1359817 九、發明說明： 【發明所屬之技術領域】 本發明係有關於一種人類抗體之新穎胺基酸序列構 造’所揭露的序列不含有支鏈或特別定義的胺基酸，尤指 一種具有人類胚系基因構造之新穎人類抗體，其可與人類 CD152抗原專一地結合，用以診斷或治療有關人類CD152 抗原異常之疾病時’可有效地降低異種抗體之免疫性，增 加抗體利用率及減少患者之副作用，同時可達與自我抗原 CD 152反應之目的。 【先前技術】 免疫球蛋白（又稱為抗體）是存在於企清或體液中一 種醣蛋白（glycoproteins)。早期抗體之研製過程必須使用 抗原（antigen)注射實驗小白鼠後取得其抗體製造細胞 後，再與鼠類骨髓瘤細胞株融合來創造能長期分泌特定單 株免疫球蛋白的融合瘤（hybridomas)。然而，以具有鼠類 蛋白序列之抗體投藥卻經常引發病患的免疫反應（人類抗 小鼠反應）。因此，必須製備出盡量排除非人類序列的完 全人類抗體，才有臨床應用價值。為了達成此目的，美國 專=第5,585,089號揭露將人類免疫球蛋白基因轉殖至鼠 類單株抗體，來獲得人類化抗體的方法。如第一圖所示， /、係種$知之抗體結構示意圖，該抗體結構包括有變異 區（la)及恆定區（lb)，該抗體變異區〇a)内部各有三個「高 度變異區（If)」’該高度變異區上佈滿能與抗原結合之胺 基酸。習知之人類化鼠源抗體係將鼠源之變異區或高度變 異區以遺傳工程之方式安插至人類抗體之骨架中。此技術 ^首創之僅含少數非人類序列的抗體，是當時最先進的產 品，並成為往後治療性單株抗體競相追隨之標準。 人類化的鼠源抗體已經成功地作為各種疾病的治療 6 1359817 樂物’特別疋如呼吸融合病毒（respiratory syncytial virus，RSV)等的傳統感染性疾病。但近來有越來越多的 抗體被用於治療其它疾病如自體免疫疾病與惡性腫瘤，後 者如轉移性乳癌、非何杰金氏淋巴瘤 '慢性淋巴細胞性白 ▲•癌與急性骨髓性白血病等。抗體亦被用於預防器官排斥 或阻止動脈整型時之凝血。但是此種人類化抗體仍多少含 有鼠類序列’患者使用後經常發生副作用：輕則引起發 燒、内寒、頭暈痛、嘔吐、噁心、腹瀉 '低血壓、全身疼 痛、身心疲憊等，重則引發嚴重的排斥現象使患者死亡。1359817 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a novel amino acid sequence structure of a human antibody. The sequence disclosed does not contain a branched or specially defined amino acid, especially a human. A novel human antibody of germline gene structure that can specifically bind to human CD152 antigen to diagnose or treat diseases related to human CD152 antigen abnormality, which can effectively reduce the immunity of heterologous antibodies, increase antibody utilization and reduce patients The side effects can be achieved with the purpose of reacting with the self antigen CD 152. [Prior Art] Immunoglobulins (also known as antibodies) are glycoproteins present in Qiqing or body fluids. In the early development of antibodies, it is necessary to use an antigen to inject an experimental mouse to obtain an antibody-producing cell, and then fuse with a murine myeloma cell line to create a hybridoma (hybridomas) capable of secreting a specific monoclonal immunoglobulin for a long period of time. However, administration of an antibody having a murine protein sequence often elicits an immune response (human anti-mouse response). Therefore, it is necessary to prepare a complete human antibody that excludes non-human sequences as much as possible, and has clinical application value. In order to achieve this, U.S. Patent No. 5,585,089 discloses the method of transducing a human immunoglobulin gene to a murine monoclonal antibody to obtain a humanized antibody. As shown in the first figure, /, the structure of the known antibody structure, the antibody structure includes a variant region (la) and a constant region (lb), and the antibody variant region 〇a) has three "highly variable regions" ( If)"' The highly variable region is covered with an amino acid that binds to the antigen. The conventional humanized mouse-derived system inserts the murine variable region or highly variable region into the skeleton of human antibodies in a genetically engineered manner. This technology, the first antibody to contain only a few non-human sequences, was the most advanced product at the time and became the standard for subsequent therapeutic individual antibodies. Humanized murine antibodies have been successfully treated as various diseases. 6 1359817 Musical objects, such as traditional infectious diseases such as respiratory syncytial virus (RSV). Recently, however, more and more antibodies have been used to treat other diseases such as autoimmune diseases and malignant tumors, such as metastatic breast cancer, non-Hodgkin's lymphoma, chronic lymphocytic white ▲ cancer and acute myeloid Leukemia and the like. Antibodies are also used to prevent organ rejection or to prevent coagulation during arterial shaping. However, this kind of humanized antibody still contains a mouse sequence. 'The patient often has side effects after use: it causes fever, internal cold, dizziness, vomiting, nausea, diarrhea, low blood pressure, body aches, physical and mental exhaustion, etc. The rejection phenomenon causes the patient to die.
為了達成不含非人類序列的抗體目的，也有人成功地 將實驗鼠的免疫球蛋白胚系基因以遺傳工程的方式置換 成一組人類的免疫球蛋白胚系基因，然後使用抗原免疫此 類之基因改造轉殖鼠來獲得完全人類抗體，但是實驗鼠基 因空間有限，無法包涵所有人類抗體胚系基因，如輕鏈基 因常只能放進人類之κ胚系基因（Jak〇b〇vitsA，1995)，因 而限制了所產抗體之多樣反應性。w〇 〇1/14424號即具體 揭露了以免疫具有人類免疫球蛋白基因改造轉殖鼠^獲 得有限之完全人類抗體的方法。In order to achieve the purpose of antibodies without non-human sequences, it has also been successfully replaced with a set of human immunoglobulin germline genes in a genetically engineered manner by immunogenic globulin germline genes, and then immunized with such genes using antigens. Reconstructed transgenic mice to obtain fully human antibodies, but the experimental mouse gene space is limited, can not contain all human antibody germline genes, such as light chain genes can often only be put into human κ germline genes (Jak〇b〇vitsA, 1995) Thus, the diverse reactivity of the antibodies produced is limited. W〇 〇 1/14424 specifically discloses a method for immunizing humans with immunoglobulin genetically engineered transgenic mice to obtain a limited total human antibody.
、本案發明人早在1994年就開發出「體外定位免疫 法」，以人類周邊血為材料來取得完全人類抗體（Chin lt ^ ^/. Immunol. 81:428, 1994; Eur. J. ImmunoL 25.^/ ^95)。近年來並針對上述之先前技術缺憾，以人類CDiM 抗原為例開發出一系列最新的技術，且於中華民國九十三 發明第G93128328號在案。本發明為接續^ =上開冑外疋位免疫法」及發明申請案所揭露之技術， 酸5Ϊ 抗原為材料鑑別出一種具有人類胚系胺基 ，構造之新穎人類抗體結構，此項創舉融合了不同人類血 ^固有的歧異性、獨特的體外培養技術與分子生物學方 去’因此能夠取得新穎構造之完全人類抗體，如且有人類 7 1359817 - VH3及νλ胚系基因的獨特結構幾乎無法使用基因改造轉 殖鼠取得。本發明所揭露的新賴結構可與人類CD 15 2抗 . 原專一地結合。 【發明内容】 如第一圖所示’一分子的抗體蛋白質由二條重鏈 (heavy chains)胜肽和二條輕鏈（light chains)胜肽所組成， 重鍵或輕鍵再分成變異區（variable region)和怪定區 (constant region)，重鏈和輕鏈的變異區共同組成「抗原 _ 結合位（antigen binding site)」，可與抗原的決定位（epit〇pe) 結合，達到一分子的抗體和一分子的抗原決定位結合之最 佳效果。重鍵可依恆定區更細分為#、0、丫、§、£五種， 輕鍵有λ和κ二種。抗體的基因是由許多片段所組成，以 人類抗體為例’ κ輕鍵、λ輕鏈與重鍵的基因分別位於第 2、第22及第14條染色體。如第二圖所示，與抗原结合 有關的抗體變異區由分別是V、（D)和J等二至三種胚系 基因片段組成’其中以V胚系基因貢獻最大，這些片段 藉由排列組合的方式’產生各式各樣的抗體。而根據 V-(D)-J遺傳信息並按照這種組合產生的抗體即稱為「胚 Φ 系抗體（germline antibody)」。現已公認重鏈之胚系基因 可分為VH1到VH7七大類型而輕鍵之胚系基因有人和κ 二大類型。 在生理狀態下，胚系抗體的「高度變異區」與其它變 異區仍繼續進行體細胞基因突變，不斷產生新修飾的抗體 群及新的多樣性，用以選出優化後能與抗原結合更緊密的 高親合力抗體。本發明之主要目的，在於利用上述胚系抗 體的特性’避免鼠源抗體人類化的缺點，故本發明將人類 胚系抗體胺基酸重新設計，以提高與人類CD152抗原專 一地結合之親合力。 8 1359817 ^ 為達上述之目的，本發明為二種創新之人類胚系抗體 • 胺基酸結構，該結構分別為一重鏈胺基酸序列（序列識別 號1 ; SEQIDN0:1)與一輕鏈胺基酸序列（序列識別號2 ; SEQ ID NO:2)，且如第三及第四圖所示，二者分別來自 VH3及νλ人類胚系抗體胺基酸序列。 有關本發明之重鏈結構内容（SEQ ID ΝΟ:1，代號為 VHnovel)配合第三圖式詳細說明如下：將VHnovel之胺 基酸序列和所有已知的抗體一級結構比對後發現 VHnovel是從基因庫典藏登記號（accession number)為 AB019439的VH3-3 0及VH3-33兩種人類胚系抗體品系衍 ® 生而來的，在98個胺基酸中僅有10個差異。 請參閱第四圖，由序列分析比對我們發現本發明之輕 鏈結構内容（SEQ ID NO:2，代號為VLnovel)序列對應到 典藏登記號為BAC01778、S78058及CAA38313等三個具 有人類胚系νλ輕鏈結構的抗體，因此VLnovel也是νλ 輕鏈家族的一員，在89個胺基酸中僅有7個差異。 以前述之創新序列結構不但可和重組人類CD152抗 原專一地結合（請參閱第五圖）；且前述之創新結構可與活 化（請參閱第六圖）之人類Τ細胞專一地作用，反映了 • CD152是Τ細胞活化後產生之蛋白質的事實。 【實施方式】 有關本發明之詳細說明及技術内容，現在配合圖式說 明如下： 一、抗-CD152人類抗體之產生The inventor of this case developed the "in vitro localization immunoassay" as early as 1994, and obtained human antibodies from human peripheral blood (Chin lt ^ ^/. Immunol. 81:428, 1994; Eur. J. ImmunoL 25 .^/ ^95). In recent years and in response to the above-mentioned prior art shortcomings, a series of the latest technologies have been developed using human CDiM antigen as an example, and the invention was filed in the Republic of China No. 93 invention No. G93128328. The present invention is a technique for the continuation of the = 上 上 上 疋 疋 及 及 及 及 及 及 及 及 及 及 及 及 及 , , , , , , , , , , , 酸 酸 酸 酸 酸 酸 酸 酸 酸 酸 酸 酸 酸 酸 酸 酸 酸 酸The inherent heterogeneity of different human blood, unique in vitro culture techniques and molecular biology to 'can thus obtain novel constructs of fully human antibodies, such as the unique structure of human 7 1359817 - VH3 and νλ germline genes Made using genetically modified transplanted mice. The novel structure disclosed in the present invention can be combined with the human CD 15 2 antibody. SUMMARY OF THE INVENTION As shown in the first figure, 'one molecule of antibody protein consists of two heavy chains and two light chains, and the heavy or light bonds are subdivided into variant regions. The region and the constant region, the heavy and light chain variants together form the "antigen binding site", which binds to the epitope of the antigen (epit〇pe) to reach one molecule. The best combination of antibody and antigenic epitope of one molecule. The heavy key can be further subdivided into five types according to the constant region: #, 0, 丫, §, £, and the light keys are λ and κ. The gene of an antibody is composed of a plurality of fragments, and human antibodies are exemplified. The genes of the kappa light bond, the lambda light chain and the heavy bond are located on chromosomes 2, 22 and 14, respectively. As shown in the second figure, the antibody variant region associated with antigen binding consists of two to three germline gene segments, V, (D), and J, respectively. Among them, the V germline gene contributes the most, and these fragments are arranged by combination. The way 'produces a wide variety of antibodies. The antibody produced according to the V-(D)-J genetic information and in accordance with this combination is called "germline antibody". It has been recognized that the germline genes of heavy chains can be divided into seven major types of VH1 to VH7 and two types of germline genes of light bonds and κ. Under physiological conditions, the "highly variable region" of germline antibodies and other variant regions continue to undergo somatic gene mutations, constantly producing newly modified antibody populations and new diversity, which can be selected to be more closely integrated with antigens after optimization. High affinity antibody. The main object of the present invention is to utilize the characteristics of the above-mentioned germline antibody to avoid the disadvantage of humanization of the mouse antibody, and the present invention redesigns the human germline antibody amino acid to enhance the affinity for specific binding to the human CD152 antigen. . 8 1359817 ^ For the above purposes, the present invention is a novel innovative human germline antibody amino acid structure which is a heavy chain amino acid sequence (SEQ ID NO: 1; SEQ ID NO: 1) and a light chain, respectively. The amino acid sequence (SEQ ID NO: 2; SEQ ID NO: 2), and as shown in the third and fourth panels, are derived from the VH3 and νλ human germline antibody amino acid sequences, respectively. The heavy chain structure of the present invention (SEQ ID ΝΟ: 1, codenamed VHnovel) is described in detail in conjunction with the third scheme as follows: VHnovel is obtained by aligning the amino acid sequence of VHnovel with all known antibody primary structures. The gene library accession number is derived from the VH3-3 0 and VH3-33 human germline antibody lines of AB019439, with only 10 differences among the 98 amino acids. Referring to the fourth figure, we found that the light chain structure content (SEQ ID NO: 2, codenamed VLnovel) of the present invention corresponds to the three human germline series with the accession numbers BAC01778, S78058 and CAA38313. The antibody of the νλ light chain structure, therefore VLnovel is also a member of the νλ light chain family, with only 7 differences among the 89 amino acids. The aforementioned innovative sequence structure not only specifically binds to recombinant human CD152 antigen (see Figure 5); and the aforementioned innovative structure can be specifically interacted with activation (see Figure 6) of human sputum cells, reflecting • CD152 is the fact that proteins produced by the activation of sputum cells. [Embodiment] The detailed description and technical contents of the present invention will now be described as follows: 1. Production of anti-CD152 human antibodies
以 Ficoll-Paque (GE Healthcare，Uppsala, Sweden) 密度離心（4〇〇xg)方式從血液捐贈者分離出的周邊血 單核細胞首先以CD45RO MACS微珠 （Miltenyi Biotec, Auburn CA)進行磁標定’隨後以 VarioMACS 9 (Miltenyi Biotec)儀器分離。沖洗出的CD45RO+ T細 胞培養於組織培養瓶中，其密度為2χ106細胞/毫升之 RPMI-1640(HyQTM; HyClone，Logan，UT)，並補充 1χ 非必需氨基酸（Life Technologies, Grand Island, NY)、10。/。人類血清、50 pg/ml慶大黴素/卡那黴素 (China Chemical & Pharmaceutical, Taipei, Taiwan) ' 50 μΜ 2-乙基硫醇及10 pg/ml美洲商陸***促進劑 (PWM ; Sigma Chemicals)。培養24小時後，細胞以 400xg離心並移出以收集上清液，製備CD45RO+ T細 胞替代因子，以0.45 mm濾膜過濾並於-20°C冷凍儲 存。 此外並以磁性細胞去除術去除周邊血單核細胞 内之可抑制體外免疫反應的毒殺細胞族群。膠體超順 磁性微珠結合至單株抗小鼠CD8與抗-CD56抗體 (Miltenyi Biotech)之用法如前面所述。毒殺細胞去除 之周邊血單核細胞於體外以二步驟免疫法進行免疫 作用。初級免疫作用之進行，係將細胞培養於含10 nM之CD 152抗原之培養基6天，其中含50 μΜ 2-乙基 硫醇、10〇/〇熱去活化人類血清、0.05 ng/ml rIL2 (Calbiochem，San Diego, CA)及25% (v/v) CD45RO+ T 細胞替代因子。在第7天，收取初級免疫細胞並以40% Ficoll-Paque離心。針對次級免疫反應，將3 X 107個 細胞與CD152抗原混合於培養瓶，其事先以5 pg/ml 的 CD40L (CD154 ; Vinci-Biochem，Vinci, Italy)固定 隔夜。將細胞培養3-5天，其培養基補充有5%人類血 清、50 μΜ 2-乙基硫醇及10 nM免疫抗原。 體外免疫之細胞隨後以EB病毒感染。簡單而 言，將107個淋巴細胞和1 ml含EB病毒之上清液於 37°C下培養2小時，此上清液源自產生EB病毒之狨猴 細胞株B95-8(美國種質保存_心’ ATCCCRL 1612 ; 由台北三軍總醫院提供）。將感染之細胞以1〇5/孔之 數量種於96孔盤並加入絲裂黴素（Kyowa HakkoPeripheral blood mononuclear cells isolated from blood donors by Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density centrifugation (4〇〇xg) were first magnetically labeled with CD45RO MACS microbeads (Miltenyi Biotec, Auburn CA) It was subsequently isolated using a VarioMACS 9 (Miltenyi Biotec) instrument. The washed CD45RO+ T cells were cultured in tissue culture flasks at a density of 2χ106 cells/ml of RPMI-1640 (HyQTM; HyClone, Logan, UT) supplemented with 1χ non-essential amino acids (Life Technologies, Grand Island, NY), 10. /. Human serum, 50 pg/ml gentamicin/kanamycin (China Chemical & Pharmaceutical, Taipei, Taiwan) '50 μΜ 2-ethyl mercaptan and 10 pg/ml Pokeweed split promoter (PWM; Sigma Chemicals). After 24 hours of culture, the cells were centrifuged at 400 x g and removed to collect the supernatant, and CD45RO + T cell replacement factor was prepared, filtered through a 0.45 mm filter and stored frozen at -20 °C. In addition, magnetic cell removal is performed to remove the toxic cell population that inhibits the immune response in vitro in peripheral blood mononuclear cells. The use of colloidal superparamagnetic microbeads to bind to monoclonal anti-mouse CD8 and anti-CD56 antibodies (Miltenyi Biotech) was as previously described. Peripheral blood mononuclear cells from the venom killing cells were immunized in vitro by a two-step immunoassay. The primary immunization was carried out by incubating the cells in a medium containing 10 nM of CD 152 antigen for 6 days, containing 50 μM 2-ethylthiol, 10 〇/〇 heat to deactivate human serum, and 0.05 ng/ml rIL2 ( Calbiochem, San Diego, CA) and 25% (v/v) CD45RO+ T cell replacement factor. On day 7, primary immune cells were harvested and centrifuged at 40% Ficoll-Paque. For the secondary immune response, 3 X 107 cells were mixed with CD152 antigen in a culture flask which was previously fixed overnight at 5 pg/ml of CD40L (CD154; Vinci-Biochem, Vinci, Italy). The cells were cultured for 3-5 days, and the medium was supplemented with 5% human serum, 50 μM 2-ethylthiol, and 10 nM immunizing antigen. The cells immunized in vitro are then infected with Epstein-Barr virus. Briefly, 107 lymphocytes and 1 ml of Epstein-Barr virus containing supernatant were cultured for 2 hours at 37 ° C. The supernatant was derived from the EB virus-producing simian cell line B95-8 (American germplasm preservation) _心' ATCCCRL 1612; provided by Taipei Military General Hospital). Infected cells were seeded in 96-well plates at a dose of 1〇5/well and added to mitomycin (Kyowa Hakko)
Kogyo，Toyoko，Japan)處理之PBMC以作為館養細胞 (104/孔）。抗體專一性經酶免疫分析法確定。 、本發明之創新結構與重組人類CD152抗原之結合性 請參閱「第四圖」，係本發明之獨特人類胚系抗 體胺基酸結構與重組人類CD152抗原結合性之示意 圖。使用酶免疫分析法，係首先於室溫下以1 Kg/inl 由BHK細胞表現之重組人類CD152 (CTLA-4)-muIg 融合蛋白（購自美國 Ancell Corporation, Bayport, MN)、1 pg/ml 單株鼠類 IgG2a (購自 Ancell)、10 μδ/ 孔的牛血清白蛋白（BSA;購自Sigma, St. Louis, MO) 或破傷風類毒素（購自 ADImmune Corporation, Taichung，Taiwan)隔夜塗覆於微量滴定盤《將創新結 構抗體以含0.5 Μ氣化鈉及0.1% Tween-20之10 mM 磷酸鈉緩衝液，pH 8.0稀釋至濃度為1 pg/ml。塗覆 處理後之滴定盤以稀釋之培養基上清液進行培養，洗 滌後以過氧化酶標定之辨識人類IgG或λ輕鏈之山 羊抗體（購自 Zymed Laboratories，So. San Francisco, CA)，並加入100 μΐ色原基質鄰-苯二胺（〇pD)(購自 Sigma)顯影。反應於30分鐘後加入1M硫酸而停止， 且其產生之吸光於490 nm下判讀。如圖所示：本發 明之人類胚系抗體胺基酸結構可和重組人類CD152 抗原專一地結合’對於非相關之抗原如單_鼠類 IgG2a、牛血清白蛋白或破傷風類毒素皆測不到明顯 的結合反應。 、新穎結構胺基酸序列之鑑別 抗體新穎結構是由cDNA序列依遺傳密碼反推 1359817 _ 成胺基酸序列而得。簡言之，將2x 104個具有與重 組人類CD 1 52抗原專一反應的單株抗體分泌細胞以 Dynabeads® mRNA DIRECTtm Micro Kit(購自 Dynal Biotech，Oslo, Norway)依製造商提供之步驟抽取 mRNA，分離出的mRNA直接加入反轉錄聚合酶鍵 反應（RT-PCR)中作為反應模板（template)，所使用之 套組為 Titan One Tube RT-PCR System (購自 Roche Diagnostics Corporation，Indianapolis, IN)。如 SEQ ID NO:3到SEQIDNO:6所示，反應所使用的二組引子 (primer)分別是能放大人類抗體基因重鏈變異區核 胃 酸序列中之第一組HuVHBack與HuJHFOR，及能放 大人類抗體基因輕鏈變異區核酸序列中之第二組 HuVIBack 與 HuVXFOR » RT-PCR 共採用 37 個循環 放大步驟：分別為94°C二分鐘之第一循環；包含94°C 三分鐘、51°C30秒及68°C —分鐘的第二至第三十六 循環；最終循環則使用68^(:十分鐘的反應條件。 經電泳確定屬單一族群之DNA片段，利用核苷 酸類似物（analog)沒有3'-hydroxyl group而可特定地 終止DNA鍊複製的原理進行定序反應：在類似物上 • 標定不同的螢光，用電泳將長短不一的片段分開，利 用雷色光偵測不同的螢光，即可在電腦中讀出DNA 的序列。所得序列再經確認（Molecular Clinical Diagnostic Laboratory，DR. Chip Biotechnology, Inc·，Taipei，Taiwan)並轉換 成胺基酸序列》 四、創新結構與活化之人類T細胞作用 請參閱「第五圖」，係本發明之獨特人類胚系抗 體胺基酸結構與活化之人類Τ細胞作用之流式細胞 分析示意圖。健康血液經傳染病篩選為陰性，並含有 正常量之丙胺酸轉移酶之血液，來自台灣血液基金會 12 1359817 抗體胺基酸結構比較圖。 第五圖’#、本發明之人類抗體結構經連續十倍稀釋 (chlution)後以酵素免疫分析法所量測對人類 CD152抗原反應性吸光度示意圖。 第六圖本發明之人類抗體結構以流式細胞分析儀所 量測對人類活化T細胞反應性示意圖。Kogyo, Toyoko, Japan) treated PBMC as a cultured cell (104/well). Antibody specificity was determined by enzyme immunoassay. The binding structure of the novel structure of the present invention to the recombinant human CD152 antigen is shown in Fig. 4, which is a schematic diagram showing the binding of the unique human germline amino acid structure of the present invention to the recombinant human CD152 antigen. Enzyme immunoassay was used to first express recombinant human CD152 (CTLA-4)-muIg fusion protein (purchased from Ancell Corporation, Bayport, MN), 1 pg/ml, expressed as BK cells at 1 Kg/inl at room temperature. Single mouse IgG2a (purchased from Ancell), 10 μδ/well of bovine serum albumin (BSA; purchased from Sigma, St. Louis, MO) or tetanus toxoid (purchased from ADImmune Corporation, Taichung, Taiwan) overnight coated In a microtiter plate, the innovative structural antibody was diluted to a concentration of 1 pg/ml in a 10 mM sodium phosphate buffer containing 0.5 Μ sodium hydride and 0.1% Tween-20 at pH 8.0. The coated titration tray was cultured with the diluted culture supernatant, and after washing, the human IgG or lambda light chain goat antibody (purchased from Zymed Laboratories, So. San Francisco, CA) was labeled with peroxidase. 100 μL of the original substrate o-phenylenediamine (〇pD) (purchased from Sigma) was added for development. The reaction was stopped after 30 minutes by the addition of 1 M sulfuric acid, and the resulting absorbance was read at 490 nm. As shown in the figure: the human germline antibody amino acid structure of the present invention can specifically bind to recombinant human CD152 antigen. 'Undetectable antigens such as single-mouse IgG2a, bovine serum albumin or tetanus toxoid are not detected. Significant binding reaction. Identification of Novel Structure Amino Acid Sequences The novel structure of the antibody is derived from the cDNA sequence according to the genetic code to reverse the 1359817 _ amino acid sequence. Briefly, 2 x 104 monoclonal antibody-secreting cells with specific reactivity with recombinant human CD 1 52 antigen were extracted from the mRNA using the Dynabeads® mRNA DIRECTtm Micro Kit (purchased from Dynal Biotech, Oslo, Norway) according to the manufacturer's protocol. The isolated mRNA was directly added to a reverse transcription polymerase bond reaction (RT-PCR) as a reaction template, and the kit used was a Titan One Tube RT-PCR System (available from Roche Diagnostics Corporation, Indianapolis, IN). As shown in SEQ ID NO: 3 to SEQ ID NO: 6, the two sets of primers used in the reaction are the first group of HuVHBack and HuJHFOR which can amplify the nuclear acid sequence of the heavy chain variant region of the human antibody gene, and can amplify humans. The second set of HuVIBack and HuVXFOR » RT-PCR in the nucleic acid sequence of the antibody gene light chain variant region uses 37 cycles of amplification steps: 94 ° C for two minutes, the first cycle; including 94 ° C for three minutes, 51 ° C 30 Second and 68 ° C - minutes of the second to the thirty-sixth cycle; the final cycle uses 68 ^ (: ten minutes of reaction conditions. Electrophoresis to determine a DNA fragment belonging to a single group, using nucleotide analogs (analog) There is no 3'-hydroxyl group to specifically terminate the principle of DNA strand replication. In the analogy: • Different fluorescent light is calibrated, electrophoresis is used to separate fragments of different lengths, and different light ray is detected by lightning light. Light, the sequence of the DNA can be read in a computer. The obtained sequence is confirmed (Molecular Clinical Diagnostic Laboratory, DR. Chip Biotechnology, Inc., Taipei, Taiwan) and converted into an amino acid sequence. For the action of activated human T cells, please refer to "fifth map", which is a schematic diagram of flow cytometry analysis of the action of the unique human germline antibody amino acid structure and activated human sputum cells of the present invention. The healthy blood is negatively screened by infectious diseases. And the blood containing a normal amount of alanine transferase, from the Taiwan Blood Foundation 12 1359817 antibody amino acid structure comparison chart. The fifth figure '#, the human antibody structure of the present invention after a ten-fold serial dilution (chlution) A schematic diagram of the reactivity of human CD152 antigen by enzyme immunoassay. Fig. 6 is a schematic diagram showing the reactivity of human activated T cells measured by a flow cytometer in the human antibody structure of the present invention.
【主要元件符號說明】 11 : 變異區 12 ： 輕鏈 13 恆定區 14 ： 重鏈 15 高度變異區 17 : 雙硫鍵 20 24 (K)輕鏈 重鍵 22 ： 輕鏈 30 重鏈第一骨架區 31 ： 重鏈第一高度變異區 32 重鏈第二骨架區 33 ： 重鏈第二高度變異區 34 重鏈第三骨架區 !〜98: 重鏈胺基酸順序 氺1 與上列胺基酸型式相 同 40 輕鏈第一骨架區 41 ： 輕鏈第一高度變異區 42 輕鏈第二骨架區 43 ： 輕鏈第二高度變異區 44 輕鏈第三骨架區 胺基酸缺失 1 〜89·· 輕鏈胺基酸順序 • 人類CD152抗原 □: 單株鼠類IgG2a 〇 牛血清白蛋白 破傷風類毒素 60 非相關對照人類抗體 流式細胞分析圖 與未活化之人類Τ細胞作用之 62 創新結構與未活化之 析圖 人類Τ 細胞作用之流式細胞分 64 非相關對照人類抗體與活化之人類丁細胞作用之流 式細胞分析圖 14 1359817 66 ： 創新結構與活化之人類T細胞作用之流式細胞分析 圖[Main component symbol description] 11 : Variant region 12 : Light chain 13 Constant region 14 : Heavy chain 15 Highly variable region 17 : Disulfide bond 20 24 (K) Light chain heavy bond 22 : Light chain 30 Heavy chain first skeleton region 31: heavy chain first highly variable region 32 heavy chain second framework region 33: heavy chain second highly variable region 34 heavy chain third skeleton region! ~98: heavy chain amino acid sequence 氺1 with upper amino acid The same type 40 light chain first skeleton region 41: light chain first highly variable region 42 light chain second skeleton region 43: light chain second highly variable region 44 light chain third skeleton region amino acid loss 1 ~ 89 · Light chain amino acid sequence • Human CD152 antigen □: Single mouse IgG2a yak serum albumin tetanus toxoid 60 Non-correlated control Human antibody flow cytometry analysis and unactivated human sputum cell action 62 Innovative structure and Activation of the map of human Τ Cellular flow cytometry 64 Flow cytometric analysis of non-related control human antibodies and activated human butyl cells Figure 14 1359817 66 : Innovative structure and activation of human T cell interaction Cell analysis chart
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