CN112843255A - Sema4c在制备抗肿瘤药物中的应用 - Google Patents
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Abstract
本发明属于生物医药技术领域，尤其涉及SEMA4C在制备抗肿瘤药物中的应用。本申请基于对乳腺癌的研究，发现乳腺癌组织特异性高表达的SEMA4C，而且这种高表达的SEMA4C参与乳腺癌的淋巴管形成相关的肿瘤转移侵袭及肿瘤增殖。但，其在免疫调节参与抗肿瘤反应以及其药物方面的中和抗体开发尚未有研究报道。本申请证实SEMA4C与T细胞的相互作用方式，并筛选出了能阻断二者结合的中和抗体，体外共培养实验结果表明，anti‑SEMA4C可以恢复T细胞趋化和抗肿瘤功能；体内实验发现anti‑SEMA4C能显著抑制肿瘤生长，且抑瘤效果明显优于anti‑PD‑1，说明SEMA4C中和抗体在抗肿瘤方面存在极大潜力。
Description
技术领域
本发明属于生物医药技术领域，尤其涉及SEMA4C在制备抗肿瘤药物中的应用。
背景技术
肿瘤组织是一个复杂的生态微环境，包括多种细胞(肿瘤细胞、成纤维细胞、淋巴细胞等)和可溶性分子以及酸性低氧高压的物理环境。尽管在肿瘤组织中CD8+T细胞、NK细胞和NKT细胞具有直接杀肿瘤功能，CD4+T细胞可以分泌抗肿瘤细胞因子和通过抗原提呈细胞(APC)促进CD8+T细胞功能活化进而抑制肿瘤，但是肿瘤微环境中存在复杂的免疫抑制性分子和机制介导肿瘤细胞逃脱免疫细胞的攻击，如：低氧低PH环境；免疫抑制性的细胞因子IL-10，TGF-β；低葡萄糖和重要氨基酸；调节性T细胞、MDSC细胞以及具有促肿瘤功能的M2型巨噬细胞；免疫抑制性的受体和配体等。由此可见，肿瘤环境中的CD8+T细胞等抗肿瘤淋巴细胞处于免疫功能障碍状态(dysfunction)。因此，拮抗淋巴细胞功能抑制信号使之免疫正常化(immune normalization)成为肿瘤免疫治疗的重要方向。
早在1997年华人科学家陈列平教授就提出抗4-1BB抗体能够通过促进T细胞活化从而增强其抗肿瘤功能，开启了肿瘤免疫的新时代。“免疫检查点”应运而生，一系列重要的免疫检查点分子如CTLA4，PD-1/PD-L1，TIM3，LAG3，TIGIT等被发现以及美国FDA批准anti-CTLA4，anti-PD-1/PD-L1单抗药物的临床应用，同时2018年医学和生理学诺贝尔奖颁给了发现CTLA4的James P.Allison教授和发现PD-1的Tasuku Honjo教授，抗免疫检查点治疗成为肿瘤免疫治疗的热门方向。
目前，抗免疫检查点的肿瘤临床治疗主要应用于黑色素瘤、非小细胞肺癌、肾细胞癌、前列腺癌、胃癌、头颈部癌等。其中nivolumab(anti-PD-1)治疗黑色素瘤的客观反映率可达43.7％，同时nivolumab联合ipilimumab(anti-CTLA4)治疗的客观反应率高达57.6％，具有较好的临床效果。那么抗免疫检查点治疗能否应用于乳腺癌？Rita Nanda等人在入组32例给与Pembrolizumab(anti-PD-1)治疗三阴性乳腺癌病人的研究中发现，对Pembrolizumab的总体响应率为18.5％。S.Adams等人在入组170例给与Pembrolizumab治疗侵袭性三阴性乳腺癌病人的研究中的总体响应率仅为4.7％。尽管anti-PD-1/PD-L1联合紫杉醇类药物化疗具有较好的治疗效果，但是单一anti-PD-1/PD-L1的抗免疫检查点治疗收效甚微。
肿瘤微环境从来都是复杂的，肿瘤微环境中的免疫抑制性信号更是复杂多样。多种免疫检查点的发现提示：在肿瘤与淋巴细胞相互博弈中免疫抑制性受体和配体不是单兵作战，免疫检查点的联合治疗必定是个性化、精准化医疗的具体体现。
发明内容
针对现有技术存在的问题，本发明提供了SEMA4C在制备抗肿瘤药物中的应用，目的在于解决现有技术中的一部分问题或至少缓解现有技术中的一部分问题。
本发明的目的是旨在探索阻碍以anti-PD-1/L1为代表的免疫检查点治疗响应率较低的肿瘤的深层机制并开发新的治疗靶点运用于肿瘤治疗。
本发明是这样实现的，SEMA4C基因或其表达产物在制备调控T细胞的趋化及活化的试剂、或调控T细胞脱颗粒功能的试剂、或调控T细胞的代谢的试剂中的应用。
进一步地，所述调控T细胞的趋化及活化具体为SEMA4C蛋白调控T细胞的趋化受体CXCR3和/或归巢受体CD62L的表达。
进一步地，所述调控T细胞的代谢具体为抑制Hif-1α和/或c-Myc蛋白的表达。
SEMA4C基因或其表达产物在制备抑制T细胞活化信号通路中磷酸酶活性的试剂中的应用。
进一步地，所述磷酸酶包括p-Plcγ、p-P65、p-AKT473、p-ERK、p-S6中的至少一种。
SEMA4C基因或其表达产物在制备调控葡萄糖转运体或糖酵解途径中代谢酶的表达的试剂中的应用。
进一步地，所述葡萄糖转运体为Glut1，所述糖酵解途径中代谢酶包括HK1、PFK1以及PK1中的至少一种。
SEMA4C基因或其表达产物在制备调控T细胞Enolase1活性的试剂中的应用。
靶向SEMA4C/Enolase1结合的试剂在制备抗肿瘤治疗药物方面的应用。
综上所述，本发明的优点及积极效果为：
本申请基于对乳腺癌的研究，发现乳腺癌组织特异性高表达的SEMA4C，而且这种高表达的SEMA4C参与乳腺癌的淋巴管形成相关的肿瘤转移侵袭及肿瘤增殖。但，其在免疫调节参与抗肿瘤反应以及其药物方面的中和抗体开发尚未有研究报道。
本申请发明人利用多片段同源重组技术构建了7个SEMA4C截断体系统，加入外源性His-Enolase1重组蛋白处理并利用His-APC流式抗体检测，证实SEMA4C的Sema domain与Enolase1存在结合。进一步地构建并筛选出了能阻断SEMA4C与Enolase1结合的中和抗体，体外共培养实验结果表明，SEMA4C中和抗体可以逆转其对于Enolase1活性的抑制作用，恢复T细胞趋化和抗肿瘤功能；体内实验发现anti-SEMA4C能显著抑制肿瘤生长，且抑瘤效果明显优于anti-PD-1，说明SEMA4C中和抗体在抗肿瘤方面存在极大潜力。
附图说明
图1是研究SEMA4C在免疫调节方面的作用的实验结果；
图2是研究SEMA4C的效应机制的实验结果；
图3是研究SEMA4C的分子机制的实验结果；
图4是研究SEMA4C与T细胞的相互作用方式的实验结果；
图5是Enolase1表达相关实验结果；
图6是SEMA4C与Enolase1结合的中和抗体筛选实验结果；
图7是中和抗体体内实验结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例对本发明进行进一步详细说明，各实施例及试验例中所用的设备和试剂如无特殊说明，均可从商业途径得到。此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
根据本申请包含的信息，对于本领域技术人员来说可以轻而易举地对本发明的精确描述进行各种改变，而不会偏离所附权利要求的精神和范围。应该理解，本发明的范围不局限于所限定的过程、性质或组分，因为这些实施方案以及其他的描述仅仅是为了示意性说明本发明的特定方面。实际上，本领域或相关领域的技术人员明显能够对本发明实施方式作出的各种改变都涵盖在所附权利要求的范围内。
为了更好地理解本发明而不是限制本发明的范围，在本申请中所用的表示用量、百分比的所有数字、以及其他数值，在所有情况下都应理解为以词语“大约”所修饰。因此，除非特别说明，否则在说明书和所附权利要求书中所列出的数字参数都是近似值，其可能会根据试图获得的理想性质的不同而加以改变。各个数字参数至少应被看作是根据所报告的有效数字和通过常规的四舍五入方法而获得的。本发明中，“约”指给定值或范围的10％以内，优选为5％以内。
本发明披露了SEMA4C在制备抗肿瘤药物中的应用，具体如下各实施例所示。
实施例
本发明涉及的实验方法：
一、动物实验
周龄相同且体重相近的雌性huPBMC-NOG-dKO人源化小鼠，购自北京维通利华实验动物技术有限公司。5×106个MDA-MB-231乳腺癌细胞用无血清DMEM培养基重悬后，与基质胶1：1混合均匀并皮下接种于小鼠。当肿瘤的体积达到50-100mm3时，将小鼠随机分为对照组、anti-SEMA4C组以及anti-PD-L1组。每周两次给予200μg药物腹腔注射，每天测量小鼠肿瘤体积并绘制变化曲线。在Day28处死所有小鼠，完整剥离皮下肿瘤组织，称量每个肿瘤重量并进行统计学分析。
二、细胞培养
人乳腺癌细胞系MDA-MB-231从ATCC获得，并使用DMEM培养基(含有10％FBS和青霉素-链霉素溶液)培养。
收集健康人外周血20mL，加入等量Ficoll淋巴细胞分离液并采用密度梯度离心法分离PBMC，使用DynabeadsTM UntouchedTM Human T Cells Kit试剂盒分离提纯CD3+T细胞，调整细胞浓度后加入RPMI-1640培养基(10％FBS、青霉素-链霉素、非-必需氨基酸、β-巯基乙醇和L-谷氨酰胺)中继续培养。CD3+T细胞用5μg/mL TCR活化72小时后，继续用20ng/mLIL-2扩增3-5天即可用于后续试验。
三、免疫组化
将福尔马林固定和石蜡包埋过的三阴性乳腺癌组织进行连续切片。石蜡切片在65-68℃加热2小时后，立即置于环保脱蜡液中脱蜡，随后利用四种不同浓度的乙醇溶液进行递进式补水。组化PBS洗涤3次后，将切片置于抗原修复液中加热30min，自然冷却至室温。利用3％H2O2消耗内源性过氧化物酶，并在37℃下用5％BSA封闭30min。加入一抗SEMA4C(1：400)，CD4(1：100)，CD8(1：400)和IFN-γ(1：400)4℃孵育过夜。第二天与相应的HRP二抗反应后，使用DAB进行显色。随后用苏木素染核，并在显微镜下观察。
四、流式细胞术检测T细胞
取106个纯化后的T细胞，PBS洗涤两次后加入抗CD4,CD8,CXCR3,CD62L,以及CD107a的抗体进行表面染色，避光室温孵育30min。同样取106个纯化后的T细胞，利用Cytofix/Cytoperm试剂盒破膜固定20min，随后加入抗Granzyme B抗体避光孵育1小时。FACS buffer(含0.1％BSA的PBS溶液)洗脱两次，使用Beckman流式细胞仪进行检测。FlowJo用于数据分析。
五、Western blot检测蛋白表达
取适量的扩增后T细胞在基础培养基中静息4小时，用不同浓度梯度、时间梯度SEMA4C处理，再使用5μg/mL TCR重新激活T细胞，然后加入100μL含有蛋白酶抑制剂的RIPA裂解液冰上裂解30min。加入蛋白上样缓冲液混匀后用BCA试剂盒测定蛋白浓度，将蛋白样置于100℃煮沸5分钟。利用10％SDS-PAGE凝胶进行电泳，PVDF膜进行转膜，随后用含有5％BSA的TBST溶液室温封闭1小时。根据目的蛋白分子量的不同，将膜剪切为若干条，分别与一抗在4℃孵育过夜。次日条带复温后用TBST充分洗涤5次，每次5min，室温与相应二抗孵育1小时。随后TBST漂洗5次，每次5min，即可用ECL显影液显色。Bio-rad曝光仪用于采集摄片。
六、mRNA提取和RT-PCR检测
取SEMA4C处理后的T细胞，使用FastPure细胞/组织总RNA分离试剂盒提取mRNA；使用HiScript II Q RT SuperMix for qPCR试剂盒进行逆转录合成cDNA；使用ChamQUniversal SYBR qPCR Master Mix进行RT-PCR检测。
使用的引物序列如下：
①Glut1:TCTGGCATCAACGCTGTCTT,AAGGCAAGTGTCTCGACAGG；
②HK1:GGTCCTGATGCGGTTGG,TCGCCTTTGTTCTCCTTGAT；
③PFK1:GGTGCCCGTGTCTTCTTTGT,AAGCATCATCGAAACGCTCTC；
④PK1:TCTCTTCGTCTTTGCAGCGT,AGATCTTGCTGCCCACTTCC；
⑤18s:ACCCGTTGAACCCCATTCGTGA,GCCTCACTAAACCATCCAATCGG。
使用的PCR体系如下：
2*ChamQ Universal SYBR qPCR Master Mix 10μL
Primer1(10μM)0.4μL
Primer2(10μM)0.4μL
Template DNA/cDNA XμL
ddH2O To 20μL
PCR程序如下：
Step1 95℃30s
Step2 95℃10s+60℃30s循环40次Step3 95℃15s+60℃60s+95℃15s
七、免疫沉淀-质谱检测(IP-MS)
纯化的CD3+T细胞用20ng/ml IL-2处理5-8天，并用TCR刺激72小时。PBS洗涤两次后，细胞沉淀用500μL NP-40裂解液重悬，置于冰上裂解30min，每间隔10min剧烈吹打一次。12000g离心10min，将上清液转移至新的EP管中，加入1μg/ml SEMA4C在4℃旋转孵育4小时。准备新的EP管，每支加入50μL磁珠悬液，放置于磁铁上1min后弃去液体。用含有5μL抗SEMA4C抗体的200μL PBS溶液(含有0.01％TweenTM 20)重悬磁珠，室温旋转孵育10min使抗体与磁珠充分结合。用PBS溶液(含有0.01％TweenTM 20)洗涤两次，将含有SEMA4C蛋白的上清液与包被抗体的磁珠溶液混合，4℃旋转孵育过夜。次日用PBS溶液(含有0.01％TweenTM20)多次洗涤免疫沉淀复合物，然后用50μL 2x蛋白上样缓冲液重悬并置于100℃煮沸10min。将混合物置于磁铁上1min分离磁珠，收集上清液待用。随后采用90V电压进行SDS-PAGE电泳，将凝胶置于考马斯亮蓝溶液中100℃染色2min。用考马洗脱液在水平摇床上充分洗涤凝胶，直至可见清晰蛋白条带。根据分子量，切下目的蛋白条带，保存在新的1.5mL EP管中，寄送公司进行质谱分析。
八、慢病毒包装与转染
利用BamHI和EcoRI两个限制性内切酶将shRNA序列插入pLVX-shRNA2载体质粒中。所用Sh-Enolase1序列如下：forward:5’-GATCCGTACCGCTTCCTTAGAACTTTTCAAGAGAAAGTTCTAAGGAAGCGGTACGTTTTTG-3’,reverse:5’-AATTCAAAAACGTACCGCTTCCTTAGAACTTTCTCTTGAAAAGTTCTAAGGAAGCGGTACG-3’。利用opti-MEM及Lipofectamine 3000将构建成功的shRNA质粒与病毒的包装质粒共转染293T细胞。24小时后换液为完全培养基，继续培养24小时后收集细胞上清(即病毒液)。取合适数量的PBMC于12孔板中，加入0.5mL病毒液与0.5mL完全培养基，置于37℃培养箱中转染24小时。随后换液为完全培养基继续培养48-72小时，可在荧光显微镜下观察表达绿色荧光细胞百分比来估计转染效率。空载的pLVX-shRNA2质粒被用作空白对照。
九、过表达质粒的构建与转染
将Enolase1(Gene ID:2023),Plastin2(Gene ID:418852),PSTPIP(Gene ID:9051),EEF1(Gene ID:1915)基因全长cDNA克隆到pIRES2-EGFP载体质粒中。利用DH5α化学感受态细胞进行转化后，在带有卡那霉素(Kanamycin)抗性的平板上进行三区划线。12-16小时后挑取多个合适大小的单克隆加入2mL含抗性的LB培养基中，250rpm于37℃震摇4-6小时。分1mL菌液送公司测序。取测序正确的菌液100μL加入250mL含抗性的LB培养基中，37℃震摇过夜。利用质粒大提试剂盒提取细菌质粒，并测定浓度。状态良好的293T细胞以合适密度种在六孔板中，PBS洗两遍后，换液为1.5mL无抗性完全培养基。用250μL opti-MEM分别溶解2μg质粒和5μL Lipofectamine 3000(每孔)，常温孵育5min后轻柔混匀，室温继续孵育20min。将转染混合溶液轻柔逐滴加入六孔板中，37℃转染过夜，换液为2mL含抗性完全培养基继续培养。
十、SEMA4C截断体的构建
SEMA4C全长蛋白由胞内区、跨膜区以及含有三个结构域的胞外段组成，这三个结构域依次是SEMA domain(D1),PSI domain(D2)以及Ig-Like domain(D3)。根据
Ultra One Step Cloning Kit试剂盒的使用说明，采用多片段同源重组法构建缺失不同结构域的7个SEMA4C截断体产物。重组产物的转化、鉴定及质粒提取方法同“过表达质粒的构建与转染”中所述。将构建成功的SEMA4C截断体质粒转染状态良好的293T细胞，与5μg His-Enolase1重组蛋白孵育2小时后，利用His-APC抗体进行流式检测，并对平均荧光强度MFI进行统计学分析。
十一、利用Enolase Activity Assay Kit试剂盒检测Enolase1的活性。
实验结果：
1.为了研究SEMA4C在免疫调节方面的作用，发明人对临床病理组织样本进行连续切片并免疫组化染色，Image J分析组化染色强度并统计分析。结果表明：SEMA4C表达水平与CD4+,CD8+T细胞的浸润程度呈显著负相关，与IFN-γ的表达量也呈负相关(图1A-D)。由此提示SEMA4C可能参与了淋巴细胞的趋化及活化。
2.接着对SEMA4C的效应机制进行了初步探讨，发明人直接用不同浓度的SEMA4C重组蛋白(R&D，6125-S4-050)在体外处理T淋巴细胞，流式检测发现CD4+T细胞的趋化受体CXCR3、归巢受体CD62L表达明显受到抑制。CD8+T细胞也呈现出相同的表型(图2A-B)，此外，CD8+CD107A+T细胞(图2C)以及CD8+Granzyme B+T细胞(图2D)所占比例显著下降，提示T细胞脱颗粒功能同样受到抑制。
3.为了进一步探究分子机制，发明人用SEMA4C重组蛋白处理TCR活化过的T淋巴细胞。Western Blot实验结果表明SEMA4C抑制T细胞活化信号通路中多种重要磷酸酶的活性，如p-Plcγ、p-P65、p-AKT473、p-ERK、p-S6，而且SEMA4C对于T细胞活化信号的抑制存在一定程度的浓度依赖性(图3A)。SEMA4C对于T细胞的代谢过程也有影响，(1)抑制Hif-1α和c-Myc蛋白的表达(图3B)，(2)抑制葡萄糖转运体Glut1以及糖酵解途径中关键代谢酶HK1、PFK1以及PK1 mRNA的水平(图3C-F)，(3)流式检测CD4+和CD8+T细胞Glut1表达均下调(图3G)，且平均荧光强度MFI有统计学差异(图3H，I)。
4.SEMA4C与T细胞的相互作用方式尚未阐明，为此，发明人用融合Fc片段的SEMA4C重组蛋白(R&D，6125-S4-050)与T细胞共孵育，anti-Fc-PE抗体染色，流式检测发现相较于对照组，实验组荧光强度明显增强，证实SEMA4C能够与T细胞直接结合(图4A)。接着利用IP-质谱技术，筛选可能的SEMA4C受体(图4B)。通过在293T工具细胞上过表达潜在受体分子，并用流式检测其与SEMA4C-Fc融合蛋白结合情况，发明人确定了SEMA4C在T细胞膜表面的受体为Enolase1(图4C)，而非目前已知的经典受体PlexinB2。为了进一步确证SEMA4C与Enolase1相互作用的结构域，发明人利用多片段同源重组技术构建了7个SEMA4C截断体系统，加入外源性His-Enolase1重组蛋白处理并利用His-APC流式抗体检测，证实SEMA4C的Sema domain与Enolase1存在结合(图4D)。
5.接着，发明人发现TCR活化的T细胞表面Enolase1表达量随时间推移增加(图5A)，且WB结果表明Enolase1逐渐向细胞膜表面转移(图5B)。此外，烯醇化酶活性测定试剂盒检测发现SEMA4C处理后的T细胞Enolase1活性下调，并且具有统计学差异(图5C-E)。代谢组学的结果验证了这个发现，Enolase1作为糖酵解过程中关键的代谢酶，催化3/2PG转化为磷酸烯醇丙酮酸盐，当Enolase1活性受到抑制时，上游代谢产物3/2PG积累，下游产物含量减少，从而抑制整个糖代谢通路(图5F)。发明人利用慢病毒转染人原代T淋巴细胞，下调Enolase1表达，再次流式检测T细胞各项功能指标，发现T细胞的活化、趋化和抗肿瘤功能均受到显著抑制(图5G-I)。
6.为了拓展此项研究成果临床应用的可能性，发明人构建并筛选出了能阻断SEMA4C与Enolase1结合的中和抗体(图6A)，并用于后续实验。体外共培养实验结果表明，SEMA4C中和抗体可以逆转其对于Enolase1活性的抑制作用(图6C-E)，恢复T细胞趋化和抗肿瘤功能(图6G-J)。
7.体内实验方面，该中和抗体被应用于人源化CDX动物模型(图7A)。给药期间(200μg/只，每周两次)，每天记录小鼠肿瘤大小并绘制变化曲线(图7B)，处死小鼠后完整剥离肿瘤组织并进行统计学分析，发现anti-SEMA4C能显著抑制肿瘤生长，且抑瘤效果明显优于anti-PD-1(图7C-D)，说明SEMA4C中和抗体在抗肿瘤方面存在极大潜力。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
序列表
<110> 华中科技大学同济医学院附属同济医院
<120> SEMA4C在制备抗肿瘤药物中的应用
<160> 10
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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tcgcctttgt tctccttgat 20
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ggtgcccgtg tcttctttgt 20
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<213> 人工序列(Artificial Sequence)
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Claims (9)
1.SEMA4C基因或其表达产物在制备调控T细胞的趋化及活化的试剂、或调控T细胞脱颗粒功能的试剂、或调控T细胞的代谢的试剂中的应用。
2.根据权利要求1所述的应用，其特征在于：所述调控T细胞的趋化及活化具体为SEMA4C蛋白调控T细胞的趋化受体CXCR3和/或归巢受体CD62L的表达。
3.根据权利要求1所述的应用，其特征在于：所述调控T细胞的代谢具体为抑制Hif-1α和/或c-Myc蛋白的表达。
4.SEMA4C基因或其表达产物在制备抑制T细胞活化信号通路中磷酸酶活性的试剂中的应用。
5.根据权利要求4所述的应用，其特征在于：所述磷酸酶包括p-Plcγ、p-P65、p-AKT473、p-ERK、p-S6中的至少一种。
6.SEMA4C基因或其表达产物在制备调控葡萄糖转运体或糖酵解途径中代谢酶的表达的试剂中的应用。
7.根据权利要求6所述的应用，其特征在于：所述葡萄糖转运体为Glut1，所述糖酵解途径中代谢酶包括HK1、PFK1以及PK1中的至少一种。
8.SEMA4C基因或其表达产物在制备调控T细胞Enolase1活性的试剂中的应用。
9.靶向SEMA4C/Enolase1结合的试剂在制备抗肿瘤治疗药物方面的应用。
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