CN110172513A - 突变型免疫多肽临床入组的筛选方法 - Google Patents
突变型免疫多肽临床入组的筛选方法 Download PDFInfo
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- CN110172513A CN110172513A CN201910460627.8A CN201910460627A CN110172513A CN 110172513 A CN110172513 A CN 110172513A CN 201910460627 A CN201910460627 A CN 201910460627A CN 110172513 A CN110172513 A CN 110172513A
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Abstract
本发明涉及涉及突变型免疫多肽临床入组的筛选方法，对正常组织进行全外显子组测序，对肿瘤组织进行全外显子组测序和全转录组测序，比较肿瘤组织和正常组织的全外显子组测序结果，以及根据肿瘤组织的全转录组测序结筛选突变基因，将野生型和筛选出的突变型的基因序列翻译并进行序列比对合成相应氨基酸序列的免疫多肽，对免疫多肽进行免疫原性体外检测，和对免疫多肽对原代肿瘤细胞的杀伤效果验证，筛选得到具有免疫原性的免疫多肽和具有杀伤效果的免疫多肽。
Description
技术领域
本发明涉及肽的筛选，具体涉及突变型免疫多肽临床入组的筛选方法。
背景技术
在肿瘤生成过程中伴随着大量的基因突变，当这些突变位于蛋白编码区时，就可能会导致肿瘤细胞或者微环境中出现新的蛋白质。这种蛋白质或其降解后的多肽片段作为新抗原可以引起免疫应答，这些蛋白质或者其降解后的多肽也被称作肿瘤新抗原。
由于肿瘤微环境中新抗原信号扰动因素多，且肿瘤细胞会通过多种机制影响免疫系统的监察能力，引起免疫逃逸，因此肿瘤患者体内的新抗原不足以激发有效的免疫反应来对抗肿瘤。如果体外制备这种肿瘤新抗原作为疫苗，即可放大肿瘤特异性的突变信号，从而充分激活免疫系统对携带这些新抗原的肿瘤细胞的重新识别和攻击，这将会为肿瘤的免疫治疗带来新动力。
发明内容
本发明的主要目的为个性化肿瘤疫苗的突变型免疫多肽临床入组提供了一种临床前筛选方法，为个性化肿瘤疫苗治疗的有效性和安全性提供了一种更有效的临床前体外验证方法。具体步骤如下：
一种突变型免疫多肽的筛选方法，包括以下步骤：
i.对正常组织进行全外显子组测序(Whole Exome Sequencing，WES)，对肿瘤组织进行WES和全转录组测序(RNA sequence，RNA-seq)。
ii.比较肿瘤组织和正常组织的WES结果，筛选出DNA水平基因突变频率AF>0.1的突变基因。
iii.根据肿瘤组织的RNA-seq结果，优化WES筛选出的基因突变，进一步筛选出RNA水平基因突变频率TPM≥0的突变基因。
iv.将野生型和筛选出的突变型的基因序列翻译并进行序列比对，找出突变前后的氨基酸序列。
v.根据肿瘤特异性外显子突变信息合成相应氨基酸序列的免疫多肽，每条免疫肽由15-30个氨基酸组成，且含有一个突变位点，和多个抗原表位。
vi.对免疫多肽进行免疫原性体外检测，和对免疫多肽对原代肿瘤细胞的杀伤效果验证，筛选得到具有免疫原性的免疫多肽和具有杀伤效果的免疫多肽。
上述方法还具有如下优选方案：
步骤ii中，筛选出DNA水平基因突变频率AF>0.15的突变基因，步骤iii中，筛选出RNA水平基因突变频率1≤FPKM≤10或0<TPM≤1的突变基因。
步骤ii中，筛选出DNA水平基因突变频率AF>0.15的突变基因，步骤iii中，筛选出RNA水平基因突变频率FPKM≥10或TPM≥1的突变基因。
步骤ii中，筛选出DNA水平基因突变频率为移码突变的突变基因，步骤iii中，筛选出RNA水平基因突变频率FPKM≤1或TPM＝0的突变基因。
步骤ii中，筛选出DNA水平基因突变频率为移码突变的突变基因，步骤iii中，筛选出RNA水平基因突变频率FPKM≥1或TPM＞0的突变基因。
附图说明
图1和图2为脊索瘤病人#1的11条特异性免疫肽经过成熟DC负载后免疫病人自身的T细胞后的ELISpot检测结果。
图3为免疫肽对病人#1的原代脊索瘤细胞的杀伤效果。
图4为筛选的流程示意图。
具体实施方式
以下，结合实施例对于本发明作进一步说明，实施例和附图仅用于解释说明而不用于限定本发明的保护范围。
实施案例-个性化疫苗治疗脊索瘤
1.肿瘤和正常组织的WES检测
1)血液样本的收集
手术前抽取病人1管EDTA抗凝血(2ml/管)，进行WES检测。
2)肿瘤组织样本的收集
剥离坏死组织和非肿瘤组织，取绿豆大(需多点取样)肿瘤组织，进行多点肿瘤组织WES检测。
2.肿瘤RNAseq检测
1)肿瘤组织样本的收集
剥离坏死组织和非肿瘤组织，取绿豆大(需多点取样)肿瘤组织，放入冻存管中，置于液氮处理30分钟后转移至-80℃保存。
2)RNAseq检测
脊索瘤患者的肿瘤组织样本进行RNAseq检测。
3.筛选出高表达的肿瘤特异性外显子突变并设计出肿瘤新抗原
根据该病人正常细胞和肿瘤组织WES结果，以及肿瘤组织的RNAseq结果，筛选肿瘤特异性外显子突变，筛选标准如表1。
表1肿瘤特异性外显子突变筛选标准
*AF:Allele Frequency，FPKM:Fragments per Kilobase Million，TPM:Transcripts Per Million
比较肿瘤组织和正常组织的全外显子组测序结果，筛选出DNA水平基因突变频率AF>0.1的突变基因，根据肿瘤组织的全转录组测序结果，优化全外显子组测序筛选出的基因突变，进一步筛选出RNA水平基因突变频率FPKM≥0或TPM≥0的突变基因，病人#1共筛选出了85个肿瘤特异性的突变位点。
根据筛选出的待检测的突变基因的信息，利用https://www.ncbi.nlm.nih.gov/或http://genome.ucsc.edu/cgi-bin/hgTables找到待检测突变基因CDS序列，以及相应突变后的碱基序列。
利用BioXM 2.6等软件，将野生型和筛选出的突变型的基因序列翻译并进行序列比对，找出突变前后的氨基酸序列。
根据肿瘤特异性外显子突变筛选标准(如表1)以及肿瘤新抗原的设计原则，择优设计不多于20条免疫多肽，每条免疫肽由15-30个氨基酸组成，且含有一个突变位点，和多个抗原表位。脊索瘤病人免疫多肽设计结果如表2。
表2：设计合成的免疫肽序列
4.免疫肽序列及其合成。
寻找第三方合成科研级的上述免疫多肽，纯度为95％。
5.免疫多肽临床前体外有效性评估。
5.1免疫肽免疫原性体外检测。
1)根据第三方提供的免疫肽的溶解度测试结果(如下表所示)，溶解免疫肽，配成终浓度为2mg/ml的多肽溶液。
2)免疫肽预刺激T细胞的培养。
a收集病人的50ml肝素抗凝血，利用Ficoll分离获得病人的PBMC。
b加入DMSO或单个多肽(2μg mL-1)孵育PBMC，培养10-14天后，进行酶联免疫斑点(enzyme-Linked Immuno Spot,ELISpot)实验检测免疫肽的免疫原性。
3)实验结果如图1和图2：病人#1的11条特异性免疫肽经过成熟DC负载后免疫病人自身的T细胞后，能明显的检测到斑点，说明已经开始产生IFN-γ(图1)。其中1-IMP04、1-IMP06、1-IMP07的斑点数分别为256.66667±22.84975；407.33333±44.19779；240.33333±26.26997，显著高于对照组NC(144.33333±8.41295)的1.5倍(图2)。
5.2免疫肽对原代肿瘤细胞的杀伤效果验证。
利用乳酸脱氢酶检测被免疫肽激活的T细胞对从同一病人身上分离的肿瘤细胞的杀伤作用。
实验结果如图3所示：1-IMP07，1-IMP08，1-IMP10的特异性的T细胞在效靶比为10：1时对靶细胞(即病人的脊索瘤细胞)的杀伤率分别为67.056±1.959，74.729±13.013，58.097±1.737明显高于对照组47.677±2.002(p<0.05)。
6.结论。
根据以上2个实验结果可以发现，针对脊索瘤病人设计的11条多肽中，1-IMP04，1-IMP06，1-IMP07具有很明显的免疫原性；1-IMP07，1-IMP08,1-IMP09对脊索瘤病人原代分离肿瘤细胞有显著的杀伤效果，如表3所示。
表3：免疫肽免疫原性和对肿瘤细胞杀伤效果总结
0:negative；+:weakly positive；++：positive；+++：strongly positive；n.d.:not determined
因此1-IMP04，1-IMP06，1-IMP07，1-IMP08,1-IMP09为此脊索瘤病人临床前筛选出的特异性免疫多肽。
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<213> 人工序列(Artificial Sequence)
<400> 3
His Ser Gln Tyr His Gly Tyr Tyr Met Lys Leu Asn Ala Pro Gln His
1 5 10 15
Pro Pro Val
<210> 4
<211> 21
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Thr Val Pro Glu His Thr Leu Asn Leu Tyr Pro Pro Ala Gln Met His
1 5 10 15
Ser Glu Gln Ser Pro
20
<210> 5
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
His Gln Leu Met Lys Ser Ile Gly Val Lys Phe Leu Ile Asn Glu Ala
1 5 10 15
Thr Thr Leu
<210> 6
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Asn Ser Val Asp Met Trp Ala Leu Gly Val Ile Ala Tyr Ile Leu Val
1 5 10 15
Ser Gly Thr Met Pro Phe
20
<210> 7
<211> 27
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Arg Ala Ala Phe Gln Leu Trp Ser Asn Val Ser Val Leu Glu Phe Trp
1 5 10 15
Glu Ala Pro Ala Thr Gly Pro Ala Asp Ile Arg
20 25
<210> 8
<211> 26
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Ser Gln Arg Thr Arg Val Phe Gly Ser Glu Arg Ile Met Trp Phe Ser
1 5 10 15
Pro Val Thr Leu Lys Glu Leu Leu Glu Phe
20 25
<210> 9
<211> 27
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Thr Ser Tyr Ala Leu Phe Val Arg Glu Asn Asn Ser His Ala Leu His
1 5 10 15
Ile Gly Ser Ile Ser Ala Thr Asp Arg Asp Ser
20 25
<210> 10
<211> 23
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Ala Ala Lys Arg Gly Pro Gly Gly Val Trp Ala Ala Glu Val Ile Ser
1 5 10 15
Asn Ala Arg Glu Asn Ile Gln
20
<210> 11
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Ala Ala Ala Ala Glu Pro Met Gly Pro Ala Gln Val Pro Met Asn
1 5 10 15
Ser
Claims (7)
1.一种突变型免疫多肽临床入组的筛选方法，其特征在于包括以下步骤：
i.对正常组织进行全外显子组测序，对肿瘤组织进行全外显子组测序和全转录组测序，
ii.比较肿瘤组织和正常组织的全外显子组测序结果，筛选出DNA水平基因突变频率AF>0.1的突变基因，
iii.根据肿瘤组织的全转录组测序结果，优化全外显子组测序筛选出的基因突变，筛选出RNA水平基因突变频率FPKM≥0或TPM≥0的突变基因，
iv.将野生型和筛选出的突变型的基因序列翻译并进行序列比对，找出突变前后的氨基酸序列，
v.根据肿瘤特异性外显子突变信息合成相应氨基酸序列的免疫多肽，每条免疫肽由15-30个氨基酸组成，且含有一个突变位点，和多个抗原表位，
vi.对免疫多肽进行免疫原性体外检测，和对免疫多肽对原代肿瘤细胞的杀伤效果验证，筛选得到具有免疫原性的免疫多肽和具有杀伤效果的免疫多肽。
2.如权利要求1所述的的突变型免疫多肽的筛选方法，其特征在于步骤ii中，筛选出DNA水平基因突变频率AF>0.15的突变基因，步骤iii中，筛选出RNA水平基因突变频率1≤FPKM≤10或0<TPM≤1的突变基因。
3.如权利要求1所述的的突变型免疫多肽的筛选方法，其特征在于步骤ii中，筛选出DNA水平基因突变频率AF>0.15的突变基因，步骤iii中，筛选出RNA水平基因突变频率FPKM≥10或TPM≥1的突变基因。
4.如权利要求1所述的的突变型免疫多肽的筛选方法，其特征在于步骤ii中，筛选出DNA水平基因突变频率为移码突变的突变基因，步骤iii中，筛选出RNA水平基因突变频率FPKM≤1或TPM=0的突变基因。
5.如权利要求1所述的的突变型免疫多肽的筛选方法，其特征在于步骤ii中，筛选出DNA水平基因突变频率为移码突变的突变基因，步骤iii中，筛选出RNA水平基因突变频率FPKM≥1或TPM＞0的突变基因。
6.采用权利要求1~5任一所述的筛选方法得到的免疫多肽。
7.权利要求1~5任一所述的筛选方法在制备肿瘤药物中的应用。
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