CN102101885A - 低诱导抑制性t细胞的人白细胞介素ⅱ突变体及其用途 - Google Patents
低诱导抑制性t细胞的人白细胞介素ⅱ突变体及其用途 Download PDFInfo
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Abstract
本发明属于生物技术的医药新产品，特别公开了一种低诱导抑制性T细胞的人白细胞介素II突变体及其用途。该低诱导抑制性T细胞的人白细胞介素II突变体，其特征在于：由组成多肽的氨基酸序列在白介素II α受体结合点突变，突变的位点在白介素II的37，38，41，42，43，44，45，61，62，65，68和72氨基酸位点。本发明通过对氨基酸定点替代，白细胞介素II突变体降低了诱导调节性T细胞的功能，同时保留了白介素II对自然杀伤细胞免疫活性的属性，白介素II的突变体可用于刺激免疫系统，增强意志肿瘤生长功能，从而提高其临床疗效和应用范围。
Description
(一)技术领域
本发明属于生物技术的医药新产品，特别涉及一种低诱导抑制性T细胞的人白细胞介素II突变体及其用途。
(二)背景技术
白细胞介素II(interleukin-2，IL-2，白介素II)是T淋巴细胞分泌的一种淋巴因子(人白介素II由133个氨基酸组成的蛋白质多肽链，分子量为15KD)，是维持免疫系统中最重要的分子之一。白介素II能促进T细胞成熟、维持T细胞活性，还能促进细胞毒性T淋巴细胞的活性，并使其转化为肿瘤浸润淋巴细胞，促进自然杀伤(NK)细胞的活性，并诱导杀伤肿瘤的淋巴因子细胞和杀伤细胞的增长。白介素II具有增强人体免疫功能，激活自然杀伤细胞，扩增T淋巴细胞的作用，能诱导和激活淋巴因子激活的杀细胞(LAK)及肿瘤浸润淋巴细胞(TLI)，这些细胞都有杀伤病毒及细菌感染细胞以及癌变细胞的作用。由于白介素II具有显著的增强免疫作用，故它有很好的抗病毒、抗癌作用和广泛的临床应用潜力。白介素II对肾细胞癌、黑素瘤、非何杰金氏淋巴瘤、结肠直肠癌有较明显疗效，对肝癌、软巢癌、头颈部鳞癌、膀胱癌、肺癌等有不同程度的疗效。在我国白介素II已广泛应用于临床癌症治疗和病毒感染(中国重组人白介素II行业格局研究与投资前景报告)。
通过促进T细胞和NK细胞成熟和增殖，白介素II诱导抗肿瘤免疫并用于黑色素瘤和肾细胞癌的临床治疗。但是，同时白介素II也增加了外周血调节性T细胞(T抑制细胞)(CD4+CD25+细胞)并抑制抗肿瘤免疫反应。在低剂量情况，白细胞介素II促进淋巴细胞杀死肿瘤功能微小，但是它却明显地促进调节T细胞功能，进一步抑制浸润淋巴细胞的功能和增殖，因为它具有杀死肿瘤细胞和促进抑制细胞的双重功能，目前，白细胞介素II的临床应用有非常大的局限性。
(三)发明内容
本发明为了弥补现有技术的不足，提供了一种降低促进调节性T细胞的能力，保持激活自然杀伤细胞功能的低诱导抑制性T细胞的白细胞介素II突变体及其用途。
本发明是通过如下技术方案实现的：
一种低诱导抑制性T细胞的人白细胞介素II突变体，其特征在于：由组成多肽的氨基酸序列在白介素II α受体结合点突变，突变的位点在人白介素II的37，38，41，42，43，44，45，61，62，65，68和72氨基酸位点。
由于白介素II有三种不同的结合受体(α，β和γ受体)，其中α受体(CD25)主要表达在T抑制细胞，其结合位点在人白介素II的37，38，41，42，43，44，45，61，62，65，68和72氨基酸位点(α受体的口袋)(科学308：1477)，结合以后信号的转导促进抑制T细胞增殖。因此，在这些位点的氨基酸突变会降低α受体(CD4CD25)和白介素II的亲和力，从而进一步影响促进调节性T细胞的能力，所以减弱α受体结合的白介素II基因突变将有减少促进抑制T细胞增殖活性的功能，并为此提高和促进免疫细胞杀伤肿瘤细胞能力。
所述突变体包括一个完整长度的人白介素II的分子或融合蛋白。
通过基因合成反应合成人白介素II突变基因，经过纯化扩增酶切，将合成的基因连接重组至多重宿主表达载体，该重组质粒的表达突变的白介素II蛋白。一个或多个氨基酸突变的白介素II表达载体转染中国仓鼠卵巢细胞(CHO)进行蛋白的生产和纯化。测试显示，位点65突变的人白介素II大大降低或失去促进调节性T细胞的能力，同时保持激活自然杀伤细胞的功能。
本发明白介素II突变体的用途为降低其诱导抑制细胞的功能，增加其抑制体内固体肿瘤生长的效用，所述固体肿瘤为肾癌细胞或其他肿瘤细胞，与其他治疗组合，作用一种疫苗成分，治疗肿瘤和病毒感染。
本发明通过对氨基酸定点替代，白介素II突变体降低了诱导调节性T细胞的功能，同时保留了白介素II对自然杀伤细胞免疫活性的属 性，白介素II的突变体可用于刺激免疫系统，增强抑制肿瘤生长功能，从而提高其临床疗效和应用范围。
(四)附图说明
下面结合附图对本发明作进一步的说明。
图1为人原生体白介素II的氨基酸分子序列；
图2为强调的被变异成人白介素II突变体的氨基酸序列；
图3为人白介素II原生体和白介素II突变体促进自然杀伤细胞增殖的作用图；
图4为小白鼠分别注射人白介素II原生体和白介素II突变体的效果图；
图5为人白介素II原生体和白介素II突变体对肿瘤免疫治疗的效果图。
(五)具体实施方式
实施例1：
人白介素II突变体库的建设和筛选
位点 野生型 突变体
37 T K
38 R K
41T K
42F K
44F K
45Y K
61E K
62E K
65P K
68E K
72L K
根据人白介素II的氨基酸序列(基因库NM-000586)对基因序列进行突变。采用重叠延伸法合成有N-终端多组胺酸标签基因，进一步克隆到多功能表达载体。这些载体转染到中国仓鼠卵巢细胞中，细 胞置于37℃、5％CO2孵箱中培养，72小时后上清液被用来筛选白介素II活性。具有对自然杀伤细胞生物活性的上清液进一步采用固定化金属离子亲和层析对重组蛋白纯化。纯化的蛋白经过电泳检测确认，并进一步用在体外的细胞增殖和体内抗瘤功能研究。
实施例2：
自然杀伤细胞体外的增殖试验
自然杀伤细胞(NK92)被转移到含有白介素II不同突变体的培养液中。6天后，细胞数用台盼蓝染色法和血细胞计数板计数来确定，如附图3所示，白介素II突变体保留促进增加自然杀伤细胞增殖的活性，其增加的幅度相似于白介素II原生体。
实施例3：
突变体白介素II对促进抑制(CD4CD25+)T细胞活性测定
如附图4所示，小白鼠分别注射白介素II原生体或白介素II突变体，每日剂量为20微克共计3天，第4天收集血液，并使用CD25和CD4抗体染色法对外周血细胞进行染色。最后用流式细胞仪测定和数据分析。
实施例4：
突变体与原生体白介素II蛋白的固体肿瘤免疫治疗
如附图5所示，为了确定该突变体相比原生体白介素II的蛋白免疫治疗效果，肾癌细胞接种在正常6周龄雌性小白鼠皮下。从第5天开始，荷瘤小鼠每日进行高剂量(50微克/鼠)白介素II原生体或白介素II突变体注射治疗，疗程共计7天，每天用电动卡尺进行肿瘤大小的测量。相比白介素II原生体，白介素II突变体的疗效观察明显的提高(P＜0.001)，误差线反应的平均标准误差。
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TTFMCEYADETATIVEFLNRWITFCQSIISTLT
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TELKHLQCLEEELKPLEEVLNKAQSKNFHLRPRDLISNINVIVLELKGSE
TTFMCEYADETATIVEFLNRWITFCQSIISTLT
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<120>低诱导抑制性T细胞的白细胞介素II突变体及其用途
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acgttcaaat tctatatgcc taaaaaagcc accgagctga aacatctgca atgcctggag 180
gaggaactga aacctctgga ggaagttctg aatctggcac agagcaaaaa ctttcatctg 240
cgtcctcgtg atctgattag caacattaac gttatcgtgc tggagctgaa aggttctgaa 300
accaccttca tgtgtgagta tgctgacgaa accgccacta ttgtcgagtt tctgaatcgc 360
tggatcacct tttgtcagag catcatcagc accctgaca 399
<210>3
<211>399
<212>DNA
<213>人工序列
<400>3
gctcctacaa gctcttccac gaaaaaaact caactgcagc tggaacatct gctgctggat 60
ctgcaaatga tcctgaacgg catcaacaac tataaaaacc cgaaactgac ccgtatgctg 120
aaattcaaat tctatatgcc taaaaaagcc accgagctga aacatctgca atgcctggag 180
gaggaactga aacctctgga ggaagttctg aatctggcac agagcaaaaa ctttcatctg 240
cgtcctcgtg atctgattag caacattaac gttatcgtgc tggagctgaa aggttctgaa 300
accaccttca tgtgtgagta tgctgacgaa accgccacta ttgtcgagtt tctgaatcgc 360
tggatcacct tttgtcagag catcatcagc accctgaca 399
<210>4
<211>399
<212>DNA
<213>人工序列
<400>4
gctcctacaa gctcttccac gaaaaaaact caactgcagc tggaacatct gctgctggat 60
ctgcaaatga tcctgaacgg catcaacaac tataaaaacc cgaaactgac ccgtatgctg 120
acgaaaaaat tctatatgcc taaaaaagcc accgagctga aacatctgca atgcctggag 180
gaggaactga aacctctgga ggaagttctg aatctggcac agagcaaaaa ctttcatctg 240
cgtcctcgtg atctgattag caacattaac gttatcgtgc tggagctgaa aggttctgaa 300
accaccttca tgtgtgagta tgctgacgaa accgccacta ttgtcgagtt tctgaatcgc 360
tggatcacct tttgtcagag catcatcagc accctgaca 399
<210>5
<211>399
<212>DNA
<213>人工序列
<400>5
gctcctacaa gctcttccac gaaaaaaact caactgcagc tggaacatct gctgctggat 60
ctgcaaatga tcctgaacgg catcaacaac tataaaaacc cgaaactgac ccgtatgctg 120
acgttcaaaa aatatatgcc taaaaaagcc accgagctga aacatctgca atgcctggag 180
gaggaactga aacctctgga ggaagttctg aatctggcac agagcaaaaa ctttcatctg 240
cgtcctcgtg atctgattag caacattaac gttatcgtgc tggagctgaa aggttctgaa 300
accaccttca tgtgtgagta tgctgacgaa accgccacta ttgtcgagtt tctgaatcgc 360
tggatcacct tttgtcagag catcatcagc accctgaca 399
<210>6
<211>399
<212>DNA
<213>人工序列
<400>6
gctcctacaa gctcttccac gaaaaaaact caactgcagc tggaacatct gctgctggat 60
ctgcaaatga tcctgaacgg catcaacaac tataaaaacc cgaaactgac ccgtatgctg 120
acgttcaaat tcaaaatgcc taaaaaagcc accgagctga aacatctgca atgcctggag 180
gaggaactga aacctctgga ggaagttctg aatctggcac agagcaaaaa ctttcatctg 240
cgtcctcgtg atctgattag caacattaac gttatcgtgc tggagctgaa aggttctgaa 300
accaccttca tgtgtgagta tgctgacgaa accgccacta ttgtcgagtt tctgaatcgc 360
tggatcacct tttgtcagag catcatcagc accctgaca 399
<210>7
<211>399
<212>DNA
<213>人工序列
<400>7
gctcctacaa gctcttccac gaaaaaaact caactgcagc tggaacatct gctgctggat 60
ctgcaaatga tcctgaacgg catcaacaac tataaaaacc cgaaactgac ccgtatgctg 120
acgttcaaat tctatatgcc taaaaaagcc accgagctga aacatctgca atgcctggag 180
aaagaactga aacctctgga ggaagttctg aatctggcac agagcaaaaa ctttcatctg 240
cgtcctcgtg atctgattag caacattaac gttatcgtgc tggagctgaa aggttctgaa 300
accaccttca tgtgtgagta tgctgacgaa accgccacta ttgtcgagtt tctgaatcgc 360
tggatcacct tttgtcagag catcatcagc accctgaca 399
<210>8
<211>399
<212>DNA
<213>人工序列
<400>8
gctcctacaa gctcttccac gaaaaaaact caactgcagc tggaacatct gctgctggat 60
ctgcaaatga tcctgaacgg catcaacaac tataaaaacc cgaaactgac ccgtatgctg 120
acgttcaaat tctatatgcc taaaaaagcc accgagctga aacatctgca atgcctggag 180
gagaaactga aacctctgga ggaagttctg aatctggcac agagcaaaaa ctttcatctg 240
cgtcctcgtg atctgattag caacattaac gttatcgtgc tggagctgaa aggttctgaa 300
accaccttca tgtgtgagta tgctgacgaa accgccacta ttgtcgagtt tctgaatcgc 360
tggatcacct tttgtcagag catcatcagc accctgaca 399
<210>9
<211>399
<212>DNA
<213>人工序列
<400>9
gctcctacaa gctcttccac gaaaaaaact caactgcagc tggaacatct gctgctggat 60
ctgcaaatga tcctgaacgg catcaacaac tataaaaacc cgaaactgac ccgtatgctg 120
acgttcaaat tctatatgcc taaaaaagcc accgagctga aacatctgca atgcctggag 180
gaggaactga aaaaactgga ggaagttctg aatctggcac agagcaaaaa ctttcatctg 240
cgtcctcgtg atctgattag caacattaac gttatcgtgc tggagctgaa aggttctgaa 300
accaccttca tgtgtgagta tgctgacgaa accgccacta ttgtcgagtt tctgaatcgc 360
tggatcacct tttgtcagag catcatcagc accctgaca 399
<210>10
<211>399
<212>DNA
<213>人工序列
<400>10
gctcctacaa gctcttccac gaaaaaaact caactgcagc tggaacatct gctgctggat 60
ctgcaaatga tcctgaacgg catcaacaac tataaaaacc cgaaactgac ccgtatgctg 120
acgttcaaat tctatatgcc taaaaaagcc accgagctga aacatctgca atgcctggag 180
gaggaactga aacctctgga gaaagttctg aatctggcac agagcaaaaa ctttcatctg 240
cgtcctcgtg atctgattag caacattaac gttatcgtgc tggagctgaa aggttctgaa 300
accaccttca tgtgtgagta tgctgacgaa accgccacta ttgtcgagtt tctgaatcgc 360
tggatcacct tttgtcagag catcatcagc accctgaca 399
<210>11
<211>399
<212>DNA
<213>人工序列
<400>11
gctcctacaa gctcttccac gaaaaaaact caactgcagc tggaacatct gctgctggat 60
ctgcaaatga tcctgaacgg catcaacaac tataaaaacc cgaaactgac ccgtatgctg 120
acgttcaaat tctatatgcc taaaaaagcc accgagctga aacatctgca atgcctggag 180
gaggaactga aacctctgga ggaagttctg aataaagcac agagcaaaaa ctttcatctg 240
cgtcctcgtg atctgattag caacattaac gttatcgtgc tggagctgaa aggttctgaa 300
accaccttca tgtgtgagta tgctgacgaa accgccacta ttgtcgagtt tctgaatcgc 360
tggatcacct tttgtcagag catcatcagc accctgaca 399
Claims (7)
1.一种低诱导抑制性T细胞的人白细胞介素II突变体，其特征在于：由组成多肽的氨基酸序列在白介素II α受体结合点突变，一个或多个突变的位点在白介素II的37，38，41，42，43，44，45，61，62，65，68和72氨基酸位点。
2.根据权利要求1所述的低诱导抑制性T细胞的人白细胞介素II突变体，其特征在于：所述突变体包括一个完整长度的白介素II的分子或融合蛋白。
3.根据权利要求1所述的低诱导抑制性T细胞的人白细胞介素II突变体在降低其诱导抑制细胞功能的应用。
4.根据权利要求3所述的低诱导抑制性T细胞的人白细胞介素II突变体在增加其抑制体内固体肿瘤生长效用的应用。
5.根据权利要求3或4所述的低诱导抑制性T细胞的白细胞介素II的突变体的用途为与其他治疗组合，或作用一种疫苗的成分。
6.根据权利要求3或4所述的低诱导抑制性T细胞的白细胞介素II的突变体的用途为治疗病毒感染。
7.根据权利要求4所述的低诱导抑制性T细胞的白细胞介素II的突变体的用途，其特征在于：所述固体肿瘤为肾癌细胞或其他肿瘤细胞。
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| CN110642934A (zh) * | 2019-09-10 | 2020-01-03 | 中国医学科学院北京协和医院 | 靶向调节t细胞的长效白介素-2及其在治疗自身免疫病中的应用 |
| WO2020020783A1 (en) * | 2018-07-24 | 2020-01-30 | Biontech Rna Pharmaceuticals Gmbh | Il2 agonists |
| WO2020146221A1 (en) * | 2019-01-07 | 2020-07-16 | Inhibrx, Inc. | Polypeptides comprising modified il-2 polypeptides and uses thereof |
| WO2021185362A1 (zh) * | 2020-03-19 | 2021-09-23 | 信达生物制药(苏州)有限公司 | 白介素2突变体及其用途 |
| AU2019343251B2 (en) * | 2018-09-21 | 2022-06-09 | Innovent Biologics (Suzhou) Co., Ltd. | Novel interleukin 2 and use thereof |
| CN114957441A (zh) * | 2022-05-20 | 2022-08-30 | 南京涵台余生物科技有限公司 | 转基因nk细胞及其在抗肿瘤药物中的应用 |
| WO2023202035A1 (zh) * | 2022-04-20 | 2023-10-26 | 北京大学宁波海洋药物研究院 | 受体偏向的peg化il-2变体组合及其应用 |
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| WO2020020783A1 (en) * | 2018-07-24 | 2020-01-30 | Biontech Rna Pharmaceuticals Gmbh | Il2 agonists |
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| AU2019343251B2 (en) * | 2018-09-21 | 2022-06-09 | Innovent Biologics (Suzhou) Co., Ltd. | Novel interleukin 2 and use thereof |
| WO2020146221A1 (en) * | 2019-01-07 | 2020-07-16 | Inhibrx, Inc. | Polypeptides comprising modified il-2 polypeptides and uses thereof |
| CN113924311A (zh) * | 2019-01-07 | 2022-01-11 | 印希比股份有限公司 | 包含修饰的il-2多肽的多肽及其用途 |
| CN110642934A (zh) * | 2019-09-10 | 2020-01-03 | 中国医学科学院北京协和医院 | 靶向调节t细胞的长效白介素-2及其在治疗自身免疫病中的应用 |
| CN110642934B (zh) * | 2019-09-10 | 2022-08-23 | 中国医学科学院北京协和医院 | 靶向调节t细胞的长效白介素-2及其在治疗自身免疫病中的应用 |
| WO2021185362A1 (zh) * | 2020-03-19 | 2021-09-23 | 信达生物制药(苏州)有限公司 | 白介素2突变体及其用途 |
| WO2023202035A1 (zh) * | 2022-04-20 | 2023-10-26 | 北京大学宁波海洋药物研究院 | 受体偏向的peg化il-2变体组合及其应用 |
| CN114957441A (zh) * | 2022-05-20 | 2022-08-30 | 南京涵台余生物科技有限公司 | 转基因nk细胞及其在抗肿瘤药物中的应用 |
| CN114957441B (zh) * | 2022-05-20 | 2023-05-09 | 江苏康合生物科技有限公司 | 转基因nk细胞及其在抗肿瘤药物中的应用 |
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