CN112618726A - 一种抗体缀合物、增强抗体分子免疫效应功能的方法 - Google Patents

一种抗体缀合物、增强抗体分子免疫效应功能的方法 Download PDF

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CN112618726A
CN112618726A CN202011516850.9A CN202011516850A CN112618726A CN 112618726 A CN112618726 A CN 112618726A CN 202011516850 A CN202011516850 A CN 202011516850A CN 112618726 A CN112618726 A CN 112618726A
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CN112618726B (zh
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吴志猛
洪皓飞
周坤
周志昉
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Jiangnan University
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Abstract

本发明公开了一种抗体缀合物、增强抗体分子免疫效应功能的方法。所述抗体缀合物由抗体分子经半抗原衍生物分子修饰所得，所述半抗原衍生物由半抗原、连接臂和耦合域组成。本发明通过温和简便的方法将半抗原衍生物修饰在抗体分子上，所获得的抗体缀合物不仅保留了原有抗体的优良亲和水平，还可以招募相应的特异性天然抗体至肿瘤细胞上，从而达到显著提高抗体ADCC和CDC等免疫效应功能的目的。最重要的是，本发明还通过对连接臂长度的优化，优化得到了SMCC‑PEG3‑Rha这一半抗原衍生物。相比于其它已报道的抗体分子，SMCC‑PEG3‑Rha修饰的抗体，具备更加突出的免疫效应功能。

Description

一种抗体缀合物、增强抗体分子免疫效应功能的方法
技术领域
本发明属于制药工程技术领域，具体涉及一种抗体缀合物、增强抗体分子免疫效应功能的方法。
背景技术
恶性肿瘤目前依然是威胁人类健康的主要疾病，其发病和死亡人数均呈逐年上升趋势。单克隆抗体是目前最有效的抗肿瘤生物治疗剂之一，它们通过靶向肿瘤特异性抗原或者相关性抗原来发挥治疗作用，主要机制包括：诱导或阻断细胞信号，激活免疫效应功能，以及作为载体将药物特异性输送到靶细胞等。其中，免疫效应功能是指抗体所具备的，激活包括抗体依赖细胞介导的细胞毒作用(ADCC)和补体依赖的细胞毒作用 (CDC)在内的多种效应作用的能力，是多数抗肿瘤抗体药物最为基本的功能。因此，通过抗体工程手段，进一步增强它们的ADCC和CDC活性是目前抗体生物医药研发的热点。
目前，使用抗体基因工程技术改造抗体是增强抗体ADCC和CDC活性的主要手段，通过该手段已经获得一些ADCC和CDC活性增强的新一代单克隆抗体。然而，不足之处是，抗体基因工程技术比较复杂，需要经过大量的筛选才能获得免疫活性较好的抗体，工作量较大；另外，改造后的抗体具有一定的免疫原性，会引起免疫副作用。
人体内存在着一些半抗原特异性天然抗体，丰度较高的有抗Rha抗体、抗DNP抗体和抗αGal抗体。通过在商业化单克隆抗体上修饰这些半抗原分子，将有望达到改善原有抗体免疫效应功能的目的。如有学者报道，通过将αGal衍生物修饰到抗CD20抗体上，可以获得CDC水平增强型的抗CD20抗体。但一方面其修饰方法复杂，会对抗体的亲和性，以及产物的稳定性和产率产生影响；另一方面，其未对所得抗体的另一个主要免疫效应功能，即ADCC水平进行评估；也未对重要的影响因素，连接臂的长度进行优化。最终导致其产物的免疫效应功能水平提升有限。
因此，亟需一种新的增强抗体免疫效应功能的方法，以缓解或解决上述问题。
发明内容
发明目的：为了克服现有技术中存在的不足，本发明提供一种抗体缀合物、增强抗体分子免疫效应功能的方法，具体是一种基于半抗原特异性天然抗体募集的策略。并通过连接臂优化，提供一种优化筛选后的半抗原衍生物分子，使得经该分子修饰的抗体具备明显优于其它已报道抗体的免疫效应功能。
技术方案：为实现上述目的，本发明采用的技术方案为：
本发明的一个目的是，提供一种抗体缀合物，由抗体分子被半抗原衍生物分子修饰得到，所述的半抗原衍生物分子由半抗原、连接臂和耦合域构成。
进一步的，本发明所述半抗原为可募集人体内天然抗体的小分子，包括在人体内丰度较高的抗鼠李糖(Rha)抗体，抗2,4-二硝基苯(DNP)抗体和抗αGal抗体，即所述的半抗原优选为Rha、DNP或αGal等。
进一步的，本发明所述的连接臂用于连接半抗原和耦合域，包括但不限于聚乙二醇链、碳链和肽链。为了便于合成均一的产物以及减少不期望的毒副作用，所述连接臂优选为低聚乙二醇(分子量小于1000)、短碳链(分子量小于100)和肽链，更进一步的，优选为低聚乙二醇PEG3。
进一步的，本发明所述的耦合域用于将半抗原衍生物修饰在抗体上，为了便于合成均一的产物以及减少半抗原衍生物对抗体原有亲和性的影响，耦合位置优选为抗体上四个二硫键还原后所形成的8个巯基；耦合域优选为含马来酰亚胺分子的SMCC，也可换做其它的含马来酰亚胺的分子(maleimides，
Figure BDA0002848225460000021
)，以及含乙烯基砜的分子(vinyl sulfones，
Figure BDA0002848225460000022
)、含丙烯酸脂和/或丙烯酸胺的分子(acrylates&acrylamides，
Figure BDA0002848225460000023
)、含3,4-二溴丁二酰亚胺的分子和含甲基丙烯酸酯的分子 (methacrylates，
Figure BDA0002848225460000024
)，R为任意有机基团。
进一步的，本发明所述的抗体为单克隆抗体，优选为一些已经上市或商业可获得的单克隆抗体。可列举的实例包括但不限于抗CD20抗体、抗CD19抗体、抗CD30抗体、抗EGFR抗体、抗EGFRvIII抗体、抗HER2抗体、抗HER3抗体、抗PSMA抗体、抗 VEGFR抗体、抗PD-L1抗体、抗cMET抗体、抗TGF-β抗体、抗MUC1抗体和抗Trop-2 抗体。
进一步的，所述抗体分子的氨基酸序列为：如SEQ ID NO.1所示的重链氨基酸和如SEQ ID NO.2所示的轻链氨基酸。
进一步的，所述半抗原衍生物分子包括：
Figure BDA0002848225460000031
更进一步的，所述半抗原衍生物的合成路径为：
Figure BDA0002848225460000032
其中，n为正整数。
本发明的另一个目的，是还提供一种提高抗体分子免疫效应功能的方法，即通过将半抗原衍生物分子修饰到抗体分子上，利用半抗原分子的天然抗体招募能力达到增强ADCC和CDC等免疫活性的目的。并最终通过对半抗原衍生物连接臂的优化，进一步筛选得到免疫效应功能最为突出的抗体缀合物。
本发明所述的提高抗体分子免疫效应功能所涉及的半抗原衍生物、抗体分子、修饰位置和方法等如前所述。
进一步的，所述将半抗原衍生物分子修饰到抗体分子上制备抗体缀合物的方法包括以下步骤：
1)将抗体脱盐置换到硼酸钠反应中；
2)加入硫醇类还原剂TCEP避光震荡反应；
3)反应完成后加入10倍当量的半抗原衍生物分子，反应后经半胱氨酸淬灭反应；
4)超滤除去多余的小分子，得到相应的抗体缀合物。
在本发明的一种具体实施方式中，所选抗体分子为抗CD20单克隆抗体美罗华(Rituximab，KEGG Accession Number:DB00073，重链如SEQ ID NO.1所示，轻链如 SEQ IDNO.2所示)，CD20在95％以上的B细胞性淋巴瘤细胞表面高表达，这个特性使之成为靶向单克隆治疗的重要靶点。优化后的半抗原衍生物分子为SMCC-PEG3-Rha
Figure BDA0002848225460000041
其中SMCC为耦合域，PEG3为低聚乙二醇链，Rha为半抗原。耦合位置为美罗华抗体上四个二硫键还原后所形成的8个巯基。
有益效果：本发明相对于现有技术的优势：
(1)本发明所述的抗体缀合物，与现有报道抗体相比，一方面抗原亲和水平未受到抑制；另一方面，具备显著增强的ADCC和CDC活性。从而在治疗时可以降低使用剂量，最终在一定程度上减少或避免一些毒副作用的产生。
(2)本发明所提供的优化后的半抗原衍生物，其所对应的抗体缀合物具备最突出的免疫效应功能，仅采用野生型始抗体4％的用量，即可展现出显著高于野生型抗体的 CDC杀伤效果。
(3)本发明所述的增强抗体免疫效应功能的策略，一方面操作简单，修饰条件温和；另一方面由于利用的是机体自身的天然抗体，从而避免了潜在的毒性和免疫原性。
附图说明
图1：蛋白质谱图。美罗华抗体及美罗华抗体缀合物αCD20-PEG1-Rha、αCD20-PEG3-Rha和αCD20-PEG6-Rha的轻链蛋白质谱结果。
图2：流式细胞表征。(A)美罗华抗体及美罗华抗体缀合物αCD20-PEG1-Rha、αCD20-PEG3-Rha和αCD20-PEG6-Rha的亲和水平评估；(B)美罗华抗体及美罗华抗体缀合物αCD20-PEG1-Rha、αCD20-PEG3-Rha和αCD20-PEG6-Rha的抗Rha抗体募集能力评估。
图3：ADCC表征。(A)在不存在抗Rha抗体时，不同浓度美罗华抗体及美罗华抗体缀合物αCD20-PEG1-Rha、αCD20-PEG3-Rha和αCD20-PEG6-Rha的ADCC水平；(B) 在存在抗Rha抗体时，不同浓度美罗华抗体及美罗华抗体缀合物αCD20-PEG1-Rha、αCD20-PEG3-Rha和αCD20-PEG6-Rha的ADCC水平。
图4：CDC表征。(A)在不存在抗Rha抗体时，不同浓度美罗华抗体及美罗华抗体缀合物αCD20-PEG1-Rha、αCD20-PEG3-Rha和αCD20-PEG6-Rha的CDC水平；(B) 在存在抗Rha抗体时，不同浓度美罗华抗体及美罗华抗体缀合物αCD20-PEG1-Rha、αCD20-PEG3-Rha和αCD20-PEG6-Rha的CDC水平。
具体实施方式
本发明公开了一种抗体缀合物，提高抗体分子免疫效应功能的方法。所述抗体缀合物为抗体分子被半抗原衍生物分子修饰所得，所述半抗原是指可招募人体内特异性天然抗体的小分子。本发明通过温和简便的方法将半抗原修饰在抗体分子上，所获得的抗体缀合物不仅保留了原有抗体的优良亲和水平，还可以招募相应的特异性天然抗体至肿瘤细胞上，从而达到提高抗体ADCC和CDC等免疫效应功能的目的。本发明还通过对半抗原衍生物分子的连接臂长度进行优化，获得了可引起最高水平免疫效应功能的抗体缀合物。
下面结合实施例对本发明做进一步说明，应当理解，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，凡在本发明的构思前提下对本发明制备方法的简单改进都属于本发明的保护范围之内。下面实施例未注明具体条件的实验方法，通常按照本领域的公知手段。下述实施例中所用的试验材料和细胞系，如无特殊说明，均为自常规试剂公司购买得到。
实施例
根据本发明的抗体缀合物的合成方法，首先是合成半抗原衍生物分子 SMCC-PEG-Rha，然后是将SMCC-PEG-Rha修饰到抗体分子上，从而制备得到相应的抗体缀合物。本发明实施例中，耦合域选用为SMCC，连接臂选用为PEG，半抗原选用为Rha，合成的半抗原衍生物分子为SMCC-PEG-Rha，并且重点针对连接臂的长短进行了不同的实验验证，以获得最优的连接臂长度。
根据下述实施例，可以更好的理解本发明。然而，本领域的技术人员容易理解，实施例所描述的具体的耦合域、连接臂、半抗原及其结果仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明的技术方案。
实施例1:含不同连接臂半抗原衍生物SMCC-PEG-Rha的制备
(1)半抗原衍生物SMCC-PEG1-Rha的合成路径和条件如下式所示：
Figure BDA0002848225460000061
经检测，终产物的核磁和质谱结果均与预期一致，表明所合成的SMCC-PEG1-Rha正确。1H NMR(400MHz,Methanol-d4)δ6.80(s,2H),4.66(d,J＝1.7Hz,1H),3.79(dd,J＝3.5,1.7Hz,1H),3.69(dt,J＝10.0,5.7Hz,1H),3.63(dd,J＝9.5,3.5Hz,1H),3.58–3.51(m,1H),3.45(ddd,J＝10.3,6.1,4.7Hz,1H),3.40–3.31(m,6H),2.14(tt,J＝12.2,3.5Hz,1H),1.82(dd,J＝13.5,3.4Hz,2H),1.76–1.69(m,2H),1.42(qd,J＝13.1,3.3Hz,2H),1.24(d,J＝6.2Hz,3H),1.00(qd,J＝13.1,3.5Hz,2H).13C NMR(101MHz,Methanol-d4)δ177.68,171.37,133.87,100.27,72.53,70.94,70.75,68.48,65.55,44.70,43.12,38.74,36.49,29.58, 28.66,16.60.HRMS(ESI,positive)calcd for C20H30N2O8Na[M+Na]+:449.1900Found: 449.1899.
(2)半抗原衍生物SMCC-PEG3-Rha的合成路径和条件如下式所示：
Figure BDA0002848225460000062
经检测，终产物的核磁和质谱结果均与预期一致，表明所合成的SMCC-PEG3-Rha正确。1H NMR(400MHz,Methanol-d4)δ6.71(s,2H),4.62(d,J＝1.7Hz,1H),3.73–3.65 (m,2H),3.57–3.49(m,8H),3.43(t,J＝5.5Hz,2H),3.30–3.19(m,6H),2.05(tt,J＝12.1,3.4Hz,1H),1.72(dd,J＝13.5,3.5Hz,2H),1.66–1.60(m,2H),1.38–1.20(m,3H),1.16(d, J＝6.2Hz,3H),0.91(qd,J＝12.9,3.5Hz,2H).13C NMR(101MHz,Methanol-d4)δ133.88,100.41,72.59,70.99,70.82,70.26,70.05,69.27,68.41,66.30,48.27,48.05,47.84,47.63, 47.41,47.20,46.99,44.67,43.14,38.86,29.57,28.63.HRMS(ESI,positive)calcd for C24H38N2O10Na[M+Na]+:537.2424Found:537.2415.
(3)半抗原衍生物SMCC-PEG6-Rha的合成路径和条件如下式所示：
Figure BDA0002848225460000071
经检测，终产物的核磁和质谱结果均与预期一致，表明所合成的SMCC-PEG6-Rha正确。1H NMR(400MHz,Methanol-d4)δ6.81(s,2H),4.71(d,J＝1.7Hz,1H),3.82–3.75 (m,2H),3.68–3.57(m,22H),3.52(t,J＝5.5Hz,2H),3.40–3.31(m,5H),2.15(tt,J＝12.2,3.6Hz,1H),1.82(dd,J＝13.7,3.5Hz,2H),1.76–1.69(m,2H),1.42(qd,J＝13.1,3.3Hz,2H),1.25(d,J＝6.2Hz,3H),1.01(qd,J＝12.9,3.6Hz,2H).13C NMR(101MHz, Methanol-d4)δ171.39,133.89,100.43,72.61,70.98,70.81,70.28,70.22,70.20,70.18,70.16, 70.13,70.02,69.88,69.19,68.41,66.40,44.67,43.14,38.86,36.51,29.60,28.63,16.67. HRMS(ESI,positive)calcd for C30H51N2O10[M+H]+:647.3391Found:647.3389
实施例2：美罗华抗体缀合物的制备
制备美罗华抗体缀合物的实施方案为：
1)将美罗华抗体脱盐置换到25mM硼酸钠反应中；
2)加入硫醇类还原剂TCEP避光震荡，期间Ellman试剂巯基数监测反应完成情况；
3)反应完成后加入10倍当量由实施例1制备得到的SMCC-PEG1-Rha或 SMCC-PEG3-Rha或SMCC-PEG6-Rha反应一段时间，半胱氨酸淬灭反应；
4)超滤法除去多余的小分子，即可得到相应的三种美罗华抗体缀合物αCD20-PEG1-Rha、αCD20-PEG3-Rha、αCD20-PEG6-Rha。
最后蛋白质谱如图1所示，结果表明美罗华抗体轻链的分子量为23131Da，美罗华抗体αCD20-PEG1-Rha的轻链分子量为23557Da，抗体缀合物αCD20-PEG3-Rha的轻链分子量为23645Da，抗体缀合物αCD20-PEG6-Rha的轻链分子量为23777Da，均与理论相符。
实施例3：美罗华抗体缀合物的亲和水平和抗体募集能力表征
流式细胞实验的实施方案为：
1)取生长状况良好的Raji细胞(CD20阳性)或K562细胞(CD20阴性)，重悬至 4×105个/mL，移取100μL细胞悬液至预先装有100μL含50nmol/L抗体样品的无菌 EP管，冰浴30min；
2)用200μL含10μg/mL的FITC耦合山羊抗人IgG抗体处理细胞(评估特异性亲和水平)；或先后用1％抗Rha兔血清和FITC耦合山羊抗兔IgG抗体处理细胞(评估抗 Rha抗体募集能力)；
3)流式细胞仪Accuri C6进行检测。
特异性亲和水平结果如图2A所示，相对于空白对照，经美罗华抗体或抗体缀合物处理后的Raji细胞荧光明显增加，且两者增加程度相差无几。此外，CD20阴性的K562 细胞则无明显变化，这说明该策略下Rha的修饰几乎不影响抗体对细胞表面CD20的特异性结合。
抗Rha抗体募集能力结果如图2B所示，相对于空白对照，仅经抗体缀合物处理后的Raji细胞有明显的荧光增长，表明美罗华抗体在被Rha修饰后，成功具备了募集抗 Rha抗体至CD20阳性肿瘤细胞表面的能力。且图2B还表明抗体缀合物αCD20-PEG3-Rha具备最高水平的抗Rha抗体募集能力。
实施例4：美罗华抗体缀合物的ADCC和CDC水平表征
细胞毒性试验的实施方案如下所示，对抗Rha抗体存在和不存在两种情况下的ADCC和CDC活性均进行评估。
ADCC实验：1)取生长状况良好的Raji细胞，重悬至4×105个/mL，移取50μL细胞悬液至96孔培养板中；2)立即加入50μL含不同浓度美罗华或美罗华抗体缀合物的 PBS溶液，50μL 4％抗Rha兔血清(或相同体积的PBS)和50μL新鲜分离的PBMC， 37℃处理4h；3)LDH试剂盒检测细胞毒性。
ADCC的结果如图3所示。在抗Rha抗体不存在的情况下(图3A)，美罗华和其缀合物的ADCC水平相差无几，在处理浓度为2.5nM时，均在20％左右；而在抗Rha抗体的存在下(图3B)，三种抗体缀合物的ADCC杀伤水平显著增强，最高值可达30-42％。表明本发明所提供的策略，可有效提升美罗华的ADCC活性。其中，缀合物αCD20-PEG3-Rha的ADCC水平最高，在2.5nM的处理浓度下，可达41.3％。
CDC实验：1)取生长状况良好的Raji细胞，重悬至4×105个/mL，移取50μL细胞悬液至96孔培养板中；2)立即加入50μL含不同浓度美罗华或其缀合物的PBS溶液， 50μL 4％抗Rha兔血清(或相同体积的PBS)和50μL 8％兔补体血清，37℃处理4h； 3)LDH试剂盒检测细胞毒性。
CDC的结果如图4所示。在抗Rha抗体不存在的情况下(图4A)，美罗华和其缀合物的CDC水平相差无几，当处理浓度为2.5nM时，均在50％左右；而在抗Rha抗体的存在下(图4B)，三种抗体缀合物的ADCC杀伤水平显著增强，当处理浓度为2.5nM 时，可达85-100％左右。表明本发明所提供的策略，可有效提升美罗华的CDC活性。其中，缀合物αCD20-PEG3-Rha的CDC水平最高，在0.1nM的处理浓度下，即可达到 69.3％，已经显著高于2.5nM原始抗体处理后的CDC水平(53.2％)。该结果表明在抗 Rha抗体的存在下，若想达到相同的杀伤效率，αCD20-PEG3-Rha的使用量仅需原始抗体的1/25甚至更少。
由以上实施例的结果可以看出，αCD20-PEG3-Rha的ADCC、CDC水平最高，即 ADCC、CDC杀伤水平得到显著增强。相对短链的αCD20-PEG1-Rha和相对长链的αCD20-PEG6-Rha的ADCC、CDC水平则相应较低，一方面，可能因为空间位阻原因，短链的αCD20-PEG1-Rha与抗Rha抗体的募集水平受限，从而导致其ADCC、CDC杀伤水平无法得到提升；另一方面，可能因为亲和性原因，长链的αCD20-PEG6-Rha与抗 Rha抗体的亲和水平减弱，从而导致其ADCC、CDC杀伤水平反而受到抑制，并未实现如现有理论预期的正相关提成杀伤水平的效果。
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
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<120> 一种抗体缀合物、增强抗体分子免疫效应功能的方法
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Claims (10)

1.一种抗体缀合物，其特征在于，由抗体分子经半抗原衍生物分子修饰形成，所述半抗原衍生物分子由半抗原、连接臂和耦合域组成。
2.据权利要求1抗体缀合物，其特征在于，所述半抗原为可募集相应特异性天然抗体的小分子，包括但不限于鼠李糖、αGal和2,4-二硝基苯。
3.根据权利要求1抗体缀合物，其特征在于，所述连接臂包括但不限于聚乙二醇链、碳链和肽链。
4.根据权利要求3所述的抗体缀合物，其特征在于，所述的连接臂为低聚乙二醇链PEG3。
5.根据权利要求1抗体缀合物，其特征在于，所述耦合域为与抗体上巯基发生thio-ene或thio-yne反应的基团，包括但不限于SMCC及其他含马来酰亚胺的分子，还包括含乙烯基砜的分子、含丙烯酸脂/丙烯酸胺的分子、含3,4-二溴丁二酰亚胺的分子和含甲基丙烯酸酯的分子；耦合位置为抗体上二硫键还原后所形成的巯基。
6.根据权利要求1所述的抗体缀合物，其特征在于，所述抗体分子为单克隆抗体分子，包括但不限于抗CD20抗体、抗CD19抗体、抗CD30抗体、抗EGFR抗体、抗EGFRvIII抗体、抗HER2抗体、抗HER3抗体、抗PSMA抗体、抗VEGFR抗体、抗PD-L1抗体、抗cMET抗体、抗TGF-β抗体、抗MUC1抗体和抗Trop-2抗体。
7.根据权利要求1所述的抗体缀合物，其特征在于，所述半抗原衍生物分子包括：
Figure FDA0002848225450000011
8.根据权利要求7所述的抗体缀合物，其特征在于，所述半抗原衍生物的合成路径为：
Figure FDA0002848225450000021
其中，n为正整数。
9.一种增强抗体分子免疫效应功能的方法，其特征在于，将半抗原衍生物分子修饰到抗体分子上，所述半抗原衍生物分子和抗体分子如权利要求1-8任一所述。
10.根据权利要求9所述的一种增强抗体分子免疫效应功能的方法，其特征在于，所述将半抗原衍生物分子修饰到抗体分子上制备抗体缀合物的方法包括以下步骤：
1)将抗体脱盐置换到硼酸钠反应中；
2)加入硫醇类还原剂TCEP避光震荡反应；
3)反应完成后加入10倍当量的半抗原衍生物分子，反应后经半胱氨酸淬灭反应；
4)超滤除去多余的小分子，得到相应的抗体缀合物。
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MEHRI ABEDI等: "MALDI-MS: a Rapid and Reliable Method for Drug-to-Antibody Ratio Determination of Antibody-Drug Conjugates", 《IRANIAN BIOMEDICAL JOURNAL》 *
宋娟等: "半抗原的设计、修饰及人工抗原的制备", 《分析化学》 *

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WO2022135200A1 (zh) 2022-06-30

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