本發(fā)明屬于基因工程領(lǐng)域，涉及抗人cd19抗原的嵌合抗原受體及其應(yīng)用，還涉及包含抗人cd19的嵌合抗原受體的慢病毒載體和應(yīng)用。
背景技術(shù)：
：單克隆抗體的產(chǎn)生技術(shù)經(jīng)歷了三個階段：經(jīng)典免疫方法產(chǎn)生的異源多克隆抗體；細胞工程產(chǎn)生的鼠源單克隆抗體及基因工程產(chǎn)生的人源單克隆抗體。將針對某一腫瘤抗原的單克隆抗體與化療藥物或放療物質(zhì)聯(lián)合，利用單克隆抗體的導(dǎo)向作用，將藥物或放療物質(zhì)攜帶至靶器官，直接殺傷靶細胞，稱為腫瘤導(dǎo)向治療。另外，將放射性標記物與單克隆抗體連接，注入患者體內(nèi)可進行放射免疫顯像，協(xié)助腫瘤的診斷。以小鼠來源單克隆抗體用于疾病治療，直接使用小鼠抗體進行人體治療時，因為鼠抗的異質(zhì)性會引起人抗鼠抗體反應(yīng)(humananti-mouseantibodyreaction，hama)，導(dǎo)致抗體半衰期短，在循環(huán)系統(tǒng)中被很快清除，失去療效。因此，治療用鼠源單抗需要進行人源化修飾以提高抗體的人源化程度、減弱hama。人源化抗體就是指抗體的可變區(qū)部分(即vh和vl區(qū))或抗體全部由人類抗體基因所編碼。人源化抗體可以大大減少異源抗體對人類機體造成的免疫副反應(yīng)，人源化抗體的形式也從最初的嵌合抗體、改型抗體等逐步發(fā)展為人源化抗體?？贵w是免疫系統(tǒng)中最重要的分子之一，體內(nèi)體液免疫產(chǎn)生的抗體其穩(wěn)定性是自然進化篩選的結(jié)果。但是利用細胞工程和基因工程產(chǎn)生的鼠源或人源化抗體，是一種重組蛋白，在生產(chǎn)、運輸、儲存以及體內(nèi)使用過程中，容易發(fā)生多種降解；在體內(nèi)應(yīng)用于腫瘤治療時，抗體的親和力，特異性以及不同ph條件下耐受能力尤其是腫瘤內(nèi)部為弱酸性環(huán)境均影響抗體穩(wěn)定性和生物學(xué)活性?？梢娞岣呖贵w藥物穩(wěn)定性對抗體成藥性至關(guān)重要，此外優(yōu)化抗體效應(yīng)功能也對抗體臨床應(yīng)用具有重要作用。但是抗體的穩(wěn)定性和可靠性仍然長久以來困擾著抗體產(chǎn)業(yè)，也是整個生物學(xué)研究領(lǐng)域需要突破的大的瓶頸。而我們針對鼠源抗體的人源化以及穩(wěn)定性、特異性和親和性改造是很有必要的。car是模擬tcr功能的人工受體，由抗原識別域、鉸鏈區(qū)和跨膜區(qū)及胞內(nèi)信號域依次連接組成，腫瘤細胞表面的抗原(受體)與嵌合抗原受體的抗體(配體)結(jié)合時，通過鉸鏈區(qū)和跨膜區(qū)將信號傳遞至胞內(nèi)，胞內(nèi)信號域再將信號轉(zhuǎn)化為活化信號，激活效應(yīng)細胞，效應(yīng)細胞通過分泌穿孔素或者產(chǎn)生細胞因子殺傷腫瘤細胞，同時效應(yīng)細胞本身也發(fā)生擴增，進一步擴大免疫殺傷作用。cd19是b細胞表面的跨膜蛋白(clusterofdifferentiation19protein，cd19)，它與b細胞活化、信號傳導(dǎo)及生長調(diào)節(jié)密切相關(guān)，是b淋巴細胞表面的一種功能受體分子，在b細胞抗原受體(bcr)識別抗原時構(gòu)成b細胞雙重抗原結(jié)合模型，參與b細胞內(nèi)ca2+的轉(zhuǎn)運，調(diào)節(jié)b細胞的活化與增殖。cdl9作為重要標記物可被用于白血病、淋巴瘤及免疫系統(tǒng)疾病的診斷和預(yù)后判斷。但是，雖然有較多臨床應(yīng)用抗體出現(xiàn)，但是還遠不能滿足臨床應(yīng)用的需求，技術(shù)上也還存在不少問題：人源化程度、car的優(yōu)化組合與特異性、穩(wěn)定性和具有更好的清除腫瘤細胞的能力的綜合考慮，使用非人單克隆抗體(例如，鼠單克隆抗體)一方面鼠源抗體通常不能介導(dǎo)補體依賴性溶解或通過抗體依賴性細胞毒性或fc受體介導(dǎo)的吞噬作用而溶解人靶細胞。另一方面，人宿主會將非人單克隆抗體識別為外源性蛋白質(zhì)誘發(fā)引起有害過敏反應(yīng)的免疫應(yīng)答：人抗小鼠抗體(hama)反應(yīng)。因此有必要進一步研究更優(yōu)化cd19抗體。目前car技術(shù)在多種血液瘤和實體瘤均有研究，尤其是在血液病腫瘤中進展迅速，但是如何選擇特異性強穩(wěn)定性好的scfv序列并形成最優(yōu)的car組合仍然還需探索。技術(shù)實現(xiàn)要素：有鑒于此，本發(fā)明的目的在于提供一種抗人cd19抗原的嵌合抗原受體，本發(fā)明的抗人cd19抗原的嵌合抗原受體能夠更穩(wěn)定的表達于t淋巴細胞，且特異性高，具有更好的清除腫瘤細胞的能力。為實現(xiàn)上述目的，本發(fā)明的技術(shù)方案為：抗人cd19抗原的嵌合抗原受體，由識別人cd19抗原的多肽(scfv)，鉸鏈區(qū)，跨膜區(qū)和胞內(nèi)信號域依次連接組成；所述識別人cd19抗原的多肽(scfv)的氨基酸序列如seqidno.1或seqidno.2或seqidno.3或seqidno.4所示。所述的嵌合抗原受體需要逾越兩個技術(shù)障礙，一是尋找更穩(wěn)定有效的識別cd19抗原的人源化單克隆抗體(scfv)，二是獲得最佳的car的組合方式。本發(fā)明所述識別人cd19抗原的多肽(scfv)的氨基酸序列是通過人源化改造得到的，為什么要進行改造？因為我們目前使用的識別cd19抗原的嵌合抗原受體在應(yīng)用時不能夠穩(wěn)定表達于t淋巴細胞尤其是病人來源的t淋巴細胞，轉(zhuǎn)染后隨著培養(yǎng)時間的延長t細胞表面的嵌合抗原受體表達顯著降低。人源化程度越高的scfv可以有效地降低car的免疫原性，增強car-t在體內(nèi)的持續(xù)和安全性。通過隨機突變的方式我們獲得了15組能夠和cd19抗原結(jié)合的人源化抗cd19單克隆抗體，但是其它的11組因為純化、親和力測試和特異性檢測綜合實驗結(jié)果不合格，慘遭淘汰。而僅僅由本發(fā)明所保護的這幾組，具有預(yù)料不到的技術(shù)效果。比再次改造前表達更穩(wěn)定，且特異性高，具有更好的清除腫瘤細胞的能力。如何改造識別cd19抗原的抗體，使之能夠較好地保留對抗原的親和活性，并且組合為最佳的抗人cd19抗原的嵌合抗原受體，是一個難題。發(fā)明人團隊在未有更有效的技術(shù)提示的情況下通過排除法將篩選出4組識別人cd19抗原的人源化多肽(scfv)并通過不同的組合方式構(gòu)建car(嵌合抗原受體)進行篩選。改造多肽的方法，是對申請人已獲得的抗cd19抗體的scfv進行隨機單點或多點突變。方法的結(jié)果存在隨機性，只有在突變得當(dāng)?shù)那闆r下，其才能發(fā)揮功能獲得特異性更好或者親和力和原有小鼠抗體的親和力相當(dāng)?shù)目贵w。car的組合方式多樣，我們將12組不同的car組合方式與我們優(yōu)選的4株人源化單克隆抗體進行隨機組合，本部分保護的car的組合方式，經(jīng)過測試之后，可以起到穩(wěn)定表達于病人來源的t淋巴細胞，并且具有更好的清除腫瘤細胞的能力，用于針對表達cd19的腫瘤的過繼細胞治療。進一步，所述鉸鏈區(qū)的氨基酸序列如seqidno:5所示。進一步，所述跨膜區(qū)為cd8tm或cd28tm；所述cd8tm的氨基酸序列如seqidno:6，所述cd28tm的氨基酸序列如seqidno:7所示。進一步，所述胞內(nèi)信號域為cd28和/或cd137和/或cd3；所述cd28的氨基酸序列如seqidno:8，所述cd137的氨基酸序列如seqidno:9，所述cd3的氨基酸序列如seqidno:10。作為一種優(yōu)選，所述胞內(nèi)信號域依次為cd28、cd3。作為一種優(yōu)選，所述胞內(nèi)信號依次為cd137、cd3。腫瘤細胞表面的抗原(受體)與所述的嵌合抗原受體的抗體(配體)結(jié)合時，通過鉸鏈區(qū)和跨膜區(qū)將信號傳遞至胞內(nèi)，胞內(nèi)信號域?qū)⑿盘栟D(zhuǎn)化為活化信號，激活效應(yīng)細胞，效應(yīng)細胞增殖、產(chǎn)生細胞因子從而殺傷腫瘤細胞。嵌合抗原受體較tcr改造更具優(yōu)勢：(1)特異性：抗體(配體)特異性識別抗原(受體)；(2)效率高：不會出現(xiàn)轉(zhuǎn)基因tcr與患者內(nèi)源性tcr發(fā)生錯配；(3)非mhc-ⅰ限制性：不需要與mhc-ⅰ分子結(jié)合，可克服腫瘤細胞、腫瘤微環(huán)境下調(diào)mhc-ⅰ分子造成的免疫逃逸；(4)抗原選擇范圍廣：抗原可以是糖類、脂類、蛋白。進一步，所述嵌合抗原受體的氨基酸序列如seqidno.11或seqidno.12或seqidno.13或seqidno.14或seqidno.15或seqidno.16或seqidno.17或seqidno.18所示。本發(fā)明的目的之二在于提供一種所述的嵌合抗原受體的慢病毒載體的制備方法，包括以下步驟：1)合成抗人cd19抗原的嵌合抗原受體的基因序列：合成依次含前導(dǎo)肽、抗人cd19抗原的不同的單鏈抗體(scfv)、鉸鏈區(qū)、跨膜區(qū)和胞內(nèi)信號域；所述前導(dǎo)肽核酸序列如seqidno.31所示；2)構(gòu)建表達嵌合抗原受體的慢病毒載體：設(shè)計引物，正向引物的核苷酸序列如seqidno:33所示，反向引物的核苷酸序列如seqidno:34所示，以所述嵌合抗原受體的基因序列為模板進行pcr擴增，得dna片段；將所述dna片段的基因序列用限制性內(nèi)切酶nhei和xhoi雙酶切，同時用限制性內(nèi)切酶nhei和xhoi酶切慢病毒表達載體prrlsin.cppt.ef1a-gfp.wpre，然后將酶切后的目的片段和慢病毒表達載體片段通過t4連接酶進行連接，獲得表達嵌合抗原受體的慢病毒載體。進一步，步驟1)所述嵌合抗原受體的基因序列如seqidno.23或seqidno.24或seqidno.25或seqidno.26或seqidno.27或seqidno.28或seqidno.29或seqidno.30所示。進一步，在步驟2)之后，包裝并純化所述慢病毒載體。本發(fā)明的目的之三在于提供一種所述的制備方法所得的慢病毒載體。在所述的方法下得到所述的慢病毒載體，這樣的慢病毒載體的陽性表達率高，在病人細胞培養(yǎng)過程中很穩(wěn)定，并且不會隨著時間的推移會導(dǎo)致car陽性率下降。于是，用所述慢病毒載體感染的t細胞，這樣的t細胞具備殺傷靶細胞的功能。本發(fā)明的目的還在于提供一種所述的慢病毒載體感染的t細胞。本發(fā)明的目的還在于提供一種所述的t細胞在用于制備b細胞惡性腫瘤的藥物中的應(yīng)用。進一步，所述b細胞惡性腫瘤的細胞或組織能夠表達cd19。cd19是正常和惡性b淋巴細胞特異性表面蛋白，在b細胞的發(fā)育、增殖和分化以及惡性轉(zhuǎn)化中發(fā)揮重要作用。因cd19在b淋巴細胞表達的特異性和惡性腫瘤表達的廣泛性，使其成為一個頗具潛力的b淋巴細胞惡性腫瘤免疫治療的分子靶點。進一步，所述b細胞惡性腫瘤包括急性淋巴細胞白血病(b-all)、慢性b-淋巴細胞白血病(b-cll)，b細胞霍奇金氏淋巴瘤(b-hl)和非霍奇金氏淋巴瘤(b-nhl)。。本發(fā)明的目的還在于提供一種所述識別人cd19抗原的多肽(scfv)的氨基酸序列在用于制備治療b細胞惡性腫瘤的藥物中的應(yīng)用。進一步，所述的b細胞惡性腫瘤的細胞能夠表達cd19。另外，回到發(fā)明的起源端，本發(fā)明的目的還在于提供一種所述識別人cd19抗原的多肽(scfv)的氨基酸序列在用于制備精準捕獲能夠表達cd19的b細胞惡性腫瘤細胞的載體中的應(yīng)用。所述識別人cd19抗原的多肽(scfv)的氨基酸序列如seqidno.1或seqidno.2或seqidno.3或seqidno.4所示。所述改造的多肽片段，其相對于被改造前，更具有更穩(wěn)定、特異性更好的特性。本發(fā)明的目的還在于提供一種seqidno.1或seqidno.2或seqidno.3或seqidno.4所示的氨基酸序列在制備car-t骨架中的抗原識別域的應(yīng)用?？偟膩碚f，本發(fā)明所述的scfv-car嵌合抗原受體在免疫細胞中表達后，不僅可以維持靶向cd19的嵌合抗原受體(chimericantigenreceptor，car)在病人細胞培養(yǎng)過程中的陽性率并且能夠加強car-t的增殖和殺傷腫瘤的能力，并且對抗原陰性的細胞無毒副作用，能夠用于腫瘤的靶向治療。本發(fā)明的有益效果在于：1)本發(fā)明提供的嵌合抗原受體識別抗人cd19抗原，能夠更穩(wěn)定的表達于t淋巴細胞，具有更好的清除腫瘤細胞的能力，不僅可以維持靶向cd19的嵌合抗原受體在病人細胞培養(yǎng)過程中的陽性率并且能夠加強car-t的增殖和殺傷腫瘤的能力，對抗原陰性的細胞無毒副作用，能夠用于腫瘤的靶向治療。2)本發(fā)明提供的通過改造的嵌合抗原受體人源化程度高，可以有效地降低car的免疫原性，增強car-t在體內(nèi)的持續(xù)和安全性。3)本發(fā)明提供的嵌合抗原受體能夠穩(wěn)定表達于t淋巴細胞尤其是病人來源的t淋巴細胞，可以用于制備治療血液系統(tǒng)腫瘤的藥物中針對腫瘤的過繼細胞治療。附圖說明圖1是抗人cd19抗原的人源化單克隆抗體純化圖。圖2是人源化單克隆抗體親和力檢測圖。圖3是人源化單克隆抗體半衰期檢測圖。圖4是靶向cd19的嵌合抗原受體的結(jié)構(gòu)示意圖。圖5是表達靶向cd19嵌合抗原受體的t細胞經(jīng)過12天培養(yǎng)，細胞表型檢測結(jié)果。圖6是表達靶向cd19嵌合抗原受體的t細胞對cd19陽性和陰性腫瘤細胞的殺傷情況。圖7是靶向cd19嵌合抗原受體的t細胞在經(jīng)cd19陽性細胞刺激激活后的細胞因子釋放。圖8是靶向cd19嵌合抗原受體的t細胞在小鼠血液移植瘤模型中對cd19陽性移植瘤的治療情況。圖9是不同刺激信號下靶向cd19嵌合抗原受體的t細胞對荷載人raji-luc腫瘤細胞的小鼠體內(nèi)治療實驗。具體實施方式以下將參照附圖，對本發(fā)明的優(yōu)選實施例進行詳細描述。優(yōu)選實施例中未注明具體條件的實驗方法，通常按照常規(guī)條件，例如分子克隆實驗指南(第三版，j.薩姆布魯克等著)中所述的條件，或按照制造廠商所建議的條件。所舉實施例是為了更好地對本發(fā)明的內(nèi)容進行說明，但并不是本發(fā)明的內(nèi)容僅限于所舉實施例。所以熟悉本領(lǐng)域的技術(shù)人員根據(jù)上述
發(fā)明內(nèi)容對實施方案進行非本質(zhì)的改進和調(diào)整，仍屬于本發(fā)明的保護范圍。對比實施例設(shè)計抗人cd19抗原的人源化單克隆抗體(1)將鼠抗人cd19抗原的單克隆抗體株fmc63輕、重鏈可變區(qū)的cdr區(qū)氨基酸序列經(jīng)過imgt/blast數(shù)據(jù)庫經(jīng)序列分析及比對，選擇同源性最高人抗體序列作為改造模板。(2)經(jīng)過分子對接模擬，與抗原抗體對接關(guān)系最為密切的抗體氨基酸殘基如下所示，對關(guān)鍵殘基以外的骨架序列參考人源抗體骨架區(qū)序列進行人源化修飾改造，而后對人源化抗體進行隨機突變，再以抗原抗體親和力篩選和分子對接模擬的方法進行親和力評估預(yù)測，優(yōu)選得到抗人cd19抗原的人源化單鏈抗體(scfv)氨基酸序列如1、2、3所示，此為申請人前期實驗成果，專利201710301492.1。為了更進一步的獲得穩(wěn)定性更好，特異性強，在人體內(nèi)不會失控增殖安全性好的cart細胞，獲得具有更好的清除腫瘤細胞能力的嵌合抗原受體，申請人團隊對已申報專利201710301492.1的人源化序列進行了隨機點突變，通過再改造的方式獲得穩(wěn)定性好，特異性強的人源化單克隆抗體來用于car的組合優(yōu)化，進行臨床的腫瘤靶向治療。本發(fā)明實施例實施例1、篩選抗人cd19抗原的人源化單克隆抗體對鼠源抗cd19抗體fmc635人源化骨架替換通過篩選獲得已申報專利201710301492.1的人源化序列(第一部分)，通過對其進行隨機突變設(shè)計人源化igg抗體分子，如表1所展示的單克隆抗體隨機突變結(jié)果。表1實施例2、靶向人cd19抗原的人源化單克隆抗體純化構(gòu)建穩(wěn)定表達人源化抗體的穩(wěn)轉(zhuǎn)細胞株，將細胞上清收獲液過0.45um濾膜，取濾液。用等體積的平衡緩沖液稀釋，測ph值。用aktaprime純化具體步驟見ge產(chǎn)品手冊，超濾濃縮，完畢后用0.22μm針頭濾器除菌過濾，分裝，做好標識放入-80℃冰箱中保存。取樣，檢測。利用sds和westernblot檢測，具體步驟見ge抗體純化手冊。檢測結(jié)果如圖1所示優(yōu)選到四株單克隆抗體得到純度較高的抗體，分別為scfv-humanized4氨基酸序列seqidno.1，scfv-humanized5氨基酸序列seqidno.2,scfv-humanized9氨基酸序列seqidno.3，scfv-humanized11氨基酸序列seqidno.4。實施例3、靶向人cd19抗原的人源化單克隆抗體特異性檢測單克隆抗體scfv標記有his標簽后克隆進質(zhì)粒載體并轉(zhuǎn)染hek293細胞，將細胞上清收獲液過0.45um濾膜，取濾液。用等體積的平衡緩沖液稀釋，測ph值。超濾濃縮后用0.22μm針頭濾器除菌過濾，分裝，做好標識放入-80℃冰箱中保存。分別取不同的人源化抗體與cd19陽性細胞raji和cd19陰性細胞k562共孵育30min后洗去多余抗體，加入fitc-his抗體染色30min后洗去未標記抗體，進行流式檢測，檢測結(jié)果如表2所示有四株單克隆抗體能夠識別陽性細胞raji并且不識別陰性細胞k562，分別為scfv-humanized4氨基酸序列seqidno.1，陽性細胞檢測率90.82％，陰性細胞檢測率0.7％；scfv-humanized5氨基酸序列seqidno.2,陽性細胞檢測率84.04％，陰性細胞檢測率0.5％；scfv-humanized9氨基酸序列seqidno.3，陽性細胞檢測率87.75％，陰性細胞檢測率0.4％；scfv-humanized11氨基酸序列seqidno.4，陽性細胞檢測率80.60％，陰性細胞檢測率0.19％。對照為商業(yè)化抗體，陽性細胞檢測率91.47％，陰性細胞檢測率0.88％。表2實施例4、靶向cd19人源化單克隆抗體穩(wěn)定性測定1)室溫條件下抗體親和力檢測優(yōu)選的單克隆抗體scfv標記有his標簽后克隆進質(zhì)粒載體并轉(zhuǎn)染hek293細胞，純化scfv-his，scfv-his和陰性對照用pbs稀釋6個梯度，分別孵育cd19表達陽性的raji細胞，流式檢測濃度梯度下rajicd19檢測的陽性率，進行流式陽性率及mfi統(tǒng)計，對不同scfv于cd19的親和力進行分析。實驗進行了三個獨立重復(fù)，結(jié)果如圖2和表3所示，其中kd(nm)越大親和力越小。表3humanized4humanized5humanized9humanized11affinity1.993ug/ml6.216ug/ml4.757ug/ml18.496ug/mlwm(kda)27.2927.3227.3127.30kd(nm)73.0304227.5256174.1853677.27942)抗體半衰期檢測：表達cd19的腫瘤細胞分為4組分別加入獲得的4種scfv，每組設(shè)置5個梯度濃度，分別4℃孵育1小時離心去除未結(jié)合抗體，37℃進行抗體解離，檢測解離15分鐘、30分鐘、45分鐘以及60分鐘后抗體的活性。結(jié)果如圖3和表4所示，其中解離率反應(yīng)抗體半衰期，解離率絕對值越小抗體半衰期越長。表4humanized4humanized5humanized9humanized11解離率(mfi)-4.138±0.2746-2.744±0.2476-0.593±0.0809-0.976±0.2076實施例5、表達靶向人cd19抗原的嵌合抗原受體的慢病毒制備(1)制備靶向人cd19抗原的嵌合抗原受體的基因序列合成依次含前導(dǎo)肽(又稱信號肽)、抗人cd19抗原的單鏈抗體scfv，hfc鉸鏈區(qū)、跨膜區(qū)和胞內(nèi)信號段的嵌合抗原受體序列，其結(jié)構(gòu)如圖4所示。其中前導(dǎo)肽的核苷酸序列如seqidno.31所示；抗人cd19抗原的人源化單鏈抗體核苷酸序列如seqidno.19、seqidno.20、seqidno.21、seqidno.22所示；hfc鉸鏈區(qū)的氨基酸序列如seqidno.5；跨膜區(qū)cd8tm或cd28tm氨基酸序列如seqidno.6、seqidno.7所示；胞內(nèi)信號段cd28、cd137及cd3的氨基酸序列如seqidno.8、seqidno.9、seqidno.10所示。最終合成的人源化抗cd19嵌合抗原受體核苷酸序列如seqidno.23、seqidno.24、seqidno.25、seqidno.26、seqidno.27、seqidno.28、seqidno.29、seqidno.30所示，氨基酸序列如seqidno.11、seqidno.12、seqidno.13、seqidno.14、seqidno.15、seqidno.16、seqidno.17、seqidno.18、所示；未做人源化的鼠抗對照抗體命名為mcd19。pcr擴增，反應(yīng)體系按kodfxneodna聚合酶(購自toyobo公司)說明書操作。然后用回收試劑盒(promega公司)進行dna片段回收，具體方法見說明書，回收獲得嵌合抗原受體，將dna回收片段送南京金斯瑞生物科技公司測序。(2)構(gòu)建表達嵌合抗原受體的慢病毒載體根據(jù)裝載scfv的不同，將嵌合抗原受體表達載體分別簡稱為pcarm19、pcarm20、pcar011、pcar012、pcar013、pcar014、pcar015、pcar016、pcar017、pcar018、pcar019、pcar020、pcar021、pcar022、pcar023、pcar024、pcar025、pcar026慢病毒載體。其中pcarm19和pcarm20為鼠源對照，pcar003氨基酸序列如seqidno.32為申請人已有人源化抗體組合的cart細胞作為對照。酶切反應(yīng)按說明書進行。酶切產(chǎn)物經(jīng)瓊脂糖凝膠電泳分離后用瓊脂糖凝膠dna片段回收試劑盒進行dna片段回收，然后將目的片段和載體片段通過t4連接酶(購自promega公司)進行連接，獲得表達嵌合抗原受體的慢病毒載體，結(jié)構(gòu)如圖4所示4種car結(jié)構(gòu)與4種人源化抗體組合共計16種組合。質(zhì)粒抽提試劑盒(invitrogen公司)抽提質(zhì)粒，具體方法見說明書。實施例6、cd19抗原的嵌合抗原受體修飾t細胞的制備(1)慢病毒的包裝本實施例包裝慢病毒采用磷酸鈣法，具體步驟見分子克隆實驗指南(第三版，j.薩姆布魯克等著)。(2)慢病毒的純化收集病毒上清，離心過濾后轉(zhuǎn)移至新的離心管；根據(jù)病毒上清量，分別加入50％peg6000(w/v)、4mnacl，再用醫(yī)用鹽水定容到peg6000終濃度為8.5％、nacl終濃度為0.3m，4℃冰箱靜置；樣品于4℃、5000r/min條件下離心30min，棄盡上清，用200μl含10％fbs的dmem培養(yǎng)基重懸病毒，1.5mlep管分裝，-80℃保存?zhèn)溆谩?3)慢病毒滴度測定病毒感染293t細胞，感染72h后于1000r/min條件下離心5min以收集細胞，用qiaampdnabloodminikit購于qiagen公司(貨號511004)基因組抽提試劑盒抽提基因組。按試劑盒說明書操作。qrt-pcr測定病毒滴度用分析軟件分析數(shù)據(jù)，根據(jù)標準曲線計算病毒滴度，結(jié)果用tu/ml表示。(4)慢病毒感染t細胞1)人外周血單核細胞的分離用加有抗凝劑的采血管采集外周血約60ml，分裝于50ml離心管各30ml，加入7.5ml羥乙基淀粉稀釋；室溫(18～25℃)自然沉降約30min，收集上層血漿，離心15min；然后用生理鹽水重懸沉淀，按體積比為1:1加到淋巴細胞分離液上，梯度離心，離心20min；離心后，取第二層白色淋巴細胞層，并用生理鹽水洗滌2次，生理鹽水重懸細胞，加入含有10％fbs的rpmi1640完全培養(yǎng)基培養(yǎng)，得人外周血單核細胞。2)慢病毒載體感染t淋巴細胞用含10％fbs的rpmi1640完全培養(yǎng)基培養(yǎng)新制備的單個核細胞pbmc，抗cd3單克隆抗體活化后進行慢病毒感染；分別加入慢病毒載體，未感染的外周血淋巴細胞(pbmc)作為空白對照；24h后將培養(yǎng)基更換為含有500iu/ml重組人il-2的rpmi1640完全培養(yǎng)基，繼續(xù)培養(yǎng)10-20天。獲得的嵌合抗原受體t細胞命名pcarm19、pcarm20、pcar011、pcar012、pcar013、pcar014、pcar015、pcar016、pcar017、pcar018、pcar019、pcar020、pcar021、pcar022、pcar023、pcar024、pcar025、pcar026、pcar003。3)靶向人cd19抗原的嵌合抗原受體(car)表達檢測在培養(yǎng)過程中對培養(yǎng)至10天的已感染病毒的t細胞，300g/min，離心5min，棄盡上清以收集細胞；將細胞調(diào)整密度為1×106個/ml；將收集的細胞分別分裝利用流式細胞術(shù)檢測protein-l陽性率，為了方便隨時監(jiān)控car在t細胞表面表達的陽性率，car基因前啟動子后設(shè)計有protein-l共表達，檢測結(jié)果即表示培養(yǎng)第4天、10天不同cd19-car組合在t淋巴細胞表達陽性率。結(jié)果如表2所示不同病毒感染t細胞后car陽性率，其中pcar019、pcar020、pcar021、pcar022、pcar023、pcar024、pcar025、pcar026感染效率高，進一步以pcarm19、pcarm20為對照對培養(yǎng)至4天、10天的已感染pcar019、pcar020、pcar021、pcar022、pcar023、pcar024、pcar025、pcar026病毒的t細胞進行陽性率檢測，結(jié)果如圖5和表5所示，t細胞pcar021、pcar022、pcar025感染效率好并且隨著培養(yǎng)時間car陽性率穩(wěn)定自激活概率小。表5實施例7、表達靶向cd19的嵌合抗原受體的t淋巴細胞抗腫瘤效果驗證以穩(wěn)定表達螢火蟲熒光素酶的cd19陽性raji細胞(簡稱為raji-luc)以及穩(wěn)定表達螢火蟲熒光素酶及人cd19抗原的k562細胞(簡稱為k562-hcd19-luc)作為靶細胞，按照1:1效靶比鋪效應(yīng)細胞。使用luciferaseassaysystem(promegacat.#e2520)試劑盒提供的標準方法檢測殺傷效果，殺傷率用下列公式計算：殺傷結(jié)果如圖6a/b和表6所示，結(jié)果表明優(yōu)選的人源化單克隆抗體scfv組合的cart淋巴細胞對cd19陽性腫瘤細胞具有顯著殺傷作用。表6實施例8、表達靶向cd19的嵌合抗原受體的t淋巴細胞細胞因子分泌能力檢測細胞因子ifn-γ檢測采用elisa的方法，使用bd公司試劑盒進行。檢測試劑盒貨號：555142，生產(chǎn)批號6266958，具體步驟見試劑盒說明書。測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度(od值)，測定應(yīng)在加終止液后15分鐘以內(nèi)進行。結(jié)果如圖7和表7所示，顯示效靶比為1:1car-t細胞殺傷靶細胞24小時后ifn-γ的分泌。優(yōu)選的人源化單克隆抗體scfv組合的cart共刺激信號均能正常工作，分泌ifn-γ。通過比較不同car-t細胞的殺傷效果，發(fā)現(xiàn)優(yōu)選的單克隆抗體scfv組合的cart細胞pcar019、pcar020、pcar021、pcar022、pcar023、pcar024、pcar025、pcar026殺傷活性強于位選擇的8種cart組合，對于cd19陽性細胞殺傷效果顯著。表7實施例9、表達靶向cd19的嵌合抗原受體的t淋巴細胞在動物模型中的抗腫瘤效果驗證建立人cd19陽性腫瘤細胞系的小鼠移植瘤模型用于驗證表達靶向cd19的嵌合抗原受體的t淋巴細胞在動物模型中的抗腫瘤效果。體內(nèi)驗證使用小鼠為nod.cg-prkdcscidii2rgtm1sug/jiccrl，簡稱nog小鼠，由日本實驗動物研究所(ciea)的mamoruito培育而成，為國際上car-t體內(nèi)相關(guān)成瘤實驗的最常見品系。體內(nèi)驗證使用的成瘤靶向細胞為前期體外驗證使用的穩(wěn)定表達螢火蟲熒光素酶的cd19陽性細胞系raji(簡稱raji-luc)。治療注射的效應(yīng)細胞為慢病毒載體pcar019、pcar020、pcar021、pcar022、pcar023、pcar024、pcar025、pcar026感染的car-t細胞，對照為生理鹽水組、未感染病毒的pbmc細胞以及未改造的fmc635抗體scfv組合的car-t細胞pcarm19和pcarm20；已申報專利201710301492.1中獲得的單克隆抗體改造的car命名為pcar003作為體內(nèi)殺傷效果對照。成瘤后3d尾靜脈注射效應(yīng)car-t細胞5*10^5細胞/只鼠。注射cart細胞后每隔7天通過perkinelmer公司的ivs活體成像系統(tǒng)拍照成像，顯示腫瘤生長情況。期間每天觀察小鼠存活情況并記錄，結(jié)果見表8和表9所示pcar021、pcar022、pcar023、pcar025、pcar026相較于鼠源對照pcarm19、pcarm20和改造前抗體組合的carpcar003顯示了更好的治療效果，熒光值越小存活小鼠越多治療效果越好。表8平均值2d9d16d23d30d37d44d分組存活291623303744salin0/63.27e+061.23e+091.32e+10controlt0/72.78e+069.51e+088.15e+09pcarm190/77.74e+051.84e+085.04e+095.03e+09pcar0031/73.14e+061.59e+072.72e+083.36e+094.77e+094.56e+08pcar0191/72.87e+061.14e+071.95e+081.03e+095.97e+093.94e+09pcar0201/72.84e+063.19e+061.86e+073.05e+086.67e+082.04e+09pcar0214/73.12e+062.48e+068.32e+062.01e+071.56e+096.64e+082.05e+09pcar0222/53.40e+064.88e+063.67e+072.91e+072.12e+092.20e+093.40e+09表9平均值7d21d35d49d63d77d分組存活72135496377salin0/71.30e+061.49e+084.98e+092.08e+08pcarm201/71.29e+068.15e+084.28e+091.51e+105.13e+081.42e+09pcar0235/79.19e+056.66e+065.35e+072.24e+089.13e+083.98e+09pcar0241/71.44e+061.42e+083.77e+097.85e+091.37e+104.70e+07pcar0256/77.97e+052.70e+067.21e+073.39e+091.28e+102.94e+09pcar0263/61.35e+061.52e+082.07e+096.01e+083.35e+096.47e+09成像結(jié)果及小鼠生存曲線如圖8和圖9所示，結(jié)果顯示pcar021、pcar022、pcar023和pcar025顯示出優(yōu)于其他組別的良好抗腫瘤效果，實驗小鼠在30d時全部存活。綜合以上結(jié)果，相對未做人源化改造的鼠抗人cd19嵌合抗原受體t細胞，應(yīng)用本發(fā)明所述的人源化改造方案得到的抗人cd19抗原的嵌合抗原受體t細胞在體外細胞實驗中殺傷特異性更好，而在體內(nèi)動物實驗中呈現(xiàn)出更好的治療效果，尤其是第二次優(yōu)化后獲得的人源化抗體scfv-humanized4氨基酸序列seqidno.1，scfv-humanized9氨基酸序列seqidno.3和scfv-humanized11氨基酸序列seqidno.4組合的pcar021、pcar022、pcar023、pcar025和pcar026氨基酸序列分別為seqidno.13或seqidno.14或seqidno.15或seqidno.17或seqidno.18所示在動物體內(nèi)具有很好地治療效果，我們的pcar系列嵌合抗原受體具有臨床上治療兒童和成人復(fù)發(fā)b細胞急性淋巴細胞白血病(b-all)，慢性淋巴細胞白血病(cll)，和b細胞非霍奇金淋巴瘤(b-nhl)等cd19陽性血液系統(tǒng)腫瘤的應(yīng)用價值。最后說明的是，以上實施例僅用以說明本發(fā)明的技術(shù)方案而非限制，盡管參照較佳實施例對本發(fā)明進行了詳細說明，本領(lǐng)域的普通技術(shù)人員應(yīng)當(dāng)理解，可以對本發(fā)明的技術(shù)方案進行修改或者等同替換，而不脫離本發(fā)明技術(shù)方案的宗旨和范圍，其均應(yīng)涵蓋在本發(fā)明的權(quán)利要求范圍當(dāng)中。<110>重慶精準生物技術(shù)有限公司<120>抗人cd19抗原的嵌合抗原受體及其應(yīng)用<160>34<170>patentinversion3.3<210>1<211>245<212>prt<213>artificial<220><223>scfv-humanized4<400>1aspileglnmetthrglnserproserserleuseralaserval51015glyaspargvalthrilethrcysargalaserglnaspileser202530lystyrleuasntrptyrglnglnlysproglylysalaproarg354045leuleuiletyrhisthrserargleuhisserglyvalproser505560argpheserglyserglyserglythrasptyrthrleuthrile657075serserleuglnprogluaspphealathrtyrtyrcysglngln808590glyasnthrleuprotyrthrpheglyglyglythrargleuglu95100105ilelysglyserthrserglyserglylysproglyserglyglu110115120glyserthrlysglyglnvalglnleuglngluserglyprogly125130135leuvallysproserglnthrleuserleuthrcysthrvalser140145150glyvalserleuproasptyrglyvalsertrpileargglnpro155160165proglylysalaleuglutrpleuglyvaliletrpglyserglu170175180thrthrtyrtyrasnseralaleulysthrargleuthrileser185190195lysaspasnserlysasnglnvalvalleuthrmetthrasnmet200205210aspprovalaspthralathrtyrtyrcysalalyshistyrtyr215220225tyrglyglysertyralametasptyrtrpglyglnglyserser230235240valthrvalserser245<210>2<211>245<212>prt<213>artificial<220><223>scfv-humanized5<400>2aspileglnmetthrglnserproserserleuseralaserval51015glyaspargvalthrilethrcysargalaserglnaspileser202530lystyrleuasntrptyrglnglnlysproglylysalaproarg354045leuleuiletyrhisthrserargleuhisserglyvalproser505560argpheserglyserglyserglythrasptyrthrleuthrile657075serserleuglnprogluaspphealathrtyrtyrcysglngln808590glyasnthrleuprotyrthrpheglyglyglythrargleuglu95100105ilelysglyserthrserglyserglylysproglyserglyglu110115120glyserthrlysglyglnvalglnleuglngluserglyprogly125130135leuvallysproserglnthrleuserleuthrcysthrvalser140145150glyvalserleuproasptyrgl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