152係純化自腹水液。 圖7示出Ramos細胞CD95染色活性，係在（A)人類IgG (實 線）或與F2-103，F5-157或F5-77(虛線）抗體，及⑻CD40L 及人類IgG (實線）或與F2-l〇3，F5-157或F5-77(虛線）抗體 培育一夜之後。F2-103，F5-157及F5-77均在瞬時轉感之 Cos細胞中表現。 詳細説明 本發明是以因CD40產生之人類抗體爲基礎，至少部份如 此。即，本發明之CD40抗體爲人類免疫球蛋白（Ig)胺基酸 序列。此意味，本發明之抗體當引入人體内無法謗生拮抗 彼之任何免疫反應。因此，本發明之CD40抗體由重覆投 予中似乎不會產生過敏性，而由於無強的免疫反應故可保 持在人體内較長時間。 本發明也是以拮抗人類CD40之人類抗體之產生爲基礎， 至少部份如此，其可調控一種以上的，CD4〇活性。例 如，本發明抗體包括可抑制CD4〇活性之CD4〇抗體，係減 V----1-----------^----； —訂·--------線 (請先閱讀背面之注意事項再填寫本頁)Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Printing 5, Inventions (4) Inhibition of CD40 activity can reduce graft-to-host disease (GVHD), graft rejection, and autoimmune diseases such as multiple sclerosis (MS), whole body Sexual lupus erythematosus (SLE), and certain types of arthritis. The formulation can also be used as an anti-inflammatory compound and can therefore be used to treat a variety of inflammatory and allergic conditions, such as qi. However, rheumatoid arthritis, autologous transplant rejection, inflammatory bowel disease, various skin conditions and psoriasis. Inhibitors of CD40 may also act as antitumor agents, as well as other inhibitors of hyperproliferative status. Promoters or stimulators of CD40 activity can also be used to increase humoral immunity against infectious agents such as viral or bacterial pathogens. Accelerators can also be used to stimulate or potentiate humoral immunity to antagonize tumors. In addition, CD40 activity enhancers or stimulators can also be used to promote memory by improving the speed or robustness of the immune response. Monoclonal antibodies that directly antagonize CD40 or CD40L in animal models have shown that inhibition of CD40 stimulation has therapeutic benefit for GVHD, autograft rejection, rheumatoid arthritis, SLE, MS and B-cell lymphoma (Buhlmann and Noelle, J. Clin. Immunol, 16:83 (1996)). Therefore, the antibody of CD40 has the potential to be an effective therapeutic agent for the south. The antagonistic anti-CD40 antibody can be used as an immunosuppressive/anti-inflammatory agent, and the agonistic anti-CD40 antibody can be used as an immunostimulant. To supplement the immune response, such as in individuals with impaired immune systems. Many anti-CD40 antibodies are currently under development, which may be murine, heterozygous or humanized. However, non-human or humanized antibodies are limited in their effectiveness because they develop an immune response to non-human counterparts. The present invention addresses this problem and presents related advantages. Summary of the invention This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 public). 2-----------Μ---:丨定--------- (Please read the notes on the back and fill out this page.) Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumer Cooperatives 1264467 Α7 V. INSTRUCTION DESCRIPTION (5) The present invention provides isolated human anti-CD40 antibodies and fragments thereof (such as scFv, Fab, which can specifically bind to CD40 (such as human CD40) and can regulate more than one CD40 activity. Fab, or F(ab,) 2). The antibody may be a plurality of strains or a single plant. Specific specific examples include antibodies shown as no. 11 and 7 2, and fusion tumors shown as 卩1-!〇2, F5-152, F2-103, F5-77, F5-157 and F4-465. Specific fragments thereof include antibody fragments shown as no. 11 and 7 2, or antibodies produced by fusion tumors shown as FM02, F5-152, F2-103, F5-77, F5-157 and F4-465. Human CD40 antibody specific light and heavy chains or fragments thereof are shown in SEQ ID NOs: 10, 11, 12, 13, 14 and 15. Additional examples include antibodies having a CD40 binding specificity designation of no. 11 or · 7 2 or by the symbols Fi-i〇2, F5-152, F2-103, F5-77, F5-157 and F4- 465 of fusion tumors produced by antibodies. Still other examples include antibodies having a CD 40 concentration activity tf of η 〇. 11 or 7 2 or by the labels Fl-102, F5_152, F2-103, F5-77, F5-157 and F4- 465 of fusion tumors produced by antibodies. CD40 activity (i.e., increased or decreased) that can be modulated by human CD40 antibodies includes, for example, CD40 signaling activity. Specific activities include regulation of cell proliferation (such as B-cells) or protein expression (such as CD95, CD80 or CD86). Human CD40 antibodies include antibodies that reduce or increase CD40 ligand (e.g., CD40L) to CD40 binding. Human CD40 antibodies are regulatable for CD40 activity with or without a CD40 ligand, such as CD40L. • Human C D 4 0 antibodies include antibodies that contain a lambda or K light bond sequence or a heavy bond sequence. Human CD40 antibodies include modified versions, such as amino acid substitutions, additions or deletions, and heterogeneous functional regions and different molecular entities, while -8- this paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X). 297 mm) —✓--------装——;—订--------- (Please read the notes on the back and fill out this page) 1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Employee Consumption Cooperatives Printed Clothing V. Inventive Note (6) Provides complementary and different functionalities on human CD40 antibodies. Therefore, a CD40 antibody that is detectably labeled is also proposed. Pharmaceutically conjugates including human CD40 antibodies are also proposed. Host cells (e.g., fusion tumors) that exhibit human CD40 antibodies are also proposed. Nucleic acids encoding human CD40 antibodies, as well as host cells containing nucleic acids encoding human CD40 antibodies, are also proposed. A method for producing a human CD40 antibody capable of regulating CD40 activity is proposed. The method comprises, for example, administering CD40 or an immunogenic fragment thereof to an animal capable of expressing human immunoglobulin (such as a mouse); screening the administered animal for the expression of a human CD40 antibody; and selecting an animal capable of producing a human CD40 antibody, An antibody is isolated from an animal that produces a human CD40 antibody; and it is determined whether a human CD40 antibody can modulate CD40 activity by producing a human CD40 antibody that modulates CD40 activity. A method for producing a human monoclonal CD40 antibody capable of regulating CD40 activity has also been proposed. The method comprises, for example, administering human CD40 or an immunogenic fragment thereof to an animal capable of expressing human immunoglobulin (such as a mouse); isolating splenocytes from an animal capable of producing a human CD40 antibody; and fusing the splenocytes with myeloma cells; A fusion tumor is produced; and a human CD40 antibody capable of regulating CD40 activity is screened for fusion tumors, and a human monoclonal CD40 antibody capable of regulating CD40 activity is produced. Antibodies produced by this method, including monoclonal antibodies isolated from fusion tumors produced by the methods of the invention, are also contemplated. A method of modulating CD40 signaling activity is provided, comprising contacting a cell exhibiting CD40 with a modulatory amount of a human CD40 antibody. CD40 Communication Activity Control-9 - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) • Γ - - 1^ —————————— ϋ I ϋ IIIII ϋ — — — — — — — (Please read the notes on the back and fill out this page) 1264467 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed? A7 B7 5. Inventive Note (7) includes increasing or decreasing the proliferation of cells (such as B-cells) or the performance of proteins (such as 0095, 0080 or 〇086). The method includes the addition of 0040 ligand as required, such as CD40L. Specific examples include wherein CD40 is a human in a cell. In another embodiment, the method is carried out using an antibody having binding specificity, having CD40 regulatory activity, or having an affinity to CD40 with an antibody shown as no. 11 or 72, a*Fl-102, F5-152 An antibody produced by a fusion tumor of F2-103, F5-77, F5-157 or F4-465 can be practiced in an individual (e.g., a human) to modulate more than one CD40 activity in an individual. Therefore, methods and detection methods for increasing the activity of CD40 communication in individuals are also proposed. In one embodiment, the method comprises administering to the individual a dose of a human anti-CD40 antibody that increases CD40 signaling activity. In another embodiment, the method comprises administering to the individual a human anti-CD40 antibody at a dose that reduces CD40 signaling activity. The method can be practiced in an individual (e.g., a human) to ameliorate the disorder or treat conditions associated with CD40 activity. That is, it seems that the cd4〇 activity can be increased or decreased, such as #失调# or other conditions (such as #失调# or another condition related to m矣 group being reduced, individual improvement, etc.), which can be treated with human CD40 antibody. Therefore, methods for treating an individual disorder or condition are also proposed. In one embodiment, the condition comprises a senile immune disorder or an undesired immune response, the method comprising administering to the individual a human anti-CD40 antibody that reduces the dose of CD40 activity, thereby relaxing the immune disorder or inhibiting the desire immune response. In another embodiment, the condition comprises an individual's immune disorder, and the method comprises administering to the individual a human anti--10-30 dose that reduces the CD40 active dose. The Chinese National Standard (CNS) A4 specification (210 X 297) applies. )))··-f-------装--------Book--------- (Please read the notes on the back and fill out this page) 1264467 A7 B7 Economy Ministry of Intellectual Property Bureau employee consumption cooperatives printed five, invention instructions (8) CD40 antibody, is to relieve immune disorders. Specific immune disorders and undesired immune responses treatable by the human CD40 antibodies of the present invention include, for example, host rejection of transplanted cells, tissues or organs, inflammation, autoimmunity, lymphoma, leukemia or myeloma. Additional immune disorders include, for example, immunodeficiency, cell proliferation disorders (e.g., benign hyperplasia or cancer/tumor). Additional methods include activating or stimulating an immune response in an individual. In a specific example, the sputum method includes an increase in the administration of the individual. _ Active dose of human anti-CD40 antibody, either by agitation or stimulating an immune response. Specific immune responses stimulated by human round antibodies include, for example, immune responses that antagonize cell colonization disorders (such as benign hyperplasia or cancer/tumor) or immune responses that antagonize pathogen infection. Therapeutic methods include prophylactic treatment with human CD40 antibodies. For example, individuals who are at the target of preventive treatment include individuals with disorders or individual risk, and if any symptoms associated with a particular disorder are rarely present, they are susceptible to this deficiency. Methods for detecting the presence of CD4 在 in samples or cells are also proposed. Methods include, for example, contacting a sample with or suspected of having CD40, or a cell that exhibits or is suspected of being CD40, in contact with a human 〇:1;)4〇 antibody, and detecting the presence of CD40 in the sample or cell. Samples that can be analyzed include, for example, tissue, fluids, or other specimens from individuals. Methods for detecting the presence or absence of an imbalance associated with increased or decreased CD40 performance in an individual have also been proposed. Methods include, for example, a sample that has or is suspected of having a CD40 or a cell that exhibits or is suspected of exhibiting CD4〇, wherein the sample -11 is of the Chinese National Standard (CNS) A4 specification (210 X 297 public) ^ ^--------- (Please read the notes on the back and fill out this page) 1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperatives Printed Clothes V. Inventions (9) Products or cells come from or exist Contacting human CD40 in an individual (eg, human) and detecting the presence or absence of increased or decreased CD40 expression in the sample or cell under control comparison, as related to increased or decreased CD40 performance in detectable individuals The existence of the disorder. A detailed description of one or more of the specific embodiments of the invention is set forth in the accompanying drawings. Other features, objects, and advantages of the invention are apparent from the description and drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the following Ramos cell staining activities: (A) anti-human CD40-FITC positive control group and anti-human CD40 antibody, nos·(B) 11 ; (C) 3 66 ; (D) 72 (E) 30. The antibody isolated by limiting dilution, the staining activity was determined as described in Example 1. Figure 2 shows the effect of anti-human CD40 antibodies: (A) no. 30, 72, 366 and (B) no· 11, 5C3 and G28, in the performance of Ramos B cell CD95, and compared to CD40L. Antibody or soluble CD40 ligand was added and the concentration is shown on the horizontal axis. The performance of CD95 was detected by the FACS method of Example 1. The vertical axis represents the peak number of CD95 performance. Figure 3 shows the ability of human anti-human CD40 antibody nos. 30, 72 and 366 to block CD95 expression in Ramos B cells. The antibody is added at a concentration as indicated with the soluble CD40 ligand. The performance of CD95 was detected by the FACS method of Example 1. The horizontal horizontal axis shows the concentration of soluble CD40 ligand, and the vertical axis shows the peak of CD95 performance. Figure 4 shows the effect of human anti-human CD40 antibody on human peripheral B cell proliferation, in the presence of (A) CD40L or (B) absence (1 μg/ml) -12- This paper scale applies to Chinese national standards (CNS) A4 size (210 X 297 mm) ϋ ϋ II n ϋ ϋ I ϋ I > II nn ϋ ϋ ϋ Order --------- line (please read the notes on the back and fill in the form) Page) 1264467 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (10) 3 days. The horizontal axis represents the protein antibody or soluble CD40L concentration. The vertical axis indicates the [3h]-breast taste included by human B cells, as described in Example 1. Figure 5 shows Ramos cell CD95 staining activity in (A) human IgG (solid line) or Fl-l〇2 (dashed line) antibody; (B) CD40L and human IgG (solid line) or F 1 -10 2 ( After the overnight incubation of the antibody, f 1 _ 1 〇 2 was purified from the ascites fluid. Figure 6 shows Ramos cell CD95 staining activity, which will be in (A) human I gG (solid line) or F4-465 or F5-152 (dashed line) antibody · and (b) CD40L, and human IgG (solid line) or After incubation with F4-465 or F5-152 (dashed line) antibodies overnight. F4-465 and F5-152 were purified from ascites fluid. Figure 7 shows Ramus cell CD95 staining activity in (A) human IgG (solid line) or with F2-103, F5-157 or F5-77 (dashed line) antibodies, and (8) CD40L and human IgG (solid line) or F2-l〇3, F5-157 or F5-77 (dashed line) antibodies were incubated overnight. F2-103, F5-157 and F5-77 were all expressed in transient transgenic Cos cells. DETAILED DESCRIPTION The present invention is based, at least in part, on human antibodies produced by CD40. Namely, the CD40 antibody of the present invention is a human immunoglobulin (Ig) amino acid sequence. This means that the antibody of the present invention does not antagonize any of the immune responses when introduced into the human body. Therefore, the CD40 antibody of the present invention does not appear to be allergenic by repeated administration, and can remain in the human body for a long period of time due to no strong immune response. The present invention is also based, at least in part, on the production of human antibodies that antagonize human CD40, which can modulate more than one, CD4〇 activity. For example, the antibody of the present invention comprises a CD4 〇 antibody which inhibits the activity of CD4 ,, and is reduced by V----1-----------^----; --Line (please read the notes on the back and fill out this page)

1264467 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（11 ) 低Ramos B細胞中由CD40L-謗生之CD95之表現（如no. 30， 72及366，以及F4-465)。本發明抗體也包括可抑制CD40活 性之CD40抗體，係減低CD40L -謗生之細胞增殖。本發明 抗體另外包括可刺激CD40活性之CD40抗體，係增加Ramos B細胞中CD40L -謗生之CD95表現（如no· 11及由融合瘤？1-102，F2-103，F5-77，F5-152 及F5-157 所產生之抗體）或細 胞之增殖（no. 11)。本發明抗體進一步包括具有SEQ ID Nos: 10，11，12，13，14或15所示序列之重或輕鏈之CD40 抗體。 因此，依據本發明，在此提出可與CD40結合之人類 CD40抗體（如人類CD40)。在一個具體實例中，CD40抗體 可調控CD40 —種以上活性（如傳訊活性）。在一方面，本發 明的CD40抗體可降低CD40活性（如將CD40L謗生之CD95表 現減緩或阻斷，或抑制細胞之增殖）。在另一方面，本發 明之CD40抗體可增加CD40活性（如CD40L謗生之CD95表現 予以增加或刺激或增加或刺激細胞之增殖）。 如此中所用的，所謂“ CD40抗體”或“抗-CD40抗體”表示 可與CD40特異結合之抗體。“人類CD40抗體”或“人類抗-CD40抗體”表示可與CD40特異結合且由人類免疫球蛋白胺 基酸序列組成之抗體。冬類CD40抗體可結合人類CD40者 是含有人類免疫球蛋白胺基酸序列之抗體，其可與人類 CD40特異結合，然而抗體也可與具有可爲人類CD40抗體 確認之表位之非人類序列結合。 本發明的CD40抗體包括多株或單株抗體，以及如此中所示 -14 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） n n ϋ n n n «1— in I f n ϋ 一· ϋ ϋ I (請先閱讀背面之注意事項再填寫本頁) #· 1264467 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（12 ) 之片段及修飾型。也提出滙集之多株及單株抗體，其含有 具不同結合特異性，結合親和力或功能之二種以上不同 CD40抗體。 本發明的CD40抗體含有k或λ鏈序列。各抗體分子含有 二個k或二個λ輕鏈。k及λ輕鏈間主要的差異在於其固定 區域之序列。於人類，k鏈可變區序列之多樣性更甚於λ 鏈可變區序列，其因而產生了較不同的（多樣的）抗體。由 融合瘤F4-465產生之CD40抗體實例含有一個人類λ輕鏈。 示範之抗體示以 nos. 11 (ATCC ΡΤΑ-2308)，30，72 (ATCC PTA-2309)及 3 6 6，或由 Fl-102，R2-103，F5-77， F5-152，F5-157，及F4-465之融合瘤所產生。雖不爲理論所 縛，可抑制CD40活性之示範CD40抗體，如nos. 30，72， 366及由融合瘤F4-465產生之抗體，似乎會減低CD40L與 CD40之結合，且不謗導CD40之傳訊。相反的，可刺激 CD40活性之CD40抗體，如no. 11，及由融合瘤？1-102，戸2-103，F5-77，F5-152及F5-157產生之抗體，似乎可謗導 CD40之傳訊，及加強CD40L活性。 如此中所用的“調控”，當用於某術語之修飾劑使用時， 表示所修飾術語之一種以上活性或功能被增加或減少。因 此，調控CD40活性表示增加或減少一種以上CD40之活性 或功能。CD40活性包括由CD40傳達的細胞内傳訊，以及 CD40傳訊任何的下游作用，其包括基因或蛋白質表現之 變化（如CD95，CD80或CD86)，或特定細胞反應如細胞增 殖之謗導，同型之轉換或其中CD40參與或扮演角色之狀 -15- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） !-----------^^裝---：----Γ — 訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 A7 -, ...... -—— B / 五、發明說明（13 ) 況或失調症之發展。因此，示範的0〇4〇活性包括調控蛋 白質表現或細胞增殖。 (請先閱讀背面之注意事項再填寫本頁) CD40活性另外的實例包括調控細胞存活(如作用如細胞 存活訊號），抗體產製，抗體同型之轉換，細胞動素之產 生（如 IL-2，IL-6，IL-8，ILM2，TNF-a，IL-4，比^及比一 ίο)，金屬蛋白酶之產生（如ΜΜΡ-W膠原酶&MMP-9/明膠 酶B)及免疫記憶力之發展。CD4〇活性進一步包括調控^ 及於細胞-細胞接觸或黏附之蛋白質之產製（如E _選擇素， VCAM-1 及ICAM-1)。 經濟部智慧財產局員工消費合作社印製 其中有CD40傳訊參與其中之生理狀況或失調症，或其可 反應以改變一種以上CD40活性（如減mCD4〇活性）之狀況 且因此可視爲是CD40活性，包括活體内非欲求之免疫反 應，如自體免疫力，過敏，發炎或移殖排斥。自體免疫力 之特殊實例包括類風濕性關節炎，狼瘡（如SLE，狼瘡腎 炎），自f豆抗體之產製，過敏及克隆氏病。由調控CD4〇活 性可治療之過敏包括對抗原，抗體等之過敏反應。CD4〇 活性之其他實例包括移植排斥，或對移植之抗原過度之免 疫反應（如由已私植器官所表現的），及相關之血液動力學 失常，如白血球減少，移植的器官/組織爲細胞或B _細 胞之浸潤及機會感染，發炎或IgE類之轉換。又另外的實 例包括拮抗治療劑或病毒載體之中和抗體之產製以供基因 治療。另一實例是血栓之形成，動脈粥樣硬化或個體血管 内膜之增厚。 增加CD40活性可造成生理作用之實例包括刺激對感染劑 -16- 本纸張尺度適用中國國家標準（CNS)A4規格（210 1264467 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 ) (如不良免疫原性之病原體）或癌症之免疫反應，或經由改 善細胞調介之免疫力或體液免疫力之記憶，增加反應抗原 之能力。經由刺激CD40，刺激免疫細胞之存活或增殖’ 也可造成治療。 本發明之CD40抗體包括具有此中CD40抗體結合特異性 之抗體。因此，在另一具體實例中，本發明提出具有和所 示 no. 11，30，72及366抗體，或由融合瘤 F 1 -102，F5-1 52， F2-103，F5-77，F5-157及F4-465所產生之抗體結合特異性 之CD40抗體。在一方面，CD40抗體具有如SEQ ID NOs:10，11，12，13，14及15所示之重或輕鏈序歹U。 如此中所用的“結合特異性”，當用於參照至抗體，表示 抗體可確認、相同之抗原表位以爲比較抗體。因此，具有如 no. 11所示抗體之結合特異性之CD40抗體，可確認如no. 11 CD40抗體之相同表位；具有如no. 72所示抗體結合特異 性之CD40抗體，可確認如no. 72 CD40抗體相同之表位； 具有由融合瘤F1-102所產生之抗體之結合特異性之CD40抗 體，可確認如F 1 -1 02融合瘤所產生抗體相同之表位；及依 此類推。 通常表位是短的胺基酸序列，如長約5個胺基酸。鑑定 表位之系統性技術是技#中已知的，且述於如U.S. Pat. No. 4,708,871中。簡言之，可合成由CD40抗原衍生之一組重 疊的寡肽，並結合至一列釘之固相上，各釘上有一種獨特 的寡肽。釘之排列包括9 6孔洞之微滴定盤，令吾等可同時 分析所有9 6種寡肽，如結合至抗-CD40單株抗體。另外， -17- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -------------裝---- (請先閱讀背面之注意事項再填寫本頁) 訂--------- 1264467 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 ) 嗤菌體呈現之肽庫套組（New England BioLabs)可用買的， 以行表位舆圖之定訂。利用這些方法，可決定每一子集可 能的連續胺基酸之結合親和力，以鑑定特定抗體可結合之 表位。當以表位長度肽序列來免疫接種取得抗體之動物 時，也可由推論鑑定出表位。 本發明人類CD40抗體包括具有一種以上CD40抗體功能 之抗體，如此中所示（如CD40調控活性）。因此，在另外的 具體實例中，本發明提出有一種以上功能的CD40抗體（如 增加或減低與CD40有關之活性，如CD95，CD80或CD86表 現或細胞增殖），如nos· 11，30，72及366所示之抗體，及 由融合瘤 Fl-l〇2，F5-152，F2-103，F5-77，F5-157 及 F4-465 所產生之抗體。 如此中所用的，所謂“功能”當用於抗體與參考用抗體之 比較時，表示抗體具有至少一種功能或活性是和參考用抗 體實質上相同的。因此，具有no. 11抗體功能之CD40抗 體，具有至少一種功能或活性和no. 11之CD40抗體實質地 相同；具有no. 72抗體功能之CD40抗體，具有和no. 72 CD40抗體實質上相同的至少一種功能和活性；具有F1-102 所示融合瘤產生之抗體之功能的CD40抗體，具有和F1-102 所示融合瘤產v生之抗體實質上相同之至少一種功能或活 性；及依此類推。 所謂“實質上相同的”，當一種抗體與另一種抗體功能或 活性之比較時，表示抗體具有與比較抗體所有的或部份功 能或活性，即使功能或活性之程度可能不同。例如，no. -18- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -·"··- ，‘---l·-------裝--------訂--------- (請先閱讀背面之注意事項再填寫本頁) «丨 1264467 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（16 ) 11所示之CD40抗體當與CD40L共培育時可增加B -細胞增 殖（如見實例3 )。因此，具有no. 11抗體功能的CD40抗體 也可刺激B -細胞增殖，雖然細胞增殖之程度由抗體所刺激 的，在相同抗體濃度下，可能較大或較小。類似的，由 F 1-102融合瘤產生之CD40抗體，可刺激Ramos細胞上CD95 之表現及刺激B -細胞之增殖。因此，具有F -10 2融合瘤產 生之抗體功能之CD40抗體，或可刺激CD95表現或B-細胞 增殖，然而CD95表現或B細胞增殖之程度，可能較由F 1 -102融合瘤產生之抗體，在相同抗體濃度下會較大或較小 些〇 具有所示範的人類CD40抗體必要的功能或活性之抗體， 可利用如實例1，3及4所示之Ramos B細胞分析法，細胞 增殖分析法，其技藝中已知之其他CD40活性分析法來鑑 定。例如，細胞增殖也可利用布氏（Burkitt)淋巴瘤細胞株 分析。布氏淋巴瘤細胞株實例包括Raji (ATCC CCL 86)， Daudi (ATCC CCL 213)及Namalwa (ATCC CRL 1432)。偵測 CD40活性的另一分析法是在反應CD40L活化作用下，於特 殊CD40抗體存在及不存在下，偵測由B細胞產生之免疫球 蛋白。細胞培育在培養基中一段時間可偵測免疫球蛋白之 分泌。免疫球蛋白產製可依此中所述的或技藝中所述之 ELISA 分析法偵測（如見 Maliszewski et al.，J. Immunol. 144:3 028 (1990))。見CD40刺激性或抑制性抗體也可由偵測 混合淋巴球中，IL-4謗生之IgE分泌而鑑知。CD40之傳訊 也可增加共一刺激物分子表現，如CD80或CD86，或黏附 -19- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） J-----------裝--------:—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制衣 1264467 ” A/ B7 五、發明說明（17 ) 分子如ICAM-1或LFA-1，或與細胞預期死亡之相關之分 子，如 CD95(如見 Durie et al·，Immunol. Today, 9:406 (1994))。這些分子不同的表現可以FACS染色測及。 本發明的CD40抗體也包括和CD40抗體（此中所示範的） 具實質上相同結合親和力之抗體。因此，在另外的實例 中，本發明提出與no. 11，30，72及366所示抗體實質上相 同結合親和力之CD40抗體，及Fl-102，F5-152，F2-103， F5-77，F5-157及F4-465融合瘤產生之抗體實質上相同結合 親和力之抗體。在一方面，CD40抗體/具有如SEQ ID NOs:10，11，12，13，14或15所示之重或輕鏈序歹|J。 所謂“實質上相同”當用於參考抗體結合親和力時，表示 解離常數（KD)在參抗體之約10-100倍内。例如，ηο·30之抗 -CD40抗體，如BiaCore分析所決定的，具有0.8-4ηΜ之KD 値。因此，具有如no.30抗-CD40抗體實質上相同結合親和 力之抗體，具有約0·008-400ηΜ之KD値。 本發明CD40抗體進一步包括，於CD40配體，如CD40L， 存在或不存在下，可調控CD40活性之抗體。因此，本發 明提出，在CD40配體，如CD40L，存在或不存在下，可調 控CD40活性之CD40抗體。在一個具體實例中，CD40抗體 可在CD40L存在下調控CD40活性。在另一具體實例中， CD40抗體可在無CD40L下調控CD40活性。在一方面， CD40抗體可在CD40L存在下刺激CD40活性，刺激程度較 無CD40抗體下更大。在另一方面，CD40抗體在CD40L存 在下可減低CD40之活性。 -20- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） Λ.----1---------裝---^----Γ — 訂---------^9^1 (請先閱讀背面之注意事項再填寫本頁) 1264467 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 ) 如此中所用的，所謂“ CD40配體”表示可結合CD40及調 控一種以上CD40活性之分子。CD40配體的特殊實例是多 肽CD40L，CD40L肽次序列如可溶性CD40L多肽缺乏穿膜 區或細胞内區域，人類CD40L之哺乳動物同系物（如鼠類 CD40L )，哺乳動物CD40L之結構或功能同系物，或哺乳 動物CD40L之衍生物（如全長CD40L之肽次序列）。特殊的 CD40L 序歹U 述於如：Spriggs et al·，J. Exp. Med. 176:1543 (1992)及Armitage et al·，Nature 357:80 (1992)。CD40配體另 外的實例包括可結合及調控CD40 —種以上活性之藥物。 爲了產生人類CD40抗體，於是產生人類CD40融合蛋白 質（hCD40:hFc)，其中含有人類CD40之細胞外區域稠和至 人類IgGl之Fc區域。融合蛋白質表現在桿病毒表現構體 上，並自受感染之Τ η 5昆蟲細胞中分離。CD40融合蛋白質 利用蛋白質G親和力管柱純化，再用來免疫接種於人類轉 染色體之（Tc)老鼠，其在染色體中含有人類IgG之k或λ鏈 (Tomizuka K et al., Proc. Natl. Acad. Sci. USA 97:722 (2000) 及Tomizuka K·，et al·，Nat Genet 16:133 (1997))。以顯示出 最南抗體移價之老鼠用於細胞融合。再自動物中利用 Kohler 及Mil stein，Nature 256:495 (1975)之經修飾方法，製 備單株抗體。移出脾臟 '並分離成單一細胞，再與骨髓瘤 細胞融合以形成融合瘤。生成之融合瘤塗佈並分析重或k 鏈之產製，及CD40抗體之產製。再以有限稀釋選殖細 胞，並進CD40抗體產製之再篩選。 所選出之分泌性融合瘤再於試管内（如在組織培養中）或 -21 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） » n I in —^1 ϋ· ·ϋ ϋ i^i —ϋ · ·ϋ n ί ^1 ϋ 1_1 ^ ^ I II ϋ m (請先閱讀背面之注意事項再填寫本頁) % 1264467 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（19 ) 於活體内（如老鼠之腹水中）培養，並純化人類CD40抗體。 抗體利用市售之蛋白質G親和力樹脂純化。抗體也可以技 藝中為知之其他技術分離或純化（見Antibodies： A LabiLra^〇rx_^nual9 Harlow and Lane (eds.)? Cold Spring Hatbof Laboratory Press，1988)。適合的技術包括 CD40親和 力純化’非變性凝膠之純化，HPLC，或RP-HPLC，在蛋白 質A官柱上純化，或這些技術任何的組合。經純化的抗 體’利用ELISA分析中老鼠Ig_吸收之抗-人類1§決定知是 人類I g。 適合產生抗體之CD40蛋白質，可以技藝中已知之各種標 準蛋白質純化或重組體表現技術中任一者來產製。例如， 如此中所示範的，CD40肽可在細胞中表現，且由細胞產 生之蛋白質可予以純化。CD40蛋白質可以重組體方法表 現’呈較大蛋白質之部份之型式。另外，可以標準的肽合 成技術，如固相合成，產生CD40抗原。部份蛋白質可含 有胺基酸序列如T 7標幟，或聚組織胺序列，以助已表現 的或合成的CD40之純化。 拮抗CD40而適於產生免疫反應之CD40型式，包括CD4〇 之可溶型式，或全長CD40之肽次序列（如長度通常是5個 胺基酸以上）。CD40另外的型式包括含有CD40之製劑或萃 取物，CD40之部份純化型式，以及CD40細胞或病毒，其 可表現CD40，或此細胞或病毒之製劑。 單株抗體也可以其他技術容易地產生（如見U.S. Pat. MOs 4,902,614， 4,543,439 及 4,411，993 ;也見 -22- 本纸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) 12644671264467 Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives A7 B7 V. INSTRUCTIONS (11) The performance of CD95 by CD40L-synthesis in low Ramos B cells (eg no. 30, 72 and 366, and F4-465). The antibodies of the present invention also include a CD40 antibody which inhibits CD40 activity and which reduces CD40L-proliferating cell proliferation. The antibody of the present invention additionally includes a CD40 antibody which can stimulate CD40 activity, and increases the CD95 expression of CD40L-hybrid in Ramos B cells (such as no. 11 and by fusion tumors? 1-102, F2-103, F5-77, F5-). Proliferation of antibodies produced by 152 and F5-157) or cells (no. 11). The antibody of the present invention further comprises a CD40 antibody having a heavy or light chain having the sequence of SEQ ID Nos: 10, 11, 12, 13, 14 or 15. Thus, in accordance with the present invention, a human CD40 antibody (e.g., human CD40) that binds to CD40 is presented herein. In one embodiment, the CD40 antibody modulates CD40-like activity (e.g., signaling activity). In one aspect, a CD40 antibody of the invention reduces CD40 activity (e.g., slows or blocks CD90L-producing CD95 expression, or inhibits proliferation of cells). In another aspect, the CD40 antibody of the invention increases CD40 activity (e.g., CD95L axillary CD95 expression increases or stimulates or increases or stimulates cell proliferation). As used herein, the term "CD40 antibody" or "anti-CD40 antibody" means an antibody which specifically binds to CD40. "Human CD40 antibody" or "human anti-CD40 antibody" means an antibody which specifically binds to CD40 and is composed of a human immunoglobulin amino acid sequence. A winter CD40 antibody that binds to human CD40 is an antibody that contains a human immunoglobulin amino acid sequence that specifically binds to human CD40, whereas antibodies can also bind to non-human sequences that have an epitope that is recognized by human CD40 antibodies. . The CD40 antibody of the present invention includes a plurality of strains or monoclonal antibodies, and as shown therein - 14 - The paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) nn ϋ nnn «1—in I fn ϋ一· ϋ ϋ I (Please read the notes on the back and fill out this page) #· 1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Employees' Consumer Cooperatives Print 5, Inventions (12) Fragments and modifications. A multiplicity of strains and monoclonal antibodies have also been proposed which contain two or more different CD40 antibodies having different binding specificities, binding affinities or functions. The CD40 antibody of the present invention contains a k or λ chain sequence. Each antibody molecule contains two k or two lambda light chains. The main difference between the k and λ light chains lies in the sequence of their fixed regions. In humans, the k-chain variable region sequence is more diverse than the lambda chain variable region sequence, which results in a different (diverse) antibody. An example of a CD40 antibody produced by fusion tumor F4-465 contains a human lambda light chain. Exemplary antibodies are shown as nos. 11 (ATCC ΡΤΑ-2308), 30, 72 (ATCC PTA-2309) and 366, or by Fl-102, R2-103, F5-77, F5-152, F5-157 , and F4-465 fusion tumors produced. Although not bound by theory, exemplary CD40 antibodies that inhibit CD40 activity, such as nos. 30, 72, 366 and antibodies produced by fusion tumor F4-465, appear to reduce the binding of CD40L to CD40 and do not guide CD40 Communication. Conversely, CD40 antibodies that stimulate CD40 activity, such as no. 11, and by fusion tumors? The antibodies produced by 1-102, 戸2-103, F5-77, F5-152 and F5-157 appear to be able to mediate CD40 signaling and enhance CD40L activity. As used herein, "modulation", when used with a modifier of a term, means that more than one activity or function of the modified term is increased or decreased. Thus, regulation of CD40 activity indicates an increase or decrease in the activity or function of more than one CD40. CD40 activity includes intracellular communication communicated by CD40, and any downstream effects of CD40 signaling, including changes in gene or protein expression (eg, CD95, CD80, or CD86), or specific cellular responses such as cell proliferation, isoform conversion Or where CD40 participates or plays the role of -15- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) !------------^^---: ----Γ — 订--------- (Please read the notes on the back and fill out this page) 1264467 A7 -, ...... -—— B / V, invention description (13 The development of the condition or disorder. Thus, exemplary activity of 〇4〇 includes regulation of protein expression or cell proliferation. (Please read the notes on the back and fill out this page.) Additional examples of CD40 activity include regulation of cell survival (eg, effects such as cell survival signals), antibody production, antibody isotype switching, and cytokines production (eg IL-2). , IL-6, IL-8, ILM2, TNF-a, IL-4, ratio ^ and ratio ίο), production of metalloproteinase (such as ΜΜΡ-W collagenase & MMP-9 / gelatinase B) and immunization The development of memory. CD4〇 activity further includes regulation of protein production by cell-cell contact or adhesion (eg, E-selectin, VCAM-1 and ICAM-1). The Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives, publishes a physiological condition or disorder in which CD40 is involved, or it may respond to changes in the condition of more than one CD40 activity (eg, mCD4 activity) and thus may be considered CD40 activity. Includes immune responses that are not desired in the body, such as autoimmune, allergic, inflammatory or transplant rejection. Specific examples of autoimmune immunity include rheumatoid arthritis, lupus (e.g., SLE, lupus nephritis), production of f-antibodies, allergies and Crohn's disease. Allergies that can be treated by modulation of CD4 sputum activity include allergic reactions to antigens, antibodies, and the like. Other examples of CD4〇 activity include transplant rejection, or excessive immune response to transplanted antigens (as manifested by organs that have been implanted), and related hemodynamic disorders such as leukopenia, transplanted organs/tissues are cells Or B _ cell infiltration and opportunistic infection, inflammation or conversion of IgE. Still other examples include the production of antagonistic therapeutic agents or viral vector neutralizing antibodies for gene therapy. Another example is the formation of a thrombus, atherosclerosis or thickening of the intima of an individual. Examples of physiological effects that increase CD40 activity include stimulation of infectious agents-16- This paper scale applies Chinese National Standard (CNS) A4 specifications (210 1264467 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Print A7 B7 V. Invention Notes ( 14) (such as a pathogen of a poor immunogenicity) or an immune response to cancer, or increase the ability to respond to antigens by improving the memory of immunity or humoral immunity of cells. Stimulate the survival or proliferation of immune cells by stimulating CD40 ' can also cause treatment. The CD40 antibody of the present invention includes an antibody having the specificity of binding of the CD40 antibody herein. Thus, in another embodiment, the present invention proposes antibodies having and shown no. 11, 30, 72 and 366, Or the antibody produced by the fusion tumors F 1 -102, F5-1 52, F2-103, F5-77, F5-157 and F4-465 binds to a specific CD40 antibody. In one aspect, the CD40 antibody has the SEQ ID NOs: heavy or light chain sequence 10U as shown in 10, 11, 12, 13, 14 and 15. The "binding specificity" as used herein, when used in reference to an antibody, means that the antibody can be confirmed, the same antigenic table. Bit ratio Therefore, the CD40 antibody having the binding specificity of the antibody as shown in No. 11 can confirm the same epitope as the no. 11 CD40 antibody; and the CD40 antibody having the antibody binding specificity as shown in No. 72 can be used. Confirming the same epitope as the no. 72 CD40 antibody; the CD40 antibody having the binding specificity of the antibody produced by the fusion tumor F1-102 can confirm the same epitope as the antibody produced by the F 1 -1 02 fusion tumor; Typically, the epitope is a short amino acid sequence, such as about 5 amino acids in length. Systematic techniques for identifying epitopes are known in the art and are described, for example, in US Pat. No. 4,708,871. Briefly, one of the overlapping oligopeptides derived from the CD40 antigen can be synthesized and bound to a solid phase of a column of nails with a unique oligopeptide. The arrangement of the nails includes a 96-well microtiter plate. So that we can simultaneously analyze all 9 oligopeptides, such as antibodies to anti-CD40 monoclonal antibodies. In addition, -17- this paper scale applies to Chinese National Standard (CNS) A4 specifications (210 X 297 mm) -- -----------Install---- (Please read the notes on the back and fill in this page) Order --------- 12 64467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperatives Printed V. Inventions (15) The New England BioLabs are available for purchase, and these are available for purchase. In a method, the binding affinity of a possible contiguous amino acid of each subset can be determined to identify an epitope to which a particular antibody can bind. When an antibody-obtaining animal is immunized with an epitope-length peptide sequence, an epitope can also be identified by inference. The human CD40 antibody of the present invention includes an antibody having more than one CD40 antibody function, as shown therein (e.g., CD40 regulatory activity). Thus, in another embodiment, the invention provides a CD40 antibody having more than one function (eg, increasing or decreasing activity associated with CD40, such as CD95, CD80 or CD86 expression or cell proliferation), such as nos· 11, 30, 72 And the antibody represented by 366, and the antibody produced by the fusion tumors Fl-1, F5-152, F2-103, F5-77, F5-157 and F4-465. As used herein, the term "functional" when used in comparison with an antibody for reference means that the antibody has at least one function or activity which is substantially identical to the reference antibody. Thus, a CD40 antibody having no. 11 antibody function, having at least one function or activity substantially identical to the CD40 antibody of no. 11; a CD40 antibody having no. 72 antibody function, having substantially the same identity as the no. 72 CD40 antibody At least one function and activity; a CD40 antibody having the function of an antibody produced by a fusion tumor represented by F1-102, having at least one function or activity substantially identical to an antibody produced by a fusion tumor represented by F1-102; analogy. By "substantially identical", when an antibody is compared to the function or activity of another antibody, it is meant that the antibody has all or part of the function or activity of the antibody, even though the degree of function or activity may vary. For example, no. -18- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -·"··- , '---l·-------Installation-- ------Set--------- (Please read the notes on the back and fill out this page) «丨1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (16 The CD40 antibody shown in 11 increases B-cell proliferation when co-incubated with CD40L (see, eg, Example 3). Therefore, a CD40 antibody having the function of no. 11 antibody can also stimulate B-cell proliferation, although the degree of cell proliferation is stimulated by the antibody, and may be larger or smaller at the same antibody concentration. Similarly, CD40 antibodies produced by F 1-102 fusion tumors stimulate the expression of CD95 on Ramos cells and stimulate the proliferation of B-cells. Therefore, a CD40 antibody having an antibody function produced by an F -10 2 fusion tumor may stimulate CD95 expression or B-cell proliferation, whereas the degree of CD95 expression or B cell proliferation may be higher than that produced by the F 1 -102 fusion tumor. An antibody having the same function or activity as the exemplified human CD40 antibody at the same antibody concentration, using Ramos B cell assays as shown in Examples 1, 3 and 4, cell proliferation assay The method is identified by other CD40 activity assays known in the art. For example, cell proliferation can also be analyzed using Burkitt's lymphoma cell line. Examples of Brucella lymphoma cell lines include Raji (ATCC CCL 86), Daudi (ATCC CCL 213) and Namalwa (ATCC CRL 1432). Another assay for detecting CD40 activity is to detect immunoglobulin produced by B cells in the presence and absence of a particular CD40 antibody under reaction CD40L activation. The cells are cultured in the medium for a period of time to detect secretion of immunoglobulins. Immunoglobulin production can be detected by ELISA assays as described herein or as described in the art (see, for example, Maliszewski et al., J. Immunol. 144:3 028 (1990)). See also CD40 stimulatory or inhibitory antibodies can also be detected by detecting IgE secretion from IL-4 in mixed lymphocytes. CD40 communication can also increase the performance of a total of stimulant molecules, such as CD80 or CD86, or adhesion -19- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) J------- ----装--------:-book--------- (please read the note on the back and then fill out this page) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed clothing 1264467 A/B7 V. INSTRUCTIONS (17) Molecules such as ICAM-1 or LFA-1, or molecules associated with expected cell death, such as CD95 (see, for example, Durie et al., Immunol. Today, 9:406 (1994) The different expression of these molecules can be measured by FACS staining. The CD40 antibodies of the invention also include antibodies having substantially the same binding affinity as the CD40 antibody (exemplified herein). Thus, in another example, the invention A CD40 antibody having substantially the same binding affinity as the antibodies shown in No. 11, 30, 72 and 366, and Fl-102, F5-152, F2-103, F5-77, F5-157 and F4-465 fusion tumors were produced. The antibody is substantially identical to the antibody that binds affinity. In one aspect, the CD40 antibody has a heavy or as shown in SEQ ID NOs: 10, 11, 12, 13, 14 or 15. Chain sequence 歹|J. By "substantially identical" when used in reference antibody binding affinity, it means that the dissociation constant (KD) is within about 10-100 times of the reference antibody. For example, ηο·30 anti-CD40 antibody, such as The BDCore assay has a KD 0.8 of 0.8-4 η 値. Therefore, an antibody having substantially the same binding affinity as the no. 30 anti-CD40 antibody has a KD 约 of about 008-400 η 値. The CD40 antibody of the present invention further includes An antibody that modulates CD40 activity in the presence or absence of a CD40 ligand, such as CD40L. Thus, the present invention contemplates a CD40 antibody that modulates CD40 activity in the presence or absence of a CD40 ligand, such as CD40L. In one embodiment, the CD40 antibody can modulate CD40 activity in the presence of CD40L. In another embodiment, the CD40 antibody can modulate CD40 activity in the absence of CD40L. In one aspect, the CD40 antibody can stimulate CD40 activity in the presence of CD40L, The degree of stimulation is greater than that of the CD40-free antibody. On the other hand, CD40 antibody can reduce the activity of CD40 in the presence of CD40L. -20- This paper scale is applicable to China National Standard (CNS) A4 specification (210 X 297 mm). Λ.----1---------装---^----Γ — order---------^9^1 (please read the notes on the back first) Fill in this page) 1264467 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7 V. INSTRUCTIONS (18) As used herein, the term "CD40 ligand" means a molecule that binds to CD40 and regulates more than one CD40 activity. A particular example of a CD40 ligand is the polypeptide CD40L, a CD40L peptide subsequence such as a soluble CD40L polypeptide lacking a transmembrane or intracellular region, a mammalian homolog of human CD40L (eg, murine CD40L), a structural or functional homolog of mammalian CD40L , or a derivative of mammalian CD40L (such as the peptide sequence of full length CD40L). A special CD40L sequence is described in, for example, Spriggs et al., J. Exp. Med. 176:1543 (1992) and Armitage et al., Nature 357:80 (1992). Additional examples of CD40 ligands include drugs that bind to and modulate CD40-like activity. To generate a human CD40 antibody, a human CD40 fusion protein (hCD40:hFc) was produced in which the extracellular region of human CD40 was fused to the Fc region of human IgG1. The fusion protein is expressed on the rod virus expression construct and is isolated from the infected η5 insect cells. The CD40 fusion protein was purified using a protein G affinity column and used to immunize human transchromosomal (Tc) mice containing the k or λ chain of human IgG in the chromosome (Tomizuka K et al., Proc. Natl. Acad) Sci. USA 97:722 (2000) and Tomizuka K., et al., Nat Genet 16:133 (1997)). The mice showing the shift of the most southern antibody were used for cell fusion. The monoclonal antibodies were prepared by reprogramming using Kohler and Milstein, Nature 256:495 (1975). The spleen is removed and separated into single cells, which are then fused with myeloma cells to form a fusion tumor. The resulting fusion tumor is coated and analyzed for the production of heavy or k chains, and the production of CD40 antibodies. The cells were then selected by limiting dilution and screened for CD40 antibody production. The selected secreted fusion tumor is then placed in a test tube (eg in tissue culture) or - 21 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) » n I in —^1 ϋ· ·ϋ ϋ i^i —ϋ · ·ϋ n ί ^1 ϋ 1_1 ^ ^ I II ϋ m (Please read the note on the back and fill out this page) % 1264467 Ministry of Economic Affairs Intellectual Property Office Staff Cooperatives Print A7 B7 V. INSTRUCTIONS (19) Culture in vivo (such as the ascites of mice) and purify human CD40 antibodies. The antibody was purified using a commercially available protein G affinity resin. Antibodies can also be isolated or purified by other techniques known in the art (see Antibodies: A LabiLra^〇rx_^nual9 Harlow and Lane (eds.)? Cold Spring Hatbof Laboratory Press, 1988). Suitable techniques include purification of CD40 affinity purified 'non-denaturing gels, HPLC, or RP-HPLC, purification on a protein A column, or any combination of these techniques. The purified antibody was determined to be human Ig by the anti-human 1 § of the Ig_absorption of the mouse in the ELISA assay. The CD40 protein suitable for the production of antibodies can be produced by any of a variety of standard protein purification or recombinant expression techniques known in the art. For example, as exemplified herein, the CD40 peptide can be expressed in cells and the protein produced by the cells can be purified. The CD40 protein can be expressed in a recombinant manner as a part of a larger protein. Alternatively, CD40 antigens can be produced by standard peptide synthesis techniques, such as solid phase synthesis. Some of the proteins may contain amino acid sequences such as the T7 marker, or a polyhistamine sequence to aid in the purification of the expressed or synthesized CD40. A CD40 version that antagonizes CD40 and is suitable for generating an immune response, including a soluble form of CD4〇, or a peptide sequence of full length CD40 (e.g., typically 5 or more amino acids in length). Additional versions of CD40 include preparations or extracts containing CD40, partially purified versions of CD40, and CD40 cells or viruses which can express CD40, or a preparation of such cells or viruses. Individual antibodies can also be readily produced by other techniques (see, for example, US Pat. MOs 4,902,614, 4,543,439 and 4,411,993; see also -22- 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) 1264467

Antibodies, Hvbridomas: A New_Dimension in Biological Analyses, Plenum Press，Kennett，McKearn，and Bechtol (eds·)，1980及Harlow et al. (198 8)，上文）。多株抗體之製 備及其純化也是精藝者熟知的（如見Green et al. (1992)於： Immunochemical Protocols, pages 1-5，Manson ed·，Humana Press ; Harlow et al· (1988)，上文；及 Coligan et al. (1994) 於 Current Protocols in Immunology· Wiley ; KBarnesetal. (1992)於 Methods in Molecular Biology· Vol· 10，pages 79-104，Humana Press) o 可被免疫接種之動物包括：兔子，大鼠，山羊，或天竺 鼠；此動物可經修飾以包括人類I g G基因區域。另外，許 多增加免疫反應之技術是已知的，如將可溶性CD40偶合 至另一蛋白質，如卵白蛋白，或鑰孔蜮血藍素（KLH)，甲 狀腺球蛋白及破傷風類毒素，或可經由佐劑之使用，如 Freund1 s完全或不完全佐劑。最初及視所需接續之任何免 疫接種，可經由腹膜内，肌内，眼内或皮下路徑。接續之 免疫接種可和抗原製劑相同或不同濃度，且在規律或不規 律時間間隔下。 經 濟 部 智 慧 財 產 局 消 費 合 社 印 製 (請先閱讀背面之注意事項再填寫本頁) 因此’在另一具體實例中，本發明提出產製人類CD40抗 體（多株抗體）之方，包括可調控CD4〇活性之抗體。在一個 具體實例中，方法包括對可表現人類免疫球蛋白之動物 (如老氣）投予CD40或其免疫原片段；對動物進行人類 CD40抗體表現之筛選；選出可產生人類。〇4〇抗體之動 物，自可產生人類CD40抗體之動物中分離抗體；並決定 23 1 X 297公釐） 1264467 A7 B7 經濟部智慧財產局員工消費合作社印制π 五、發明說明（21 ) 人類CD40抗體是否可調控CD40之活性，由是鑑定出可調 控CD40活性之人類CD40抗體。在另一具體實例中，方法 包括對可表現人類免疫球蛋白之動物（如老鼠）投予人類 CD40或其免疫原片段；自可產生人類CD40抗體之老鼠中 分離脾細胞；脾細胞與骨髓瘤細胞融合，以產生融合瘤； 及針對可調控CD40活性之人類CD40抗體之表現，進行融 合瘤之篩選，由是可鑑定出可產生人類CD40抗體之融合 瘤。 本發明進一步提出已經修飾之人類CD40抗體。修飾之實 例包括CD40抗體一種以上胺基酸之置換，加入或刪除， 其係具有未經修飾CD40之所有或至少部份的功能。經修 飾之人類CD40抗體在人體中應該是相當無免疫原性的， 即抗體在人體中不會謗生強的免疫反應。在特殊具體實例 中，人類CD40抗體片段包括scFv，Fab，Fab1或卩（&1)，）2片 段。在特殊方面，scFv，Fab，Fab·或F(ab')2片段具有實質 上相同的結合特異性，至少是全長人類CD40抗體之一種 活性，或和未修飾的CD40抗體相同的結合親和力。在較 特殊方向，抗體爲nos. 11，30，72及366所示抗體之scFv， Fab，Fab·，或 F(ab· )2片段，及由融合瘤 Fl-102，F5-152， F2-103，F5-77，F5-157及F4-465所產生之抗體之片段。另 外具體實例中，具有一個以上胺基酸置換，加入或刪除之 人類CD40抗體，具有和未修飾之人類CD40抗體實質上相 同的結合特異性，至少一種活性，或和未修飾之人類 CD40抗體實質上相同之結合親和力。在特殊方面，經修 -24- 本纸張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ：---^--------裝---^----:—訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（22 飾型式爲示於SEQ ID N〇s:1〇，n，12，13，㈣卜之重或 輕鏈序列。 本發明之CD4〇抗體片段（如Fab，Fab，，F(ab· )2及scFv)可 由抗體t蛋白水解作用而製備，如完整抗體經由胃蛋白酶 或木瓜蛋白酶之水解。特言之…蛋白酶行酵素解離而 產生之抗體片段可生成示爲？（讣，）2之58片段。此片段可利 用硫醇返原劑進一步解離，產生3,5S Fab，單價片段。另 外，利用胃蛋白酶行酵素水解，可直接產生二個單價的 Fab，及 Fc 片段（如見 Goldenberg，u s pat N〇s· 4,〇36,945 及 4,331，647及此巾之參考；Edelman et al^ Enymology 1:422 (1967)及 Coligan et al，在上文 2 8」-2 8 i〇 及2· 10.1-2· 10.4段落中）。也可使用其他解離抗體之方法， 如分離重鏈以形成單價輕-重鏈片段，進一步解離片段或 其他的酵素或化學方法。遺傳技術包括CD4〇抗體基因至 宿主細胞中所有的或部份的表現，如C 〇 s細胞或E c〇u。 重組體宿主細胞可合成完整的或單一抗體鏈，如scFv(見 如 Whitlow et al·，In: Methods: A Companion to Methods in Enzymology 2:97 (1991), Bird et al.5 Science 242:423 (1988);及U.S. Pat· No· 4,946,778)。 置換作用可以是原構的或非原構的，且可在抗體之固定 或可變區中。在固定或可變區中一個或一些固有胺基酸之 置換似乎是可忍受的。固有胺基酸置換之特殊實例有： lie，VaL，Leu或Ala互相間；Lys及Arg互相關；Glu及Asp互 相間；及Glu及Asn互相間。在高度可變區域中多個胺基酸 25- 本纸張尺度適用中國國家標準（CNS)A4規格（2】0 X 297公釐） ;—^--------裝—·----Ti 訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 A7 B7 經濟部智慧財產局員工消費合作社印制π 五、發明說明（23 I典固有的置換，似乎可影響結合活性，特異性或抗體功 能或活性。因此在高度可變區之置換，可進行其作用之分 析以鑑定出保有至少部份的結合活性，特異性或未置換抗 之功能或活性者。此具有胺基酸置換之抗體包括在内， 只要相對於未置換抗體，經置抗體可保有未修之人類 CD40抗體至少部份的結合特異性，一種活性或結合親和 力即可。 具有胺基酸置換之經修飾CD40抗體之另一特殊實例是其 中第二異質序列，即異質的功能性區域，可對抗體提供不 同的或互補的功能者。例如，胺基酸標幟如T 7或聚組胺 酸可黏附至抗體，以促進純化或偵測。又另一特殊實例是 加上一個肽毒素，如肉毒毒素可黏附至CD40抗體上以對 準可表現CD40之細胞而殺死之。因此，在另個具體實例 中，本發明提出嵌合的CD40抗體，其中CD40抗體具有可 提供抗體不同功能之一種以上胺基酸，即一種異質的功能 區域。 異質的功能區域並不限於胺基酸序列。因此，異質的功 能區域可含有任何各樣的不同型式之小的或大的功能部 份。此部份包括核酸，肽，碳水化合物，脂質或小的有機 化合物，如藥物（如化療藥物，如長春新鹼，胺甲碟呤 等” 在抗體序列及異質功能區域間也可嵌入連接子序列，如 此二者可至少保有部份的功能或活性。連接子序列可有一 種以上的特性，包括具撓性之構型，無法形成有序之二級 -26- 本纸張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱） :-----------裝---：----Γ — 訂--------- (請先閒讀背面之注意事項再填寫本頁) 1264467Antibodies, Hvbridomas: A New_Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980 and Harlow et al. (198 8), supra). The preparation and purification of polyclonal antibodies are also well known to the artisan (see, for example, Green et al. (1992) in: Immunochemical Protocols, pages 1-5, Manson ed., Humana Press; Harlow et al. (1988), supra And Coligan et al. (1994) in Current Protocols in Immunology· Wiley; KBarnesetal. (1992) in Methods in Molecular Biology· Vol. 10, pages 79-104, Humana Press) o Animals that can be immunized include: Rabbit, rat, goat, or guinea pig; this animal can be modified to include the human I g G gene region. In addition, many techniques for increasing the immune response are known, such as coupling soluble CD40 to another protein, such as ovalbumin, or keyhole limpet hemocyanin (KLH), thyroglobulin and tetanus toxoid, or Use of the agent, such as Freund1 s complete or incomplete adjuvant. Any immunization, initially and as needed, may be via the intraperitoneal, intramuscular, intraocular or subcutaneous route. Successive immunizations may be the same or different concentrations as the antigen preparation, and at regular or irregular intervals. Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Consumer Cooperative (please read the note on the back and fill out this page). Therefore, in another specific example, the present invention proposes a method for producing a human CD40 antibody (multiple antibodies), including An antibody that regulates the activity of CD4〇. In one embodiment, the method comprises administering CD40 or an immunogenic fragment thereof to an animal exhibiting human immunoglobulin (e.g., old gas); screening the animal for human CD40 antibody expression; and selecting to produce human. 〇4〇 antibody animal, isolated from animals that can produce human CD40 antibody; and determined 23 1 X 297 mm) 1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing π V. Invention description (21 ) Human Whether the CD40 antibody regulates the activity of CD40 is the identification of a human CD40 antibody that modulates CD40 activity. In another embodiment, the method comprises administering human CD40 or an immunogenic fragment thereof to an animal capable of expressing a human immunoglobulin, such as a mouse; isolating splenocytes from a mouse capable of producing a human CD40 antibody; splenocytes and myeloma The cells are fused to produce a fusion tumor; and for the expression of a human CD40 antibody that can regulate CD40 activity, a fusion tumor is screened, and a fusion tumor capable of producing a human CD40 antibody can be identified. The invention further provides human CD40 antibodies that have been modified. Examples of modifications include the substitution, addition or deletion of more than one amino acid of the CD40 antibody, which has the function of all or at least a portion of the unmodified CD40. The modified human CD40 antibody should be relatively non-immunogenic in the human body, ie, the antibody does not produce a strong immune response in the human body. In a particular embodiment, the human CD40 antibody fragment comprises scFv, Fab, Fab1 or 卩 (&1), 2 fragments. In a particular aspect, the scFv, Fab, Fab or F(ab')2 fragment has substantially the same binding specificity, at least one of the activity of a full length human CD40 antibody, or the same binding affinity as an unmodified CD40 antibody. In a more specific orientation, the antibodies are scFv, Fab, Fab·, or F(ab·)2 fragments of the antibodies shown in nos. 11, 30, 72 and 366, and by fusion tumors Fl-102, F5-152, F2- Fragments of antibodies produced by 103, F5-77, F5-157 and F4-465. In another embodiment, a human CD40 antibody having more than one amino acid substitution, addition or deletion has substantially the same binding specificity as the unmodified human CD40 antibody, at least one activity, or the unmodified human CD40 antibody substantial The same binding affinity. In special aspects, the repaired-24- paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm):---^--------Install---^--- -:-订--------- (Please read the note on the back and fill out this page) 1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (22 decoration type is shown in SEQ ID N 〇 s: 1 〇, n, 12, 13, (4) heavy or light chain sequence. The CD4 〇 antibody fragment of the present invention (such as Fab, Fab, F(ab.) 2 and scFv) can be antibody t Prepared by proteolysis, such as hydrolysis of intact antibodies via pepsin or papain. In particular, antibody fragments produced by dissociation of protease enzymes can produce a fragment of 58 (?, 2). This fragment can utilize sulfur. The alcohol reversion agent is further dissociated to produce 3,5S Fab, a monovalent fragment. In addition, by pepsin enzymatic hydrolysis, two monovalent Fabs, and Fc fragments can be directly produced (see, for example, Goldenberg, us pat N〇s·4, 〇36,945 and 4,331,647 and references to this towel; Edelman et al^ Enymology 1:422 (1967) and Coligan et al, above 2 8”-2 8 I〇 and 2· 10.1-2· 10.4). Other methods of dissociating antibodies can also be used, such as isolation of heavy chains to form monovalent light-heavy chain fragments, further dissociation of fragments or other enzymes or chemical methods. The expression of all or part of the CD4〇 antibody gene into the host cell, such as C 〇s cells or E c〇u. The recombinant host cell can synthesize a complete or single antibody chain, such as scFv (see, eg, Whitlow et al, In: Methods: A Companion to Methods in Enzymology 2:97 (1991), Bird et al. 5 Science 242:423 (1988); and US Pat. No. 4,946,778). The replacement may be an original or a non-primitive And in the fixed or variable region of the antibody. The substitution of one or some of the intrinsic amino acids in the fixed or variable region appears to be tolerable. Specific examples of intrinsic amino acid substitutions are: lie, VaL, Leu or Ala are interdependent; Lys and Arg are cross-correlated; Glu and Asp are inter-related; and Glu and Asn are inter-related. Multiple amino acids in highly variable regions 25- This paper scale applies to Chinese National Standard (CNS) A4 Specifications (2) 0 X 297 mm) ;—^--------Installation—·--- -Ti Order--------- (Please read the note on the back and fill out this page) 1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative print π V. Invention description (23 I code inherent replacement , appears to affect binding activity, specificity or antibody function or activity. Thus, in the highly variable region, an analysis of its effect can be performed to identify a function or activity that retains at least a portion of the binding activity, specificity or non-displacement resistance. The antibody having an amino acid substitution is included as long as the immobilized antibody retains at least a part of the binding specificity, an activity or binding affinity of the unmodified human CD40 antibody relative to the unsubstituted antibody. Another specific example of a modified CD40 antibody having an amino acid substitution is one in which a second heterogeneous sequence, i.e., a heterogeneous functional region, provides a different or complementary function to the antibody. For example, an amino acid label such as T7 or polyglycolic acid can be attached to the antibody to facilitate purification or detection. Yet another particular example is the addition of a peptide toxin, such as botulinum toxin, which adheres to the CD40 antibody to kill cells that express CD40. Thus, in another embodiment, the invention provides a chimeric CD40 antibody, wherein the CD40 antibody has one or more amino acids that provide different functions of the antibody, i.e., a heterogeneous functional region. Heterogeneous functional regions are not limited to amino acid sequences. Therefore, the heterogeneous functional area can contain any of a variety of different types of small or large functional parts. This part includes nucleic acids, peptides, carbohydrates, lipids or small organic compounds, such as drugs (such as chemotherapy drugs, such as vincristine, amine methotrexate, etc.). Linker sequences can also be embedded between antibody sequences and heterogeneous functional regions. Therefore, the two can retain at least part of the function or activity. The linker sequence can have more than one characteristic, including a flexible configuration, and cannot form an ordered secondary -26- This paper scale applies to Chinese national standards. (CNS) A4 specification (210 X 297 public):-----------Install---:----Γ-book--------- (please read it first) Please note the following on the back page) 1264467

五、發明說明（a ) 經濟部智慧財產局員工消費合作社印製 、、、口構或疏水性或荷電特性，其可促進任一功能區域或與之 又互作用。常見於撓性蛋白質區域之胺基酸包括Gly，Asn 及Ser ’預期其可滿足上述對於連接子序列之準則。另一進 乎中性之胺基酸，如Thr及Ala，也可用於連接子序列中。 連接子序列之長度可有變化，但爲不致顯著影響融合蛋白 貝之功此或活性之長度（如見us· pat· N〇. 6,〇87,329)。 抗體修飾之另一實例是可被測及之標幟。因此，在另一 具m貫例中’本發明提出可被測及地標記之人類cd4〇抗 體。 標幟之特殊實例包括：發螢光團，發色團，放射活性同 位素（如1125)，電子雲試劑，酵素及配體。酵素通常可由 其活性中測及。如辣根過氧化酶通常可由其將受質（如 3,3’，5,5’ -四甲基聯苯胺（TMB))轉化成藍色色素（其可被定 量）之能力而測及。配體可結合另外的分子，如生物素， 其可結合抗生物素蛋白或鏈抗生物素蛋白，I g G，其可結 合蛋白質A ’及技藝中已知的許多受體-配體複合物。應 了解’ CD40抗體可有二種以上的修飾或標幟。例如，單 株抗體可偶合至生物素，以利用抗生物素蛋白偵測其存 在’以及以I 2 5標冗之以提供訊號。其他的過突變及可能 性是精藝者顯而易見的，且也視爲本發明範圍内。 本發明進一步提出編碼人類CD40抗體（本發明）之核酸， 包括經修飾之型式’片段，礙合體等。在特殊具體實例 中，核酸係编碼no· 11，30，72及366所示之完整的或鏈 CD40抗體（或由 Fl-102，F5-152，F2-103，F5-77，F157 及 -27- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 裝---^----^—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 1264467 A/ B7 五、發明說明（25 ) F4-465所產生抗體。在特殊方面，核酸包括seqid NOs:10，11，12 ’ 13，14或15。在另一方面，核酸係編碼 no· 11 ’ 30，72 及 366 抗體或融合瘤 fi_i〇2，F5-152，F2 103，F5-77，F5-157及F4-465所產生抗體之完整的單一鏈之 次序列，如SEQ ID NOs: 10，11，12，13，14或15所示之次 序列。 所渭“核酸”或“聚核嘗酸”可互換使用指所有型式的核 酸’包括去氧核糖核酸（DNA)及核糖核酸（rnA)。核酸可 以是雙，單股，或參股，線狀或環狀。核酸包括基因體 DNA，cDNA及抗意識股。RNA核酸可爲經剪接或未經剪 接之mRNA，rRNA，eRNA或抗意識股（如RNAi)。本發明的 核酸包括自然生成的，合成的，以及核茹酸類似物及衍生 物。此種改變的或經修飾之聚核苷酸包括可提供如核酸酶 抗性之類似物。核酸可有任何的長度，如此卩I。 N〇s:10，11，12，13，14或15中任一者。核酸長度也可較 示範之SEQ ID NOs:10，11，12，13，14或 15的少。例如， SEQ ID N〇s: 10 ’ 11 ’ 12 ’ 13 ’ 14或15任一者之次序列可編 碼CD40抗體重或輕鏈，或k或入鏈片段，其有SEq ID NOs: 1 〇，11，12，13，14或1 5全長之至少部份的結合特異 性’結合親和力或功能。 由於遺傳密碼簡併性之故，核酸包括在SEq ID N〇s:10， 11 ’ 12 ’ 13，14或15所示序列中具簡併性之序列。因此， 本發明包括具有SEQ ID NOs:10，11，12，13，14或15所示 序列中有核苷酸變化之核酸，其仍可編碼完整的或單鏈的 -----------裝---：----Γ丨訂--------- (請先閱讀背面之注意事項再填寫本頁) -28-V. INSTRUCTIONS INSTRUCTIONS (a) The Intellectual Property Office of the Ministry of Economic Affairs, the Consumer Cooperatives, prints, orchestrate or hydrophobic or charged characteristics that promote or interact with any functional area. Amino acids commonly found in flexible protein regions, including Gly, Asn and Ser', are expected to meet the above criteria for linker sequences. Another neutral amino acid, such as Thr and Ala, can also be used in the linker sequence. The length of the linker sequence may vary, but does not significantly affect the length of the fusion protein or the activity (see, for example, us. pat. N〇. 6, 〇 87, 329). Another example of antibody modification is the flag that can be measured. Therefore, in another embodiment, the present invention proposes a human cd4 〇 antibody which can be detected and labeled. Specific examples of the label include: fluorophore, chromophore, radioactive isotope (such as 1125), electron cloud reagents, enzymes and ligands. Enzymes are usually measured by their activity. For example, horseradish peroxidase is typically measured by its ability to convert a substrate (e.g., 3,3',5,5'-tetramethylbenzidine (TMB)) to a blue pigment (which can be quantified). The ligand may bind to another molecule, such as biotin, which binds to avidin or streptavidin, IgG, which binds to protein A' and many receptor-ligand complexes known in the art. . It should be understood that 'CD40 antibodies can have more than two modifications or labels. For example, a monoclonal antibody can be coupled to biotin to detect its presence by avidin and to be redundant with I 2 5 to provide a signal. Other over-mutations and possibilities are apparent to those skilled in the art and are considered to be within the scope of the invention. The invention further provides nucleic acids encoding human CD40 antibodies (inventions), including modified versions, fragments, and the like. In a specific embodiment, the nucleic acid encodes a complete or stranded CD40 antibody as indicated by no. 11, 30, 72 and 366 (or by Fl-102, F5-152, F2-103, F5-77, F157 and - 27- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm).---^----^----------- (Please read the notes on the back first) Fill in this page again) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1264467 A/ B7 V. Description of Invention (25) Antibodies produced by F4-465. In special aspects, nucleic acids include seqid NOs: 10,11,12 ' 13, 14 or 15. On the other hand, the nucleic acid encodes antibodies produced by no 11 '30, 72 and 366 antibodies or fusion tumors fi_i〇2, F5-152, F2 103, F5-77, F5-157 and F4-465. a complete sequence of single strands, such as the sequence shown in SEQ ID NOs: 10, 11, 12, 13, 14 or 15. The reference to "nucleic acid" or "nucleic acid" is used interchangeably to refer to all types. Nucleic acids include both deoxyribonucleic acid (DNA) and ribonucleic acid (rnA). Nucleic acids can be double, single stranded, or shareholding, linear or circular. Nucleic acids include genomic DNA, cDNA and anti-sense stocks. The RNA nucleic acid can be a spliced or unspliced mRNA, rRNA, eRNA or anti-aware strand (such as RNAi). The nucleic acids of the invention include naturally occurring, synthetic, and nuclear ruthenium analogs and derivatives. The modified or modified polynucleotide includes an analog which provides resistance such as nuclease. The nucleic acid can have any length, such as 卩I. N〇s: 10, 11, 12, 13, 14 or 15 The nucleic acid length may also be less than that of the exemplary SEQ ID NOs: 10, 11, 12, 13, 14 or 15. For example, SEQ ID N〇s: 10 ' 11 ' 12 ' 13 ' 14 or 15 The subsequence may encode a heavy or light chain of the CD40 antibody, or a k or a stranded fragment having the binding specificity of the SEq ID NOs: 1 〇, 11, 12, 13, 14 or 15 Due to the degeneracy of the genetic code, the nucleic acid comprises a sequence with degeneracy in the sequence shown in SEq ID N〇s: 10, 11 ' 12 ' 13, 14 or 15. Thus, the invention includes having the SEQ ID NOs: a nucleic acid having a nucleotide change in the sequence shown in 10, 11, 12, 13, 14 or 15, which can still encode a complete or single-stranded ------- ----Install---:----Γ丨定--------- (Please read the notes on the back and fill in this page) -28-

1264467 經濟部智慧財產局員工消費合作社印製 Λ7 B7 五、發明說明（26 ) CD40抗體，或其次序列。 核酸可利用各種熟知的標準選殖及化學合成方法產製， 且可以k置-4曰令的突變作用或精藝者熟知之其他重組技 術有思地改變。聚核苷酸之純度可經由定序，凝膠電泳等 決定。 本發明的核酸可嵌入核酸構體内，其中核酸的表現爲 表現控制要件”影響或調控，在此稱爲“表現匣”。所謂 “表現控制要件，，指可調控或影響與之操作鏈結之核酸序列 之表現之一種以上核酸序列要件。表現控制要件可操作性 鏈結至核酸序列，以控制轉綠作用及適當時核酸序列之轉 〃睪作用。表現控制要件可包括（適合地）：啓動子，加強 子，轉綠終結子，基因沈默者，起始密碼子（如ATG)在蛋 白質-編碼基因之前，等。 所謂“操作性鏈結，，指並置，其中所述之組份其相互關係 令彼等可以欲求方式作用。典型而言，此要件係並置在基 因之5,或3,末端，但也可以是不表現子。特殊實例是編碼 CD40抗體之核酸，操作性鏈結至表現控制元件，如此核 酸可在元件之控制下表現。 表現控制元件包括可原構地活化轉錄作用之元件，其是 可謗導的（即於活化作用時需一個外在訊號），或可抑制= (即需訊號以停掉轉錄作用；當訊號不再存在時，轉錄作 用或被活化或“被抑制”）。本發明表現匣中也包括控制元 件，其足以提供基因表現而是特殊細胞型式或組織所可控 制的（即，組織-特異的控制元件）。典型而言，此元件係 -29- 本紙張尺度適时關家標準（CNS)A4規格⑵G x 297公爱) ------一 *-----------裝 *--·----；—訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 經濟部智慧財產局員工消費合作社印制π A7 B7 五、發明說明（27 ) 在選殖序列之上游或下游（即5，及3，）。啓動子通常位在編 碼序列之5 ’。啓動子，由重組體DN a或合成技術所產 生’可用來提供本發明聚核棼酸之轉錄作用。“啓動子，，意 指足以指令轉綠作用之最小序列元件。 本發明之核酸可嵌入質體内，以在宿主細胞中增殖，且 若欲求時可用於接續之遺傳操作中。質體是可在宿主細胞 中穩定增殖之核酸，質體可視所需含有表現控制元件，以 在宿主細胞中驅動編碼CD40抗體之核酸之表現。在此載 體可與質體同義地使用，且也可包括有表現控制元件，以 可在宿主細胞中表現。質體及載體通常含有至少一個複製 源’以供在細胞中增繁，及一個啓動子。因此質體及載體 可用於編碼CD40抗體之核酸之遺傳操作，產製CD4〇抗體 或抗思識股’並在宿主細胞或有機體中表現CD4〇抗體。 當在細菌系統中選殖時，原構性的啓動子如T7等，以及 可謗導之啓動子^^噬菌體人之卟^扣^啤^加⑻^ lac融合啓動子）均可使用，以及可謗導的啓動子（如對四環 素具反應的）。當在昆蟲細胞系統中選殖時，也可使用原 構的或可謗生之啓動子（如蛻皮激素）。當在哺乳動物細胞 系統中選殖時，可使用原構的啓動子，如sv40，RSV等， $由哺乳動物細胞基因衍生之可誘生啓動子（如金屬硫新 質II A啓動子，熱休克啓動子）或由哺乳動物病毒來之啓動 子（如腺病毒後期啓動子，可誘生之老鼠乳腺腫瘤病毒表 末端重覆子）。也可使用以牛乳頭狀瘤病毒（Bpv)爲基礎之 載體，其具有以染色體的元件型式複製之能力（Sarvef以 ,-----------裝—.----^丨訂--------- (請先閱讀背面之注意事項再填寫本頁) -30 -1264467 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed Λ7 B7 V. Description of invention (26) CD40 antibody, or its subsequence. Nucleic acids can be produced by a variety of well-known standard selection and chemical synthesis methods, and can be altered by mutations of the k--4 command or other recombinant techniques well known to those skilled in the art. The purity of the polynucleotide can be determined by sequencing, gel electrophoresis or the like. The nucleic acid of the present invention can be embedded in a nucleic acid construct, wherein the expression of the nucleic acid is the influence or regulation of the expression control element, which is referred to herein as "performance 匣". The so-called "performance control element" refers to a controllable or influential operational link. The nucleic acid sequence is characterized by more than one nucleic acid sequence. The performance control element operability is linked to the nucleic acid sequence to control the transfusion effect and, if appropriate, the transgene action of the nucleic acid sequence. Performance control elements can include (suitably): a promoter, a booster, a green terminator, a gene silencer, a start codon (such as ATG) before the protein-coding gene, and the like. By "operating chain," it is a juxtaposition in which the components are related to each other so that they can function in a desired manner. Typically, this element is juxtaposed at the 5, or 3, end of the gene, but it can also be No particular expression. A specific example is a nucleic acid encoding a CD40 antibody, operably linked to a performance control element, such that the nucleic acid can be expressed under the control of the element. The expression control element comprises an element that amorphically activates transcription, which is a Guided (ie, an external signal is required for activation), or can be suppressed = (ie, signal is required to stop transcription; when signal is no longer present, transcription is either activated or "suppressed"). The performance sputum also includes control elements that are sufficient to provide gene expression but are controllable by a particular cell type or tissue (ie, tissue-specific control elements). Typically, this element is -29- the paper size is timely Guanjia Standard (CNS) A4 Specifications (2) G x 297 publicity) ------One *-----------Installed *-------;------- ---- (Please read the notes on the back and fill out this page) 1264467 Ministry of Economics Intellectual Property Employee Affiliation Cooperative Printed π A7 B7 V. Inventive Note (27) upstream or downstream of the selection sequence (ie 5, and 3). The promoter is usually located at 5 ' of the coding sequence. Promoter, by recombinant DN 'Or produced by synthetic techniques' can be used to provide transcription of the polynucleic acid of the invention. "Promoter," means the smallest sequence element sufficient to command a greening effect. The nucleic acids of the invention can be inserted into the plastid to proliferate in the host cell and, if desired, can be used in subsequent genetic manipulations. A plastid is a nucleic acid that stably proliferates in a host cell, and the plastid may optionally contain an expression control element to drive the expression of the nucleic acid encoding the CD40 antibody in the host cell. The vector can be used synonymously with the plastid and can also include a performance control element for expression in the host cell. The plastids and vectors typically contain at least one source of replication 'for proliferation in the cell, and a promoter. Thus, plastids and vectors can be used for the genetic manipulation of nucleic acids encoding CD40 antibodies, producing CD4 〇 antibodies or anti-sense stocks and expressing CD4 〇 antibodies in host cells or organisms. When colonized in a bacterial system, a prokaryotic promoter such as T7, and a promoter that can be used as a promoter, a phage, a sputum, a sputum, a succulent promoter, and a (8) lac fusion promoter can be used, and A promoter that can be mediated (eg, reactive to tetracycline). In the case of colonization in insect cell systems, an inducible or viable promoter (e.g., ecdysone) can also be used. When colonized in mammalian cell systems, prokaryotic promoters such as sv40, RSV, etc., can be used to induce promoters derived from mammalian cell genes (eg, metal sulphur II II promoter, heat) A shock promoter or a promoter derived from a mammalian virus (such as an adenovirus late promoter, which can induce a mouse mammary tumor virus end-repetition). A bovine papilloma virus (Bpv)-based vector can also be used, which has the ability to replicate in the form of a chromosomal element (Sarvef, -------------)------^ -定--------- (Please read the notes on the back and fill out this page) -30 -

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五、發明說明（28 ) 經濟部智慧財產局員工消費合作社印製 al.，Mol· Cell· Biol. 1:486 (1981))。另外，反轉錄病毒基因 體可遺傳地修飾，以可在哺乳動物宿主細胞中引入及指令 CD40抗體之表現。 7 表現系統進一步包括特別設計之載體，以供活體内使 用，包括：腺病毒載體（us· Pat. N〇s. 5,7〇〇,47〇及 5,73 1，172)，腺病毒相關之載體（u s. pat. N〇. 5,6〇4,〇9〇)， 單純疱疹病毒載體（U.S· Pat· No· 5,501,979)及反轉綠病毒 載骨豆（U.S. Pat· Nos· 5,624,820，5,693,508及5,674,703及WIPO 刊物WO92/05266及W092/14829)。牛乳頭狀瘤病毒（BPV)也 可應用於基因治療中（U.S· Pat· Ν〇· 5,719,054)。此基因治 療載體也包括CMV爲基礎之載體（u s. Pat. N〇. 5,561,063) 〇 在酵母中，可使用許多含有原構性或可謗生啓動子之載 月亘（如見 Ausubel et al.，In: Current Protocols in MolecularV. INSTRUCTIONS (28) Printed by the Consumers' Cooperative of the Intellectual Property Office of the Ministry of Economic Affairs al., Mol·Cell Biol. 1:486 (1981)). In addition, retroviral genes can be genetically modified to introduce and direct the expression of CD40 antibodies in mammalian host cells. 7 The expression system further includes specially designed vectors for in vivo use, including: adenoviral vectors (us· Pat. N〇s. 5,7〇〇, 47〇 and 5,73 1,172), adenovirus-associated Vector (u s. pat. N〇. 5,6〇4, 〇9〇), herpes simplex virus vector (US·Pat No 5,501,979) and inverted green virus-loaded bean (US Pat· Nos· 5,624,820 , 5,693,508 and 5,674,703 and WIPO publications WO92/05266 and W092/14829). Bovine papilloma virus (BPV) can also be used in gene therapy (U.S. Pat. 5, 719, 054). This gene therapy vector also includes a CMV-based vector (u s. Pat. N〇. 5, 561, 063). In yeast, many of the sputum containing the constitutive or agitation promoter can be used (see, for example, Ausubel et al). .,In: Current Protocols in Molecular

Biol〇gy，ν〇1· 2, Ch. 13, ed·，Greene Publish，Assoc· & Wiley Interscience, 1988; Grant et al. Methods in Enzvmologv. 153:5 16 (1987), eds. Wu & Grossman Bitter Methods in 3En_zymology? 152:673 (1987), eds. Berger & Kimmel, Acad. Press, N.Y., and, Strathern et al., The Molecular Biology of the_Yeast Saccharomvces (1982) eds. Cold Spring Harbor Press，Vols· I及II)。原構性的酵母啓動子，如ADH或 LEU2，或可謗生之啓動子如GAL，均可使用（R· Rothstein In: DNA Cloning, A Practical Approach. Vol. 11，Ch· 3，ed. D.M. Glover，IRL Press，Wash·，D.C.，1986)。另夕卜，可促進 -31 - 本纸張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） - 裝—·----^—訂·-------- (請先閒讀背面之注意事項再填寫本頁) 1264467 經濟部智慧財產局員工消費合作社印制π A7 、發明說明（29 ) 核酸序列整合酵母染色體，且經由同質重組作用之載 月豆疋技藝中已知的，且可被採用。當嵌入之聚核苷酸對轉 傳統載體而言是太大的時候，通常會採用酵母人工染色體 (YAC)(如大於約12 kb以上）。 本發明也提出編碼本發明人類CD40抗體之核酸，以供後 入佰王細胞中。在一個具體實例中，宿主細胞是原核細 月已在另具骨豆貫例中，宿主細胞是眞核細胞。在各種方 面，眞核細胞是酵母或哺乳動物（如人類，靈長類等）細 胞。 如此中所用的“宿主細胞，，是核酸可被引入之細胞，其中 其可被增殖，轉錄，或表現所編碼之CD40。此術語也包 括主題宿主細胞任何的子代。宿主細胞包括子代細胞，其 可此與親代細胞不同，因爲在複製中可能有突變的發生。 然而，此細胞仍可視爲是本發明之宿主細胞。 宿王細胞包括以下微生物，但不限於此：細菌或酵母； 及植物，昆蟲及哺乳動物細胞。例如，爲重組體噬菌體核 酸，質體核酸或黏接質體核酸表現載體所轉形之細菌；爲 重組體酵母表現載體轉形之酵母；爲重組體病毒表現載體 (如花椰菜花葉病毒，CaMV ;煙草花葉病毒，TMV)所感 染之植物細胞系統，或爲重組體質體表現載體轉形之植物 細胞系統（如：Ti質體）；爲重組體病毒表現載體（如桿狀 病毒）感染之昆蟲細胞系統；或爲重組體病毒表現載體（如 反轉錄病毒，腺病毒，牛痘苗病毒）感染之動物細胞系 統，或經遺傳操作可穩定表現之經轉形的動物細胞系統。 ------------裝---.----！—訂--------- (請先閱讀背面之注意事項再填寫本頁) 32- 1264467Biol〇gy, ν〇1· 2, Ch. 13, ed·, Greene Publish, Assoc· & Wiley Interscience, 1988; Grant et al. Methods in Enzvmologv. 153:5 16 (1987), eds. Wu & Grossman Bitter Methods in 3En_zymology? 152:673 (1987), eds. Berger & Kimmel, Acad. Press, NY, and, Strathern et al., The Molecular Biology of the_Yeast Saccharomvces (1982) eds. Cold Spring Harbor Press, Vols · I and II). A prokaryotic yeast promoter, such as ADH or LEU2, or a viable promoter such as GAL, can be used (R· Rothstein In: DNA Cloning, A Practical Approach. Vol. 11, Ch·3, ed. DM Glover, IRL Press, Wash·, DC, 1986). In addition, can promote -31 - the paper size applies to the Chinese National Standard (CNS) A4 specifications (210 X 297 mm) - loaded -·----^-booking-------- ( Please read the following notes on the back of the page and fill in this page. 1264467 Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumer Cooperatives, Printed π A7, Inventions (29) The nucleic acid sequence integrates yeast chromosomes and is carried out by homogenous recombination. It is known and can be employed. Yeast artificial chromosomes (YAC) (e.g., greater than about 12 kb) are typically employed when the embedded polynucleotide is too large for a conventional vector. The present invention also proposes a nucleic acid encoding a human CD40 antibody of the present invention for subsequent entry into a scorpion cell. In a specific example, the host cell is a pronuclear squam, which is in another case, and the host cell is a nucleus. In various aspects, the nucleus cells are yeast or mammalian (e.g., human, primate, etc.) cells. As used herein, a "host cell" is a cell into which a nucleic acid can be introduced, wherein it can be propagated, transcribed, or behaves as encoded by CD40. This term also includes any progeny of the subject host cell. The host cell includes daughter cells. This may be different from the parental cell, since mutation may occur in the replication. However, the cell may still be regarded as the host cell of the present invention. The Kingcell cell includes the following microorganisms, but is not limited thereto: bacteria or yeast; And a plant, an insect, or a mammalian cell, for example, a recombinant phage nucleic acid, a plastid nucleic acid or a plastid nucleic acid, which expresses a transformed plasmid; a recombinant yeast expressing a vector-transformed yeast; A plant cell system infected with a vector (such as cauliflower mosaic virus, CaMV; Tobacco mosaic virus, TMV), or a plant cell system (eg, Ti plastid) transformed into a recombinant plastid; a recombinant virus An insect cell system infected with a vector (such as a baculovirus); or a recombinant viral expression vector (eg, retrovirus, adenovirus, vaccinia) Virus) Infected animal cell system, or transformed animal cell system that can be stably expressed by genetic manipulation. ------------Install---.----!--- ------- (Please read the notes on the back and fill out this page) 32- 1264467

經濟部智慧財產局員工消費合作社印製 表現載體也含有編碼可選擇標幟之核酸，以對選擇性壓 力或可鑑知之標幟（如β-半乳糖甚酸）提供抗性，由是 載體欲鑑定之細胞可生長及擴大。另夕卜，可選擇的標：可 以疋種第一載體’其與含有本發明聚核苷酸之第—載體The Ministry of Economic Affairs, the Intellectual Property Office, the Consumer Cooperatives, and the printed performance carrier also contain nucleic acids encoding selectable markers to provide resistance to selective pressure or detectable markers (such as β-galactose and acid). The identified cells can grow and expand. In addition, a selectable target: a first vector can be produced and a vector containing the polynucleotide of the present invention

共同轉感至宿主細胞。 E 有許多的選擇系統可採用，包括單純録病毒胸誓激酶 基因/Wigler et al.，Cell丨i :223 (1977))，以黃嘌呤鳥嘌呤轉 磷酸核糖基酶（Szybalska et al·，Proc. Natl· Acad Sci USA 48:2026 (1962))及腺苷轉磷酸核糖基酶（L〇wy et ai， 22:817 (1980))基因’可分別應用於tk_，_义或啊細胞 中。另外，h代謝抗性可充作dhfr之選擇基礎，其可對胺 甲碟吟 4疋供抗性（〇· Hare et al·，Proc· Natl. Acad。Sci. USA 78:1527 (1981)) ; gpt基因，可提供對霉紛酸之抗性 (Mulligan et al., Proc. Natl. Acad. Sci. USA 78:2072 (1981));新黴素基因，可提供對胺基葡糖苷g-4 18之抗性 (Colberre-Garapin et al·，J. Mol· Biol. 150:1(1981));嘌呤黴 素；及潮霉素基因，可提供對潮霉素之抗性（Santerre et al·，Gene 30:147 (1984))。另外的可選擇基因包括trpB，其 令細胞可利用p朵替代色胺酸；hisD，令細胞可利用組胺 醇替代組胺酸（Hartman et al·，Proc. Natl. Acad. Sci. USA 85:8047 (1988));及〇DC(鳥胺酸去羧酶），可提供對鳥胺酸 去羧酶抑制劑，2-(二氟甲基）-DL-鳥胺酸，DFMO之抗性 (McConlogue (1987)在 ： Current Communications in Molecular Biology. Cold Spring Harbor Laboratory) ° -33- 本纸張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） .-----------裝—·----„—訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 A7 B7 經濟部智慧財產局員工消費合作社印制衣 五、發明說明（） 如此中所揭示的，本發明的人類CD40抗體包括可調控一 種以上CD40活性之抗體。因此，本發明的CD40抗體，包 括經修飾型式，片段及編碼CD40抗體之核酸，其核甞酸 變型及次序列，均可在細胞，組織，器官，或全有機體中 於試管内，活體内或活體外調控一種以上的CD40活性。 例如，當欲減低CD40活性時，可以減低CD40活性之CD40 抗體（如nos. 30，72，366及由融合瘤F4-465所產生之抗體） 來減低CD40活性。當需增加CD40活性時，可以可增加 CD40活性之CD40抗體（如no. 11及由融合瘤卩1-102，？2-103，F5-77，F5-152及F5-157產生之抗體）來增加CD40活 性0 因此，本發明提出在細胞，組織，器官或全有機體中， 於試管内，活體内或活體外調控CD40活性之方法。在一 個具體實例中，本發明方法包括：在試管内，活體内或活 體外將細胞，組織，器官或全有機體與調控劑量之人類 CD40抗體接觸。在一方面，CD40被調控的是人類的。在 另一方面，在CD40配體（如CD40L )存在或不存在下， CD40傳遞活性是增加的（如可由增加之CD95，CD80或 CD86表現或增加的細胞增殖作用來反映出來）。又在另一 方面，於CD40配體（如CD40L)之存在或不存在下，CD40傳 訊活性是減低的（如由減低的CD95，CD80或CD86表現或減 低的細胞增殖所反映）。又在另一方面，CD40抗體具有no. 11，30，72或3 66之CD40抗體，或由融合瘤Fl-102，F5-152，F2-103，F5-77，F5-157或F4-465所產生抗體之結合特 -34- (請先閱讀背面之注意事項再填寫本頁) 裝 訂--- 錚 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1264467 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（32 ) 異性’結合親和力或一種以上的功能。在特殊方面， CD40抗體包括如η〇· 11，30，72或366之CD40抗體，或由融 合瘤 Fl-102，F5-152，F2-103，F5-77，F5-157或F4-465所產 生之抗體。在較特殊方面，抗體具有如SEq ID NOs:l〇， 11，12，13，14，或15示之重或輕鏈序列。 CD40在免疫細胞功能及傳訊上扮演重要角色，包括B _ 細胞及T -細胞爲抗原呈現細胞之活化作用，如巨嗤細胞及 樹突細胞。在此狀況下，CD40之作用似乎是細胞存活之 訊號。CD40之活化作用可刺激抗體之產製，同型之轉換 及記憶力之確立。CD40之活化作用可刺激細胞動素之產 製’如 IL-2 ’ IL-6 ’ IL-8，IL-12，TNF-α，IL-4，IL-S 及IL-10 ;及金屬蛋白酶，如MMP_I/膠原酶及MMP-9/明膠酶B。 CD40活化作用可刺激涉及細胞-細胞接觸或黏附中蛋白質 之產製’如E -選擇素，vcam-Ι及ICAM-1。CD40在標的細 胞上之確忍可對N K細胞胞毒活性提供活化路徑。因此， 增加的或減少的細胞存活，抗體產製，同型轉換，記憶力 之崔立、、’田胞動素’金屬蛋白酶或涉及於細胞-細胞接觸 或黏附中心蛋白質之產製，或N K細胞胞毒性活性，經由 適合細胞與本發明CD40抗體之接觸均可達成。 因此，本發明進一步提出調控細胞存活，抗體產製，同 型轉換，圮憶力之確立，細胞動素，金屬蛋白酶或涉及於 細胞-細胞接觸或黏附中蛋白質之產製，或NK細胞胞毒活 性足方法’方法包括將細胞與調控劑量之cd4〇抗體接 觸。此方法可在個體中實行以在個體中達成作用。 裝---.----Γ — 訂--------- (請先閱讀背面之注意事項再填寫本頁) -35-Co-transfer to host cells. There are many selection systems available, including the purely virus-threatening kinase gene/Wigler et al., Cell丨i: 223 (1977)), which is a yellow-ostrich-transfer phosphoribosylase (Szybalska et al., Proc Natl·Acad Sci USA 48:2026 (1962)) and adenosine phosphoribosyltransferase (L〇wy et ai, 22:817 (1980)) genes can be applied to tk_, _ or ah cells, respectively. In addition, h-metabolism resistance can be used as a basis for selection of dhfr, which can confer resistance to A. sinensis (〇· Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)) ; gpt gene, can provide resistance to myric acid (Mulligan et al., Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neomycin gene, can provide amino-glucoside g- 4 18 resistance (Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981)); puromycin; and hygromycin genes, which provide resistance to hygromycin (Santerre et al ·, Gene 30: 147 (1984)). Additional selectable genes include trpB, which allows cells to replace p-tryptophan with p; hisD, which allows cells to replace histidine with histamine (Hartman et al., Proc. Natl. Acad. Sci. USA 85: 8047 (1988)); and 〇DC (ornithine decarboxylase), which provides resistance to avidinate decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO ( McConlogue (1987) at: Current Communications in Molecular Biology. Cold Spring Harbor Laboratory) ° -33- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) .-------- ---装—·----„-订--------- (Please read the note on the back and fill out this page) 1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed Clothing BRIEF DESCRIPTION OF THE INVENTION As disclosed herein, the human CD40 antibody of the present invention includes an antibody that modulates more than one CD40 activity. Thus, the CD40 antibody of the present invention includes a modified version, a fragment, and a nucleic acid encoding the CD40 antibody, the nucleus thereof. Both citrate variants and subsequences can be in vitro in cells, tissues, organs, or whole organisms. More than one CD40 activity is regulated in vitro or in vitro. For example, when CD40 activity is to be reduced, CD40 antibodies (such as nos. 30, 72, 366 and antibodies produced by fusion tumor F4-465) can be reduced to reduce CD40 activity. CD40 activity. When it is necessary to increase CD40 activity, CD40 antibody which can increase CD40 activity (such as no. 11 and produced by fusion tumors 卩1-102, ?2-103, F5-77, F5-152 and F5-157) Antibodies) to increase CD40 activity. Thus, the present invention contemplates methods for regulating CD40 activity in vitro, in vivo, or in vitro in cells, tissues, organs, or whole organisms. In one embodiment, the methods of the invention include: Cells, tissues, organs or whole organisms are contacted with a regulated dose of human CD40 antibody in vitro, in vitro or in vitro. In one aspect, CD40 is regulated by humans. On the other hand, in CD40 ligands (eg CD40L) In the presence or absence, CD40 transmission activity is increased (as reflected by increased cell proliferation of CD95, CD80 or CD86 expression or increased). On the other hand, in CD40 ligand (eg CD40L) Save The absence or, CD40 information transfer activity reduced (e.g., by the reduction of CD95, CD80 or CD86 expression, or a low cell proliferation minus reflected). In yet another aspect, the CD40 antibody has a CD40 antibody of no. 11, 30, 72 or 3 66, or a fusion tumor Fl-102, F5-152, F2-103, F5-77, F5-157 or F4-465 Combination of produced antibodies -34- (Please read the note on the back and fill out this page) Binding --- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1264467 Ministry of Economics Property Bureau Staff Consumer Cooperatives Print A7 B7 V. Invention Description (32) Heterosexual 'binding affinity or more than one function. In a particular aspect, the CD40 antibody comprises a CD40 antibody such as η〇· 11, 30, 72 or 366, or by a fusion tumor Fl-102, F5-152, F2-103, F5-77, F5-157 or F4-465 The antibody produced. In a more particular aspect, the antibody has a heavy or light chain sequence as set forth in SEq ID NOs: l, 11, 12, 13, 14, or 15. CD40 plays an important role in immune cell function and communication, including B_ cells and T-cells, which are antigen-presenting cells, such as giant sputum cells and dendritic cells. Under this condition, the role of CD40 appears to be a signal of cell survival. The activation of CD40 stimulates the production of antibodies, the conversion of isoforms and the establishment of memory. Activation of CD40 stimulates the production of cytokines such as IL-2 'IL-6' IL-8, IL-12, TNF-α, IL-4, IL-S and IL-10; and metalloproteinases, Such as MMP_I / collagenase and MMP-9 / gelatinase B. CD40 activation stimulates the production of proteins involved in cell-cell contact or adhesion, such as E-selectin, vcam-Ι and ICAM-1. CD40 does provide a pathway for activation of N K cell cytotoxic activity on the target cells. Thus, increased or decreased cell survival, antibody production, homotypic switching, memory of Cui Li, 'T-cellin' metalloproteinase or production involving cell-cell contact or adhesion center proteins, or NK cell Toxicity activity can be achieved by contacting a suitable cell with a CD40 antibody of the invention. Therefore, the present invention further proposes to regulate cell survival, antibody production, homotypic conversion, establishment of sputum, cytokines, metalloproteinases or proteins involved in cell-cell contact or adhesion, or NK cell cytotoxic activity. The foot method' method involves contacting the cells with a modulatory dose of a cd4 antibody. This method can be practiced in an individual to achieve an effect in the individual. Pack---.----Γ-book--------- (Please read the notes on the back and fill out this page) -35-

1264467 A7 經濟部智慧財產局員工消費合作社印製 栓 官 五、發明說明（33 ) 凋控CD40活性之方法可用來治療與CD4〇相關之失調 症。本發明因此提出治療與CD40相關之失調症之方法。 如此中所用的，所謂‘‘與CD4〇相關的失調症，，表示其中調 控CD40活性可改善或減低一種以上狀況或失調症中非欲 求之症狀之任何非欲求之生理狀況或病理性失調症。 例如’本發明可降低CD40活性之人類CD40抗體，可用 來治療與CD40有關之失調症，其中降低的€1)4〇活性可改 善或減低失碉症一種以上非欲求的症狀。因此，當扣 與活體内非欲求之免疫反應或過程有關時，如自體免疫， 過敏’發炎或移植排斥，因此可降低CD40活性的本發明 CD40抗體，可投予至有自體免疫力，過敏，發炎或移植 排斥或有這些狀況高危險群之個體，以抑制個體中之自體 免疫力，過敏，發炎或移植排斥。 以本發明的人類CD40抗體可治療之自體免疫失調症特殊 實例包括··類風濕性關節炎，狼瘡（如SLe，狼瘡腎炎）， 自體抗體之產製，其係在抗體拮抗骨髓磷脂基礎蛋白質造 成多發性硬化之例子中，及在抗體拮抗胰島素造成糖尿 病’及克隆氏病之例子中。以本發明之CD40抗體可治療 之過敏包括對抗原，抗生素等之過敏反應。 & ^ 以本發明的人類CD40抗體可治療之特殊發炎實例包括： 血官發炎性疾病（如動脈粥樣硬化傷害，斑之瓦解及血 之形成），發炎性細胞動素之產生（如LIF，GM-CSF，及匕 6) ’肺纖維化及與發炎有關之多發性硬化，或組織或器 的移植。CD40活性可與由病毒性心肌炎引起之發炎有 •-----------裝---„----丨訂--------- (請先閱讀背面之注意事項再填寫本頁) -36 12644671264467 A7 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed Suspended Officer V. Invention Description (33) The method of controlling CD40 activity can be used to treat disorders associated with CD4〇. The present invention therefore proposes a method of treating a disorder associated with CD40. As used herein, the term "' associated with CD4〇 indicates that any uncontrolled physiological condition or pathological disorder in which CD40 activity is modulated may ameliorate or reduce the undesired symptoms of more than one condition or disorder. For example, the human CD40 antibody of the present invention which reduces CD40 activity can be used to treat CD40-related disorders, wherein a reduced activity of €1) can improve or reduce one or more undesired symptoms of aphasia. Therefore, when the deduction is related to an undesired immune response or process in the living body, such as autoimmunity, allergy 'inflammation or transplant rejection, the CD40 antibody of the present invention which can reduce the activity of CD40 can be administered to autoimmune, Allergic, inflammatory or transplant rejection or individuals with a high risk group of these conditions to inhibit autoimmune, allergic, inflammatory or transplant rejection in an individual. Specific examples of autoimmune disorders treatable by the human CD40 antibody of the present invention include rheumatoid arthritis, lupus (such as SLe, lupus nephritis), production of autoantibodies, which are based on antibodies antagonizing the basis of myelin Examples of proteins causing multiple sclerosis, and examples of antibodies that antagonize insulin causing diabetes and Crohn's disease. Allergies which can be treated with the CD40 antibody of the present invention include allergic reactions to antigens, antibiotics and the like. & ^ Examples of specific inflammation treatable by the human CD40 antibody of the present invention include: blood inflammatory diseases (such as atherosclerotic lesions, plaque disintegration and blood formation), production of inflammatory cytokines (such as LIF) , GM-CSF, and 匕6) 'Pulmonary fibrosis and multiple sclerosis associated with inflammation, or tissue or device transplantation. CD40 activity can be associated with inflammation caused by viral myocarditis. --------------------------- Please read the back Please fill out this page again) -36 1264467

經濟部智慧財產局員工消費合作社印製 關，且如此可抑制CD40活性之CD40抗體，即 與病毒感染有關之發炎。 用來抑帝 以本發明人類CD40抗體可治療之特殊移植 性或慢性）包括血管，腎，肝，心，肺，胰臟及皮戶= 植包括由個體之組織或器官移植至相同或不同個 地方。移植也涉及由身體某區至另一區組織或^官之a， 植。在相同種類但遺傳上不同動物間組織或器官間5 ^ ^ 稱爲同種異基因移植。動物器官移植至人體稱爲異^ ^ 植。 、夕 因此，本發明的人類CD4〇抗體可單獨使用，或配合可和 制移植器官排斥之治療劑使用。此作用劑之實例有··访^ 嘌呤：皮質固醇類，及環胞靈。CD4〇抗體治療也可：輕 或預防在進行免疫抑制治療之移植受者中常見的副作用， 如發燒，厭食，血液動力學失常，白血球過少，經移植器 官/組織被T -細胞或B _細胞所浸潤及機會感染。 σ 有額外的狀況會存在，其中可能欲抑制免疫反應。例 如，產製中和抗體以拮抗治療劑，如在重覆投予胰島素之 糖尿病患中之抗-胰島素抗體，或在可產生抗-病毒抗體之 個體中（如腺病毒或與腺有關之病毒）其曾以基因治療^病 毒載體治療過，均可利用本發明之CD4〇抗體抑制之。汽 可增加CD40活性之人類CD40抗體，也可用來治療其中 CD40活性之增加可改善或減低失調症一種以上非欲求症 狀之失調症。因此，可增加CD40活性之CD4〇抗體可用來 治療欲增加或刺激免疫反應之個體。此中有許多狀況是希 -37- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ------------^^裝---«----τι 訂--------- (請先閱讀背面之注意事項再填寫本頁} 1264467 經濟部智慧財產局員工消費合作社印制衣 A7 B7 五、發明說明（35 ) 望可刺激免疫反應的。例如，在癌症之例子中，可刺激 CD40活性之CD40抗體可用來加強免疫反應以拮抗癌症。 至於感染性疾病，可刺激CD40活性之CD40抗體可用來加 強免疫反應以拮抗感染。 CD40活性被視爲涉及於細胞調介之抗病毒免疫力中記憶 力之產生。因此可刺激CD40活性之人類CD40抗體可用來 促進記憶力，其接續地可促進較快速的免疫反應以拮抗病 毒。刺激CD40活性之CD40抗體，可改進免疫反應以拮抗 不良免疫原性之病原體，如流感嗜血菌，肺炎鏈球菌及腦 膜炎奈瑟氏球菌。 CD40也存在於其他各種組織及細胞中。如，CD40存在 於上皮細胞，血管内皮及平滑肌細胞，且CD40配體（CD 1 54)爲由凝血酶-活化之血小板所表現。這些發現顯示 CD40活性在血管血栓-動脈粥樣化病理生理學上之角色。 因此，本發明的人類CD40抗體，可降低CD40活性可用於 個體中抑制血栓之形成或動脈粥樣硬化。 CD40也在人類腎小管，胸腺上皮及神經細胞中表現。令 人感興趣的，CD40活化作用似乎可在神經細胞中謗生細 胞預期死亡，和其在免疫系中之細胞存活角色相反。因 此，本發明可用來降低CD40活性之人類CD40抗體，可於 神經細胞中用於抑制細胞預期死亡。因此，CD40可用於 治療特徵爲細胞退化或非欲求的或過度細胞死亡之神經失 調症，如巴金森氏病，阿滋海默爾氏病，亨丁頓氏病，脊 髓腦運動失調/萎縮等。 -38- 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) 1264467The CD40 antibody, which inhibits CD40 activity, is inflamed in connection with viral infections. It is used to inhibit the special transplantability or chronicity of the human CD40 antibody of the present invention including blood vessels, kidneys, liver, heart, lung, pancreas and skin. The plant includes transplanting from the tissues or organs of the individual to the same or different ones. local. Transplantation also involves the organization of a region from one body to another, or a plant. 5 ^ ^ between the same species but genetically different inter-animal tissues or organs is called allogeneic transplantation. Transplantation of animal organs into the human body is called heterogeneous planting. Further, the human CD4 〇 antibody of the present invention can be used alone or in combination with a therapeutic agent which can be used for rejection of transplanted organs. Examples of such agents are: visits to 嘌呤: corticosteroids, and cyclosporine. CD4〇 antibody therapy can also: lightly or prevent side effects commonly seen in transplant recipients undergoing immunosuppressive therapy, such as fever, anorexia, hemodynamic disorders, leukopenia, transplanted organs/tissues by T-cells or B-cells Infiltration and opportunistic infections. σ There are additional conditions that may exist to suppress the immune response. For example, the production of neutralizing antibodies to antagonize therapeutic agents, such as anti-insulin antibodies in diabetic patients who are repeatedly administered insulin, or in individuals who can produce anti-viral antibodies (such as adenovirus or gland-associated virus) It has been treated with a gene therapy virus vector and can be inhibited by the CD4 〇 antibody of the present invention. Vapor can increase CD40 activity in human CD40 antibodies and can also be used to treat disorders in which an increase in CD40 activity can ameliorate or reduce one or more undesired symptoms of the disorder. Thus, CD4 〇 antibodies that increase CD40 activity can be used to treat individuals who wish to increase or stimulate an immune response. Many of the conditions are: Greek-37- This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------------^^装---«-- --τι 订--------- (Please read the notes on the back and fill out this page) 1264467 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperatives Printed Clothing A7 B7 V. Invention Description (35) Hope to stimulate For example, in the case of cancer, a CD40 antibody that stimulates CD40 activity can be used to potentiate an immune response to antagonize cancer. As for infectious diseases, a CD40 antibody that stimulates CD40 activity can be used to potentiate an immune response to antagonize an infection. Activity is thought to be involved in the production of memory in the antiviral immunity of cell mediation. Thus human CD40 antibodies that stimulate CD40 activity can be used to promote memory, which in turn can promote a faster immune response to antagonize the virus. Stimulate CD40 activity The CD40 antibody can improve the immune response to antagonize pathogens of poor immunogenicity, such as Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis. CD40 is also present in various other tissues and cells. For example, CD40 is present in Skin cells, vascular endothelium and smooth muscle cells, and CD40 ligand (CD 1 54) is expressed by thrombin-activated platelets. These findings indicate the role of CD40 activity in the pathophysiology of vascular thrombosis-atherosclerosis. The human CD40 antibody of the present invention can reduce CD40 activity and can be used for inhibiting thrombus formation or atherosclerosis in an individual. CD40 is also expressed in human renal tubules, thymic epithelium and nerve cells. Interesting, CD40 activation It appears that twin cells are expected to die in nerve cells, and their cell survival role in the immune system is reversed. Therefore, the present invention can be used to reduce CD40 activity of human CD40 antibodies, which can be used in nerve cells to inhibit expected cell death. Therefore, CD40 can be used to treat neurological disorders characterized by cell degeneration or undesired or excessive cell death, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, spinal cord dysmotility/atrophy, etc. -38- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) —, --------^ —-—<----------- (please first Read the notes on the back and fill out this page) 1264467

經濟部智慧財產局員工消費合作社印制衣 CD40也表現在腫瘤上，如黑色素瘤，卡波西氏肉瘤，骨 肉瘤及Ewing，肉瘤。在惡性黑色素瘤中CD4〇似乎先負面預 後之表徵。CD40在人類膀胱腫瘤細胞中抑制由調介之 細胞預期死亡。在腎腫瘤團塊之腫瘤血管中已可測及 CD40。B -細胞淋巴瘤中CD4〇之刺激可刺激生長。因此 CD40在某些腫瘤中可作用爲細胞存活或生長因子，且可 促進血管生成。其他數據顯示CD4〇可在經轉形細胞中謗 使細胞死亡。 因此，很明顯的CD40在癌細胞中有雙重功能，在某些中 其可促進存活或細胞之增殖/生長，而在其他卻是刺激細 胞預期此it。因此’可降低與腫瘤存活有關之cd4〇活性 之本發明的人類CD40抗體，可用於治療細胞增殖性失調 症（如腫瘤），其中CD40作用是細胞存活訊號或生長啓進 劑’或直接地（如在腫瘤細胞中）或間接的（即經由腫瘤團 塊内血管生成之刺激作用）。本發明可增加與增殖減低或 細胞預期死亡之增加有關之CD40活性之人類CD40抗體， 將可用於治療細胞增殖性失調症（如腫瘤），其中CD40作用 是一種因子，其可促進或刺激細胞預期死亡，細胞死亡或 生長遏止，或直接地（如在腫瘤細胞中）或間接地（如經由 刺激可拮抗腫瘤之免疫反應）。 其他有CD40參與之生物路徑及生理狀況述於下： Biancone, et al. (Int. J. Mol. Med. 3:343 (1999)), Laman et al (Dev. Immunol. 6:215 (1998)), Kooten and Bachereau (J. Leukoc. Biol 67:2 (2000)), Noelle et al. (Ann. NY Acad. Sci. -39- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 、-----------裝—.—^—訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（37 ) 815:384 (1997))，Noelle (Immunity 4:415 (1996))，Grewal et al. (Curr. Opin. Immunol. 9:491 (1997)); Grewal et al. (Ann. Rev. Immunol. 16:1 1 1 (1998); Grewal et al. (Immunol. Rev. 153:85 (1996)); Gruss et al. (Leuk. Lymphoma 24(5-6):393 (1997)); van Kooten et al. (Curr. Opin. Immunol 9:330 (1997))。此路徑以及其他技藝中已知的，可順應本 發明人類CD40抗體之調控，如此處或技藝中已知之與 CD40活性有關之生理狀一般。 當然，本發明的人類CD40抗體可配合其他治療使用，其 可增加或降氐CD40活性，或可互補CD40之抗體功能。例 如，可減低CD40活性之本發明人類CD40抗體，可配免疫 遏止性樂物（如固醇類）或其他治療策略使用，以治療自體 免疫失調症，發炎或可抑制移植排斥。另外，可增加 CD40活性之本發明人類CD40抗體，可配合免疫加強藥物 或其他治療策略使用，以對病原體或癌症增加免疫反應 ，或可改進免疫記憶力。 本發明方法，包括治療個體之CD40相關失調症，似乎可 造成個體狀況之改善，減低症狀或減低個體發展與CD40 相關失調症之症狀危險性。因此，改善包括與自體免疫 力，過敏，移植物對宿主病等相關症狀有一種以上症狀減 低。改善也可以是治療有CD40相關失調症或其危險性個 體所用之藥物給藥頻率或用量之減低。例如，以固醇類治 療之自體免疫患者，當配合人類CD40抗體一起治療時， 前者之用量可減少。因此改善也包括和以人類CD40抗體 -40- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） '-----------裝—.—*1 訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 A7 五 、發明說明（38 B7 經濟部智慧財產局員工消費合作社印划农 治療前比較，固醇類之蛉 改善可能只兩^ Λ "、率或劑量有所減低。 長的時間，如數月或年。改善未必=時，天或週，或較 症狀之完全消除。例如， 疋失凋症任何的或所有 較不嚴重程^ 的類風濕性關節炎減緩i 至狂度也疋一種改善。 及玍 或個體狀-或相關狀況有部份二；：：有漸增的改進 下^可謂達到令人滿意之臨床終點;^疋在短或長時間 失=:體中所述或技藝中已知之_相關 險性者因也:括有發展成CD4。相關失調症危 失調編=細⑽0相關 此也包括預防性方法。現失調症顯明症狀者。因 、、’、生療之危險群個體可鑑定爲有發展成CD40相關失調 紅具遣傳素因或家族史者。危險群之個體因此可以遣傳 篩選之例行方法鑑知’尋求遺傳傷害是否存在或研究個體 足家族病史，以確定其是否爲失調症之高危險群。危險群 個體之特殊實例爲具家族史者，或有其他的遺傳特性可 π有k成癌症之傾向，其中的贅瘤或具抗藥性之贅瘤細 係可表現CD40。遺傳疾病的一個特殊實例是X_性聯 IgM過鬲症狀，已知其同缺乏CD40L-CD40交互作用所 (Allen et al·，Science，259:990 (1993))。 CD40抗體可以單一或多次劑量投予，如每週一次，約 至1 〇週，或依適度下儘可能長期投藥，以達到CD40相 失調症一種以上症狀嚴重度之減低。劑量可依下列而 -41 - 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) 裝 訂--- 顯 之 致 關 變 0 1264467 Λ7 五、發明說明（39 ) 化·奴治療之失調症，失 床終點，先前的或同時的4 度，欲求之臨 齡，性別，或種族，及心：的一般健康狀況，年 解，可影響爲提供足以達到治療；素；精藝者了 里及時間。劑量可由實驗決 …d 所需之人類臨床試驗。在本發明=動=模式或視 性處理，方法之劑量或策略可依藥丄及治療 飾或變化之。 m柒子基因數據而特異地修 長=,，，是指動物，通常是哺乳動物，如非人狀靈 描），農場動物（馬，牛，山羊 ）!田（狗及 (老鼠，大鼠，兔子，天諸），貫驗用動物 ^ 一机）及人類。個體包括動物疾病 換式（如自體免疫老鼠， 伙他The Ministry of Economic Affairs, the Intellectual Property Office, the employee consumption cooperative, printed clothing, CD40 is also manifested in tumors, such as melanoma, Kaposi's sarcoma, osteosarcoma and Ewing, sarcoma. CD4〇 appears to be a negative predictor in malignant melanoma. CD40 inhibits the expected death of cells mediated by human bladder tumor cells. CD40 has been detected in tumor blood vessels of renal tumor masses. Stimulation of CD4〇 in B-cell lymphoma stimulates growth. Therefore, CD40 acts as a cell survival or growth factor in certain tumors and promotes angiogenesis. Other data show that CD4〇 can cause cell death in transformed cells. Therefore, it is apparent that CD40 has a dual function in cancer cells, in some of which promotes survival or cell proliferation/growth, while others stimulate cells to expect this. Thus, the human CD40 antibody of the present invention which reduces the activity of cd4〇 associated with tumor survival can be used for the treatment of cell proliferative disorders such as tumors, wherein the CD40 effect is a cell survival signal or a growth promoter' or directly ( As in tumor cells) or indirectly (ie via stimulation of angiogenesis in tumor masses). The human CD40 antibody of the present invention which increases CD40 activity associated with decreased proliferation or increased expected cell death will be useful for the treatment of cell proliferative disorders such as tumors, wherein CD40 action is a factor that promotes or stimulates cellular expectations. Death, cell death or growth arrest, either directly (as in tumor cells) or indirectly (eg, by stimulation, can antagonize the immune response of the tumor). Other biological pathways and physiological conditions involving CD40 are described below: Biancone, et al. (Int. J. Mol. Med. 3:343 (1999)), Laman et al (Dev. Immunol. 6:215 (1998) ), Kooten and Bachereau (J. Leukoc. Biol 67:2 (2000)), Noelle et al. (Ann. NY Acad. Sci. -39- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297) )),-----------装—.—^—订--------- (Please read the notes on the back and fill out this page) 1264467 A7 B7 Ministry of Economics Printed by the Bureau of Property and Staff Consumer Cooperatives. V. Inventions (37) 815:384 (1997)), Noelle (Immunity 4:415 (1996)), Grewal et al. (Curr. Opin. Immunol. 9:491 (1997) Grewal et al. (Ann. Rev. Immunol. 16:1 1 1 (1998); Grewal et al. (Immunol. Rev. 153:85 (1996)); Gruss et al. (Leuk. Lymphoma 24 (5) -6): 393 (1997)); van Kooten et al. (Curr. Opin. Immunol 9:330 (1997)). This route, as well as known in the art, is responsive to the regulation of human CD40 antibodies of the invention, Physiological conditions known in the art or related to CD40 activity. Of course, the human CD4 of the present invention The 0 antibody can be used in combination with other therapies, which can increase or decrease the activity of CD40, or can complement the function of the antibody of CD40. For example, the human CD40 antibody of the present invention can reduce the activity of CD40, and can be combined with immunosuppressive music (such as sterols). Or other therapeutic strategies for the treatment of autoimmune disorders, inflammation or inhibition of transplant rejection. In addition, the human CD40 antibody of the invention, which increases CD40 activity, can be used in conjunction with immunopotentiating drugs or other therapeutic strategies to target pathogens or Cancer increases the immune response, or may improve immune memory. The methods of the invention, including treating a CD40-associated disorder in an individual, appear to cause an improvement in the individual's condition, reducing symptoms or reducing the risk of developing a symptom associated with a CD40-related disorder. Improvements include more than one symptom reduction with autoimmune, allergic, graft-to-host disease and other related symptoms. The improvement may also be a reduction in the frequency or amount of administration of the drug for the treatment of a CD40-related disorder or a dangerous individual. For example, in autoimmune patients treated with sterols, the amount of the former can be reduced when treated with human CD40 antibodies. Therefore, the improvement also includes the Chinese National Standard (CNS) A4 specification (210 X 297 mm) for human CD40 antibody-40-this paper size '-------------------*1 --------- (Please read the notes on the back and fill out this page) 1264467 A7 V. Invention Description (38 B7 Ministry of Economic Affairs Intellectual Property Bureau employees consumption cooperatives before printing and treatment, sterols蛉 Improvement may only be two Λ ", rate or dose is reduced. Long time, such as several months or years. Improvement may not be = when, days or weeks, or more complete elimination of symptoms. For example, 疋 疋 任何Or all less severe cases of rheumatoid arthritis slow down i to madness is also an improvement. And 玍 or individual traits - or related conditions have part two;:: there is an incremental improvement ^ can be said to achieve Satisfactory clinical end point; ^ 疋 in short or long time loss =: known in the body or known in the art _ related risk factors also include: development of CD4. Related disorders, dysregulation = fine (10) 0 related Including preventive methods. Those with symptoms of dysregulation may be identified as Developed into a CD40-related disordered red person with a vegetarian or family history. Individuals in the risk group can therefore send a routine screening method to identify 'seeking the existence of genetic damage or studying the individual family history to determine if it is a disorder. High risk groups. Special cases of individuals in the risk group are family history, or have other genetic characteristics that may lead to cancer, and the tumor or drug resistant tumor line can express CD40. A particular example of a disease is X-linked IgM over-symptoms, which are known to be incompatible with CD40L-CD40 interactions (Allen et al., Science, 259:990 (1993)). CD40 antibodies can be administered in single or multiple doses. Inject, for example, once a week, about 1 week, or as long as possible, as long as possible to achieve a reduction in the severity of more than one symptom of CD40 phase disorders. The dose can be as follows -41 - 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) Binding--- Show the change 0 1264467 Λ7 V. Invention description (39) Loss Symptoms, end of bed loss, previous or simultaneous 4 degrees, desire for age, gender, or race, and heart: general health, annual solution, can be affected to provide enough to achieve treatment; vegetarian; And the time. The dose can be determined by the human clinical trial required. In the present invention = motion = mode or visual treatment, the dosage or strategy of the method can be based on the drug and the treatment or change. Specific slenderness =,,, refers to animals, usually mammals, such as non-humanoids), farm animals (horses, cattle, goats)! Tian (dogs and (rats, rats, rabbits, heavens), Through the use of animals ^ one machine) and humans. Individuals include animal diseases, such as autoimmune mice,

GvHD動物卜 如，結腸炎，移植及 體體包括經修飾型’片段及編碼。_ ^核I，核讀變型及其次序列，均可納入藥學級人物 cmnt樂學組合物可活體内或活體外投予至個體，並對與 目關·^失調症提供治療，以實行本發明。 藥學組合物包括“藥學上可接|的，，及“生理上可接受的” 載劑，稀釋劑或賦形劑。如此中所用的，“藥學上可接A 的及“生理上可接受的，，包括溶劑（水性或非水性卜溶 片J礼Μ ’分散介質’衣劑，等渗及吸收促進劑或延遲 劑’可與藥學投藥相容。此調和物可含於錠劑（有包衣或 未包衣）’膠囊劑（硬或軟），微珠，乳劑，教劑，顆粒—， -42- 本纸張尺度_巾關家鮮（CisTi_)A4祕⑵Q χ 297公 訂 i 經 濟 部 智 慧 財 產 局 消 費 合 作 社 印 製 1264467 A7 五、發明說明（4〇 ) 晶體，懸液劑，糖漿劑或酏劑。補充的活性化合物（如保 藏劑，抗菌劑，抗病毒劑及抗眞菌劑）也可納入組合物 中〇 藥學組合物可調和成與特定投藥路徑相容的。因此，藥 學組合物包括適於各種投藥路徑之載劑，稀釋劑或賦形 劑。 供腸外，皮内或皮下投藥之藥學組合物可包括無菌稀釋 劑，如水，食鹽水，固定油類，聚乙二醇，甘油，丙二醇 或其他合作的溶劑；抗菌劑如苄醇或對羥苯甲酸甲酯；抗 氧化劑如抗壞血酸或亞硫酸氫鈉；嵌合劑如乙二^四= 酸；緩衝溶液如醋酸鹽，擰檬酸鹽或磷酸鹽，及調整等滲 度（作用物，如氯化鋼或右旋糖。腸外製劑可密封在安叛 中，可分散之注射器或由玻璃或塑質製成之多重劑量小瓶 供/王射〈藥學組合物，包括無菌水溶液（纟溶性）或分气 劑及供無菌可注射溶液或分散液暫時製劑之無菌粉末。二 靜脈内投藥時，適合的載劑包括生理食鹽水，制菌水： Cremophor EL™ (BASF，parsippany，叫或鱗酸鹽缓衝食骑 水（PBS)。載劑可以是溶劑或分散介f，含有如水= :’多兀醇(如甘油，丙二醇，及液態聚乙二醇等），及並 適合的混合物。液動性可利用衣劑維持 二 散劑例子中係維持所需之赶子士,π 对細在刀 幵"而之粒子大小及可使用界面活性劑。 杬囷制及抗眞菌劑包括如：對苯胺基苯甲酸醋，， 齡’杬壞血鉍’硫柳汞。在許多例子中，等滲劑如糖類， -43- 本紙張尺度適用中國國家標GvHD animals such as colitis, transplantation and body include modified 'fragments and codes. _ ^Nuclear I, nuclear read variants and subsequences thereof, all of which can be incorporated into a pharmaceutical grade, the cmnt music composition can be administered to an individual in vivo or ex vivo, and provide treatment for the treatment of the disorder, to practice the present invention. . Pharmaceutical compositions include "pharmaceutically acceptable," and "physiologically acceptable" carriers, diluents or excipients, as used herein, "pharmaceutically pharmaceutically acceptable and "physiologically acceptable" , including solvent (aqueous or non-aqueous tablets), 'dispersing medium' coating, isotonic and absorption enhancer or retarder' can be compatible with pharmaceutical administration. This blend can be contained in tablets (with package) Clothing or uncoated) 'Capsules (hard or soft), microbeads, emulsions, teachants, granules -, -42- The paper size _ towel Guan Jia Xian (CisTi_) A4 secret (2) Q χ 297 public i economy Ministry of Intellectual Property, Consumer Cooperatives, Printing 1264467 A7 V. Description of Invention (4〇) Crystals, suspensions, syrups or elixirs. Supplementary active compounds (such as preservatives, antibacterials, antivirals and antibacterial agents) The pharmaceutical composition may also be incorporated into the composition to be compatible with the particular route of administration. Thus, the pharmaceutical compositions include carriers, diluents or excipients suitable for various routes of administration. Or pharmaceutical composition for subcutaneous administration may include none Diluent, such as water, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol or other co-solvent; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; Mixtures such as Ethylene^4 = acid; buffer solutions such as acetate, citrate or phosphate, and adjustment of isotonicity (activities such as chlorinated steel or dextrose. Intestinal preparations can be sealed in rebel , a dispersible syringe or a multi-dose vial made of glass or plastic. The pharmaceutical composition comprises a sterile aqueous solution (sputum soluble) or a gassing agent and a sterile preparation for a sterile injectable solution or dispersion. Powder. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water: Cremophor ELTM (BASF, parsippany, or sulphate buffered water (PBS). The carrier can be solvent or dispersed. The medium f contains, for example, water = : 'polyhydric alcohol (such as glycerin, propylene glycol, and liquid polyethylene glycol, etc.), and a suitable mixture. The hydraulic fluid can be used to maintain the two powders in the example. Sister, π is fine幵"The particle size and the use of surfactants. Tanning and antibacterial agents include, for example, p-anilinobenzoic acid vinegar, age '杬 铋 铋 铋 thiomercury. In many cases, isotonic agents Such as sugar, -43- This paper scale applies to Chinese national standard

蠢· -III. c請先閱讀背面之注意事項再填寫本頁) 裝---.------ I 訂----- 1264467 A7 經濟部智慧財產局員工消費合作社印製 五、發明說明（' 夕元醇如甘露醇，山梨醇，氣化鈉可包括在組合物中。也 包括可延缓吸收之作用劑，如單硬脂酸鋁及明膠均可延緩 可注射組合物之吸收。 無菌可注射溶液劑之製備係將活性化合物以必要劑量納 入適合溶劑中，加有一種或上述數種組份之組合，繼而過 濾減菌。一般而言，分散劑之製備是將活性化合物納入無 菌4媒中，其中含有基本的分散介質及上述其他的組份。 在可製成無菌可注射溶液劑之無菌散劑例子中，較佳之製 法爲眞空乾燥及冷凍乾燥，可生成活性組份加上任何額外 欲求組份（來自先前無菌過濾溶液）之散劑。 於經黏膜或穿皮投藥時，適合欲穿透障壁之穿透劑可用 於調和物中。此穿透劑大體上是技藝中已知的，且包括如 於穿黏膜投藥時之清潔劑，膽鹽及梭鏈孢酸衍生物。於 皮投藥時，活性化合物可調和至油膏，刮髮膏，凝膠或 劑等技藝中已知的。 可使用生物可降解且生物可相容的聚合物，如乙烯醋 乙烯酯，聚酐類，聚乙醇酸，膠原蛋白，多原酸酯，及， 乳酸。製備此調和物之方法爲精藝者顯而易知的。材料也 可睛自 Alza Corporation and Nova Pharmaceuticals，Inc。 質體懸液劑（包括利用抗释或病毒衣殼蛋白質可對準細 或組織之脂質體）也可充作藥學上可接受之載劑。這些 依據精藝者已知之方法製備，如 抗體可共價共軛作用化學修飾至聚合物，以增加其循 半衰期。特殊的聚合物，及黏附彼等至肽之方法，述 -44 本纸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) 裝 穿 乳 多 脂 胞 可 環 於 1264467 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 ) U.S· Pat. Nos. 4,766，106; 4，179,337; 4,495,285 ;及4,609,546 中。聚合物以聚氧乙基化多元醇及聚乙二醇（PEG)爲其實 例0 另外的藥學碉和物及遞送系統爲技藝中已知的，且可應 用於本發明之方法及組合物中（如見Remington’s pharmaceutical Sciences (1990) 18th ed.5 Mack Publishing Co.5 Easton, PA; The Merck Index (1996) 12th ed.? Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Prinripleq 紅 Solid Dosage Forms· Technonic Publishing Co·，Inc·， Lancaster，Pa·，（1993); and Poznansky et al.5 Drug DeliveryStupid -III. c Please read the notes on the back and fill out this page) Pack---.------ I Book----- 1264467 A7 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed V. DESCRIPTION OF THE INVENTION ('Uranol such as mannitol, sorbitol, sodium sulphate may be included in the composition. Also included are agents which delay absorption, such as aluminum monostearate and gelatin may delay absorption of the injectable composition. The preparation of a sterile injectable solution is carried out by adding the active compound to a suitable solvent in a suitable amount, and adding one or a combination of several of the above components, followed by filtration and sterilizing. In general, the preparation of the dispersing agent is to incorporate the active compound. Sterile 4 medium containing the basic dispersion medium and the other components mentioned above. In the case of a sterile powder which can be made into a sterile injectable solution, the preferred method is hollow drying and freeze drying to form an active ingredient plus Any additional component of the desired component (from a prior sterile filtration solution). For transmucosal or transdermal administration, penetrants suitable for penetrating the barrier may be used in the blend. This penetrant is generally known in the art. And include detergents such as bile salts and fusidic acid derivatives when administered through the mucosa. When administered in the form of a skin, the active compound can be adjusted to the art of ointments, shaving creams, gels or agents. Biodegradable and biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and lactic acid. The method of preparing this blend is fine art. It is also well known. Materials can also be obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Plasmin suspensions (including liposomes that can be aligned to fine or tissue using anti-release or viral capsid proteins) can also be used as pharmacy. Acceptable carrier. These are prepared according to methods known to the artisan, such as antibodies can be chemically modified to the polymer by covalent conjugation to increase their half-life. Special polymers, and methods of adhering them to peptides , -44 The 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). Wear the latex and the fat can be ringed at 1264467. property Bureau employee consumption cooperatives printed A7 B7 V. Invention description (42) US Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546. Polymers are polyoxyethylated polyols and polyethylene glycols. (PEG) as its Example 0 Additional pharmaceutical compositions and delivery systems are known in the art and are applicable to the methods and compositions of the present invention (see, for example, Remington's Pharmaceutical Sciences (1990) 18th ed. 5 Mack Publishing. Co.5 Easton, PA; The Merck Index (1996) 12th ed.? Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Prinripleq Red Solid Dosage Forms· Technonic Publishing Co., Inc., Lancaster, Pa·, (1993); Poznansky et al.5 Drug Delivery

Systems, R. L. Juliano, ed.? Oxford, N.Y. (1980), pp. 253-315)。 藥學調和物可包裝成單位劑型，使易於投藥及劑量之均 一化。此中所用之單位劑型，指適合充作單位劑量之物理 上不連續單位’以應用於欲治療之個體；各單位含有預定 量之活性化合物，經估計可產生欲求之治療作用，並加上 藥用載劑或賦形劑。 本發明提出套組，含有人類CD40抗體，編碼人類CD40 抗體之核酸或其藥學調和物，包裝至適合的包裝材質内。 套組通常包括有標籤，或.包裝便條，包括組分説明或其試 官内’活體内或活體外之使用指示。套組也可含有此組份 《集合，如二種以上的人類CD40抗體。 所謂“包裝材質”指將組份包納在套組内之物理結構。包 裝材料可維持組份在無菌狀況下，且可以用於此目的之材 -45- 本紙張尺度適用中國國家標準（CNS)A4規格⑵G x 297公爱） iL-------#-裝 (請先閱讀背面之注意事項再填寫本頁) I n ϋ 一、* n ϋ n n n n ϋ I · 1264467 L、發明說明（43 签成（如紙，波狀的纖維，破璃，塑質，錫箔 。標幟或包裝便條可包括在適合的書寫指示中。 之發明套組另外包括在本發明方法中利用套組組子 、^幟或指示。説明書包括料此處所述本發明任何方七 2明、’包括處理，偵測，追縱或診斷方法。因此，套烏 亦八控一種以上0040活性之人類cd40抗體，在包I 二77 h内’並加上在本發明治療方法中投予抗體之指牙 也月^己CD40抗體之材質可視所需包括在套組中，^ :對照或標準樣品，其中含有已知量之c謂或反應 物，提供進行分析之適合的條件。 此才曰TF可以是在“印刷品上”，4包括或固定在套組中之 ^張或卡片’或固定在套組或包裝材料上之標籤，或黏附 含有套組組份之小瓶或管内。指示説明另外可包括在電 腦可讀取之媒體上，如磁片（軟式磁碟或硬碟），視所需之 CD如CD-或DVD-R〇M/RAM，磁帶，電子貯存媒體如ram 及ROM，及其融合體如磁性/光學貯存媒體。 經濟部智慧財產局員工消費合作社印制π 本發明套組可另外包括有生長培養基(如可產製CD40抗 體I融合瘤），緩衝劑，或保藏劑或穩定劑，於含有人類 CD40抗體之藥學調和物中。套組之各組份可密封在個別 容器中，且各種容器均可納入單一包裝之内。本發明之套 組可5又计成供冷處貯存。本發明套組可進一步設計成含有 可產製人類CD40抗體之融合瘤或其他宿主細胞。套組中 之細胞可維持在適合的貯存條件下，直到細胞即可使用爲 止。例如，含有-種以上融合瘤或其他細胞之套組，可含 -46- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱） 1264467 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（44 ) 有適合的細胞貯存培養基（如10-20% DMSO於組織培養生 長基中，如DMEM，α-ΜΕΜ等），如此細胞可解;東及生長。 本發明之人類CD40抗體，可用於偵測或純化CD40多 肽。此方法包括將疑似含有CD40之樣品（在溶液，固相， 於試管内或活體内，或於完整細胞或有機體中）與CD40抗 體在令其結合之條件下接觸，並偵測CD40之存在，或純 化經結合之CD40多肽。 因此，本發明也提出偵測樣品中是否有CD40存在之方 法。在一個具體實例中，方法包括將有或疑似有CD40之 樣品與人類CD40抗體在條件下接觸，偵測樣品中之CD40 並決定樣品中是否有CD40之存在。 CD40之偵測可以傳統方法進行，如免疫沈澱，西方墨點 法，免疫組織學染色或流體細胞計數。例如，經標記之人 類CD40抗體可與樣品共培育，再利用蛋白質Α或蛋白質G 在條件下純化，以令抗體與CD40結合。經結合之CD40自 抗體中解離並以凝膠電泳及接續之染色（銀或考馬斯藍染 色）而定量之。另外，樣品可經由凝膠電泳分級分離，並 吸潰在膜上，以轉移經分級分離之蛋白質。抗-CD40抗體 與膜共培育再加上第二抗體（經修飾便可測及）可用來偵測 與存在於膜上之CD40結合:之抗-CD40抗體。 CD40偵測法可用於偵測CD40之診斷策略。例如，當 CD40水平之增加或降低和病理學之發展，存在或演進有 關聯時，本發明抗體可用來偵測任何CD40之增加或減 少。此外，當在增加或減低CD40水平之治療後欲追蹤 -47- 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) 裝---：---,--訂---------· 1264467 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（45 ) CD40之水平時，本發明抗體可在治療之前，之中或之後 用來偵測CD40水平之增加或減少，歷長或短時間下。 因此，本發明也提出可在個體樣品中（含有生物流體， 細胞，或組織或器官樣品，〜如活組織檢）偵測CD40存在之 方法。在一個具體實例中，方法包括將得自個體含有或疑 似含有CD40之樣品與人類CD40抗體接觸，所在之條件可 令CD40被偵測，並決定個體受試樣品中是否有CD40之存 在。 此外，本發明的人類CD40抗體可用來偵測人體中與 CD40增加的或減少的表現有關之失調症之存在。因此本 發明提出診斷失調症之方法，其至少有部份的特徵是在於 增加的或減少的CD40表現。在一個具體實例中，方法包 括將具有或疑似具有CD40之樣品，其中樣品得自或存在 於人體中，與人類CD40抗體接觸，並偵測樣品中相較於 對照組，增加的或減低的CD40表現之存在，由是可偵測 人體中與增加的或減低的CD40表現有關之失調症之存 在。樣品可得自無此病理狀況之相符合的個體，以作 CD40水平之比較。對正常且符合之個體統計上有意義數 目下取樣，可決定出CD40水平之正常範圍。利用抗體偵 測經結合抗原存在及含量之熟知方法爲技藝中已知的，且 可用來偵測CD40之存在（如見Harlow and Lane，上文 1988)。 與病理學有關之CD40之本質，使得本發明CD40抗體得 以介入治療，或單獨的或組合以其他適於治療此病理狀況 -48- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） .-----------裝---*----^—訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（46 ) 之治療型式。 人類CD40抗體也可用來追蹤循環的可溶性CD40之存 在，其係投予至個體，或可偵測個體中CD40之活體内水 平。例如，疑似含有可溶性CD40之血清與CD40抗體共培 育，如上述，在可令結合發生之狀況下，再決定可溶性 CD40之存在。CD40之存在顯示個體血清中有CD40之存 在。血清可在個體投予CD40前得自個體，以作比較。 除非另有所定義，此中所用的所有技術及科學術語均有 一般精藝者所了解之相同意義。雖然和此中所述的相似或 相當之方法及材料均可用來實行或測試本發明，此中所述 的仍是適合的方法及材料。 所有刊物，專利及其他參文獻，GenBank引證及ATCC引 證，在此均以全文列爲本案參考。若有衝突時，説明書 (包括定義）可控制。 如此中所用的，單數“一個”，“及”及“該”包括複數符 號，除非内容有清楚顯示。因此，關於“ CD40抗體’’包括 許多的此抗體，而關於“ CD40活性”包括一種以上的CD40 活性或功能，及其他。 本發明的許多具體實例已有所述。然而應了解只要不偏 離本發明之精義及範圍，此中可有各種的變化。因此，以 下實例用以説明而非限制在申請專利範圍中所述之範疇。 實例 實例1 此實例描述用於產製人類抗體之細胞及CD40表現構體， -49- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -----------•裝-------ί 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 1264467 A7 B7 五、發明說明（47 ) 其係可結合至人類CD40，及CD95表現及DNA合成分析 法。 細月包及抗體 EL-4細胞爲Dr. Stephen Schoenberger所贈。Ramos B細胞 及G28.5融合瘤購自ATCC。人類周邊B細胞購自AllCells， LLC (Berkeley，CA). P-RE共軛的及HRP共軛的-山羊抗-人類。特異抗體，及HRP共軛的-山羊抗人類k特異抗體購 自 Southern Biotechnology Associates/ Inc. (Birmingham， AL)。FITC-共軛的-抗-人類CD40抗體及P-RE共軛的-抗-人類 CD95抗體，購自 Pharmingen (San Diego，CA)。 人類CD40 ·· F c融合蛋白質之表現 以人類CD40 cDNA充作模板，進行PCR以擴大涵蓋人類 CD40細胞夕卜區域之片段，利用的引子爲5’-CCCAGATCT GTCCATCCAGAACCACCCACTGCATGCAGAG-3’； SEQ ID N0:1 及5，-ACAAGATCTGGGCTCTACGTATCTCAGCCGATCCT GGGGAC-3，； SEQ ID N0:2)在 9 5〇C 下 5 秒，55〇C 下 30秒， 及7 2 °C下3 0秒歷2 0個周期。經擴大的cDNA嵌入pFastBac 供者質體（Gibco BRL)在蜜蜂melityin訊號肽之3 ’ -端，及在 人類IgGl或老鼠IgG2b Fc序列之5 ’端。依廠商操作指示， 產生攜有人類CD40-FC基因融合體之重組體桿病毒。Tn5 昆蟲細胞以病毒感染，再培養4天。昆蟲細胞上清液混合 以蛋白質 G Sepharose (Amersham Pharmacia)。經4°C 下緩和 震盪培育一夜後，Sepharose充填至管柱内，再以20倍體 積之PBS洗滌。人類CD40-Fc融合蛋白質以20mM甘胺酸- -50- 1— —- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） .---.--------Μ,---.----Tit--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 經濟部智慧財產局員工消費合作社印制衣 A7 B7 五、發明說明（48 ) HC1 (pH 3.0)溶離。 於人類CD40之細胞表面表現時，自Randolph，J. Noelle取 得攜有人類CD40基因全長cDAN之pEF-Bos載體。全長 cDNA在Xbal處切割，再嵌入pCDNA3載體内，並轉感至 EL4細胞。以0 · 5毫克/毫升G418 (Gibco BRL)選出穩定的 轉感子。以FACS分析證實CD40之表現，利用FITC-共軛的 抗-人類 CD40抗體（Pharmingen)。Systems, R. L. Juliano, ed.? Oxford, N.Y. (1980), pp. 253-315). The pharmaceutical blends can be packaged in unit dosage forms for ease of administration and uniformity of dosage. The unit dosage form used herein refers to a physically discrete unit suitable for use as a unit dose to be applied to the individual to be treated; each unit contains a predetermined amount of the active compound, which is estimated to produce the desired therapeutic effect, and is administered Use a carrier or excipient. The present invention proposes a kit comprising a human CD40 antibody, a nucleic acid encoding a human CD40 antibody or a pharmaceutical composition thereof, packaged into a suitable packaging material. The kit usually includes a label, or a package note, including instructions for the component or instructions for use in vivo or in vitro. The kit may also contain this component "collection, such as two or more human CD40 antibodies. The so-called "packaging material" refers to the physical structure that encloses the components in the kit. The packaging material can maintain the components under aseptic conditions and can be used for this purpose-45- This paper scale applies to China National Standard (CNS) A4 specifications (2) G x 297 public interest) iL-------#- Loading (please read the precautions on the back and fill out this page) I n ϋ I, * n ϋ nnnn ϋ I · 1264467 L, invention description (43 sign (such as paper, corrugated fiber, broken glass, plastic, Tinfoil. A label or packaged note may be included in a suitable written instruction. The inventive kit additionally includes the use of a set of sets, instructions or instructions in the method of the invention. The instructions include any of the aspects of the invention described herein.七二明, 'includes processing, detection, tracing or diagnostic methods. Therefore, the set of more than one 0040 active human cd40 antibody, within the package I II 77h' and added in the treatment method of the present invention The material of the antibody to which the antibody is administered may also be included in the kit, as needed, or a standard sample containing a known amount of c- or reactant to provide suitable conditions for analysis. This 曰TF can be on “printed matter”, 4 included or fixed in the set a card or card' or a label attached to the kit or packaging material, or a vial or tube containing the kit component. Instructions may also be included on a computer readable medium such as a magnetic disk (soft) Disk or hard disk), depending on the required CD such as CD- or DVD-R〇M/RAM, magnetic tape, electronic storage media such as ram and ROM, and their fusions such as magnetic/optical storage media. Employee Consumption Cooperative Printed π The kit of the invention may additionally comprise a growth medium (such as a CD40 antibody I fusion tumor), a buffer, or a preservative or stabilizer, in a pharmaceutical blend containing a human CD40 antibody. The components of the group can be sealed in individual containers, and the various containers can be included in a single package. The kit of the present invention can be counted as a cold storage. The kit of the present invention can be further designed to contain a production system. A fusion tumor or other host cell of a human CD40 antibody. The cells in the kit can be maintained under suitable storage conditions until the cells are ready for use. For example, a kit containing more than one type of fusion tumor or other cells may contain - 46- Paper scale applies to China National Standard (CNS) A4 specification (210 X 297 public) 1264467 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Description of invention (44) Suitable cell storage medium (eg 10-20%) DMSO in tissue culture growth groups, such as DMEM, α-ΜΕΜ, etc., so that the cells are solvable; East and growth. The human CD40 antibody of the present invention can be used for detecting or purifying CD40 polypeptide. This method includes suspected CD40-containing The sample (in solution, solid phase, in vitro or in vivo, or in intact cells or organisms) is contacted with the CD40 antibody under conditions which bind it, and detects the presence of CD40, or purifies the bound CD40 polypeptide. Therefore, the present invention also proposes a method of detecting the presence of CD40 in a sample. In one embodiment, the method comprises contacting a sample with or suspected of CD40 with a human CD40 antibody under conditions, detecting CD40 in the sample and determining if a CD40 is present in the sample. Detection of CD40 can be performed by conventional methods such as immunoprecipitation, Western blotting, immunohistological staining or fluid cell counting. For example, a labeled human CD40 antibody can be co-incubated with a sample and purified under conditions using protein mash or protein G to bind the antibody to CD40. The bound CD40 is dissociated from the antibody and quantified by gel electrophoresis and subsequent staining (silver or Coomassie blue staining). Alternatively, the sample can be fractionated by gel electrophoresis and aspirated onto the membrane to transfer the fractionated protein. The anti-CD40 antibody is co-cultured with the membrane plus a secondary antibody (measured by modification) that can be used to detect binding to CD40 present on the membrane: an anti-CD40 antibody. The CD40 detection method can be used to detect the diagnostic strategy of CD40. For example, an antibody of the invention can be used to detect an increase or decrease in any CD40 when the increase or decrease in CD40 levels is correlated with the development of pathology, presence or evolution. In addition, when you want to follow the treatment of increasing or decreasing the CD40 level -47- This paper size is 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) ---:---,----------- 1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (45) CD40 level, the antibody of the present invention can Used to detect an increase or decrease in CD40 levels before, during, or after treatment, for a long or short period of time. Thus, the present invention also contemplates methods for detecting the presence of CD40 in an individual sample (containing biological fluids, cells, or tissue or organ samples, such as biopsy). In one embodiment, the method comprises contacting a sample from an individual containing or suspected of containing CD40 with a human CD40 antibody under conditions such that CD40 is detected and determining whether an individual is present in the test sample for the presence of CD40. Furthermore, the human CD40 antibodies of the invention can be used to detect the presence of a disorder associated with increased or decreased expression of CD40 in the human body. The present invention therefore proposes a method of diagnosing a disorder characterized in that it is at least partially characterized by increased or decreased CD40 performance. In one embodiment, the method comprises administering a sample having or suspected of having CD40, wherein the sample is obtained or present in the human body, is contacted with a human CD40 antibody, and detects increased or decreased CD40 in the sample compared to the control group. The presence of performance is due to the presence of disorders associated with increased or decreased CD40 performance in the human body. Samples can be obtained from individuals who do not have this pathological condition for comparison of CD40 levels. Sampling a statistically significant number of normal and consistent individuals determines the normal range of CD40 levels. Well-known methods for detecting the presence and amount of bound antigen using antibodies are known in the art and can be used to detect the presence of CD40 (see, for example, Harlow and Lane, supra, 1988). The nature of CD40 associated with pathology allows the CD40 antibody of the invention to be treated intervening, either alone or in combination with other suitable conditions for treating this pathological condition - 48- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297) )).-----------装---*----^-book--------- (please read the notes on the back and fill out this page) 1264467 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (46) treatment type. Human CD40 antibodies can also be used to track the presence of circulating soluble CD40, which is administered to an individual or can detect the in vivo level of CD40 in an individual. For example, a serum suspected of containing soluble CD40 is co-cultured with a CD40 antibody, and as described above, the presence of soluble CD40 is determined in the case where binding can occur. The presence of CD40 indicates the presence of CD40 in the individual's serum. Serum can be obtained from individuals prior to administration of CD40 for comparison. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by the ordinary art. Although methods and materials similar or equivalent to those described herein can be used to practice or test the invention, the methods and materials described herein are still suitable. All publications, patents and other references, GenBank citations and ATCC citations are hereby incorporated by reference in their entirety. In the event of a conflict, the instructions (including definitions) can be controlled. As used herein, the singular "", "," Thus, reference to "CD40 antibody" includes a number of such antibodies, and "CD40 activity" includes more than one CD40 activity or function, and others. Many specific examples of the invention have been described. However, it should be understood that The invention is intended to be illustrative and not restrictive of the scope of the scope of the invention. Examples Example 1 This example describes cells and CD40 for producing human antibodies. Performance structure, -49- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) -----------•装------- ί 订- ------- (Please read the notes on the back and fill out this page.) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1264467 A7 B7 V. Invention Description (47) The system can be combined with human CD40, and CD95 Performance and DNA synthesis analysis. The fine moon pack and antibody EL-4 cells were given by Dr. Stephen Schoenberger. Ramos B cells and G28.5 fusion tumors were purchased from ATCC. Human peripheral B cells were purchased from AllCells, LLC (Berkeley, CA). ). P-RE conjugated and HRP conjugated - goat anti-human Specific antibodies, and HRP-conjugated-goat anti-human k-specific antibodies were purchased from Southern Biotechnology Associates/ Inc. (Birmingham, AL). FITC-conjugated-anti-human CD40 antibody and P-RE conjugated- Anti-human CD95 antibody, purchased from Pharmingen (San Diego, CA). Expression of human CD40·· F c fusion protein Using human CD40 cDNA as a template, PCR was performed to expand fragments covering human CD40 cells. The primer is 5'-CCCAGATCT GTCCATCCAGAACCACCCACTGCATGCAGAG-3'; SEQ ID NO: 1 and 5, -ACAAGATCTGGGCTCTACGTATCTCAGCCGATCCT GGGGAC-3,; SEQ ID NO: 2) 5 seconds at 9 5 ° C, 30 seconds at 55 ° C, and 7 30 cycles at 2 °C for 20 cycles. The expanded cDNA was inserted into the pFastBac donor plastid (Gibco BRL) at the 3'-end of the honeymelinin signal peptide and 5' in the human IgG1 or mouse IgG2b Fc sequence. The recombinant rod virus carrying the human CD40-FC gene fusion was generated according to the manufacturer's instructions. The Tn5 insect cells were infected with the virus and cultured for another 4 days. The insect cell supernatant was mixed with protein G Sepharose (Amersham Pharmacia). After a night of incubation at 4 ° C with shaking, Sepharose was filled into the column and washed with 20 times volume of PBS. The human CD40-Fc fusion protein is applied to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) on a paper scale of 20 mM glycine - 50-1 - (---.------- -Μ,---.----Tit--------- (Please read the notes on the back and fill out this page) 1264467 Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Printed Clothing A7 B7 V. Description of the invention (48) HC1 (pH 3.0) is dissolved. On the cell surface of human CD40, a pEF-Bos vector carrying the full length cDAN of the human CD40 gene was obtained from Randolph, J. Noelle. The full-length cDNA was cut at Xbal, embedded in the pCDNA3 vector, and transfected into EL4 cells. A stable transsensor was selected at 0. 5 mg/ml G418 (Gibco BRL). The expression of CD40 was confirmed by FACS analysis using FITC-conjugated anti-human CD40 antibody (Pharmingen).

Ramos B細胞上CD95之表現 2.1 5 X 106 Ramos B細胞塗佈在4 8孔洞之盤上。加入經純 化的 IgG 或可溶性人類 CD40L (ALEXIS San Diego，CA)。 經培育2 4小時後，細胞收集再以P-RE共軛的抗-人類CD95 抗體染色，並以FACS掃描分析。 DNA合成 以3H-胸苷之納入偵測D N A合成。人類周邊血液B細胞 (5 X 104細胞/孔洞）塗佈在9 6孔洞盤中，並加上IOUCi/毫 升[3H]胸甞，10毫微克/毫升人類IL-4，抗-CD40抗體及/或 可溶性CD40L。經培養3天後，細胞利用Tomtec細胞收穫 器（EG&G)回收，且以Betaplate液體閃爍計數器（Pharmacia) 偵測。 實例2 . 此實例描述利用人類染色體轉移的老鼠，產製人類CD40 之人類單株抗體。 人類染色體轉移老氣（Tomizuka et al·, Proc. Natl. Acad. Sci. USA 97:722 (2000)及 Tomizuka et al·，Nat. Genet. -51 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -----------裝---r---.--訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（49 ) 16:133 (1997))係在免疫球蛋白區域中帶有人類染色體片 段，以1 00微克的hCD40-hFc於CFA皮下免疫接種。在1 〇 及20天後，老鼠以100微克hCD40-hFc於IFA皮下追加注 射。在第37天時，給予100微克hCD40-hFc無佐劑之最終 靜脈内注射。 以ELISA決定抗體校價。簡言之，人類CD40-老氣F c融 合蛋白質以1.4微克/¾升濃度塗佈在盤上，利用碳酸鹽緩 衝溶液，在3 7 °C下歷1小時。以PBS/0.1%吐溫20洗條3次 後，盤以PBS/1%BSA，3 7 °C阻斷。在孔洞中加入稀的抗 體或血清，盤再於3 7 °C下培育1小時。經洗滌3次後，在 孔洞中加入稀的H R P共軛之山羊抗·人類γ鏈特異抗體，或 H R Ρ共軏之山羊抗-人類k鏈特異抗體，並在3 7 °C下培育1 小時。洗蘇3次後，在孔洞中加入TMB受質溶液 (DAKO)，並在室溫下培育3 〇分鐘。以微盤計價器偵測 4 5 0毫微米下之光密度。在第3次追加係在6隻老鼠中均偵 測到校價的增加。 自顯出最高抗體校價的6隻老鼠中選出2隻，以產製單株 抗體。回收脾，融合至骨髓瘤細胞株（SP2/〇-Agl4)，且將 融合物塗佈在9 6孔洞盤上（共1 8盤）。以FACS分析及 ELISA篩選約15〇〇個融合瘤孔洞，利用經稀釋的11111>共輕 之山羊抗-人類γ特異抗體，或HRP共軛的山羊抗-人類k 特異柷體爲二次抗體。重鍵或k鏈陽性之細胞轉移至2 4孔 洞盤’並擴大之。 於抗體純化時，融合瘤培養在CELLine (IBS)中，並利用 -52- 本紙張尺度朗巾關家鮮（CNS)A4規格（21Q χ挪公爱） γ ^^1 ί 1 βϋ ^^1 —ϋ emmmf I mmte ί I— I- 1 n « ^ I ϋ ί ϋ— ϋ —^1 11__ ·ϋ I (請先閱讀背面之注意事項再填寫本頁) 1264467 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（5〇 ) 蛋白質G親和力樹脂純化抗體。來自融合瘤之上清液，在 ELISA中呈陽性，也以細胞染色分析之，利用EL-4細胞其 可在表面上表現人類CD40。CD40之表現以FITC共軛之抗-人類CD40抗體證實。融合瘤上清液加至5 X 105 EL4細 胞，並在4 °C下培育3分鐘。細胞洗三次，利用 PBS/5%FBS，再加入0 · 1微克的R-PE共軛的抗人類γ抗體， 並在4 °C下培育3 0分鐘。經洗滌3次後，細胞以FACS掃描 染色分析之。分析顯示有4 5個融合瘤呈陽性。 陽性融合瘤純系接受有限稀釋，至少三次，可分離出13 個單一純系。自1 3個單一純系中，可分離出4種抗體（Nos. 11，20，72及366)。細胞染色分析顯示，4種抗體染色之細 胞，均可表現細胞表面人類CD40(圖1 )。雖然人類Fc在Tc 老鼠中應是無免疫原性的，仍以ELISA進行F c反應性之負 面篩選，利用人類CD40-老鼠Fc融合蛋白質來進行。 實例3 此實例描述以CD40抗體調控CD40抗體。此實例也描述 CD40抗體之CD40結合親和力。Performance of CD95 on Ramos B cells 2.1 5 X 106 Ramos B cells were plated on a 48-well plate. Purified IgG or soluble human CD40L (ALEXIS San Diego, CA) was added. After 24 hours of incubation, the cells were harvested and stained with P-RE conjugated anti-human CD95 antibody and analyzed by FACS scanning. DNA synthesis The synthesis of D N A was detected by the inclusion of 3H-thymidine. Human peripheral blood B cells (5 X 104 cells/well) were plated in 96-well plates with IOUCi/ml [3H] chest, 10 ng/ml human IL-4, anti-CD40 antibody and / Or soluble CD40L. After 3 days of culture, cells were recovered using a Tomtec cell harvester (EG&G) and detected by a Betaplate liquid scintillation counter (Pharmacia). Example 2. This example describes a human monoclonal antibody that produces human CD40 using a mouse that has been transferred by human chromosomes. Human chromosome transfer is old (Tomizuka et al., Proc. Natl. Acad. Sci. USA 97:722 (2000) and Tomizuka et al., Nat. Genet. -51 - This paper scale applies to the Chinese National Standard (CNS) A4 specification. (210 X 297 mm) -----------Install---r---.--Book--------- (Please read the notes on the back and fill in this Page 1264467 Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, Printed A7 B7 V. Inventive Note (49) 16:133 (1997)) Human chromosome fragment with immunoglobulin region at 100 micrograms of hCD40-hFc Immunization was subcutaneously administered to CFA. After 1 〇 and 20 days, the mice were additionally injected subcutaneously with IFA at 100 μg of hCD40-hFc. On day 37, a final intravenous injection of 100 micrograms of hCD40-hFc without adjuvant was administered. Antibody pricing is determined by ELISA. Briefly, human CD40-old gas F c fusion protein was coated on a disk at a concentration of 1.4 μg/3⁄4 liter, using a carbonate buffer solution for 1 hour at 37 °C. After washing the strips three times with PBS/0.1% Tween 20, the plates were blocked with PBS/1% BSA at 37 °C. Dilute antibodies or serum were added to the wells and the plates were incubated for 1 hour at 37 °C. After washing for 3 times, a rare HRP conjugated goat anti-human γ chain specific antibody, or HR Ρ conjugated goat anti-human k chain specific antibody was added to the well, and incubated at 37 ° C for 1 hour. . After 3 washes, TMB substrate solution (DAKO) was added to the wells and incubated for 3 minutes at room temperature. The optical density at 4500 nm was detected by a microdisk counter. In the third addition, the increase in the school price was detected in all six mice. Two out of six mice showing the highest antibody price were selected to produce monoclonal antibodies. The spleens were recovered, fused to a myeloma cell line (SP2/〇-Agl4), and the fusion was spread on a 96-well plate (total of 18 disks). Approximately 15 融合 fusion tumor pores were screened by FACS analysis and ELISA, and a diluted 11111> conjugated goat anti-human gamma specific antibody or HRP conjugated goat anti-human k specific steroid was used as a secondary antibody. Cells with positive or k-strand positives are transferred to the 24-well plate and expanded. When the antibody is purified, the fusion tumor is cultured in CELLine (IBS), and the -52- paper scale is used to close the home fresh (CNS) A4 specification (21Q χ公公爱) γ ^^1 ί 1 βϋ ^^1 —ϋ emmmf I mmte ί I— I- 1 n « ^ I ϋ ϋ ϋ — ϋ —^1 11__ ·ϋ I (Please read the notes on the back and fill out this page) 1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Office Staff Consumption Co-operative printing 5, invention description (5 〇) Protein G affinity resin purification antibody. The supernatant from the fusion tumor was positive in the ELISA and also analyzed by cell staining, and the EL-4 cells were used to express human CD40 on the surface. The performance of CD40 was confirmed by FITC-conjugated anti-human CD40 antibody. The fusion supernatant was applied to 5 X 105 EL4 cells and incubated at 4 °C for 3 minutes. The cells were washed three times, and PBS/5% FBS was added, and 0.1 μg of R-PE conjugated anti-human γ antibody was added and incubated at 4 ° C for 30 minutes. After washing 3 times, the cells were analyzed by FACS scanning staining. Analysis showed that there were 45 positive tumors. The positive fusion tumors were subjected to limiting dilution, at least three times, and 13 single pure lines were isolated. Four antibodies (Nos. 11, 20, 72 and 366) were isolated from 13 single pure lines. Cell staining analysis showed that the cells stained by the four antibodies all showed cell surface human CD40 (Fig. 1). Although human Fc should be non-immunogenic in Tc mice, negative screening for F c reactivity was performed by ELISA using human CD40-mouse Fc fusion protein. Example 3 This example describes the regulation of CD40 antibodies with CD40 antibodies. This example also describes the CD40 binding affinity of CD40 antibodies.

Ramos B細胞，是一種人類B淋巴瘤細胞株，其在可溶性 CD40L或激動性抗- CD40抗體之刺激下可表現CD95 (Schattner et al·，J. Exp· Med· 1182:1557 (1995))以此來分析 四種抗體之分析（Nos. 11，30，72及366)。簡言之，Ramos B細胞與經純化的抗體培育，且在培養2 4小時後偵測CD95 之表現（圖2 A及2 B )。結果顯示，CD95之表現可由對照的 可溶性CD40配體及G28-5，一種激動的老鼠抗-人類CD40 -53- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -----------裝—'—^—訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（51 ) 抗體，所謗生。相反的，人類抗體nos. 11，30，72及366無 法謗生可測及二CD95表現。 爲了決定CD40抗體阻斷CD40L與CD40結合之能力，將 Ramos B細胞與1微克/毫升純化的抗體及可溶性CD40L培 育。在培養2 4小時後，可偵測CD95之表現。三種抗體 (Nos·30,72，及3 66)可完全抑制可溶性CD40L對CD95表現 之謗導作用。（圖3 ) 如上述決定抗-人類CD40抗體在人類周邊B細胞增殖作 用上之作用。在無可溶性CD40L下，四種抗體無一者可謗 導人類B -細胞之增殖作用。然而，在含有1微克/毫升可溶 性CD40L之培養中，No. 11抗體可強烈地加強人類B -細胞 之增殖作用，其方式類似鼠類抗體G8-5或5 C3，一種先前 已知可謗導B細胞增殖作用之抗體（圖4 A )。另三種人類抗 體（Nos· 30，72，及366)可完全抑制可溶性CD40L之作用（圖 4B)。 人類抗-人類CD40抗體之產製，其在組合以CD40L下可 加強人類周邊B細胞之增殖，也是有治療應用性。在此方 面，其他團體也報告指出，老鼠抗-人類CD40抗體之激動 活性，如 5 C ?或 G28-5 (Pound et al., Int. Immunol· 11:11 (1999))。人類抗體No. 11並不抑制可溶性CD40L與CD40之 結合，但其可強烈地加強在組合CD40L下B -細胞之增殖作 用。依據這些特性，人類抗體No. 11和5C3爲相同類型的抗 體，此爲一種不會阻斷天然配體結合之激動性抗體。此抗 體適於抗-腫瘤或抗-病毒治療。 -54- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -----------裝-------T--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 1264467 A/ B7 五、發明說明（52 ) 以BiaCore分i斤決定抗-CD40抗體No. 3 0之親和力。人類 CD40-老鼠F c融合蛋白質與感受器碎片交聯，並以廠商之 操作指示偵測親和力。Kd値爲0·8-4ηΜ。 如上述在Raoms細胞分析中測試抗- CD40抗體融合瘤F1-102，F5-152 及 F4-465。F1-102 及 F5-152 得自人類 CD40-FC 免 疫接種之Tc老鼠，且因此可表現人類k輕鏈。F4-465得自 HAC 老鼠（Kuroiwa et al·，Nature Biotech.，10:1086 (2000)) 且因此表現人類λ輕鏈。 結果示於圖5及6顯示，由F1-102及F5-152產生之抗體可 刺激Ramos細胞上CD95之表現，並可加強CD40L之刺激作 用，顯示這些抗體是具激動性的。相反的，F4-465無法正 向調控CD95表現，並阻斷CD40L之刺激作用。F4-465是由 λ鏈組成的第一種具拮抗性之抗體。 實例4 此實例説明在哺乳動物細胞株產製重組抗體，CD40抗體 之方法。此實例也描述重組產製之CD40抗體之CD40調控 活性。 重組抗體之產製是自可產生抗-人類CD40抗體之融合瘤 中選殖免疫球蛋白（Ig)基因，並在哺乳動物細胞中表現。 簡言之，利用Tri-試劑依據废商操作指示（Molecular Research Center，Inc.，Cincinnati，Ohio)自各融合瘤 F2-103， F5-77及F5-157中純化出總RNA。自總RNA中利用SMART RACE cDNA擴大套組（Clontech Laboratories，Inc.，Palo Alto, CA)及 Superscript II RT (GibcoBRL)可合成全長 cDNA。人 -55- 本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） -----------裝—.—τ—訂--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 Λ A/ B7 五、發明說明（53 ) (請先閱讀背面之注意事項再填寫本頁) 類重及人類輕鏈之5 ’可變區以P C R分離，利用如廒商指示 之 5’-RACE PCR (Clontech Laboratories，Inc.)。於 5 ’ -引導 中使用由廠商供應之通用的引子混合物，配合下列基因特 異引子之一用於3、引導。其他所有重鏈擴大時，基因特 異的引子是 5’--GTGCACGCCGCTGGTCAGGGCGCCTG-3’ (SEQ ID N0:3)。爲擴大k鏈時，基因特異的引子序列是5、 GTTGAAGCTCTTTGTGACGGGCGAGC-3’（SEQ ID N0:4) 〇 全長的P C R產物以凝膠純化，且鈍端連接至經SrfI切割之 PCR-Script (Stratagene，La Jolla，CA)或PCR-Blunt (Invitrogen， Carlsbad，CA)再以 CFAR 定序，Molecular Biology Core Facility (University of California，San Diego) 0 經濟部智慧財產局員工消費合作社印製 人類重及輕鏈可變序列，接下來選殖至N 5 K G 1 - V a 1 LARK (IDEC)，利用PCR。重鏈序列選殖至Sail及Nhel位 置，利用 5,引子5’-ACCGTGTCGACGGTGATCAGGACTGAACAG-3, (SEQ ID N0:5)於 F5-77(K1H1)及 F5-157(K3H3)或 5，、 ACCGTGTCGACGCTGATCAGGACTGCACA-3’（SEQ ID Ν0··6)於 F2-103(K1H1)及3’引子5，-AGTGCTAGCTGAGGAGACGGTGAC-3, (SEQ ID N0:7)。k鏈可變序列選殖至Bglll及BsiWI位置，利用 5’ 引子5，-AACTCCAGATCTAGGGCAAGCAGTGGTAAC-3, (SEQ ID N0:8)及3,引子5，-丁八丁(：(^0丁八000111}八丁01(：€八(：(：丁丁00丁0 3’（SEQ ID Ν0··9)。以所示融合瘤產生的抗體重及輕鏈序列 如下： -56- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱） 1264467 A7B7 五、發明說明（54 ) $2-103-重 _ | gctgatc^^xtgc^cacagagaactc^ccatggagtttggktgagctgggttttccttgttgctatttt j AAAAGGTGTCCAG-rGTGAGGTQCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAG I ACTCTCCTGTGCAGTCTCTGGXTTCACCTTCAGTACCTACTGGATGCACTGGGTCCGCCAAGCTCCAGGGAA ί ggggctggtgtgggtctcacgtattaat^stgatgggagtagcacaacctacgcggactccgtgaagggccg i ATTCACCATCTCCAGAGACAACGCCAAGAXCAeGCTGTATCTGCAAATGAACAGTCrrOAGAGeCGAGG九CAC * GGCTGTGTA-rTACTGTGCAAGAGATAGAGTACTATGGATCGGGGAGTTATCCTACTACGGTATGGACGTCTG i GGGCCAAGGGACC^CGGTCACCGTCTCCTCAGCTAGC^CC^AGGGCCCATCGGTCTTCCCCCTGGCACCCTC j CTCCAAGAGCACCTCT (SEQ ID JT〇：X〇)F2-103-輕 GGGGAGTCAGACCCAfiTCAGGACACAGCATQGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCT CTG-CKrrCCCAGCTGCC^JlTGm^CATCCAGATGACCCAGTCTCCTTCa\CCCTGTCTGCATCTGTAGG^GA CAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAiLCTGCTTGCCCTGGTATCAGCAjSAAACCAGG GJ^GCCCCTAAGCrrCCTGCTCTATAAGGCATCTGGTTTAGAAAGTG<3GGTCCCATCAAGGTTCAGCGQCAG TGGATCTGGGA〇^^\TTCACTCTCACC^TC^JvCAGCCTGCAGCCTGATGATTTT〇CAACXTATU'ACTGCCA acagtctaat^ttattcgtggacgttcgcccacgggaccaaggtggaaatcaaacgtacggtggctgcacc atctgtcttcatcttcccgccatctgatgagcagttgaaatctgcaactgcctctgttgtgtgcctgctg^ taacttctatcccagagaggccaaagtxcagtggaaggtggataacgccctccaatcgggtaactcccagga GAjSTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGC^ CTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA (SEQ ID NO: 11)Ramos B cell, a human B lymphoma cell line, exhibits CD95 under the stimulation of soluble CD40L or an agonistic anti-CD40 antibody (Schattner et al., J. Exp. Med. 1182: 1557 (1995)) This was used to analyze the analysis of four antibodies (Nos. 11, 30, 72 and 366). Briefly, Ramos B cells were incubated with purified antibodies and the expression of CD95 was detected after 24 hours of culture (Figures 2 A and 2 B). The results showed that the performance of CD95 can be determined by the control of soluble CD40 ligand and G28-5, an agonistic mouse anti-human CD40-53- this paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -- ---------Installation-'-^-book--------- (Please read the note on the back and fill out this page) 1264467 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative System 5, invention instructions (51) antibodies, produced. In contrast, human antibodies nos. 11, 30, 72, and 366 were unable to produce measurable and secondary CD95 performance. To determine the ability of CD40 antibodies to block the binding of CD40L to CD40, Ramos B cells were incubated with 1 μg/ml of purified antibody and soluble CD40L. After 24 hours of culture, the performance of CD95 was detected. Three antibodies (Nos·30, 72, and 3 66) completely inhibited the CDKL-induced CD95 expression. (Fig. 3) The effect of the anti-human CD40 antibody on the proliferation of human peripheral B cells was determined as described above. In the absence of soluble CD40L, none of the four antibodies can induce the proliferation of human B-cells. However, in cultures containing 1 μg/ml of soluble CD40L, the No. 11 antibody strongly potentiated the proliferation of human B-cells in a manner similar to the murine antibody G8-5 or 5 C3, a previously known guideline An antibody against B cell proliferation (Fig. 4A). Three other human antibodies (Nos. 30, 72, and 366) completely inhibited the action of soluble CD40L (Fig. 4B). The production of human anti-human CD40 antibody, which can enhance the proliferation of human peripheral B cells in combination with CD40L, is also therapeutically applicable. In this regard, other groups have also reported agonistic activity of mouse anti-human CD40 antibodies, such as 5 C or G28-5 (Pound et al., Int. Immunol 11:11 (1999)). Human antibody No. 11 did not inhibit the binding of soluble CD40L to CD40, but it strongly potentiated the proliferative effect of B-cells in combination with CD40L. Based on these characteristics, human antibodies No. 11 and 5C3 are the same type of antibody, an agonistic antibody that does not block the binding of natural ligands. This antibody is suitable for anti-tumor or anti-viral treatment. -54- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) -----------Installation-------T--订----- ---- (Please read the notes on the back and fill out this page.) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1264467 A/ B7 V. Description of Invention (52) Determine the anti-CD40 antibody No. by BiaCore. 30 0 affinity. The human CD40-mouse Fc fusion protein was cross-linked with receptor fragments and the affinity was detected using the manufacturer's instructions. Kd値 is 0·8-4ηΜ. Anti-CD40 antibody fusion tumors F1-102, F5-152 and F4-465 were tested in Raoms cell assay as described above. F1-102 and F5-152 were obtained from human CD40-FC immunovaccinated Tc mice and thus exhibited human k light chains. F4-465 was obtained from HAC mice (Kuroiwa et al., Nature Biotech., 10: 1086 (2000)) and thus exhibited the human lambda light chain. The results are shown in Figures 5 and 6. The antibodies produced by F1-102 and F5-152 stimulated the expression of CD95 on Ramos cells and enhanced the stimulation of CD40L, indicating that these antibodies are agonistic. In contrast, F4-465 was unable to positively regulate CD95 expression and block the stimulatory effects of CD40L. F4-465 is the first antagonistic antibody consisting of a lambda chain. Example 4 This example illustrates the method of producing a recombinant antibody, CD40 antibody, in a mammalian cell line. This example also describes the CD40 regulatory activity of recombinantly produced CD40 antibodies. Recombinant antibodies are produced by cloning immunoglobulin (Ig) genes from fusion tumors that produce anti-human CD40 antibodies and are expressed in mammalian cells. Briefly, total RNA was purified from each of the fusion tumors F2-103, F5-77 and F5-157 using Tri-reagent according to the waste commercial instructions (Molecular Research Center, Inc., Cincinnati, Ohio). Full-length cDNA was synthesized from total RNA using the SMART RACE cDNA amplification kit (Clontech Laboratories, Inc., Palo Alto, CA) and Superscript II RT (GibcoBRL).人-55- This paper scale applies to China National Standard (CNS) A4 specification (210 x 297 mm) -----------Installation..—τ—订--------- (Please read the notes on the back and fill out this page) 1264467 Λ A/ B7 V. Description of the invention (53) (Please read the note on the back and then fill out this page) 5 'variable area of heavy weight and human light chain Isolation by PCR, using 5'-RACE PCR (Clontech Laboratories, Inc.) as indicated by the manufacturer. A generic primer mix supplied by the manufacturer is used in the 5'-guide, and one of the following gene-specific primers is used for 3. Guidance. When all other heavy chains are expanded, the gene-specific primer is 5'--GTGCACGCCGCTGGTCAGGGCGCCTG-3' (SEQ ID NO: 3). To expand the k-strand, the gene-specific primer sequence is 5, GTTGAAGCTCTTTGTGACGGGCGAGC-3' (SEQ ID NO: 4) 〇 full-length PCR product is gel purified and blunt-ended to SrfI-cleaved PCR-Script (Stratagene, La Jolla, CA) or PCR-Blunt (Invitrogen, Carlsbad, CA) followed by CFAR sequencing, Molecular Biology Core Facility (University of California, San Diego) 0 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed human heavy and light chain The variable sequence was next cloned to N 5 KG 1 - V a 1 LARK (IDEC) using PCR. The heavy chain sequence was cloned to the Sail and Nhel positions using 5, the primer 5'-ACCGTGTCGACGGTGATCAGGACTGAACAG-3, (SEQ ID NO: 5) to F5-77 (K1H1) and F5-157 (K3H3) or 5, ACCGTGTCGACGCTGATCAGGACTGCACA-3 '(SEQ ID Ν0··6) at F2-103 (K1H1) and 3' primer 5, -AGTGCTAGCTGAGGAGACGGTGAC-3, (SEQ ID NO: 7). The k-chain variable sequence was cloned to the Bglll and BsiWI positions, using the 5' primer 5, -AACTCCAGATCTAGGGCAAGCAGTGGTAAC-3, (SEQ ID NO: 8) and 3, the primer 5, - Ding Ba Ding (: (^0丁八000111) 01(:€8(:(:丁丁00丁0 3'(SEQ ID Ν0··9). The antibody heavy and light chain sequences produced by the fusion tumors shown below are as follows: -56- This paper scale applies to Chinese national standards ( CNS) A4 size (210 X 297 Kimiyoshi) 1264467 A7B7 V. invention is described in (54) 2-103- heavy $ _ | gctgatc ^^ xtgc ^ cacagagaactc ^ ccatggagtttggktgagctgggttttccttgttgctatttt j AAAAGGTGTCCAG-rGTGAGGTQCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAG I ACTCTCCTGTGCAGTCTCTGGXTTCACCTTCAGTACCTACTGGATGCACTGGGTCCGCCAAGCTCCAGGGAA ί ggggctggtgtgggtctcacgtattaat ^ stgatgggagtagcacaacctacgcggactccgtgaagggccg i ATTCACCATCTCCAGAGACAACGCCAAGAXCAeGCTGTATCTGCAAATGAACAGTCrrOAGAGeCGAGG nine CAC * GGCTGTGTA-rTACTGTGCAAGAGATAGAGTACTATGGATCGGGGAGTTATCCTACTACGGTATGGACGTCTG i GGGCCAAGGGACC^CGGTCACCGTCTCCTCAGCTAGC^CC^AGGGCCCATCGGTCTTCCCCCTGGCACCCTC j CTCCAAGAGCACCTCT (SEQ ID JT〇:X〇)F2-103-Light GGGGAGTCAGACCCAfiTCAGGACACAGCATQGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCT CTG-CKrrCCCAGCTGCC ^ JlTGm ^ CATCCAGATGACCCAGTCTCCTTCa \ CCCTGTCTGCATCTGTAGG ^ GA CAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAiLCTGCTTGCCCTGGTATCAGCAjSAAACCAGG GJ ^ GCCCCTAAGCrrCCTGCTCTATAAGGCATCTGGTTTAGAAAGTG < 3GGTCCCATCAAGGTTCAGCGQCAG TGGATCTGGGA〇 ^^ \ TTCACTCTCACC ^ TC ^ JvCAGCCTGCAGCCTGATGATTTT〇CAACXTATU'ACTGCCA acagtctaat ^ ttattcgtggacgttcgcccacgggaccaaggtggaaatcaaacgtacggtggctgcacc atctgtcttcatcttcccgccatctgatgagcagttgaaatctgcaactgcctctgttgtgtgcctgctg ^ taacttctatcccagagaggccaaagtxcagtggaaggtggataacgccctccaatcgggtaactcccagga GAjSTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGC ^ CTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA (SEQ ID NO: 11)

.GA 經濟部智慧財產局員工消費合作社印製 F5-157-重 GGTGATC^GGACrGAACAGGGAGAACTC^CCATGGAGTTTGGGCTGGGCTGGCrTTTTCTTGTGGCTATTTT AAAAGGTGTCCAGTGTGAGGTGCAGCTGTTCGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTOAG actctcctgtgcagcctctggattcgcctttagcagctatgccatgagctgggtccgccaggctccagggaa ggggctggagtgcgtctcagctatxagtggtagtggtggtagcacataotacgcagactccgtgaagggccg I gttcaccatctccaqagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacac I GGCCGTATATTACTGTGCGAAACATGOGOGGTACTATGGTTCGGGGAGTTATGGGTACTTTGACTACrrGGGG I CCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTrCCCCCTGGCACCCTCCTC I caagagcacctctgggggcacaocggccctgggctgcctggtcaaggactacttccccgaaocggt^cggt I GTCG ISBQ ID NO：12)I F5-157-輕 ί CAACGCAGAfiTACGCGGGGAGGAGTCAGACCCAGTCAGGACACAGCATGGACATGAGGGTCCCCGOTCAGCT j CC^GGGCCTCCTGCTGCTCTGGTTCCC^GGTTCCAGATGCGACA.TCCAGATGACCCAGTCTCCATCTTCCGT I GTCTGCATCrrGC^GAGACAGAGTCACCATCXCTTGTCGGaCGAGTCAGOGTATTAGCAGCTGGTrAGCCTG ;GTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGGATCCAGTTTGCAAAGTGGGGTCCC j ATCAAGGTTCAfiCGGCAGTGGATT*rGGGACAGATTTCACTCT〇^CCATCGGCAGCCTGCACCCTGAAGATTT tgc^cttactattctcaaca^gctagcagtttccctcggacgttcggccaagggaccaaggtggagatcaa ACGTACGGTGGCTGCACCivTCTGTCrXCATCrTCCCGCCATCTGATGAGCAGTTGA^TCTGGAACTGCCTC tgttgtgtgcctgctgaataacttctatcccagagacgccaaagtacagtggaagctggataacgccctcca atcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccct GACGCTGAGCAAAGCAGACrTACGAGAAACACAAAGTCTACGCCTGCGAAfiTCACCCATCAGGGCCTGA (SEQ ID NO :13) 57 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 裝--------„—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 1264467 A7 -----B7_ 五、發明說明（55 ) ! F5-77-重.GA Ministry of Economic Affairs Intellectual Property Office employees consumer cooperatives printed F5-157- heavy GGTGATC ^ GGACrGAACAGGGAGAACTC ^ CCATGGAGTTTGGGCTGGGCTGGCrTTTTCTTGTGGCTATTTT AAAAGGTGTCCAGTGTGAGGTGCAGCTGTTCGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTOAG actctcctgtgcagcctctggattcgcctttagcagctatgccatgagctgggtccgccaggctccagggaa ggggctggagtgcgtctcagctatxagtggtagtggtggtagcacataotacgcagactccgtgaagggccg I gttcaccatctccaqagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacac I GGCCGTATATTACTGTGCGAAACATGOGOGGTACTATGGTTCGGGGAGTTATGGGTACTTTGACTACrrGGGG I CCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTrCCCCCTGGCACCCTCCTC I caagagcacctctgggggcacaocggccctgggctgcctggtcaaggactacttccccgaaocggt ^ cggt I GTCG ISBQ ID NO: 12) I F5-157- light ί CAACGCAGAfiTACGCGGGGAGGAGTCAGACCCAGTCAGGACACAGCATGGACATGAGGGTCCCCGOTCAGCT j CC ^ GGGCCTCCTGCTGCTCTGGTTCCC ^ GGTTCCAGATGCGACA.TCCAGATGACCCAGTCTCCATCTTCCGT I GTCTGCATCrrGC ^ GAGACAGAGTCACCATCXCTTGTCGGaCGAGTCAGOGTATTAGCAGCTGGTrAGCCTG; GTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGGATCCAGTTTGCAAAGTGGGGTCCC j A RGGGACAGATTTCACTCT〇 TCAAGGTTCAfiCGGCAGTGGATT * ^ CCATCGGCAGCCTGCACCCTGAAGATTT tgc ^ cttactattctcaaca ^ gctagcagtttccctcggacgttcggccaagggaccaaggtggagatcaa ACGTACGGTGGCTGCACCivTCTGTCrXCATCrTCCCGCCATCTGATGAGCAGTTGA ^ TCTGGAACTGCCTC tgttgtgtgcctgctgaataacttctatcccagagacgccaaagtacagtggaagctggataacgccctcca atcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccct GACGCTGAGCAAAGCAGACrTACGAGAAACACAAAGTCTACGCCTGCGAAfiTCACCCATCAGGGCCTGA (SEQ ID NO: 13) 57 applicable to the present paper China National Standard Scale (CNS) A4 size (210 X 297 mm) mounted ----- ---„-订·--------(Please read the notes on the back and fill out this page) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1264467 A7 -----B7_ V. Invention Description (55 ) ! F5-77-Heavy

j GGTCTATATAAGCAfiAfiCTGGGTACGTCCTCACATTa^GTGATCAGCACTGAACACAGACCCGTCGACGQTG I ATCAGGACTGAACAGArSAG^CTCACCATGGAGTTTGOGCTC^CTGGCTTri^CrTGTGGCrATTTTAA^A j gotgtccagtgtgaggtgcagctgttggagtctgggggagocttggtacagcctgggcggtccctgagactc I tcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggj GGTCTATATAAGCAfiAfiCTGGGTACGTCCTCACATTa ^ GTGATCAGCACTGAACACAGACCCGTCGACGQTG I ATCAGGACTGAACAGArSAG ^ CTCACCATGGAGTTTGOGCTC ^ CTGGCTTri ^ CrTGTGGCrATTTTAA ^ A j gotgtccagtgtgaggtgcagctgttggagtctgggggagocttggtacagcctgggcggtccctgagactc I tcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaagggg

：CrGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCC^TGA^GGGOCGGTTC I accatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagcogaggacacggcc:CrGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCC^TGA^GGGOCGGTTC I accatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagcogaggacacggcc

! GTATATTACTGTGCGAAAiGATGGGGGGTACTATGGrrCQGGGAGTrATGGGTACTTTGACTACTGGGGCCAG ;ggaaccctggtcaccgtctcctcagctagcaccaaoggoccatcggtcttccccctgocaccctcctccaag ：AGCACCTCTGGCGGCACAGCGGCCCTGGGCTGCCTGGTCA^GGACTACTTCCCC (S2Q ID N0:14) j F5-77-輕 ， I CAAjGCAGTGGTAACAACGCAGAGTACCOCiGGGGQAGTCAGACCCAGTCixGGACACAGCATGGACATGAGGGT j CCCCGCTCAGCTCCTGOOQCTCCTGCTQCTCTGGrrCCCAiSGXTCCAGATGCGACATCaA^ATGACCCAGTC TCCATCTTCCGTGTCTGGATCTGTAGGAGACJcGAGTQACCATC^CTTGTCGGCOGAGTCAGGGTATTAGCAG ,CTGGTTAGCCTG0TATC^JSC^GA^\CCAGGGXAJ^CCCTAA5CTCCTGATC,rATGCTGQATCCAGTTT3CA ^G^GGCGTCCCATCAAGGTl'CAGCGGCAGTO^TTTGGGACAGATTTCAt^rCTC^CCATCAGCAGCCTGCA j GCCTCL\^GATTTTGCAACTTACTAWGTCAACAiB<3CTAGCAGTTTCCCTCG'3ACATTCGGCCAAGa<3ACCAA I GvSTGGAGATCAAACGTACGGTGGCTGCACC^TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC I TGGAACTCCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAfiGCCAAAiSTACAGTGGAAGGTGGA ;T^CGCCCTCCAATCGC^TAACrrCCCAGGAGAGTOTCACAGAqCAGGACiKGCAXGG^CAGCACCTACAGCCT I CAG<：AGCACCCTGACGCTGAGa^\GCAGACTACGAGAAXa^J^GTCTACGCCTGCGAAGTCACCCATCA :GGGCCTGA (SEQ ID K〇：lS) 表現質體以電泳脈動，瞬時轉感至C〇S- 1細胞。簡言之， 3 X 106細胞懸浮在0.7毫升無血清iDMEM*，其中含有 3 0微克質體DNA，再置於〇·4,公耸BioRad石英管#165-2088 内。細胞在Gene Pulser II (BioRad)上電泳動脈，設定在 240伏特’ 0.950的電容，15-25毫秒之固定時間。在電流 輸送後，細胞轉移至10公分皿中，其中含有1〇毫升 DMEM，10% FBS。72小時後回收含有人類Ig之適調培養 人類抗月豆利用 Protein A sepharose 4 Fast Flow (Amersham #17-0618-02)純化自培養基。簡言之，調適培養基填加至 0 · 5耄升管柱内。收集流過之流份再填加至管柱二次以 上。管柱以10毫升PBS洗滌，且抗體以2.5毫升的20mM 甘胺酸，pH 3.0溶離。溶離·出之流份收集在〇.5毫升中， -58- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱） «狀衣--------*-—11--------- (請先閱讀背面之注意事項再填寫本頁) 1264467 A7 B7 五、發明說明（56 ) 並以2 5微升的1M Tris，pH 9.0立即中和。匯集含有蛋白 質之流份，利用NAP-5管柱在廠商指示下（NAP-5, Pharmacia Biotech)交換至1毫升PBS中。抗體濃度在280 毫微米吸光度下，或以標準的Bradford分析法決定。各經 純化抗體與人類CD40結合之能力可以FACS證實，利用EL-4細胞如本文件先前所述。可決定出樣品之熱原水平爲每 1 0 微克少於 0.03 EU，依 Limulus Amebocyte Lystate (LAL) 分析法進行（Associates of Cape Cod，Inc.，Falmouth，ΜΑ)。 圖7的結果顯示，由融合瘤F2-103，F5-77及F5-157各自 產生之人類抗體，可刺激Ramos細胞之CD95表現。就如同 CD40 抗體，no. 11 及 F1-102 及 F5-152，抗體 F2-103，F5-77 及F5-157也可用於刺激CD40活性。 結果顯示，染色體轉移的老鼠可用來產生高親和力的分 級人類CD40抗體，其可調控一種以上的CD40活性。可抑 制Ramos細胞上由CD40L謗導之CD95表現或可抑制人類周 邊B細胞增殖作用之抗體，在可以減低CD40活性而治療之 CD40-相關失調症中似乎是有效率的治療劑。而可刺激 Ramos細胞上由CD40L謗導之CD95表現或可刺激人類周邊 .B細胞增殖作用之抗體，在可以增加CD40活性而治療之 CD40-相關失調症中似乎是有效率的治療劑。 -----------φ裝——.---ί訂---------Φ. (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 -59- 本紙張尺度適用中國國家標準（CNS)A4規格（2】0 X 297公釐）! GTATATTACTGTGCGAAAiGATGGGGGGTACTATGGrrCQGGGAGTrATGGGTACTTTGACTACTGGGGCCAG; ggaaccctggtcaccgtctcctcagctagcaccaaoggoccatcggtcttccccctgocaccctcctccaag: AGCACCTCTGGCGGCACAGCGGCCCTGGGCTGCCTGGTCA ^ GGACTACTTCCCC (S2Q ID N0: 14) j F5-77- light, I CAAjGCAGTGGTAACAACGCAGAGTACCOCiGGGGQAGTCAGACCCAGTCixGGACACAGCATGGACATGAGGGT j CCCCGCTCAGCTCCTGOOQCTCCTGCTQCTCTGGrrCCCAiSGXTCCAGATGCGACATCaA ^ ATGACCCAGTC TCCATCTTCCGTGTCTGGATCTGTAGGAGACJcGAGTQACCATC ^ CTTGTCGGCOGAGTCAGGGTATTAGCAG, CTGGTTAGCCTG0TATC ^ JSC ^ GA ^ \ CCAGGGXAJ ^ CCCTAA5CTCCTGATC, rATGCTGQATCCAGTTT3CA ^ G ^ GGCGTCCCATCAAGGTl'CAGCGGCAGTO ^ TTTGGGACAGATTTCAt ^ rCTC ^ CCATCAGCAGCCTGCA j GCCTCL \ ^ GATTTTGCAACTTACTAWGTCAACAiB < 3CTAGCAGTTTCCCTCG'3ACATTCGGCCAAGa < 3ACCAA I GvSTGGAGATCAAACGTACGGTGGCTGCACC ^ TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC I TGGAACTCCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAfiGCCAAAiSTACAGTGGAAGGTGGA; T ^ CGCCCTCCAATCGC ^ TAACrrCCCAGGAGAGTOTCACAGAqCAGGACiKGCAXGG ^ CAGCACCTACAGCCT I CAG <: AGCACCCTGACGCTGAGa ^ \ GCAGACTACGAGAAXa ^ J ^ GTCTACGCCTGCGAAGTCACC CATCA: GGGCCTGA (SEQ ID K〇: lS) The plastids were electrophoresed and pulsed, and transiently transfected into C〇S-1 cells. Briefly, 3 X 106 cells were suspended in 0.7 ml of serum-free iDMEM* containing 30 μg of plastid DNA and placed in 〇·4, in a BioRad quartz tube #165-2088. Cells were electrophoresed on a Gene Pulser II (BioRad) and set at 240 volts for a 0.950 capacitance for a fixed period of 15-25 milliseconds. After current delivery, the cells were transferred to a 10 cm dish containing 1 mL of DMEM, 10% FBS. A well-conditioned culture containing human Ig was recovered after 72 hours. Human anti-moon bean was purified from the medium using Protein A sepharose 4 Fast Flow (Amersham #17-0618-02). Briefly, the conditioning medium was added to a 0.5 liter column. The flow through is collected and added to the column twice or more. The column was washed with 10 ml of PBS, and the antibody was eluted with 2.5 ml of 20 mM glycine, pH 3.0. Dissolved and separated fractions were collected in 〇5 ml, -58- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 public). «衣衣--------*-- 11--------- (Please read the note on the back and fill out this page) 1264467 A7 B7 V. Inventive Note (56) and neutralize immediately with 2 5 μl of 1M Tris, pH 9.0. The fractions containing the protein were pooled and exchanged into 1 ml of PBS using a NAP-5 column under the manufacturer's instructions (NAP-5, Pharmacia Biotech). Antibody concentrations were determined at 280 nm absorbance or by standard Bradford analysis. The ability of each purified antibody to bind to human CD40 can be confirmed by FACS using EL-4 cells as previously described herein. The pyrogen level of the sample can be determined to be less than 0.03 EU per 10 micrograms, according to the Limulus Amebocyte Lystate (LAL) assay (Associates of Cape Cod, Inc., Falmouth, ΜΑ). The results in Figure 7 show that human antibodies produced by each of the fusion tumors F2-103, F5-77 and F5-157 stimulate the CD95 expression of Ramos cells. Just like CD40 antibodies, no. 11 and F1-102 and F5-152, antibodies F2-103, F5-77 and F5-157 can also be used to stimulate CD40 activity. The results show that chromosome-transferred mice can be used to generate high affinity graded human CD40 antibodies that modulate more than one CD40 activity. An antibody that inhibits CD95 expression by CD40L or inhibits proliferation of human peripheral B cells on Ramos cells appears to be an effective therapeutic agent in CD40-related disorders treated to reduce CD40 activity. Antibodies that stimulate CD95L-mediated CD95 expression on Ramos cells or that stimulate human peripheral B cell proliferation may appear to be effective therapeutic agents in CD40-associated disorders that can increase CD40 activity. ----------- φ装——.--- 订定---------Φ. (Please read the notes on the back and then fill out this page) Ministry of Economic Affairs Intellectual Property Bureau Employee Consumption Cooperative Printed -59- This paper scale applies to China National Standard (CNS) A4 specification (2) 0 X 297 mm)

Claims (1)

126^1(65? 10333號專利申請案 A8 B8 C8 中文申請專利範圍替換本(95年5月） D8 十背專利範園- 公告本 ^年月 ' 曰修(策)正本 ----------- 其中該抗體係由融合瘤細胞株或其繼代純系所產生，且 其中該融合瘤示為F卜1〇2，F2-103，F5-77，F5-157或F4-465 〇 2, —種人類抗-CD40單株抗體或其可與CD40結合之片段， 其中該抗體或其片段具有？1-102^5-152，卩2-103，卩5-77，F5-157或F4-465所示融合瘤產生之抗體之CD40結合 特異性。 3. —種人類抗-CD40單株抗體或其可與CD40結合之片段， 其中該抗體或其片段具有卩卜1〇2，F2-103，F5-77，或F5-157所不融合瘤產生之抗體之CD40调控活性。 4· 一種人類抗_ CD40單株抗體或其可與CD40結合之片段， 其中該抗體或其片段具有F4-465所示融合瘤產生之抗體 之CD40調控活性。 5·根據申請專利範圍第1至4項中任一項之人類抗-CD40單 株抗體或其可與C D 4 0結合之片段，其中該片段包括 scFv，Fab，Fab1，或F(ab，）2片段。 6.根據申請專利範圍第4項之人類抗-CD40單珠抗體或其可 與C D 4 0結合之片段，其中該抗體可減低CD40配體與 CD40之結合。 7·根據申請專利範圍第3項之人類抗- CD40單株抗體或其可 與C D 4 0結合之片段，其中該抗體可增加CD40配體與 CD40之結合。 8.根據申明專利範圍第4項之人類抗-C D 4 0早株抗體或其可 O:\67\67726-950503.DOC 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 1264467 έβ8 C8 D8 六、申請專利範圍 與C D 4 0結合之片段，其中該抗體可減低CD40活性。 9. 根據申請專利範圍第8項之人類抗- CD40單株抗體或其可 與C D 4 0結合之片段，其中該抗體可減低表現CD40之細 胞之增殖。 10. 根據申請專利範圍第9項之人類抗-CD40單株抗體或其可 與C D 4 0結合之片段，其中該細胞是B -細胞。 11. 根據申請專利範圍第8項之人類抗-CD40單株抗體或其可 與C D 4 0結合之片段，其中該抗體可減低蛋白質之表 現。 12. 根據申請專利範圍第1 1項之人類抗- CD40單株抗體或其 可與CD40結合之片段，其中該蛋白質包括CD95，CD80 或CD86。 13. 根據申請專利範圍第3項之人類抗-CD40單株抗體或其可 與CD40結合之片段，其中該抗體可增加CD40之活性。 14. 根據申請專利範圍第1 3項之人類抗- CD40單株抗體或其 可與CD40結合之片段，其中該抗體可增加表現CD40之 細胞之增殖。 15. 根據申請專利範圍第1 4項之人類抗- CD40單株抗體或其 可與C D 4 0結合之片段，其中該細胞是B -細胞。 16. 根據申請專利範圍第1 3項之人類抗- CD40單株抗體或其 可與C D 4 0結合之片段，其中該抗體可增加蛋白質之表 現。 17. 根據申請專利範圍第1 6項之人類抗- CD40單株抗體或其 可與CD40結合之片段，其中該蛋白質包括CD95，CD80 或CD86。 O:\67\67726-950503.DOC - 2 - 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 8 8 8 8 A B c D 1264467 六、申請專利範圍 18. —種人類單株抗體或其可與c d 4 0結合之片段，其中該 抗體包括選自由 Fl-102，F4-465，F2-103，F5-77 及F 5 - 1 5 7所示融合瘤所製造之抗體之重鏈可變序列及 輕鏈可變序列。 19. 一種人類單株抗體或其可與c D 4 0結合之片段，其中該 抗體包括選自由序列辨識編號1 〇及序列辨識編號： 1 1 ;序列辨識編號：1 2及序列辨識編號：1 3或序列辨 識編號：1 4及序列辨識編號：1 5之序列對所編碼之重 鏈可變序列及輕鏈可變序列。 O:\67\67726-950503.DOC 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐)126^1(65?10333 Patent Application A8 B8 C8 Chinese Patent Application Substitution (May 95) D8 Ten Back Patent Fan Park - Announcement ^年月月 '曰修(策)本本----- Wherein the anti-system is produced by a fusion tumor cell strain or a sub-supplemental line thereof, and wherein the fusion tumor is shown as Fb1, F2-103, F5-77, F5-157 or F4- 465 〇2, a human anti-CD40 monoclonal antibody or a fragment thereof which binds to CD40, wherein the antibody or fragment thereof has ?1-1025-152, 卩2-103, 卩5-77, F5- The CD40 binding specificity of the antibody produced by the fusion tumor represented by 157 or F4-465. 3. A human anti-CD40 monoclonal antibody or a fragment thereof which binds to CD40, wherein the antibody or fragment thereof has a 〇1〇2 , F2-103, F5-77, or F5-157 does not bind to the CD40-regulated activity of an antibody produced by the tumor. 4. A human anti-CD40 monoclonal antibody or a fragment thereof that binds to CD40, wherein the antibody or fragment thereof The CD40-regulating activity of the antibody produced by the fusion tumor represented by F4-465. The human anti-CD40 monoclonal antibody according to any one of claims 1 to 4 or which can bind to CD 40 a fragment, wherein the fragment comprises a scFv, Fab, Fab1, or F(ab,) 2 fragment. 6. A human anti-CD40 monobe antibody according to claim 4 of the patent application or a fragment thereof that binds to CD40, Wherein the antibody reduces the binding of the CD40 ligand to CD40. 7. The human anti-CD40 monoclonal antibody according to claim 3 or a fragment thereof which binds to CD40, wherein the antibody increases CD40 ligand and Combination of CD40. 8. Human anti-CD 40 early strain antibody according to item 4 of the scope of patent application or its O:\67\67726-950503.DOC This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1264467 έβ8 C8 D8 6. A fragment of the patent application that binds to CD 40, wherein the antibody can reduce CD40 activity. 9. Human anti-CD40 monoclonal antibody according to claim 8 of the patent application or a fragment that binds to CD40, wherein the antibody reduces proliferation of cells expressing CD40. 10. A human anti-CD40 monoclonal antibody according to claim 9 or a fragment thereof that binds to CD40, wherein The cells are B-cells. 11. According to the patent application A human anti-CD40 monoclonal antibody according to Item 8 or a fragment thereof which binds to CD 4 , wherein the antibody reduces the expression of the protein. 12. A human anti-CD40 monoclonal antibody or a fragment thereof which binds to CD40 according to claim 11 of the patent application, wherein the protein comprises CD95, CD80 or CD86. 13. A human anti-CD40 monoclonal antibody or a fragment thereof which binds to CD40 according to claim 3, wherein the antibody increases the activity of CD40. 14. A human anti-CD40 monoclonal antibody or a fragment thereof which binds to CD40 according to claim 13 of the patent application, wherein the antibody increases proliferation of cells expressing CD40. 15. A human anti-CD40 monoclonal antibody or a fragment thereof which binds to CD40 according to claim 14 of the patent application, wherein the cell is a B-cell. 16. A human anti-CD40 monoclonal antibody or a fragment thereof which binds to CD20 according to claim 13 of the patent application, wherein the antibody increases the expression of the protein. 17. A human anti-CD40 monoclonal antibody or a fragment thereof that binds to CD40 according to claim 16 of the patent application, wherein the protein comprises CD95, CD80 or CD86. O:\67\67726-950503.DOC - 2 - This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 8 8 8 8 AB c D 1264467 VI. Patent application scope 18. Human beings a monoclonal antibody or a fragment thereof that binds to cd40, wherein the antibody comprises an antibody selected from the group consisting of Fl-102, F4-465, F2-103, F5-77 and F5-157. Heavy chain variable sequences and light chain variable sequences. 19. A human monoclonal antibody or a fragment thereof that binds to CD20, wherein the antibody comprises a sequence number selected from Sequence Identification Number 1 and Sequence Identification Number: 1 1; Sequence Identification Number: 1 2 and Sequence Identification Number: 1 3 or sequence identification number: 14 and sequence identification number: the sequence of 15 is encoded by the heavy chain variable sequence and the light chain variable sequence. O:\67\67726-950503.DOC This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm)
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