CN116396383A - 血液分离的免疫细胞在治疗疾病中的应用 - Google Patents
血液分离的免疫细胞在治疗疾病中的应用 Download PDFInfo
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Abstract
本发明涉及血液分离的免疫细胞在治疗疾病中的应用。本发明从血液中提取制备得到了DC‑CIK免疫细胞,将所述的免疫细胞与靶向S100A9的单克隆抗体联合或单独使用后能够有效的抑制神经胶质瘤细胞的存活率,将二者制备成为药物组合物能够有效的用于神经胶质瘤的治疗。并且,所述的单抗能够高特异性的抑制S100A9蛋白的表达活性。
Description
技术领域
本申请涉及生物领域,具体的涉及一种血液分离的免疫细胞在治疗疾病中的应用。
背景技术
癌症治疗领域的发展一直在探索新的治疗方法。近年来,免疫细胞疗法逐渐成为癌症治疗的新兴领域,该方法通过分离患者血液中的免疫细胞,经过体外扩增和改造后,再回输至患者体内,从而有针对性地攻击癌细胞。免疫细胞疗法具有针对性强、副作用相对较小的特点,已成功应用于多种癌症的治疗,如白血病、淋巴瘤等。
免疫细胞疗法主要包括两种类型:自然杀伤细胞(NK细胞)疗法和T细胞受体改造(CAR-T)细胞疗法。NK细胞具有直接杀伤肿瘤细胞的能力,不需要经过免疫识别过程。在NK细胞疗法中,首先从患者血液中分离出NK细胞,然后进行体外扩增,最后将扩增后的NK细胞回输至患者体内,以增强其抗肿瘤作用。CAR-T细胞疗法则是通过基因工程技术将具有特异性抗原识别功能的嵌合抗原受体(CAR)导入T细胞,从而赋予T细胞识别和攻击肿瘤细胞的能力。经过体外扩增后,将改造过的T细胞回输至患者体内,发挥抗癌作用。
尽管免疫细胞疗法在癌症治疗领域取得了一定的成功,但仍面临一些挑战,如疗效的个体差异、副作用的控制及治疗成本的问题。为了进一步提高免疫细胞疗法的疗效和安全性,科研人员正不断优化相关技术,如改善细胞制备方法、开发新型CAR结构以降低副作用,以及探索联合其他免疫治疗手段以增强疗效。随着技术的不断发展,免疫细胞疗法有望为更多癌症患者带来希望。
S100A9蛋白是一种小分子钙结合蛋白,属于S100蛋白家族成员。近年来,研究表明S100A9与多种癌症的发生、发展和转移密切相关。S100A9在许多恶性肿瘤中表达增高,如乳腺癌、结肠癌、胃癌等。研究发现,S100A9可能通过调控多种信号通路影响肿瘤细胞的生长、凋亡、侵袭和转移。例如,S100A9可以激活NF-κB、MAPK、STAT3等信号通路,促进肿瘤细胞的增殖和抵抗凋亡。此外,S100A9还参与调控肿瘤微环境,通过影响免疫细胞的浸润和功能,促进炎症反应以及细胞外基质重塑等过程,从而为肿瘤的发展和转移创造有利条件。
由于S100A9在多种癌症中的异常表达和显著的生物学作用,其已成为肿瘤研究领域的重要靶点。针对S100A9的抑制剂、拮抗剂以及中和抗体等治疗策略正在积极开发中。部分研究表明,S100A9抑制剂可以显著抑制肿瘤生长、侵袭和转移,且具有较好的安全性。同时,S100A9在肿瘤中的表达水平也被认为是一种潜在的生物标志物,有望为癌症的诊断和预后评估提供重要信息。尽管S100A9与癌症关系的研究已取得一定进展,但仍需深入研究其作用机制、筛选更有效的治疗药物,以期将其应用于癌症的临床诊治。
神经胶质瘤是一种常见的中枢神经系统肿瘤,具有高度侵袭性和复发率。由于S100A9在多种癌症中的异常表达及显著的生物学作用,近年来,科研人员开始关注S100A9在神经胶质瘤中的作用及其治疗潜力。一些研究发现,S100A9在神经胶质瘤组织中的表达显著增高,且其表达水平与肿瘤的恶性程度和预后密切相关。基于此,研究者们开始探讨针对S100A9的治疗策略,如利用S100A9单克隆抗体进行治疗。
S100A9单克隆抗体通过特异性结合S100A9蛋白,抑制其在神经胶质瘤中的生物学作用。研究表明,S100A9单克隆抗体可以有效抑制神经胶质瘤细胞的增殖、侵袭和转移。此外,S100A9单克隆抗体还能调节肿瘤微环境,抑制炎症反应、免疫细胞的浸润和功能等过程,从而降低肿瘤的恶性程度。尽管S100A9单克隆抗体在神经胶质瘤治疗中的应用尚处于早期研究阶段,但其具有很大的潜在价值。未来,需要开展更多的实验研究和临床试验,以验证S100A9单克隆抗体治疗神经胶质瘤的有效性和安全性,为神经胶质瘤患者提供新的治疗选择。
但是目前,针对S100A9单克隆抗体提供可以选择的类型和种类还不够多,特别是S100A9单克隆抗体联合免疫细胞用于神经胶质瘤细胞的抑制实验的研究还不够多,提供可选择的形式也有待进一步的提高。
发明内容
本发明提供了一种针对神经胶质瘤治疗的抗体疗法。神经胶质瘤是一种常见的中枢神经系统恶性肿瘤,具有高度侵袭性和复发率。当前,神经胶质瘤的治疗手段主要包括手术切除、放疗和化疗,但治疗效果有限,预后较差。因此,亟需研发新型治疗策略以提高神经胶质瘤患者的生存率和生活质量。本发明设计了一种针对神经胶质瘤相关分子靶点的抗体疗法,旨在实现高效、安全和持久的治疗效果。
一方面,本发明提供了特异性针对S100A9的单克隆抗体,通过测序,鉴定得到其重链可变区序列如下所示(SEQ ID NO:1):
EVQLQESGPGLVAPPQSLSITCTVSGFSLFMIAYVWVRQPPGRGLEWLGHHAFHSWAKWGWCWHQRLSISKDNSKSQVFLGMNSLQTDDTAIYYCARVDWIMQKHTWGAGTTVTVSS
轻链可变区如下所示(SEQ ID NO:2):
DILMTQSPASLSASVGETVSITCGSKWIMTHAAGWYQQKQGKSPQFLVYGFNCFQQGVPSRFQGSGSGTQYSLKIRSLQPEDFGNYYCDYVTHFAFIFGAGTKLEIK
具体的,在一些实施方案中,所述的分离的抗S100A9抗体、其抗原结合片段、其变体或其衍生物的重链可变区序列与SEQ ID No.1所示的序列具有至少95%但不是100%的序列同一性或与SEQ ID No.4所示序列相比具有至少一个氨基酸取代,例如至少约96%、97%、98%或99%,例如至少2、3、4、5个或更多个氨基酸取代,优选保守取代,和/或其轻链可变区序列与SEQ ID No.2所示的序列具有至少95%但不是100%的序列同一性或与SEQID No.5所示序列相比具有至少一个氨基酸取代,例如至少约96%、97%、98%或99%,例如至少2、3、4、5个或更多个氨基酸取代,优选保守取代。所述氨基酸取代不会破坏所述分离的抗S100A9抗体、其抗原结合片段、其变体或其衍生物与其抗原的结合。
关于本文确定的抗体序列的“百分比(%)氨基酸序列同一性”或“同源性”定义为,在序列比对后,候选序列中与被比较的抗体中的氨基酸残基一致的氨基酸残基的百分比,所述比对将任何保守替换作为序列同一性一部分。例如,可以使用公开可用的计算机软件,如BLAST、BLAST-2、ALIGN、Megalign(DNASTAR)或MUSCLE软件,以本领域技术人员已知的各种方法进行比对,以确定氨基酸序列同一性的百分比。本领域熟练技术人员可以确定用于测量比对的适当参数,包括在整个待比较序列的全长上达到最大比对所需的任何算法。
进一步的,本发明还提供了一种药物组合物,所述的药物组合物含有本发明的特异性针对S100A9的单克隆抗体,其重链可变区序列如SEQ ID NO:1所示,轻链可变区如SEQID NO:2所示。
进一步的,所述的药物组合物还含有药学上可接受的载体。
在实际应用中,所述的载体可以是需要根据药物的性质、给药途径和剂型要求来选择合适的赋形剂或载体。例如微晶纤维素、轻质碳酸钙、磷酸氢钙、玉米淀粉、乳糖、纤维素、羧甲基纤维素钠、羧甲基纤维素钙、聚乙烯吡咯烷酮(PVP)、聚山梨酯80(Tween 80)、硬脂酸镁、蔗糖、甘油、丙二醇、聚乙二醇(PEG)、山梨醇、、甲基纤维素、乙酸纤维素、聚丙烯酸钠、玻璃状碳酸钙、黄原胶、青蒿素、聚丙烯酸酯、粘豆胶、羧甲基纤维素钾、珍珠岩、胶原蛋白、黄酮类、月桂酸钠、脱氢醋酸、聚乙烯醇、聚糖、葡萄糖、玻璃状硅酸钠、蒙脱石、聚氯乙烯、聚对苯二甲酸乙二醇酯(PET)、聚乙烯醚、纳米硅、硅胶、聚丙烯酸盐、纤维素琼脂、石蜡、胶体硅酸钠、聚醚、聚丙烯酸、硼砂、硼等。
可接受的载体、赋形剂或稳定剂在使用的剂量和浓度下对接受者无毒,并包含缓冲剂如磷酸盐、柠檬酸盐和其他有机酸;抗氧化剂,包括抗坏血酸和蛋氨酸;防腐剂(如十八烷基二甲基苄基氯化铵;氯化六甲铵;苯扎氯铵,苄索氯铵;苯酚、丁醇或苯甲醇;对羟基苯甲酸烷基酯,如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯;儿茶酚;间苯二酚;环己醇;3-戊醇;和间甲酚);低分子量(少于约10个残基)多肽;蛋白质,如血清白蛋白、明胶或免疫球蛋白;亲水性聚合物,如聚乙烯吡咯烷酮;氨基酸,如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸;单糖、二糖和其他碳水化合物,包括葡萄糖、甘露糖或糊精;螯合剂,如EDTA;糖,如蔗糖、甘露醇、海藻糖或山梨糖醇;成盐的反离子如钠。
本申请还提供了将所述抗体试剂施用于个体以治疗疾病或病况(如癌症)的方法,其中所述方法还包括施用第二药剂或疗法。在一些实施方案中,所述第二药剂或疗法是用于治疗疾病或病况的标准或常用药剂或疗法。在一些实施方案中,所述第二药剂或疗法包括免疫细胞。
进一步的,所述的免疫细胞是本领域常见的DC-CIK细胞。
进一步的,本发明还提供了S100A9的单克隆抗体和DC-CIK细胞在制备用于杀伤神经胶质瘤细胞的药物组合物中的用途。
其中,单抗的用量为100-200μg/mL,所述胶质瘤细胞与DC-CIK细胞的效靶比为1:10-1:20。
在一些实施方案中,上述方面中的药物组合物还可以用于治疗其他的肿瘤,优选地,所述肿瘤选自黑色素瘤、胰腺癌、结直肠癌、乳腺癌、肺癌、鼻咽癌、肝癌、胃癌、食道癌、乳腺癌、肾癌、喉癌、胆囊癌、膀胱癌、前列腺癌、宫颈癌、乳腺癌、卵巢癌、子宫癌、头颈癌、皮肤癌、甲状腺癌、舌癌、胸腺癌、囊性脑肿瘤、神经胶质瘤、淋巴瘤,优选地,选自黑色素瘤、胰腺癌、结直肠癌和乳腺癌。
有益效果
本发明从血液中提取制备得到了DC-CIK免疫细胞,将所述的免疫细胞与靶向S100A9的单克隆抗体联合或单独使用后能够有效的抑制神经胶质瘤细胞的存活率,将二者制备成为药物组合物能够有效的用于神经胶质瘤的治疗。并且,所述的单抗能够高特异性的抑制S100A9蛋白的表达活性。
附图说明
图1本发明单克隆抗体的亚型鉴定
图2S-2B4单克隆抗体的Western blotting鉴定结果图
图3各治疗组对细胞S100A9蛋白的表达情况的影响
具体实施方式
下面将参照附图更详细地描述本发明的具体实施例。虽然附图中显示了本发明的具体实施例,然而应当理解,可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反,提供这些实施例是为了能够更透彻地理解本发明,并且能够将本发明的范围完整的传达给本领域的技术人员。
实施例1靶向S100A9的单克隆抗体的制备
用重组人S100A9蛋白,Unitprot ID:P06702购买自纽普生物对8周龄的BALB/c小鼠进行免疫,共免疫4次,在第一次免疫中使用完全弗氏佐剂与蛋白等比例混合后进行乳化,其他免疫中使用不完全弗氏佐剂与蛋白等比例混合后进行乳化,免疫剂量为每只60μg。免疫完成后3周,处死小鼠,制备脾细胞悬液,取对数生长期的小鼠骨髓瘤细胞SP2/0,两者按照1:6的比例进行细胞融合。融合后的细胞悬液加入含有饲养细胞的96孔板,37℃、5%CO2培养。待细胞克隆出现后,取细胞上清以酶联免疫吸附实验(ELISA)检测抗体滴度,挑选阳性克隆。对选取的阳性细胞进采用有限稀释法进行5次克隆化,直至细胞孔中的培养上清阳性率达100%。通过有限稀释法筛选并获得一株能够稳定分泌抗S100A9的杂交瘤细胞,命名为S-2B4。
大量培养获得的杂交瘤细胞,接种小鼠腹腔制备腹水。收集小鼠腹水,采用中压液相色谱仪纯化单抗。用平衡液(20mmol/L磷酸盐缓冲液,p,7.4)平衡HilTrap Protein A HP柱,腹水,0.22μm滤膜过滤后上柱结合,平衡液洗涤,然后以甘氨酸缓冲,0.1mol/L,pH2.7)洗脱抗体.分段收集洗脱液,用中和液(1mol/Ltris-HCl,pH9.0)调整抗体溶液pH至7.0。12%SDS-PAGE电泳分析煮沸的抗体显示存在轻链和重链二条条带,纯化的单抗加入15%甘油,BCA定量限定抗体的浓度为3.4mg/mL,分装,-80℃保存。
实施例2单抗特性鉴定
通过抗体亚型、抗体效价的检测对所制备的单克隆抗体进行初步鉴定。使用间接ELISA法进行测定抗体效价,将重组人S100A9蛋白按1μg/mL包被于聚苯乙烯微孔板,使用PBS对初始浓度为1mg/mL的单克隆抗体进行稀释作为一抗,浓度从1:500梯度稀释至1:512000,HRP标记的二抗来反应,以OD450nm值是阴性对照2.1倍,即P/N>2.1检测孔的最低抗体浓度的定为抗体效价。单抗亚型的鉴定则以纯化的单抗作为一抗,HRP标记的抗小鼠亚类的抗体作为二抗来分别反应。结果如图1所示,S-2B4单克隆抗体亚型为IgG1;效价检测结果显示:S-2B4单克隆抗体的效价为1:2580000。
实施例2S-2B4单克隆抗体的Western blotting鉴定
取重组人S100A9蛋白、BSA进行SDS-PAGE分析,通过半干转膜仪转至PVDF膜。5%脱脂奶粉封闭、洗涤后,以S-2B4单克隆抗体(l:2000稀释)单克隆抗体作为一抗,37℃孵育1h;以HRP标记的羊抗鼠IgG(l:3000稀释)为二抗,37°C孵育1h,ECL发光显色鉴定单抗的特异性情况。结果如图2所示。
从图2的结果可以看出,针对S-2B4单克隆抗体能够特异性的和重组人S100A9蛋白结合,而不与BSA结合,表献出了较好的特异性。
实施例3S-2B4单克隆抗体的亲和力鉴定鉴定
在Octet系统中,通过生物层干涉测量(BLI)技术对抗体与S100A9蛋白的结合进行分析。按照生产商推荐的方法,使用抗人IgG涂覆的生物传感器(Fc特异性)来俘获S-2B4单克隆抗体抗体。通过带负载的生物传感器置于含有10μg/mL在10倍稀释的动力学缓冲液(Fortebio)中稀释的S100A9蛋白的孔中,将S100A9蛋白加载到固定的S-2B4抗体之上。在约10分钟时间内,实时测量由S100A9蛋白结合引起的生物传感器表面上光反射的差异(Δλ,nm),并将其用来通过Octet软件(V4.0,Fortebio)计算缔合常数(ka[1/Mxs])。接下来,将带负载的生物传感器置于只含有动力学缓冲液(10倍稀释于PBS)的孔中以测定解离常数(kd[l/s])。使用模型1∶1(langmuir),进行动力学分析,测定亲和力(KD[M])。结果如表1所示。
表1S-2B4单克隆抗体的亲和力性质
抗体名称 | Ka[1/Ms] | Kd[1/s] | KD[M] |
S-2B4 | 3.83E+05 | 1.75E-04 | 4.57E-10 |
表1显示S-2B4单克隆抗体具有较好的亲和力。
实施例4DC-CIK细胞的制备
该方法为本领域已知的较为成熟的方法:用淋巴细胞分离液分离健康自愿捐献血液中的白细胞,收集单个核细胞中的贴壁细胞,2h后细胞呈圆形,呈小型集落聚集,为DC细胞。加入1000U/mLrhIL-4、1000U/mLrhGM-CSF到含10%新生小牛血清的RPMI1640培养基中培养7d,DC细胞成熟,细胞表面有大量的突起,为典型的树突细胞形态,即成熟的DC细胞。
用淋巴细胞分离液分离健康成人白细胞,收集单个核细胞中的非黏附细胞。加500U/mLrhIL-2、5ng/mLIL-1α及50ng/mL CD3、50ng/mL rhIFN-γ到含用10%小牛血清的RPMI1640完全培养基中培养。第8天将DC和CIK细胞在37℃、5%CO2培养箱中混合培养2d,即制备得到DC-CIK细胞。
实施例5DC-CIK细胞联合单抗对神经胶质瘤细胞杀伤作用的检测
采用乳酸脱氢酶(LDH)释放测定法,按照Cyto tox 96non-radioactivecytotoxicity assay说明进行操作,以U251人神经胶质瘤细胞为靶细胞,调整靶细胞浓度为1×105个/mL,实验共分如下各组:
(1)DC-CIK低效靶比组,DC-CIK细胞为效应细胞,浓度为1×106个/mL;
(2)DC-CIK高效靶比组,DC-CIK细胞为效应细胞,浓度为3×106个/mL;
(3)联合治疗组,DC-CIK细胞浓度为1×106个/mL,加入S-2B4单克隆抗体,终质量浓度100μg/mL;
(4)低浓度单克隆抗体治疗组,S-2B4单克隆抗体,终质量浓度100μg/mL;
(5)高浓度单克隆抗体治疗组,S-2B4单克隆抗体,终质量浓度200μg/mL;
(6)阳性对照组,5-FU 100μg/mL;
加到96孔板中,在所述效靶比下,均设3个复孔。混匀上述细胞,在5%CO2、37℃的CO2培养箱中培养12h,收集培养后的上清液,在490nm波长下,用酶标仪检测其A值。重复实验5次。计算细胞杀伤率。杀伤率=(A实验-A效应细胞自然释放-A靶细胞自然释放)/(A靶细胞最大释放-A靶细胞自然释放)。
表2DC-CIK细胞联合单抗对U251细胞的杀伤作用
各组 | 杀伤率(%) |
DC-CIK低效靶比组 | 35.23±1.35 |
DC-CIK高效靶比组 | 44.68±2.07# |
联合治疗组 | 93.56±3.42* |
低浓度单克隆抗体治疗组 | 75.32±2.14¥ |
高浓度单克隆抗体治疗组 | 80.63±1.78¥ |
阳性对照组 | 68.73±1.86¥ |
与同组效靶比10:1比较#<0.05;与DC-CIK低效靶比组比较8<0.05;与DC-CIK低效靶比组比较¥<0.05;
从表2可以看出,本发明的单克隆抗体以及DC-CIK均可以有效的对U251细胞杀伤,随着效靶比的增加,DC-CIK细胞的杀伤作用均增强,单克隆抗体也随着浓度的升高而对U251细胞的杀伤率而提高。联合治疗组的杀伤率高于DC-CIK细胞组以及单克隆抗体治疗组,差异有显著性(P<0.05)。见表2。
将以上各组处理后的U251细胞进行收集分离,保持细胞浓度相同,向细胞沉淀中细胞裂解液(RIPA:PMSF=100:1),置于冰上裂解30min,用超声破碎仪破碎细胞,4℃,13000rpm离心15min;用移液枪将各组蛋白上清液转移至新的1.5mlEP管中,并记录上清液体积;用细胞裂解液将各组细胞总蛋白浓度调整到一致,之后再加入蛋白上清液1/4体积的SDS蛋白上样缓冲液,煮10-15min;采用Western Blot检测各组细胞S100A9蛋白的表达情况,以未处理的U251正常细胞作为对照基础表示为1,结果如图3所示。
从图3的结果可以看出,单克隆抗体处理组以及联合处理组的S100A9蛋白相对于对照均显著的被抑制,而阳性对照以及DC-CIK细胞处理组对S100A9蛋白的影响较小,这也说明,本发明的单克隆抗体能够较好的抑制S100A9蛋白的活性进而抑制U251细胞的活性。
本文通过一些实施方式和具体的实施例对本发明进行了说明,而且还针对许多细节进行了描述。对于本领域技术人员而言,本发明可以通过采用一些其它的具体实施方式来实现,也可以在不偏离本发明主旨的情况下针对所披露的内容进行调整和变化。本发明的内容应当包括针对本文所披露内容的调整或变化,或者说应当包括由所附权利要求书所定义的以及与之等同的范围。
Claims (5)
1.一种单克隆抗体,其特征在于,该抗体能与S100A9蛋白特异性结合;所述抗体的重链可变区由如SEQ ID NO:1所示;所述抗体的轻链可变区如SEQ ID NO:2所示。
2.如权利要求1所述的抗体,其特征在于,该抗体是IgG1。
3.一种如权利要求1-2任意一项所述的抗体的用途,其特征在于,所述抗体用于制备治疗与S100A9高表达相关的疾病的药物组合物。
4.S100A9单克隆抗体和DC-CIK细胞的组合在制备用于杀伤神经胶质瘤细胞的药物组合物中的用途,所述抗体的重链可变区由如SEQ ID NO:1所示;所述抗体的轻链可变区如SEQ ID NO:2所示。
5.如权利要求4所述的用途,其特征在于单克隆抗体的用量为100-200μg/mL,所述神经胶质瘤细胞与DC-CIK细胞的效靶比为1:10-1:20。
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