TWI821956B - 免疫調節及抗腫瘤相關奈米抗體暨其核酸編碼序列及其應用 - Google Patents
免疫調節及抗腫瘤相關奈米抗體暨其核酸編碼序列及其應用 Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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Abstract
本發明提供一種免疫調節及抗腫瘤相關奈米抗體,其與一人類白血球抗原-G、一程序性細胞死亡配體1及一CD3 ε專一性結合。本發明亦提供該免疫調節及抗腫瘤相關奈米抗體的核酸編碼序列、該免疫調節及抗腫瘤相關奈米抗體用於治療癌症及免疫相關疾病的用途。
Description
本發明是有關於一種免疫調節及抗腫瘤相關奈米抗體暨其核酸編碼序列及其應用。
癌症又名為惡性腫瘤,為細胞不正常增生,且這些增生的細胞可能侵犯身體的其他部分,為由控制細胞分裂增殖機制失常而引起的疾病。全世界罹患癌症的人口有不斷增加的趨勢,癌症係國人十大死因之一,且已連續多年居十大死因之榜首。
常規的腫瘤治療方法包括手術治療、放射線治療、化學治療及標靶治療等。腫瘤免疫治療為上述治療方法以外的另一種治療腫瘤的方法,是透過激活患者自身免疫系統,利用腫瘤細胞或腫瘤抗原物質誘導機體的特異性細胞免疫及體液免疫反應,增強機體的抗癌能力,阻止腫瘤的生長、擴散及復發,以達到清除或控制腫瘤的目的。然而,目前的腫瘤治療方法仍存在效果不彰及副作用強烈的問題,甚至會衍生出其他免疫相關疾病。
人類白血球抗原-G (human leukocyte antigen-G, HLA-G)已被發現高度表現於多種實體腫瘤上,且有抑制免疫細胞的特性。因此,已有研究人員致力於研發以HLA-G作為辨識腫瘤的標靶分子並找出這些標靶分子是否具有成為抗癌藥物的潛力。
程序性細胞死亡配體1 (programmed cell death ligand 1, PD-L1)被發現表現於多種實體腫瘤的細胞表面。因此,已有研究人員致力於研發以PD-L1作為辨識腫瘤的標靶分子並找出這些標靶分子是否具有成為抗癌藥物的潛力。
CD3ε (CD3 epsilon)為在T細胞上發現的一種跨膜蛋白,已被發現與腫瘤及調節免疫功能有關聯性。因此,已有研究人員致力於研發以CD3ε作為辨識腫瘤及調節免疫功能的標靶分子並找出這些標靶分子是否具有成為抗癌藥物或免疫調節藥物的潛力。
為了解決上述問題,本領域的技術人員亟需研發出新穎且更有效治療癌症及免疫相關疾病、免疫調節及活化免疫細胞的醫藥品以造福有此需求的廣大族群。
有鑑於此,本發明之目的為提供一種免疫調節及抗腫瘤相關奈米抗體,其與一人類白血球抗原-G (human leukocyte antigen-G, HLA-G)、一程序性細胞死亡配體1 (programmed cell death ligand 1, PD-L1)及一CD3 ε (CD3 epsilon)專一性結合,該免疫調節及抗腫瘤相關奈米抗體包含一下列胺基酸序列的組合:序列識別號:1、序列識別號:2,及序列識別號:3。
在本發明的一實施例中,該胺基酸序列是該免疫調節及抗腫瘤相關奈米抗體的一重鏈可變域(heavy chain variable domain, VHH)的一胺基酸序列。
在本發明的一實施例中,該免疫調節及抗腫瘤相關奈米抗體進一步包含一可結晶片段區域(fragment crystallizable region, Fc region)。
在本發明的一實施例中,該免疫調節及抗腫瘤相關奈米抗體與一第二抗體綴合以形成三特異性T-細胞連接(triple specific T-cell engager, TriTE)。
在本發明的一實施例中,該免疫調節及抗腫瘤相關奈米抗體活化及/或聚集CD3陽性細胞。
在本發明的一實施例中,該免疫調節及抗腫瘤相關奈米抗體具有殺死腫瘤細胞的作用。
在本發明的一實施例中,該腫瘤細胞是選自於下列所組成的群組:肺腺癌細胞、乳癌細胞、神經膠質母細胞瘤細胞、卵巢癌細胞、口腔癌細胞,及其組合。
本發明之另一目的為提供一種經分離的核酸,其編碼一如前所述的免疫調節及抗腫瘤相關奈米抗體之胺基酸序列,該經分離的核酸包含一下列核苷酸序列的組合:序列識別號:4、序列識別號:5,及序列識別號:6。
本發明之另一目的為提供一種醫藥組成物,包含一如前所述的免疫調節及抗腫瘤相關奈米抗體及一醫藥學上可接受的載劑。
本發明之另一目的為提供一種如前所述的免疫調節及抗腫瘤相關奈米抗體用於製備一治療癌症及免疫相關疾病之醫藥品的用途。
在本發明的一實施例中,該癌症是選自於下列所組成的群組:肺腺癌、乳癌、神經膠質母細胞瘤、卵巢癌、口腔癌,及其組合。
綜上所述,本發明免疫調節及抗腫瘤相關奈米抗體的功效在於:藉由表面電漿子共振結合分析(surface plasmon resonance binding assay, SPR binding assay)證明可專一性結合HLA-G、PD-L1及CD3 ε、藉由T細胞(即周邊血液單核細胞)增殖及活化分析(T cell (i.e., peripheral blood mononuclear cell, PBMC) proliferation and activation assay)證明可促進T細胞群聚增生及活化、增強PBMC中CD3陽性T細胞增殖、藉由免疫細胞化學(immunocytochemistry, ICC)染色證明可與腫瘤細胞結合並殺死腫瘤細胞、藉由酵素結合免疫吸附分析法(enzyme linked immunosorbent assay, ELISA)證明可增強腫瘤細胞中細胞激素(cytokine)分泌、藉由動物實驗證明可抑制癌細胞生長,藉此可達到治療癌症及免疫相關疾病的效用。特別地,相較於習知的抗體必須藉由載體將基因轉染至細胞中表現抗體功能而有低產量及效果不彰的缺點,本發明免疫調節及抗腫瘤相關奈米抗體可在體外大規模製備後直接投藥至需要的個體內進行治療。
以下將進一步說明本發明的實施方式,下述所列舉的實施例係用以闡明本發明,並非用以限定本發明之範圍,任何熟習此技藝者,在不脫離本發明之精神和範圍內,當可做些許更動與潤飾,因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。
定義
本文中所使用數值為近似值,所有實驗數據皆表示在
20%的範圍內,較佳為在
10%的範圍內,最佳為在
5%的範圍內。
如本文中所使用的,用語“免疫調節及抗腫瘤相關奈米抗體”與、“以PD-L1、HLA-G及CD3ε奈米抗體為基礎的三特異性T-細胞連接(PD-L1 x HLA-G x CD3ε nanobody-based triple specific T-cell engager, PD-L1 x HLA-G x CD3ε nanobody-based TriTE)”、“Nb-TriTE”及“V
HH奈米抗體”可交換使用。
如本文中所使用的,用語“CD3e”、“CD3 ε”及“CD3 epsilon”可交換使用。
如本文中所使用的,用語“第二抗體”意指可與奈米抗體綴合以形成三特異性T-細胞連接(triple specific T-cell engager, TriTE)的抗體。較佳地,第二抗體可包括,但不限於:抗CD3
、CD3、人類白血球抗原-G (human leukocyte antigen-G, HLA-G)、程序性細胞死亡配體2 (programmed cell death ligand 2, PD-L2)、T細胞免疫球蛋白結構域及黏蛋白結構域3 (T-cell immunoglobulin domain and mucin domain 3, Tim3)、表皮生長因子受體(epidermal growth factor receptor, EGFR)、EGFRvIII、人類表皮生長因子受體2 (human epidermal growth factor receptor 2, Her2)、B細胞成熟抗原(B-cell maturation antigen, BCMA)、CD19、CD20、CD34、CD16、Fc、上皮細胞黏附分子(epithelial cell adhesion molecule, EpCAM)、間皮素(mesothelin)、紐約食道鱗狀上皮細胞癌-1 (New York esophageal squamous cell carcinoma-1, NY-ESO-1)、糖蛋白100 (glycoprotein 100, gp100)及黏蛋白1 (Muc1)抗體。
如本文中所使用的,“治療(treating)”或“治療(treatment)”意指緩解(alleviating)、減少(reducing)、改善(ameliorating)、減輕(relieving)或控制(controlling)一疾病(disease)或障礙(disorder)的一或多個臨床徵兆(clinical sign),以及降低(lowering)、停止(stopping)或逆轉(reversing)一正在被治療中的病況(condition)或症狀(symptom)之嚴重性(severity)的進展(progression)。
依據本發明的醫藥品可利用熟習此技藝者所詳知的技術而被製造成一適合於非經腸道地(parenterally)投藥的劑型(dosage form),這包括,但不限於:注射品(injection)[例如,無菌的水性溶液(sterile aqueous solution)或分散液(dispersion)]、無菌的粉末(sterile powder)、錠劑(tablet)、片劑(troche)、口含錠(lozenge)、丸劑(pill)、膠囊(capsule)、分散性粉末(dispersible powder)或細顆粒(granule)、溶液、懸浮液(suspension)、乳劑(emulsion)、糖漿(syrup)、酏劑(elixir)、濃漿(slurry)以及類似之物。
依據本發明的醫藥品可以一選自於由下列所構成的群組中的非經腸道途徑(parenteral routes)來投藥:腹膜內注射(intraperitoneal injection)、皮下注射(subcutaneous injection)、表皮內注射(intraepidermal injection)、皮內注射(intradermal injection)、肌肉內注射(intramuscular injection)、靜脈內注射(intravenous injection)以及病灶內注射(intralesional injection)。
依據本發明的醫藥品可包含有一被廣泛地使用於藥物製造技術之醫藥學上可接受的載劑。例如,該醫藥學上可接受的載劑可包含一或多種選自於由下列所構成之群組中的試劑:溶劑(solvent)、乳化劑(emulsifier)、懸浮劑(suspending agent)、分解劑(decomposer)、黏結劑(binding agent)、賦形劑(excipient)、安定劑(stabilizing agent)、螯合劑(chelating agent)、稀釋劑(diluent)、膠凝劑(gelling agent)、防腐劑(preservative)、潤滑劑(lubricant)、吸收延遲劑(absorption delaying agent)、脂質體(liposome)以及類似之物。有關這些試劑的選用與數量是落在熟習此項技術之人士的專業素養與例行技術範疇內。
依據本發明的該醫藥學上可接受的載劑包含有一選自於由下列所構成之群組中的溶劑:水、生理鹽水(normal saline)、磷酸鹽緩衝生理鹽水(phosphate buffered saline, PBS)、含糖溶液、含有醇的水性溶液(aqueous solution containing alcohol),以及它們的組合。
如本文中所用的,“核酸”、“核酸序列”或“核酸片段”等術語意指呈單股或雙股形式的去氧核糖核苷酸序列或核糖核苷酸序列,且當中包含有已知的天然存在的核苷酸(naturally occurring nucleotides)或人造化學仿效物。如本文中所用的,“核酸”此術語可與“基因”、“cDNA”、“mRNA”、“寡核苷酸”和“聚核苷酸”交換使用。
實施例 1. 免疫調節及抗腫瘤相關奈米抗體的製備
在本實施例中,免疫調節及抗腫瘤相關奈米抗體(immunomodulation and anti tumor-related nanobody)(即以PD-L1、HLA-G及CD3ε奈米抗體為基礎的三特異性T-細胞連接(PD-L1 x HLA-G x CD3ε nanobody-based triple specific T-cell engager, PD-L1 x HLA-G x CD3ε nanobody-based TriTE),下稱Nb-TriTE)的製備流程如下。Nb-TriTE基因在表現載體pcDNA3.4 (安比西林(Amp)抗性)中構築。質體藉由限制酶消化及定序驗證進行鑑定。藉由螢光金奈米團簇3000 (lipofectamine 3000)將質體轉導到293F細胞中,並在37°C及8% CO
2下作用3天。藉由離心獲取上清液。藉由流通(flow-through)將上清液與蛋白A珠粒(1mL)結合。用含有合適梯度咪唑(imidazole)(10 mM、20 mM、50 mM、100 mM、250 mM及500 mM)的緩衝液洗滌及洗脫蛋白A珠粒。洗脫部分進行十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)分析,根據蛋白質的純度及產量確定後續純化方案(離子交換層析(ion exchange chromatography)或凝膠過濾層析(gel filtration chromatography))。符合要求的蛋白質藉由凝膠過濾層析分離純化,且緩衝液換成磷酸鹽緩衝生理鹽水(phosphate buffered saline, PBS)緩衝液。蛋白質組分經SDS-PAGE分析,符合要求的組分合併濃縮,用0.22 μm濾膜過濾及分裝。將蛋白質儲存於-20
或更低溫度。
關於Nb-TriTE的結構及組成顯示於圖1A,其中Anti-HLA-G表示抗-人類白血球抗原-G (human leukocyte antigen-G, HLA-G)奈米抗體,其胺基酸序列為序列識別號:1,編碼抗-HLA-G奈米抗體的胺基酸序列之核苷酸序列為序列識別號:4;Anti-PDL1表示抗-程序性細胞死亡配體1 (programmed cell death ligand 1, PD-L1)奈米抗體,其胺基酸序列為序列識別號:2,編碼抗-PD-L1奈米抗體的胺基酸序列之核苷酸序列為序列識別號:5;Anti-CD3e表示抗-CD3ε奈米抗體,其胺基酸序列為序列識別號:3,編碼抗-CD3ε奈米抗體的胺基酸序列之核苷酸序列為序列識別號:6;Signal peptide表示訊息胜肽,其胺基酸序列為序列識別號:7,編碼訊息胜肽的胺基酸序列之核苷酸序列為序列識別號:8;VHH1表示重鏈可變域1 (heavy chain variable domain 1, VHH1),即為抗-PD-L1奈米抗體;Flexible linker表示可撓性連接子,即為連接子(Linker),其胺基酸序列為序列識別號:9,編碼可撓性連接子的胺基酸序列之核苷酸序列為序列識別號:10;VHH2表示重鏈可變域2 (heavy chain variable domain 2, VHH2),即為抗-HLA-G奈米抗體;VHH3表示重鏈可變域3 (heavy chain variable domain 3, VHH3),即為抗-CD3ε奈米抗體;Hinge表示鉸鏈;CH2及CH3表示人類IgG1可結晶片段區域(fragment crystallizable region, Fc region),即為人類IgG1 Fc-域(Human IgG1 Fc-domain),其胺基酸序列為序列識別號:11,編碼人類IgG1 Fc-域的胺基酸序列之核苷酸序列為序列識別號:12。
關於Nb-TriTE的限制酶消化結果顯示於圖1B,其中徑1 (lane 1)表示質體,徑2 (lane 2)表示以XbaI-BamHI消化的質體,徑M (lane M)表示DNA標記(DNA marker)。
關於純化的Nb-TriTE的凝膠電泳分析結果顯示於圖1C。
實施例 2. Nb-TriTE 的表面電漿子共振結合分析 (surface plasmon resonance binding assay, SPR binding assay) 結果
在本實施例中,Nb-TriTE的表面電漿子共振結合分析(surface plasmon resonance binding assay, SPR binding assay)的操作流程如下。將CM5或NTA晶片由BIAcore T200 (Biacore-GE Healthcare, Piscataway, NJ)進行SPR分析。簡言之,在10 mM緩衝溶液(pH 4.0、5.5 或6.0)中以20 μg/mL的濃度範圍稀釋蛋白質(PD-L1、HLA-G或CD3重組蛋白質)樣品,以獲得最大的表面保留用於固定在晶片上,遵循製備過程並選擇晶片上較高表面濃度的配體(25、12.5、6.25、3.125、1.5625及0.78125 nM)條件。然後是再生探索(regeneration scouting)及表面性能測試(surface performance test),在再生探索及表面性能測試之後,然後選擇再生方法來運行實驗。然後選擇結合分析(binding analysis)及直接結合(direct binding)來研究蛋白質結合。選擇動力學分析(kinetic analysis)並選擇質量轉移(mass transfer)進行結合實驗的動力學分析。之後,進行數據分析及確定動力學常數。
Nb-TriTE的表面電漿子共振結合分析結果顯示於圖2A至圖2C,其中圖2A顯示Nb-TriTE與PD-L1結合的SPR動力學分析,配體為PD-L1,分析濃度為100、50、25、12.5、6.25、3.125、1.5625、0.78125 nM,流速為30 μL/分鐘,締合時間(association time)為120秒,解離時間(dissociation time)為900秒,阿替利珠單抗(Atezolizumab)是一種用於治療尿路上皮癌、非小細胞肺癌(NSCLC)、三陰性乳腺癌、小細胞肺癌及肝細胞癌的IgG1同種型完全人源化抗PD-L1單株抗體,Anti-PDL1表示抗-PD-L1奈米抗體;圖2B顯示Nb-TriTE與HLA-G結合的SPR動力學分析,配體為HLA-G,分析濃度為305、152.5、76.25、38.125、19.0625 nM,流速為30 μL/分鐘,締合時間為180秒,解離時間為360秒,Anti-HLA-G表示抗-HLA-G奈米抗體,Anti-CD3e表示抗-CD3ε奈米抗體,87G表示商用抗-HLA-G單株抗體;圖2C顯示Nb-TriTE與CD3ε結合的SPR動力學分析,配體為CD3ε,分析濃度為28.125、56.25、112.5、225、450、900、1800、3600 nM,流速為15 μL/分鐘,締合時間為350秒,解離時間為900秒,Anti-CD3e表示抗-CD3ε奈米抗體,莫羅單抗-CD3 (Muromonab-CD3)是降低接受器官移植病人急性排斥反應的免疫抑制劑,且是針對T細胞表面CD3受體的單株抗體,Response表示反應,Time表示時間。由圖2A至圖2C可見,Nb-TriTE分別以2.6、53.3及3.47 nM內的K
D有效結合PD-L1、HLA-G及CD3ε蛋白質。
實施例 3. Nb-TriTE 的 T 細胞增殖 (T cell proliferation) 分析結果
在本實施例中,Nb-TriTE的T細胞(即周邊血液單核細胞)增殖分析(T cell (i.e., peripheral blood mononuclear cell, PBMC) proliferation assay)的操作流程如下。將1 x 10
5的PBMC細胞接種在12孔盤中,分別加入臨床用CD3ε單株抗體OKT3 (5
g, Invitrogen, Cat:MA1-10175)及Proleukin (IL-2: 200U),或是10
g/ml的Nb-TriTE。5或7天後,記錄總細胞數,然後用FITC-綴合的CD3單株抗體(OKT3, 11-0037-42, eBioscience)染色,然後藉由流動式細胞測量術進行分析。CD3陽性細胞數計算為CD3細胞百分比(%)×總細胞數,正對照組為Proleukin (IL-2: 200U)及OKT3 (5
g)。
Nb-TriTE的T細胞增殖(T cell proliferation)分析結果顯示於圖3,其中PBMC表示周邊血液單核細胞(peripheral blood mononuclear cell)。由圖3可見,Nb-TriTE可促進T細胞群聚增生及活化。
實施例 4. Nb-TriTE 的免疫細胞化學 (immunocytochemistry, ICC) 染色結果
在本實施例中,對於腫瘤細胞、T細胞及Nb-TriTE (即V
HH奈米抗體)的免疫細胞化學(immunocytochemistry, ICC)分析的操作流程如下。將腫瘤細胞(4 x 10
4或1 x 10
5)接種在6孔盤的蓋玻片上,培育過夜。在指定的處理後,將細胞培育以CellTracker Green,接而添加Nb-TriTE及PBMC歷時一小時。接著,將細胞固定在1%多聚甲醛(paraformaldehyde)中,用PBS清洗,在含有0.5% BSA的PBS中使用0.1% Triton X-100透化30分鐘,用2% BSA封阻,並與專一性抗體(配於2% BSA/含有0.05% Tween-20的PBS (PBST)中)一起作用。在清洗之後,將細胞與螢光素綴合的抗體一起作用,用PBST清洗,並使用含有抗褪色劑及4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole, DAPI)的水性封固劑封固。在Leica TCS SP8 X共焦顯微鏡(Leica)下分析影像。
對於腫瘤細胞、T細胞及Nb-TriTE (即V
HH奈米抗體)的免疫細胞化學染色結果顯示於圖4,其中使用的細胞株A549是人類非小細胞肺癌細胞株,PBMC表示周邊血液單核細胞,VHH表示Nb-TriTE,上圖的細胞數目為1 x 10
5/孔,下圖的細胞數目為4 x 10
4/孔。本實施例的結果顯示,Nb-TriTE具有結合A549細胞的能力。
實施例 5. Nb-TriTE 與 PBMC 在增強對人類癌細胞株的細胞溶解上的效用評估
在本實施例中,評估Nb-TriTE與PBMC在增強對A549-人類肺腺癌細胞株、MDA-MB-231-人類乳癌細胞株、U87-神經膠質母細胞瘤(glioblastoma)細胞株、SKOV3-人類卵巢癌細胞株及FaDu-人類口腔癌細胞株(購自美國類型培養物收集中心(American Type Culture Collection, ATCC))的細胞溶解上的效用。操作流程如下。將1
10
5腫瘤細胞接種於12-孔盤上隔夜。隔天,將5
10
5的初代PBMC添加到含有腫瘤細胞的孔中。添加Nb-TriTE。48小時後,初代PBMC對腫瘤細胞的專一性溶解藉由活/死細胞-介導的細胞毒性分析(LIVE/DEAD cell-mediated cytotoxicity assay)使用流動式細胞測量術分析確定。
關於Nb-TriTE與PBMC在增強對人類癌細胞株的細胞溶解上的結果顯示於圖5A及圖5B,其中圖5A的A549表示人類肺腺癌細胞株,231表示MDA-MB-231-人類乳癌細胞株,U87表示神經膠質母細胞瘤(glioblastoma)細胞株,SKOV3表示人類卵巢癌細胞株,FaDu表示人類口腔癌細胞株,PBMC與A549細胞的比例為3:1,Nb-TriTE濃度為10
g/ml,共培養歷時48小時;圖5B的Nb-TriTE濃度為0、1、10、100、1000、10000及100000 μg/ml,效應(effector, E)細胞為PBMC,標的(target, T)細胞為A549細胞,E:T ratio表示效應/標的比。本實施例的結果證實,Nb-TriTE增強PBMC-誘導的對腫瘤細胞的細胞毒性。
實施例 6. 藉由酵素結合免疫吸附分析法 (enzyme linked immunosorbent assay, ELISA) 測定 Nb-TriTE 對共培養系統中細胞激素分泌的影響評估
在本實施例中,使用商業ELISA套組(Thermo Fisher Scientific)測量人類細胞激素穿孔素(perforin)、顆粒酶B (granzyme B)、腫瘤壞死因子
(tumor necrosis factor alpha, TNF-
)及干擾素
(interferon gamma, IFN-
)。操作流程如下。首先,收集來自Nb-TriTE、A549細胞及PBMC的樣品並裝載於96孔盤上於4
下過夜。隔天,移除樣品,在室溫下用3%脫脂牛奶封阻2小時。接著,用PBST (0.05% Tween溶於PBS中)清洗5次。清洗5次後,於室溫加入生物素化的抗體2小時。清洗5次後,每孔與100
l含有鏈黴抗生物素蛋白-辣根過氧化酶綴合物(streptavidin-HRP conjugates)的PBST在室溫下作用2小時。以PBST清洗7次後,加入50
l的TMB受質(用於偵測HRP活性)(ThermoFisher, Cat No:N301)。之後,添加50
l的終止溶液(ThermoFisher, Cat No:N600)來終止反應,然後使用450 nm波長的ELISA讀取儀進行測量。
本實施例的結果顯示於圖6,其中A549表示人類肺腺癌細胞株,Nb表示奈米抗體,效應(effector, E)細胞為PBMC,標的(target, T)細胞為A549細胞,E:T ratio表示效應/標的比,#表示未偵測到,Perforin表示穿孔素,Granzyme B表示顆粒酶B,TNF-
表示腫瘤壞死因子
(tumor necrosis factor alpha),IFN-
表示干擾素
(interferon gamma),共培養歷時48小時。本實施例的結果顯示,Nb-TriTE增強A549與PBMC共培養的細胞激素分泌。
實施例 7. Nb-TriTE 在阻斷人源化小鼠的肺癌生長上的效用評估
本實施例的操作流程如下。6至8週齡的NOD/SCID
(NSG)小鼠購自The Jackson Laboratory。小鼠用於異種移植(xenograft)肺腫瘤模型。對於肺腫瘤,將Luc
+A549細胞重新懸浮於含有Matrigel的PBS中,然後將細胞(5x10
5/20 µL)皮下注射到小鼠的右背部。植入後7天,經由尾靜脈注射為每隻小鼠注入解凍的 PDL1xHLA-GxCD3 Nano-TriTE及PBMC (5x10
6/100 µL PBS),然後每週注入PDL1xHLA-GxCD3 Nano-TriTE。使用體內成像系統(in vivo imaging system, IVIS)(PerkinElmer)經由生物發光成像每週監測腫瘤生長。在指定的天數中,將小鼠犧牲,並將腫瘤獲取、測量及拍照。
本實施例的結果顯示於圖7A至圖7C,其中圖7A的Luc表示螢光素酶(luciferase),PBMC表示周邊血液單核細胞(peripheral blood mononuclear cell),A549表示人類肺腺癌細胞株,TriTE表示Nb-TriTE。本實施例的結果顯示,Nb-TriTE可阻斷人源化小鼠的肺癌生長。
綜上所述,本發明免疫調節及抗腫瘤相關奈米抗體藉由表面電漿子共振結合分析(surface plasmon resonance binding assay, SPR binding assay)證明可專一性結合HLA-G、PD-L1及CD3 ε、藉由T細胞(即周邊血液單核細胞)增殖及活化分析(T cell (i.e., peripheral blood mononuclear cell, PBMC) proliferation and activation assay)證明可促進T細胞群聚增生及活化、增強PBMC中CD3陽性T細胞增殖、藉由免疫細胞化學(immunocytochemistry, ICC)染色證明可與腫瘤細胞結合並殺死腫瘤細胞、藉由酵素結合免疫吸附分析法(enzyme linked immunosorbent assay, ELISA)證明可增強腫瘤細胞中細胞激素(cytokine)分泌、藉由動物實驗證明可抑制癌細胞生長,藉此可達到治療癌症及免疫相關疾病的效用。特別地,相較於習知的抗體必須藉由載體將基因轉染至細胞中表現抗體功能而有低產量及效果不彰的缺點,本發明免疫調節及抗腫瘤相關奈米抗體可在體外大規模製備後直接投藥至需要的個體內進行治療。
以上所述僅為舉例性,而非為限制性者。任何未脫離本發明之精神與範疇,而對其進行之等效修改或變更,均應包含於後附之申請專利範圍中。
無
圖1A顯示免疫調節及抗腫瘤相關奈米抗體(immunomodulation and anti tumor-related nanobody)(即以PD-L1、HLA-G及CD3ε奈米抗體為基礎的三特異性T-細胞連接(PD-L1 x HLA-G x CD3ε nanobody-based triple specific T-cell engager, PD-L1 x HLA-G x CD3ε nanobody-based TriTE),下稱Nb-TriTE)的結構及組成,其中Anti-HLA-G表示抗-人類白血球抗原-G (human leukocyte antigen-G, HLA-G)奈米抗體,其胺基酸序列為序列識別號:1,編碼抗-HLA-G奈米抗體的胺基酸序列之核苷酸序列為序列識別號:4;Anti-PDL1表示抗-程序性細胞死亡配體1 (programmed cell death ligand 1, PD-L1)奈米抗體,其胺基酸序列為序列識別號:2,編碼抗-PD-L1奈米抗體的胺基酸序列之核苷酸序列為序列識別號:5;Anti-CD3e表示抗-CD3ε奈米抗體,其胺基酸序列為序列識別號:3,編碼抗-CD3ε奈米抗體的胺基酸序列之核苷酸序列為序列識別號:6;Signal peptide表示訊息胜肽,其胺基酸序列為序列識別號:7,編碼訊息胜肽的胺基酸序列之核苷酸序列為序列識別號:8;VHH1表示重鏈可變域1 (heavy chain variable domain 1, VHH1),即為抗-PD-L1奈米抗體;Flexible linker表示可撓性連接子,即為連接子(Linker),其胺基酸序列為序列識別號:9,編碼可撓性連接子的胺基酸序列之核苷酸序列為序列識別號:10;VHH2表示重鏈可變域2 (heavy chain variable domain 2, VHH2),即為抗-HLA-G奈米抗體;VHH3表示重鏈可變域3 (heavy chain variable domain 3, VHH3),即為抗-CD3ε奈米抗體;Hinge表示鉸鏈;CH2及CH3表示人類IgG1可結晶片段區域(fragment crystallizable region, Fc region),即為人類IgG1 Fc-域(Human IgG1 Fc-domain),其胺基酸序列為序列識別號:11,編碼人類IgG1 Fc-域的胺基酸序列之核苷酸序列為序列識別號:12。
圖1B顯示Nb-TriTE的限制酶消化結果,其中徑1 (lane 1)表示質體,徑2 (lane 2)表示以XbaI-BamHI消化的質體,徑M (lane M)表示DNA標記(DNA marker)。
圖1C顯示純化的Nb-TriTE的凝膠電泳分析結果。
圖2A顯示Nb-TriTE與PD-L1結合的SPR動力學分析,配體為PD-L1,分析濃度為100、50、25、12.5、6.25、3.125、1.5625、0.78125 nM,流速為30 μL/分鐘,締合時間(association time)為120秒,解離時間(dissociation time)為900秒,阿替利珠單抗(Atezolizumab)是一種用於治療尿路上皮癌、非小細胞肺癌(NSCLC)、三陰性乳腺癌、小細胞肺癌及肝細胞癌的IgG1同種型完全人源化抗PD-L1單株抗體,Anti-PDL1表示抗-PD-L1奈米抗體,Response表示反應,Time表示時間。
圖2B顯示Nb-TriTE與HLA-G結合的SPR動力學分析,配體為HLA-G,分析濃度為305、152.5、76.25、38.125、19.0625 nM,流速為30 μL/分鐘,締合時間為180秒,解離時間為360秒,Anti-HLA-G表示抗-HLA-G奈米抗體,Anti-CD3e表示抗-CD3ε奈米抗體,87G表示商用抗-HLA-G單株抗體,Response表示反應,Time表示時間。
圖2C顯示Nb-TriTE與CD3ε結合的SPR動力學分析,配體為CD3ε,分析濃度為28.125、56.25、112.5、225、450、900、1800、3600 nM,流速為15 μL/分鐘,締合時間為350秒,解離時間為900秒,Anti-CD3e表示抗-CD3ε奈米抗體,莫羅單抗-CD3 (Muromonab-CD3)是降低接受器官移植病人急性排斥反應的免疫抑制劑,且是針對T細胞表面CD3受體的單株抗體,Response表示反應,Time表示時間。
圖3顯示Nb-TriTE的T細胞增殖(T cell proliferation)分析結果,其中PBMC表示周邊血液單核細胞(peripheral blood mononuclear cell)。
圖4顯示對於腫瘤細胞、T細胞及Nb-TriTE (即V
HH奈米抗體)的免疫細胞化學(immunocytochemistry, ICC)染色結果,其中使用的細胞株A549是人類非小細胞肺癌細胞株,PBMC表示周邊血液單核細胞,VHH表示Nb-TriTE,上圖的細胞數目為1 x 10
5/孔,下圖的細胞數目為4 x 10
4/孔。
圖5A顯示Nb-TriTE與PBMC在增強對人類癌細胞株的細胞溶解上的結果,其中A549表示人類肺腺癌細胞株,231表示MDA-MB-231-人類乳癌細胞株,U87表示神經膠質母細胞瘤(glioblastoma)細胞株,SKOV3表示人類卵巢癌細胞株,FaDu表示人類口腔癌細胞株,PBMC與A549細胞的比例為3:1,Nb-TriTE濃度為10
g/ml,共培養歷時48小時。
圖5B顯示Nb-TriTE與PBMC在增強對人類肺腺癌細胞株A549的細胞溶解上的結果,其中Nb-TriTE濃度為0、1、10、100、1000、10000及100000 μg/ml,效應(effector, E)細胞為PBMC,標的(target, T)細胞為A549細胞,E:T ratio表示效應/標的比。
圖6顯示Nb-TriTE增強A549與PBMC共培養的細胞激素分泌,其中A549表示人類肺腺癌細胞株,Nb表示奈米抗體,效應(effector, E)細胞為PBMC,標的(target, T)細胞為A549細胞,E:T ratio表示效應/標的比,#表示未偵測到,Perforin表示穿孔素,Granzyme B表示顆粒酶B,TNF-
表示腫瘤壞死因子
(tumor necrosis factor alpha),IFN-
表示干擾素
(interferon gamma),共培養歷時48小時。
圖7A至圖7C顯示Nb-TriTE可阻斷人源化小鼠的肺癌生長,其中圖7A的Luc表示螢光素酶(luciferase),PBMC表示周邊血液單核細胞(peripheral blood mononuclear cell),A549表示人類肺腺癌細胞株,TriTE表示Nb-TriTE。
<110> 聖安生醫股份有限公司
<120> 免疫調節及抗腫瘤相關奈米抗體暨其核酸編碼序列及其應用
<150> US 63/165,191
<151> 2021-03-24
<150> US 63/165,274
<151> 2021-03-24
<150> US 63/165,266
<151> 2021-03-24
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 128
<212> PRT
<213> 人工序列
<220>
<223> 抗-HLA-G奈米抗體
<210> 2
<211> 121
<212> PRT
<213> 人工序列
<220>
<223> 抗-PD-L1奈米抗體
<210> 3
<211> 128
<212> PRT
<213> 人工序列
<220>
<223> 抗-CD3 epsilon奈米抗體
<210> 4
<211> 384
<212> DNA
<213> 人工序列
<220>
<223> 抗-HLA-G奈米抗體
<210> 5
<211> 363
<212> DNA
<213> 人工序列
<220>
<223> 抗-PD-L1奈米抗體
<210> 6
<211> 384
<212> DNA
<213> 人工序列
<220>
<223> 抗-CD3 epsilon奈米抗體
<210> 7
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 訊息胜肽
<210> 8
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 訊息胜肽
<210> 9
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 可撓性連接子
<210> 10
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 可撓性連接子
<210> 11
<211> 232
<212> PRT
<213> 人工序列
<220>
<223> 人類IgG1 Fc-域
<210> 12
<211> 699
<212> DNA
<213> 人工序列
<220>
<223> 人類IgG1 Fc-域
Claims (9)
- 一種免疫調節及抗腫瘤相關奈米抗體,其與一人類白血球抗原-G(human leukocyte antigen-G,HLA-G)、一程序性細胞死亡配體1(programmed cell death ligand 1,PD-L1)及一CD3 ε(CD3 epsilon)專一性結合,該免疫調節及抗腫瘤相關奈米抗體包含以下胺基酸序列的組合:序列識別號:1、序列識別號:2,及序列識別號:3,其中該免疫調節及抗腫瘤相關奈米抗體是以2.6~53.3nM的KD結合PD-L1、HLA-G及CD3ε。
- 如請求項1的免疫調節及抗腫瘤相關奈米抗體,其中該胺基酸序列是該免疫調節及抗腫瘤相關奈米抗體的一重鏈可變域(heavy chain variable domain,VHH)的一胺基酸序列。
- 如請求項1的免疫調節及抗腫瘤相關奈米抗體,其活化及/或聚集CD3陽性細胞。
- 如請求項3的免疫調節及抗腫瘤相關奈米抗體,其具有殺死腫瘤細胞的作用。
- 如請求項4的免疫調節及抗腫瘤相關奈米抗體,其中該腫瘤細胞是選自於下列所組成的群組:肺腺癌細胞、乳癌細胞、神經膠質母細胞瘤細胞、卵巢癌細胞、口腔癌細胞,及其組合。
- 一種經分離的核酸,其編碼一如請求項1至5中任一項的免疫調節及抗腫瘤相關奈米抗體之胺基酸序列,該經分離的核酸包含一下列核苷酸序列的組合:序列識別號:4、序列識別號:5,及序列識別號:6。
- 一種醫藥組成物,包含一如請求項1至5中任一項的免疫調節及抗腫瘤相關奈米抗體及一醫藥學上可接受的載劑。
- 一種如請求項1至5中任一項的免疫調節及抗腫瘤相關奈米抗體用於製備一治療癌症及免疫相關疾病之醫藥品的用途。
- 如請求項8的用途,其中該癌症是選自於下列所組成的群組:肺腺癌、乳癌、神經膠質母細胞瘤、卵巢癌、口腔癌,及其組合。
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