TWI817350B - 與Globo系列抗原結合之嵌合抗原受體及其用途 - Google Patents
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- TWI817350B TWI817350B TW111104664A TW111104664A TWI817350B TW I817350 B TWI817350 B TW I817350B TW 111104664 A TW111104664 A TW 111104664A TW 111104664 A TW111104664 A TW 111104664A TW I817350 B TWI817350 B TW I817350B
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- antigen receptor
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Abstract
本發明係關於與Globo系列抗原(例如Globo H、SSEA-3或SSEA-4)結合的嵌合抗原受體(CAR),其包含一抗原結合片段(Fab)或一單鏈變異區片段(scFv)。 此外,本發明亦提供對一個體施用在抑制癌細胞上有效量的嵌合抗原受體的方法。
Description
本申請案主張於2021年2月9日提交的美國臨時申請案第63/147,237號及於2021年2月9日提交的美國臨時申請案第63/147,441號的優先權,其內容透過引用全部併入本文。
本發明係關於與Globo系列抗原(例如Globo H、SSEA-3或SSEA-4)結合的嵌合抗原受體(chimeric antigen receptors,CARs),其包含一抗原結合片段(antigen-binding fragment,Fab)或一單鏈變異區片段(single-chain variable fragment,scFv)。此外,本發明亦提供對一個體施用在抑制癌細胞上有效量的嵌合抗原受體的方法。
惡性腫瘤細胞表現了許多表面醣類。例如,Globo H (Fucα1→2Galß1→3N-GalNAcß1→3Galα1→4Galß1→4Glc)已被證實在多種上皮癌過度表現,並且與乳癌及小細胞肺癌的腫瘤侵襲性與不良預後有關。先前研究顯示,在乳癌細胞及乳癌幹細胞上觀察到Globo H及階段特異性胚胎抗原3 (stage-specific embryonic antigen-3,Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1)(SSEA-3,亦稱Gb5)(Chang WW
et al., (2008) PNAS, 105(33):11667-11672;Cheung SK
et al.,(2016) PNAS, 113(4):960-965)。此外,SSEA-4 (stage-specific embryonic antigen-4 (階段特異性胚胎抗原4))(Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1)已被普遍用作人類多能胚胎幹細胞的細胞表面標記,並被用於分離間質幹細胞和濃縮神經前驅細胞(Kannagi R
et al., (1983) EMBO J, 2:2355-2361)。這些發現顯示Globo系列抗原(Globo H、SSEA-3及SSEA-4)是癌症治療的獨特標靶,且可用於有效引導治療劑靶向癌細胞。
嵌合抗原受體(CAR)是被設計為賦予T細胞靶向一特定蛋白質之新能力的受體蛋白。嵌合抗原受體作為合成受體,可重新指定T細胞的特異性、功能及代謝。嵌合抗原受體係由一T細胞活化區(T-cell activating domain,通常包括CD3複合體的zeta鏈)及用以指定特異性之衍生自免疫球蛋白的細胞外重鏈和輕鏈所組成(June CH and Sadelain M., (2018) N Engl J Med, 379(1):64-73)。將嵌合抗原受體導入T細胞使得該些作用細胞能透過單鏈變異區片段(scFv)識別腫瘤相關抗原(tumor associated antigens,TAAs),並且能透過細胞內訊息傳導區(cytoplasmic signaling domains)活化T細胞,釋放穿孔蛋白(perforin)、顆粒酶(granzyme)及多種細胞激素(cytokines)以發揮強力的抗腫瘤效用。因此,嵌合抗原受體T細胞(CAR T cells)以非受限於主要組織相容複合體(MHC)的方式發揮作用,其巧妙地將細胞毒性T細胞的強大腫瘤毒殺力和抗體的特異性抗原識別力結合在一起。與單株抗體(monoclonal antibody,mAb)療法相比,CAR T細胞療法在產生持久的腫瘤反應方面更為有效,並且對實體瘤具有更強的穿透力及更低的抗藥性風險(Han Y
et al., (2018) Am J Cancer Res, 8(1):106-119)。
鑒於有效治療及/或預防癌症的需求尚未被滿足,前述發現成為開發Globo系列抗原之嵌合抗原受體的理由。為滿足此類及其他需求,本發明提供針對Globo系列抗原的嵌合抗原受體。
本發明係關於嵌合抗原受體(CAR),其包含(1)一第一胞內域(endodomain)及一單鏈變異區片段(scFv),或(2)一第一胞內域及一抗原結合片段(Fab)。進一步地,該第一胞內域包含CD3 zeta鏈(CD3zeta)或免疫球蛋白E受體I型γ鏈(FcɛRIγ),且該單鏈變異區片段或該抗原結合片段識別Globo系列抗原。此外,當該嵌合抗原受體包含該第一胞內域及該單鏈變異區片段時,該嵌合抗原受體包含與SEQ ID NO:14、16或18有80%至100%序列同一性的一胺基酸序列,而當該嵌合抗原受體包含該第一胞內域及該抗原結合片段時,該嵌合抗原受體具有與SEQ ID NO:13、15或17有80%至100%序列同一性的一胺基酸序列。
在一些實施例中,本發明提供一種嵌合抗原受體(CAR),其包含與Globo H特異性結合的一單鏈變異區片段(scFv),且該嵌合抗原受體具有一胺基酸序列,係與SEQ ID NO:14、16或18的序列同一性為至少約80%、至少約81%、至少約82%、至少約83%、至少約84%、至少約85%。至少約86%,至少約87%、至少約88%、至少約89%、至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%、至少約99%或約100%。
在一些實施例中,本發明提供一種嵌合抗原受體(CAR),其包含與Globo H特異性結合的一抗原結合片段(Fab),且該嵌合抗原受體具有一胺基酸序列,係與SEQ ID NO:13、15或17的序列同一性為至少約80%、至少約81%、至少約82%、至少約83%、至少約84%、至少約85%、至少約86%、至少約87%、至少約88%、至少約89%、至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%、至少約99%或約100%。
在一些實施例中,該嵌合抗原受體進一步包含一第二胞內域,該第二胞內域包含CD28、CD137、CD4、OX40、4-1BB、CD3Z或可誘導T細胞共刺激分子(ICOS),其中該單鏈變異區片段與該第二胞內域融合,並且該第二胞內域與該第一胞內域融合。
在一些實施例中,該單鏈變異區片段包含一胺基酸序列,該胺基酸序列與SEQ ID NO:3或6的序列同一性為至少約80%、至少約81%、至少約82%、至少約83%、至少約84%、至少約85%、至少約86%、至少約87%、至少約88%、至少約89%、至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%、至少約99%或約100%。
在一些實施例中,該嵌合抗原受體(CAR)包含一重鏈可變區(variable heavy chain region,V
H),該重鏈可變區所具有的一胺基酸序列係與SEQ ID NO:1或4的序列同一性為至少約80%、至少約81%、至少約82%、至少約83%、至少約84%、至少約85%、至少約86%、至少約87%、至少約88%、至少約89%、至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%、至少約99%或約100%。
在一些實施例中,該嵌合抗原受體(CAR)包含一輕鏈可變區(variable light chain region,V
L),該輕鏈可變區所具有的一胺基酸序列係與SEQ ID NO:2或5的序列同一性為至少約80%、至少約81%、至少約82%、至少約83%、至少約84%、至少約85%、至少約86%、至少約87%、至少約88%、至少約89%、至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%、至少約99%或約100%。
在一些實施例中,該嵌合抗原受體(CAR)包含一鉸鏈區(hinge region),該鉸鏈區所具有的一胺基酸序列係與SEQ ID NO:7的序列同一性為至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%、至少約99%或約100%。
在一些實施例中,該鉸鏈區是CD8的鉸鏈區。
在一些實施例中,該嵌合抗原受體(CAR)包含一CD28區,該CD28區所具有的一胺基酸序列係與SEQ ID NO:8的序列同一性為至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%、至少約99%或約100%。
在一些實施例中,該嵌合抗原受體(CAR)包含一4-1BB區,該4-1BB區所具有的一胺基酸序列係與SEQ ID NO:9的序列同一性為至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%、至少約99%或約100%。
在一些實施例中,該嵌合抗原受體(CAR)包含一CD3zeta (CD3z) 區,該CD3zeta區所具有的一胺基酸序列係與SEQ ID NO:10或11的序列同一性為至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、 至少約96%、至少約97%、至少約98%、至少約99%或約100%。
在一些實施例中,該嵌合抗原受體係結合至Globo系列抗原。此外,該Globo系列抗原係選自由Globo H、階段特異性胚胎抗原3 (stage-specific embryonic antigen 3,SSEA-3)及階段特異性胚胎抗原4 (stage-specific embryonic antigen 4,SSEA-4)所組成的群組。
此外,本發明亦提供一種治療患有腫瘤的個體的方法,該方法包含:
(A)從患有該腫瘤的該個體獲得T細胞;
(B)利用包含編碼一嵌合抗原受體(CAR)的一核酸序列的一載體轉導該T細胞,以產生表現該嵌合抗原受體的T細胞(CAR-T細胞);
(C)擴增該CAR-T細胞;及
(D)將該擴增的CAR-T細胞注入該個體內以引起免疫反應。此外,該嵌合抗原受體包含一單鏈變異區片段(scFv)或一抗原結合片段(Fab)。該單鏈變異區片段或抗原結合片段能識別Globo系列抗原。此外,當該嵌合抗原受體包含該單鏈變異區片段時,該嵌合抗原受體包含與SEQ ID NO:14、16或18具有80%至100%序列同一性的一胺基酸序列。當該嵌合抗原受體包含該抗原結合片段時,該嵌合抗原受體具有與SEQ ID NO:13、15或17具有80%至100%序列同一性的一胺基酸序列。
在一些實施例中,該個體為人類。
在一些實施例中,該免疫反應係由T細胞介導。
在一些實施例中,該載體包含一慢病毒(lentivirus)、一伽瑪反轉錄病毒(gamma retrovirus)或一腺相關病毒(adeno-associated virus)。
在一些實施例中,表現Globo系列抗原(例如 Globo H)的癌症包括但不限於肉瘤(sarcoma)、皮膚癌、白血病、淋巴癌、腦癌、膠質母細胞瘤、肺癌、乳癌、口腔癌、頭頸癌、鼻咽癌、食管癌、胃癌、肝癌、膽管癌、膽囊癌、膀胱癌、胰臟癌、腸癌、大腸癌、腎癌、宮頸癌、子宮內膜癌、卵巢癌、口腔癌、口咽癌、喉癌、食道癌、直腸癌、膽管癌、宮頸癌、睾丸癌、神經內分泌癌、腎上腺癌、甲狀腺癌、骨癌、基底細胞癌、鱗狀細胞癌、黑色素瘤及前列腺癌。
在一些實施例中,例示的2C2抗體(抗Globo H單株抗體)如同PCT專利公開文獻(WO2015157629A2及WO2017062792A1)所描述,其內容透過引用全部納入本文。
在一些實施例中,例示的R783抗體(抗Globo H單株抗體)如同美國臨時申請案第63/147,441號所述。
本發明還提供一種抑制表現Globo H的癌細胞的方法,包含向一有此需要的個體施用有效量的本文所述的抗體或其抗原結合部分,其中該表現Globo H的癌細胞被抑制。
本發明亦涵蓋嵌合抗原受體(CAR)的數個胺基酸序列,包含與一醣類抗原例如Globo系列抗原特異性結合的一抗原結合片段(Fab)或一單鏈變異區片段(scFv)。在一實施例中,該醣類抗原係為SSEA-3。在另一實施例中,該醣類抗原係為SSEA-4。在另一實施例中,該醣類抗原係為Globo H。
本文之嵌合抗原受體(CAR)及方法可用於所有脊椎動物,例如哺乳動物和非哺乳動物,包括人類、小鼠、大鼠、豚鼠、倉鼠、狗、貓、牛、馬、山羊、綿羊、豬、猴子、猿類、大猩猩、黑猩猩、兔子、鴨、鵝、雞、兩棲動物、爬蟲類及其他動物。
本文中所用的冠詞「一」及「一種」係指一個或多於一個(即至少一個)該冠詞在文法上的受詞。舉例來說,「一元件」係指一個元件或多於一個元件。
本文中所用的「有效量」係指足以減輕癌症的症狀和跡象的疫苗或醫藥組成物的劑量,該癌症的症狀和跡象例如體重減輕、疼痛及可觸摸到的腫塊,該腫塊是可被檢測的,不論是臨床上可觸摸到或者透過各種成像方法可被放射學檢測到。術語「有效量」及「治療有效量」可互換使用。
術語「個體(subject)」可以指患有癌症的一脊椎動物或被認為需要癌症治療的一脊椎動物。個體包括所有溫血動物,例如哺乳動物,例如靈長類動物,及較佳地為人類。個體亦可以是非人靈長類動物。術語「個體」包括家養動物,如貓、狗等,家畜(例如牛、馬、豬、綿羊、山羊等)及實驗室動物(例如小鼠、兔、大鼠、沙鼠、豚鼠等)。因此,獸醫用及醫用製劑在本文的考量範疇。
以下關於實施本發明的具體層面的例子僅用於說明目的,而非意圖以任何方式限制本發明的範圍。
實施例
實施例1. Globo H CAR-T細胞的製備
抗Globo H抗體2C2或R783被用於製備scFv CAR或Fab CAR構築體。為製備第三代嵌合抗原受體,胞內域(intracellular domain)包含CD28、4-1BB及CD3zeta。CD3zeta突變體係一具有較低酪胺酸激酶(tyrosine kinase)活性以產生持久性CAR的突變區。圖1列出Globo-H CAR的示意圖。使用人類泛T細胞分離試劑組(Miltenyi Biotec,Cat. No.130-096-535)從低溫保存的人類外周血單核細胞(PBMCs)中分離出人類T細胞。在添加10% FBS及重組人類IL-7 (10 ng/mL;PeproTech,Cat. No. 200-07)和IL-15 (10 ng/mL;PeproTech,Cat. No. 200-15)的RPMI-1640培養基中,以1:1之細胞/磁珠比例使用人類CD3/CD28 T細胞活化磁珠(Dynabeads Human T-Activator CD3/CD28;Thermo Fisher,Cat. No. 11131D)活化及擴增泛T細胞。活化2天後,用1 MOI的慢病毒轉導T細胞以表現CAR。3天後,從轉導的T細胞中移除磁珠,並且在細胞密度為0.5-1×10
6/mL的情況下每2-3天更換含IL-7/IL-15(各為10 ng/mL)的培養基。在轉導後的第10-14天,收集細胞用於體外及體內實驗。在用於體外實驗前,T細胞係在不含IL-7和IL-15的培養基中培養一天。
實施例2. Globo H CAR-T細胞的體外細胞毒性試驗
檢測Globo H CAR-T細胞對健康供體中的Globo-H陽性腫瘤細胞株(MCF-7及HCC-1428:乳癌;NCI-N87:胃癌;SW480:結腸癌)及Globo-H陰性腫瘤細胞株(SK-OV-3:卵巢癌)在連續E:T比(即作用CAR-T細胞:目標腫瘤細胞)下的細胞毒性。穩定表現冷光素酶(luciferase)的目標細胞預先附著於96孔聚苯乙烯白盤進行隔夜培養。將去除IL-7和IL-15的CAR-T細胞的連續稀釋品加入目標細胞中進行24小時共同培養後,讀取冷光值。用Bio-Glo冷光素酶檢測系統(Bio-Glo Luciferase Assay System;Promega,Cat. No. G7940)檢測冷光訊號。
圖2顯示2C2-Fab CAR T細胞、2C2-scFv CAR T細胞、R783-Fab CAR T細胞及R783-scFv CAR T細胞對Globo-H陽性腫瘤細胞株具有療效(圖2A-2D),但此療效未見於Globo-H陰性腫瘤細胞中(圖2E),這同時證明了Globo-H CAR-T構築體的特異性。圖3亦顯示2C2-Fab CAR T細胞、2C2-Fab (CD3zm) CAR T細胞、R783-scFv CAR T細胞及R783-scFv (CD3zm) CAR T細胞對Globo-H陽性腫瘤細胞株具有相似的療效(圖3A-3C),但此療效未見於Globo-H陰性腫瘤細胞株中(圖3D),這可以證明CD3zeta突變體(CD3zm) CAR沒有喪失細胞毒性。
此外,進行了另一項體外持久性試驗。將腫瘤細胞株(1×10
5個MCF-7或SK-OV3)以CellTracker
TM深紅色染料(CellTracker
TMDeep Red Dye;Thermo Fisher,Cat. No.C34565)在37℃下預染15分鐘,然後植入24孔組織培養盤中16小時,其後,將1×10
5個CAR T細胞加入腫瘤細胞(E:T = 1:1)。3天後,腫瘤細胞被完全消除(第一輪)。收集孔中的所有細胞,用PBS清洗,重新懸浮於新鮮的2% FBS RPMI-1640培養基中,而後加入已植入1×10
5個腫瘤細胞的一個新培養盤進行3天培養(第二輪)。若可能,將此過程再重複一次(第三輪)。在每一輪結束時,用CountBright
TM絕對計數微珠(CountBright
TMAbsolute Counting Beads;Thermo Fisher,Cat. No. C36950)透過流式細胞儀計算殘餘腫瘤細胞(APC
+)和CAR T細胞(CD3
+)的絕對細胞數。
圖4顯示CD3zm CAR T細胞具有更高的細胞毒性(圖4A),並且在重複毒殺試驗後有更多的CAR T細胞存留(圖4B),這可以證明帶有CD3zeta突變體(CD3zm)的2C2-Fab CAR T細胞相比帶有野生型CD3zeta (CD3z)者更持久。
實施例3. Globo H CAR-T細胞的體內療效試驗
實施例3-1. NCI-N87胃癌異種移植模型
6至8週大的ASID小鼠(NOD.Cg-Prkdc
scidIl2rg
tm1Wjl/YckNarl)係購自國家實驗動物中心(臺北,臺灣),用於所有體內模型。在第0天透過皮下注射2×10
6個N87-Luc細胞與Matrigel (1:1;BD Bioscience)的混合物於ASID小鼠的右側。在第11天以2×10
6個Globo H CAR-T細胞或載體對照T細胞對小鼠進行靜脈注射。用游標尺測量皮下腫瘤的三維尺寸(mm
3)。在體內研究期間,每週在Ami-HT光學影像系統上對小鼠進行兩次拍攝,拍照前對小鼠腹腔注射200 μL 15 mg/mL D-冷光素(D-Luciferin;Biosynth,Cat. No. L-8220)。用公式V = 1/2 (長×寬
2)計算腫瘤體積。
圖5顯示2C2-Fab CAR T細胞及R783-scFv CAR T細胞在NCI-N87腫瘤模型中表現出明顯的療效。圖5A顯示CAR-T組(n = 3)及對照組(n = 3)在腫瘤接種後第11天至第32天的代表性腫瘤生物冷光影像。在第22天,9隻ASID小鼠皆可以觀察到腫瘤的存在。然而,在注射2C2-Fab CAR-T細胞或R783-scFv CAR-T細胞後的14天(第25天)或21天(第32天),只有3隻ASID小鼠(載體對照T細胞)觀察到腫瘤的存在。圖5B顯示三個腫瘤組及一個無腫瘤空白對照組的腫瘤生物冷光動力學曲線。曲線所顯示的資料是每組三隻小鼠的平均值±SD。
實施例3-2. Globo H CAR T細胞在不同腫瘤模型中的體內療效
MCF-7及HCC-1428乳癌原位移植模型:
對6至8週大的ASID小鼠皮下植入雌激素錠劑(17β-雌二醇0.36 mg /錠劑,90天釋放;Innovative Research of America)。二天後,在小鼠的第四乳腺脂肪墊原位注射8×10
6個MCF-7或HCC-1428細胞與Matrigel (1:1;BD Bioscience)的混合物。在第12天以2×10
6個Globo H CAR-T細胞或載體對照T細胞對小鼠進行靜脈注射。用游標尺測量原位腫瘤的三維尺寸(mm
3)。用公式V = 1/2 (長×寬
2)計算腫瘤體積。
SW-480結腸癌異種移植模型:
在第0天透過皮下注射1×10
6個SW-480細胞與Matrigel (1:1;BD Bioscience)的混合物於6至8週大的ASID小鼠的右側。在第12天以2×10
6個Globo H CAR-T細胞或載體對照T細胞對小鼠進行靜脈注射。用游標尺測量皮下腫瘤的三維尺寸(mm
3)。用公式V = 1/2 (長×寬
2)計算腫瘤體積。
圖6顯示2C2-Fab CAR T細胞在MCF-7乳癌原位移植模型(圖6A)、HCC-1428乳癌原位移植模型(圖6B)及SW-480結腸癌異種移植模型(圖6C)中表現出明顯的療效。參照這些模型中注射對照T細胞或2C2-Fab CAR T細胞的二個腫瘤組(每組n = 3)的腫瘤大小的動態曲線。曲線顯示的資料是每組三隻小鼠的平均值±SD。
實施例4. Globo H CAR T細胞在NCI-N87胃癌異種移植模型中的體內持久性
在第0天透過皮下注射2×10
6個N87-Luc細胞與Matrigel (1:1;BD Bioscience)的混合物於6至8週大的ASID小鼠的右側。在第10天以2×10
6個Globo H CAR-T細胞或載體對照T細胞對小鼠進行靜脈注射。在多次腫瘤挑戰試驗中,小鼠在第40天接受2×10
6個N87-Luc的第二劑腫瘤細胞皮下注射於左側。在第69天,於右側皮下第三次接種帶有2×10
6個N87-Luc細胞的腫瘤。用游標尺測量皮下腫瘤的三維尺寸(mm
3)。在體內研究期間,每週在Ami-HT光學影像系統上對小鼠進行兩次拍攝,拍照前對小鼠腹腔注射200 μL 15 mg/mL D-冷光素(D-Luciferin;Biosynth,Cat. No. L-8220)。用公式V = 1/2 (長×寬
2)計算腫瘤體積。
圖7顯示2C2-Fab CAR T細胞及2C2-Fab (CD3zm) CAR T細胞在NCI-N87腫瘤模型中表現出明顯的療效和持久性。圖7A顯示四個腫瘤組及一個無腫瘤空白對照組從第0天至第47天的腫瘤生物冷光動力學曲線(第一次接種腫瘤之生長)。曲線所顯示的資料是每組三隻小鼠的平均值±SD。以2C2-Fab CAR T細胞或2C2-Fab (CD3zm) CAR T細胞治療的原發腫瘤在第25天已被全部消除。圖7B顯示2C2-Fab CAR T細胞及2C2-Fab (CD3zm) CAR T細胞治療組的二次接種及三次接種腫瘤從第45天至第90天的生物冷光動力學曲線。PBS對照腫瘤組為無腫瘤小鼠,其接種2×10
6個N87-Luc腫瘤細胞作為二次腫瘤挑戰的對照。2C2-Fab CAR T細胞或2C2-Fab (CD3zm) CAR T細胞治療組(n = 3)的二次接種腫瘤在第60天皆被清除。只有2C2-Fab (CD3zm) CAR T細胞治療組(n = 3)在第三次腫瘤挑戰後全部存活。
除非另有定義,本文使用的所有技術與科學術語以及任何字首縮寫詞的含義與本發明所屬技術領域中熟習技藝者的通常理解相同。儘管任何與本文所述相似或等效的用於資訊交流的組合物、方法、試劑組(kits)和手段皆可用於實現本發明,本文僅描述較佳的用於資訊交流的組合物、方法、試劑組和手段。
在法律允許的範圍內,本文引用的所有參考文獻均透過引用納入本文。對該些參考文獻的討論僅是為了總結其作者的論述,而非承認任何參考文獻(或任何參考文獻的一部分)是相關的先前技術。申請人對任何引用的參考文獻的準確性和相關性保有質疑的權利。
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圖1係Globo-H CAR的示意圖;圖中共有六個Globo H CAR構築體(2C2-Fab CAR:SEQ ID NO:13;2C2-scFv CAR:SEQ ID NO:14;R783-Fab CAR:SEQ ID NO:15;R783-scFv CAR:SEQ ID NO:16;2C2-Fab CAR (CD3zeta突變體):SEQ ID NO:17;R783-scFv CAR (CD3zeta突變體):SEQ ID NO:18)。
圖2係 Globo H CAR T細胞的體外(
in vitro)細胞毒性;(A) MCF-7:乳癌細胞株-Globo H陽性;(B) HCC-1428:乳癌細胞株-Globo H陽性;(C) NCI-N87:胃癌細胞株-Globo H陽性;(D) SW-480:結腸癌細胞株-Globo H陽性;(E) SK-OV-3:卵巢癌細胞株-Globo H陰性。
圖3係帶有CD3zeta突變體(CD3zm)的Globo H CAR T細胞的體外細胞毒性;(A) HCC-1428:乳癌細胞株-Globo H陽性;(B) MCF-7:乳癌細胞株-Globo H陽性;(C) NCI-N87:胃癌細胞株-Globo H陽性;(D) SK-OV-3:卵巢癌細胞株-Globo H陰性。
圖4係帶有CD3zeta突變體(CD3zm)的Globo H CAR T細胞的體外持久性;(A) 對MCF-7 (Globo H陽性)及SK-OV-3 (Globo H陰性)癌症細胞株的細胞毒性;(B) MCF-7 (Globo H陽性)及SK-OV-3 (Globo H陰性)癌症細胞株中的T細胞數量。
圖5係NCI-N87胃癌異種移植模型(xenograft model)中Globo H CAR T細胞的體內(
in vivo)療效;(A)從第11天至第32天的腫瘤生物冷光影像;(B)腫瘤生物冷光的動力學曲線。
圖6係不同腫瘤模型中Globo H CAR T細胞的體內療效;(A) MCF-7乳癌原位移植模型(orthotopic model);(B) HCC-1428乳癌原位移植模型;(C) SW480結腸癌異種移植模型。
圖7係NCI-N87胃癌異種移植模型中Globo H CAR T細胞的體內持久性;(A)四個腫瘤組別及一無腫瘤空白對照組從第0天至第47天的腫瘤生物冷光的動力學曲線(第一次接種腫瘤之生長);曲線顯示的資料是每組三隻小鼠的平均值±SD;以2C2-Fab CAR T細胞或2C2-Fab (CD3zm) CAR T細胞處理的原發腫瘤在第25天被全部移除;(B) 2C2-Fab CAR T細胞及2C2-Fab (CD3zm) CAR T細胞治療組的二次接種及三次接種腫瘤從第45天至第90天的生物冷光動力學曲線。
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<110> 台灣浩鼎生技股份有限公司
<120> 與Globo系列抗原結合之嵌合抗原受體及其用途
<130> OBIP-4PCT
<140> TW 111104664
<141> 2022-02-09
<150> US 63/147237
<151> 2021-02-09
<150> US 63/147441
<151> 2021-02-09
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 243
<212> PRT
<213> 人工序列
<220>
<223> 2C2重鏈(HC)
<210> 2
<211> 235
<212> PRT
<213> 人工序列
<220>
<223> 2C2輕鏈(LC)
<210> 3
<211> 272
<212> PRT
<213> 人工序列
<220>
<223> 2C2 scFv
<210> 4
<211> 241
<212> PRT
<213> 人工序列
<220>
<223> R783重鏈(HC)
<210> 5
<211> 233
<212> PRT
<213> 人工序列
<220>
<223> R783輕鏈(LC)
<210> 6
<211> 265
<212> PRT
<213> 人工序列
<220>
<223> R783 scFv
<210> 7
<211> 45
<212> PRT
<213> 人工序列
<220>
<223> CD8鉸鏈
<210> 8
<211> 68
<212> PRT
<213> 人工序列
<220>
<223> CD28跨膜及共刺激區
<210> 9
<211> 42
<212> PRT
<213> 人工序列
<220>
<223> 4-1BB共刺激區
<210> 10
<211> 114
<212> PRT
<213> 人工序列
<220>
<223> CD3zeta區
<210> 11
<211> 114
<212> PRT
<213> 人工序列
<220>
<223> CD3zeta突變體(CD3zm)區
<210> 12
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> T2A連接子
<210> 13
<211> 768
<212> PRT
<213> 人工序列
<220>
<223> 2C2-Fab CAR
<210> 14
<211> 541
<212> PRT
<213> 人工序列
<220>
<223> 2C2-scFv CAR
<210> 15
<211> 764
<212> PRT
<213> 人工序列
<220>
<223> R783-Fab CAR
<210> 16
<211> 534
<212> PRT
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<223> R783-scFv CAR
<210> 17
<211> 768
<212> PRT
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<223> 2C2-Fab CAR(CD3zm)
<210> 18
<211> 534
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<213> 人工序列
<220>
<223> R783-scFv CAR(CD3zm)
Claims (16)
- 一種結合至Globo H的嵌合抗原受體,包含:一抗原結合片段或一單鏈變異區片段,其中該抗原結合片段包含具有SEQ ID NO:1之胺基酸序列的一重鏈可變區及具有SEQ ID NO:2之胺基酸序列的一輕鏈可變區,該單鏈變異區片段包含SEQ ID NO:3之胺基酸序列;一第一胞內域,係包含CD3 zeta鏈(CD3zeta),其中該第一胞內域包含SEQ ID NO:11之胺基酸序列;以及一第二胞內域,係包含來自CD28、CD137、CD4、OX40、4-1BB、ICOS或其組合的一胞內域序列。
- 如請求項1所述的嵌合抗原受體,其中該第二胞內域包含CD28及4-1BB,其中該抗原結合片段或該單鏈變異區片段與該第二胞內域融合,並且該第二胞內域與該第一胞內域融合。
- 一種結合至Globo H的嵌合抗原受體,包含:一抗原結合片段或一單鏈變異區片段,其中該抗原結合片段包含具有SEQ ID NO:4之胺基酸序列的一重鏈可變區及具有SEQ ID NO:5之胺基酸序列的一輕鏈可變區,該單鏈變異區片段包含SEQ ID NO:6之胺基酸序列;一第一胞內域,係包含CD3zeta,其中該第一胞內域包含SEQ ID NO:11之胺基酸序列;以及一第二胞內域,係包含來自CD28、CD137、CD4、OX40、4-1BB、ICOS或其組合的一胞內域序列。
- 如請求項3所述的嵌合抗原受體,其中該第二胞內域包含CD28及4-1BB,其中該抗原結合片段或該單鏈變異區片段與該第二胞內域融合,並且該第二胞內域與該第一胞內域融合。
- 如請求項2或4所述的嵌合抗原受體,其中該嵌合抗原受體包含一鉸鏈區,該鉸鏈區具有與SEQ ID NO:7有至少95%序列同一性的一胺基酸序列。
- 如請求項5所述的嵌合抗原受體,其中該鉸鏈區是CD8的鉸鏈區。
- 如請求項2或4所述的嵌合抗原受體,其中該嵌合抗原受體包含與SEQ ID NO:8有至少95%序列同一性的一CD28胞內域序列。
- 如請求項2或4所述的嵌合抗原受體,其中該嵌合抗原受體包含與SEQ ID NO:9有至少95%序列同一性的一4-1BB胞內域序列。
- 如請求項1所述的嵌合抗原受體,其中該嵌合抗原受體包含SEQ ID NO:17之胺基酸序列。
- 如請求項3所述的嵌合抗原受體,其中該嵌合抗原受體包含SEQ ID NO:18之胺基酸序列。
- 一種如請求項1至10中任一項所述之嵌合抗原受體在製備治療患有腫瘤個體的醫用製劑之用途,其中該嵌合抗原受體係表現於一CAR-T細胞上,該CAR-T細胞是對從患有該腫瘤的該個體獲得的T細胞轉導包含編碼該嵌合抗原受體的一核酸序列的一載體而產生並且經過擴增,其中該擴增的CAR-T細胞被注入該個體內以引起免疫反應。
- 如請求項11所述的用途,其中該個體為人類。
- 如請求項11所述的用途,其中該免疫反應係由T細胞介導。
- 如請求項11所述的用途,其中該載體係選自一慢病毒(lentivirus)、一伽瑪反轉錄病毒(gamma retrovirus)及一腺相關病毒(adeno-associated virus)。
- 如請求項11所述的用途,其中該腫瘤表現Globo H。
- 如請求項11所述的用途,其中該腫瘤係選自由乳癌、肺癌、食道癌、直腸癌、膽管癌、肝癌、口腔癌、胃癌、結腸癌、鼻咽癌、腎癌、前列腺癌、卵巢癌、宮頸癌、子宮內膜癌、胰臟癌、睾丸癌、膀胱癌、頭頸癌、神經內分泌癌、腎上腺癌、甲狀腺癌、骨癌、皮膚癌、基底細胞癌、鱗狀細胞癌、黑色素瘤、及腦腫瘤所組成的群組。
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