全长人源抗VEGF抗体库的构建及展示
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摘要
肾癌是泌尿系统威胁人类健康的常见恶性肿瘤,对放疗、化疗均不敏感。由于肾癌起病隐匿,在诊断时有25～30%已发生转移。而且,即使是局限性肾癌,采取根治术后仍有近30%患者会复发。针对这部分转移及复发肾癌的治疗是泌尿外科的难点和研究方向。
随着研究深入,发现肾癌特别是透明细胞癌多存在Von Hippel-Lindau(VHL)基因突变,乏氧诱导因子(hypoxia-inducible factor, HIF)及血管内皮生长因子(vascular endothelial growth factor, VEGF)表达增高,而且VEGF增高与肿瘤分期及预后密切相关。因此,抑制或阻断VHL-HIF-VEGF信号通路,特别是阻断VEGF或其受体VEGFR,就可能抑制肾癌的生长。
贝伐单抗(Bevacizumab)是VEGF特异性的经过人源化改造的鼠源抗体,能特异性与VEGF结合并阻断其生物活性。贝伐单抗联合IFN-α可以提高转移性肾癌的客观缓解率(ORR)和无疾病进展率(PFS)。2007年12月美国FDA批准贝伐单抗联合IFN-α作为靶向治疗转移性肾癌的一线治疗方案。
然而,通过杂交瘤技术获得的单抗需经过人源化改造,一方面不可能完全去除抗体的免疫原性,另一方面抗体空间构型可能发生改变,亲和力往往会降低,很难达到预期的作用效果。
为了尽可能减少药用抗体的免疫原性,研究者尝试利用哺乳动物细胞表面抗体展示技术用于人源抗体的筛选,但目前的这些哺乳动物细胞表面展示技术所构建抗体库容量多局限于104～106,筛选抗体多样性不足,不能满足筛选高亲和力抗体的要求。
为了克服哺乳动物细胞表面展示技术筛选全长人源抗体存在的上述不足,本课题组对现有的哺乳动物细胞展示技术作了技术改良。
人的VEGF是一种自身蛋白,以来自于自身免疫病患者的外周血淋巴细胞为材料构建抗体库筛选到抗VEGF抗体的几率更大。通过提取自身免疫病患者的外周血淋巴细胞的总RNA,进行RT-PCR扩增全套抗体基因,随后使用哺乳动物细胞表达载体pDGB-HC-TM,构建了三个全长人源抗体基因库,库容量达到1010以上。将抗体基因库通过四片段连接插入哺乳动物细胞表达载体pDGB4,然后将其稳定转染FCHO细胞。由于采用基因定点整合技术,每个细胞只表达一种抗体,从而成功建成能展示全长抗体的哺乳动物细胞库。
本课题通过全基因合成的方法合成了贝伐单抗的全长基因,并根据哺乳动物细胞对密码子的选择性对抗体基因的碱基序列进行表达优化,既作为筛选抗体的阳性对照,同时也用于抗体的链置换筛选技术,以提高筛选VEGF特异性抗体的成功率；与已经构建好的三个抗体库基因进行链置换,构建了全长人源抗VEGF抗体库,并成功将其展示于哺乳动物细胞表面。
一、全长人源初级抗体库的构建及展示
目的：
构建大容量的全长人源初级抗体基因库。
方法：
1.外周血淋巴细胞的分离及总RNA的提取
从供体中采集适量静脉血,置于肝素抗凝管中,采用密度梯度离心法分离外周血单核细胞,加入适量Buffer RLT,裂解细胞,—80℃冰箱保存备用或者直接用于提取总RNA。
2. cDNA第一链的合成
以总RNA为模板,应用根据V-BASE网站信息所设计合成的全套人抗体重链可变区引物和全套人抗体轻链恒定区引物,逆转录合成cDNA第一条链。
3.全套IgG1重链可变区基因和轻链K型全长基因的扩增
以合成的cDNA为模板,用相应的特异性引物扩增全套IgG1轻链K型全长基因及全套IgG1重链可变区基因(3套轻链引物,3套重链引物)。PCR条件为：94-C预变性5分钟,然后进行35个循环的PCR扩增(94℃变性30s,55℃退火30s,72℃延伸1min),随后在延长温度下反应7min,以确保全长目的片段的合成。
PCR扩增产物的分离纯化
用PCR扩增抗体全套Kappa轻链和重链可变区基因。PCR产物用1%的琼脂糖凝胶电泳分离纯化目的片段。然后将回收得到的3组重链基因PCR扩增产物,再次经1%琼脂糖凝胶电泳后回收目的片段备用。并用同样方法处理3组轻链PCR扩增产物。
重链抗体基因库及轻链基因库的构建及鉴定
以BsmBI对重链基因扩增产物及载体pDGB-HC-TM进行酶切,电泳分离纯化目的片段,将回收的载体片段和抗体基因片段用T4DNA连接酶按1：1比例混合连接,在16℃反应24h后导入化学转化感受态大肠杆菌TOPO-10,构建重链基因库。
轻链全长基因的扩增产物经SfiI酶切后回收,与同样经SfiI酶切回收的载体pDGB-HC-TM片段用T4DNA连接酶按1：1比例混合连接,在16℃反应24h后导入化学转化感受态大肠杆菌TOPO-10,构建轻链基因库。
分别从重链抗体库和轻链抗体库中各随机挑取10个单克隆,摇菌培养过夜后抽提质粒,测序分析抗体基因库的质量及多样性。
4.抗体库在哺乳动物细胞表面的展示
分别将中提所获得的重链库pDGB-HClib-TM与轻链库pDGB-LClib的质粒DNA共转染293T细胞,将上述转染后继续培养48-72h的293T细胞进行回收,然后行免疫荧光染色及流式细胞学分析。
结果与讨论：
分离获得淋巴细胞的数量总计为1.4×108个。取一定量的细胞裂解物,提取获得约3.5u g总RNA,其A260/A280的值为1.90。PCR扩增共获得约2μg的全套IgG1轻链K型全长基因和约2μg的全套重链可变区基因。
根据在带有氨苄青霉素抗性的LB平皿上长出的细菌克隆数,计算所构建的抗体重链基因库库容量为1.89×105,背景克隆的比例为3.60%；抗体轻链基因库库容量为6.54×104,背景克隆的比例为1.65%。用同样的方法一共构建了三个大容量的全长人源抗体基因库,库容量分别为1.19×1010、2.0×1010和5.27×1011。在其中一个抗体基因库里面的重链库和轻链库中各挑取10个单克隆进行测序分析,结果表明重链库的10个单克隆中有8个含有正确的VH编码序列,编码8个特异的氨基酸序列；轻链库的10个单克隆中有7个含有正确的LCκ编码序列,编码7个特异的氨基酸序列。将此重链库和轻链库共转染哺乳动物细胞后,理论上抗体库的多样性可以达到6.67×109[(1.89×105×80%)×(6.54x104x70%)],可以满足筛选高亲和力、高特异性抗体的要求。将抗体库瞬时转染293T细胞后,在63.40%的细胞表面可以检测到全长人源抗体的表达。
结论：
本研究采用哺乳动物细胞展示技术,成功用哺乳动物细胞表面展示载体pDGB-HC-TM构建了库容量大且多样性好的全长人源抗体基因库,可以满足筛选高亲和力、高特异性抗体的要求。
二、全长人源二级抗体库的构建及展示
目的：
将第一部分构建好的三个初级抗体基因库通过四片段连接插入哺乳动物细胞表达载体pDGB4,构建三个全长人源二级抗体库,并将此三个抗体基因库合并一起稳定转染哺乳动物细胞FCHO,获得能够稳定展示全长人源抗体的哺乳动物细胞库。
方法：
1.双表达载体pDGB4的制备
以BsmBI和SfiI对载体pDGB4进行双酶切,经1%琼脂糖凝胶电泳鉴定,并分离纯化5kb和3kb的两段目的DNA片段。
2.抗体基因的制备
以第一部分已经制备的抗体重链基因库(pDGB-HClib-TM)的细菌沉淀为来源,进行质粒中提获得抗体重链基因库的质粒DNA；用BsmBI进行酶切,用1%琼脂糖凝胶电泳分离纯化0.45kb的DNA片段。
以第一部分已经制备的抗体轻链基因库(pDGB-LClib)的细菌沉淀为来源,进行质粒中提获得抗体轻链基因库的质粒DNA；用SfiI进行酶切,用1%琼脂糖凝胶电泳分离纯化0.75kb的DNA片段。
3.转化感受态大肠杆菌TOPO-10及单克隆转染
将获得的抗体轻重链基因库片段插入到pDGB4载体的相应酶切位点之间,获得pDGB-Full-Length-lib。转化化学感受态大肠杆菌TOPO-10,构建全长人源二级抗体库。用同样的方法将第一部分所构建的另外2个初级抗体基因库构建成为四片段连接的全长人源二级抗体库。在转化菌落中随机挑取十个单克隆,进行质粒DNA的小提。
4.抗体库在哺乳动物细胞表面的展示
将二级抗体库的质粒DNA瞬时转染293T细胞,用PE标鼠抗人kappa链抗体进行细胞免疫荧光染色,用流式细胞仪进行检测分析。
5.构建稳定表达单克隆抗体的细胞库
将上述构建的三个四片段连接二级抗体库质粒中提后进行1：1：1合并,与pOG44的质粒DNA共转染常规培养至对数生长期的FCHO细胞,构建稳定表达抗体的细胞库。
结果与讨论：
根据在带有氨苄青霉素抗性LB平皿上长出的克隆数,计算所构建的全长抗体二级基因库库容量为2.476x106,背景克隆的比例为0.30%。用同样的方法将第一部分构建好的第二、第三个初级抗体库基因通过四片段连接插入哺乳动物细胞表达载体pDGB4,构建了第二、第三个四片段连接二级抗体库,库容量分别为9.22×105和1.0828×106,背景克隆的比例分别为0.06%和0.17%。从二级抗体库的建库菌平皿中随机挑取十个单克隆进行质粒小提后瞬时转染293T细胞,流式细胞仪检测有9个克隆能够表达出抗体。将中提获得二级抗体库的质粒DNA进行293T细胞的瞬时转染,有61.78%的细胞表面可成功展示可被检测到的全长抗体。将三个四片段连接二级抗体库基因进行质粒中提后稳定转染FCHO细胞,成功构建了稳定细胞库,库容量为3.76×106,用流式细胞仪检测有59.62%的细胞表面可成功展示可被检测的全长抗体。
结论：
成功将第一部分构建好的三个初级抗体库基因通过四片段连接插入哺乳动物细胞双表达载体pDGB4,构建了三个全长人源二级抗体库,并将此三个基因库合并一起稳定转染哺乳动物细胞FCHO,获得能够稳定展示全长人源抗体的哺乳动物细胞库,为特异性抗体的筛选奠定了基础。
三、全长人源抗VEGF抗体库的构建及展示
目的：
合成并优化贝伐单抗的全长基因,与前面构建好的三个抗体库进行链置换,构建全长人源抗VEGF抗体库。
方法：
1.贝伐单抗序列的合成与优化
在贝伐单抗的重链可变区碱基序列前面加上BstXI和BsmBI的酶切序列以及信号肽序列,将其序列进行人类密码子偏向性优化以及基因表达的优化,以BsmBI进行酶切,1%琼脂糖凝胶电泳分离纯化0.45kb大小的目的DNA片段。
在贝伐单抗的轻链可变区碱基序列前面加上SfiI的酶切序列以及信号肽序列,将其序列进行人类密码子偏向性优化以及基因表达的优化,采用重叠延伸PCR技术进行轻链全长的制备,再以SfiI进行酶切,用1%琼脂糖凝胶电泳分离纯化0.714kb大小的目的片段。
2.双表达载体pDGB4片段的制备
以BsmBI和SfiI对载体pDGB4进行双酶切,经1%琼脂糖凝胶电泳鉴定,并分离纯化5kb大小和3kb大小的两段目的DNA片段。
3.构建质粒pDGB4-avastin
将载体pDGB4片段和优化后合成、制备的贝伐单抗重链抗体片段、轻链抗体片段分别酶切并纯化后,抗体重链基因和抗体轻链基因同时插入到pDGB4载体的相应酶切位点之间,转化化学感受态大肠杆菌TOPO-10,随机挑取单克隆,使用两套引物分别对单克隆的贝伐单抗链可变区序列和轻链可变区序列进行PCR鉴定,根据PCR鉴定的结果,将单克隆菌液进行扩增培养、小提测序分析。
4.链置换法构建抗VEGF抗体库
以BsmBI对载体pDGB4进行单酶切,分离纯化8.65kb大小的目的片段。将该载体片段与经由BsmBI进行酶切并纯化后的贝伐单抗重链抗体片段进行连接获得新质粒pDGB4-avastin-vh,转化化学感受态大肠杆菌TOPO-10,用引物247和引物248来进行PCR鉴定后进行质粒中提,以SfiI进行酶切,分离纯化约8.3kb大小的目的片段,与同样经由SifI进行酶切全长人源轻链基因库(pDGB-LClib)手纯化出的轻链抗体库基因片段进行连接,获得新质粒pDGB4-avastin-vh-Hu-LCs,转化化学感受态大肠杆菌TOPO-10,构建含贝伐单抗重链的全长人源抗VEGF抗体库,计算抗体库的库容量。
同样用链置换方法,获得新质粒pDGB4-avastin-vk-Hu-HCs,转化化学感受态大肠杆菌TOPO-10,构建含贝伐单抗轻链的全长人源抗VEGF抗体库,计算抗体库的库容量。
结果与讨论：
分别在贝伐单抗的重链可变区序列前面加上了BstXI和BsmBI的酶切序列和信号肽序列、在贝伐单抗的轻链可变区序列前面加上SfiI的酶切序列和信号肽序列,并且进行了人类密码子偏向性优化以及基因表达的优化。经过优化,贝代单(?)重链可变区碱基序列的密码子适应指数(CAI)从0.80提高到0.87,最优密码子使用频率(FOP)也得到了优化,而GC含量变化不大；轻链可变区碱基序列的密码子适应指数(CAI)从0.80提高到0.85,最优密码子使用频率(FOP)也得到了优化,而GC含量变化不大。通过与前面构建好的三个抗体库进行链置换,构建了库容量分别为7.58x105和1.33×106,背景克隆的比例分别为2.51%和1.37%的全长人源抗VEGF抗体库。将抗体库瞬时转染293T细胞后,在细胞表面可以检测到全长人源抗VEGF抗体的表达；在抗VEGF抗体库1中,有22%的细胞表面可成功展示可被检测到的全长抗体,在抗VEGF抗体库2中,有28.05%的细胞表面可成功展示可被检测到的全长抗体。
结论：
成功在贝伐单抗的重链可变区序列和轻链可变区序列的前面加上了相应的酶切序列和信号肽序列并进行优化、全基因合成与制备,与前面构建好的三个抗体库基因进行链置换,构建了全长人源抗VEGF抗体基因库,将抗体库瞬时转染293T细胞后,在细胞表面可以检测到全长人源抗VEGF抗体的表达,为进一步利用贝伐单抗的抗VEGF导向选择作用,以便筛选具有与贝伐单抗相同的抗原亲和性和抗原结合表位的全长人源抗VEGF抗体做好了准备,也为开展全长人源抗VEGF单抗靶向治疗转移性肾癌奠定了实验基础。
Renal cell carcinoma (RCC) is the most common kidney malignant tumor, which is not sensitive to radiotherapy and chemotherapy. Due to its insidious onset,25to30percent of patients had metastasis in the time of diagnosis and nearly30%of localized renal cell carcinoma will relapse even after radical resection. People are looking forward to a new therapeutic approach to solve this problem.
With in-depth study, Von Hippel-Lindau (VHL) gene mutation is found in most renal cell carcinoma, especially clear cell carcinoma, with a high level of hypoxia-inducible factor and vascular endothelial growth factor. VEGF is closely related to tumor stage and prognosis. Therefore, inhibition or blocking of VHL-HIF-VEGF signaling pathway, in particular, blocking VEGF or its receptors VEGFR, may inhibit the growth of renal cell carcinoma.
Bevacizumab is a VEGF-specific murine antibody, which is humanized and due to its biological activity of blocking VEGF, is used for the treatment of patients with solid tumors and high level of VEGF. Bevacizumab combined with IFN-a can increase the Objective Response Rate (ORR) and Progression Free Survival (PFS) of metastatic renal cell carcinoma. Thus, U.S. FDA approved bevacizumab plus IFN-alpha as first-line treatment of the targeted therapy of metastatic renal cell carcinoma in December2007.
However, humanization of monoclonal antibodies obtained by hybridoma technology, on the one hand, can not completely remove antibodies' immunogenicity. On the other hand, the antibody space configuration may be changed, the affinity tends to reduce and it is verydifficult to achieve the desired effect.
In order to minimize the immunogenicity of pharmaceutical antibodies, the researchers are trying to use mammalian cell surface display technology for the screening of human antibodies. The diversity of the antibody library contructed by mammalian display technology was limited to104to106so far and can not meet the requirements of screening high affinity antibodies.
We had made technical improvements of the existing mammalian cell surface display technology in order to overcome these shortcomings of it.
Using total RNA isolated from peripheral blood lymphocytes of patients with autoimmune disease as starting material, whole set of antibody genes were amplified by RT-PCR and then the antibody genes were inserted into the mammalian cell expression vector pDGB-HC-TM, Three full-lenth fully human antibody gene libraries have been constructed with a diversity of1010. Using four-way ligation, the antibody heavy and light genes were simultaneously inserted into mammalian display vector pDGB4to construct the sub-libraries. After transfection of the libraries into FCHO cell, a cell library which could stably display full-length antibodies on cell surface was successfully constructed.
Both heavy and light chain genes of Bevacizumab were synthesized and the codons were optimized for high expression of the antibody in mammalian cells. It will be used as a positive control during antibody screen. And it has also been used in chain exchange screen strategy to increase the success rate of screen. Replacing the heavy chain or light chain libraries in vector pDGB4with light chain or heavy chain genes of Bevacizumab, the full-length fully human anti-VEGF antibody libraries have been successfully constructed.
Part Ⅰ:Construction of Full-length Fully Human primary Antibody Display Library
Objective:
To construct3full-length human primary antibody display libraries with large capacity.
Methods:
Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of selected donors by gradient centrifugation. The total RNA was isolated from the purified PBMC using RNA Easy kit. The concentration of the total RNA was measured.
The amplification of heavy chain variable domain and full length kappa chain was carried out by two-step RT-PCR using specific forward and reverse primers. The vector pDGB-HC-TM and RT-PCR amplified DNA fragments were digested by proper restriction enzymes. The ligation was performed at16℃for24hours. The transformation efficiency was calculated and antibody gene libraries were constructed. The transfection of libraries into293T cells was carried out in12-well plate. The antibody expression on cell surface was detected by FACS and the data were analazed using FCS Express V3software.
Results and discussion:
Total6PCR reactions were carried out to amplify antibody genes,3for heavy chain variable domain (VH), and3for full length kappa chain. The sizes of these fragments are about0.45kb for VH and0.75kb for kappa chain. The PCR fragments were separated by electrophoresis and the right size fragments were purified. After digestion of VH library by BsmBI, the VH fragments were inserted into vector pDGB-HC-TM between comparable BsmBI sites. The light chain fragments were insterted into the vector pDGB-HC-TM between SfiI to replace the HC-TM in the original vector. The heavy chain library constructed has a size of1.89×105and light chain library has a size of6.54×104. Totoal3pairs of heavy and light chain liraries were constructed using this method.10heavy chain clones and10light chain clones were randomly picked up for sequence and expression analysis. Results show that8heavy chain clones and7light chain clones contain right coding regions for unique amino acid sequences. The heavy chain and light chain gen libraries were co-transfected into the293T cells. The expression of full length antibodies on293T cell surface were analyzed by FACS. The results show that63.40%cells expressed detectable antibodies.
Conclusion:
Using vector pDGB-HC-TM, three full-length human mammalian display antibody libraries with a combinatory diversity of1.19×10、2.0×1010and5.27×1011were successfully constructed, which allows the display of full-length antibodies on mammalian cell surface.
Part Ⅱ:Construction of Full-length Fully Human secondary Antibody Display Library
Objective:
To construct three full-length human secondary antibody display libraries with large combinatory diversity.
Methods:
After digestion of VH library by BsmBI and light chain library with SfiI, both VH and light chain fragments were simultaneously inserted vector pDGB4by four-way ligation. Totoal3liraries were constructed using this method. After tranformation of libraries into competent bacteria cell, the transformation efficiency and library size were calculated. The stable transfection of libraries into293T cells was carried out in a12-well plate. The antibody expression on cell surface was detected by FACS and the data were analyzed using FCS Express V3software.
Results and discussion:
A four-way ligation library was constructed with a size of2.476x106and a background of0.30%. Another two libraries were constructed using this method, with sizes of9.22×105and1.0828×106, backgrounds of0.06%and0.17%. The transfection of one of these four-way ligation libraries to293T cells was carried out in a12-well plate with61.78%cells expressed detectable antibodies.
After the four way ligation library was stably transfected into FCHO cell, a cell library which could stably display full-length antibodies was successfully constructed, with a size of3.76×106. We could detect that there were59.62%cells in the cell library could display full-length library. This cell library would be used for screening of specific antibody.
Conclusion:
We have successfully constructed three secondary antibody display libraries with large combinatory diversity and a stable cell library with a size of3.76×106, which could stably display full-length antibodies for antibody screen.
Part Ⅲ:Construction of Anti-VEGF Antibody Display Library
Objective:
To construct a full-length human anti-VEGF antibody library by using chain exchange strategy.
Methods:
Both heavy and light chain genes of antibody avastin were synthesized and the codons were optimized for high expression of the antibody in mammalian cells. The appropriate restriction enzyme recognizing sequences and signal peptide sequences were added in front of the heavy chain and the light chain genes. Then both genes were simultaneously inserted into the vector pDGB4to construct the avastin expression vector. The heavy chain or light chain libraries in secondary Antibody Display Library constructed previously was replaced with avastin light chain or heavy chain. In that way, the heavy or light chain in the library was exchanged.
The appropriate restriction enzyme sequence and signal peptide sequence was added in front of the heavy chain variable variable region and the light chain variable region sequence, which was then optimiazed in order to improve gene expression and meet the human codon bias. After digestion of avastin-VH by BsmBl and digestion of avastin-Vk by SfiI, the fragments were inserted into the vector pDGB4between comparable BsmBI sites and SfiI sites; a new vetor pDGB4-avastin was constructed. And then another new vector pDGB4-avastin-vh was constructed. The light chain fragments were insterted into the new vector pDGB4-avastin-vh between SfiI sites in order to construct vector pDGB4-avastin-vh-Hu-LCs. Finally, a full-length human anti-VEGF antibody library was built by the transformation of the new vector pDGB4-avastin-vh-Hu-LCs.
Results and discussion:
Both heavy and light chain genes of antibody avastin were synthesized and the codons were optimized for high expression of the antibody in mammalian cells. After optimization, the CAI (Codon Adaptation Index) of heavy chain was improved from0.80to0.87and the CAI of light chain from0.80to0.85.
Three new expression vetors were constructed:vector pDGB4-avastin for avastin expression; vector pDGB4-avastin-vh containing avastin heavy chain and unrelated light chain; and vector pDGB4-avastin-LC containing avastin light chain and unrelated heavy chain. Finally, a full-length fully human anti-VEGF antibody library, pDGB4-avastin-vh-Hu-LCs, was constructed with a size of7.58×105and a background of2.51%by the replacement of the unrelated light chain in the vector pDGB4-avastin-vh by light chain library. Using the similar method, another full-length fully human anti-VEGF antibody library, pDGB4-avastin-vk-Hu-HCs, was constructed with a size of1.33×106and a background of1.37%by the replacement of the unrelated heavy chain in the vector pDGB4-avastin-vk by heavy chain library.
Conclusion:
We have successfully constructed a full-length fully human anti-VEGF antibody library by using chain exchange strategy.
随着研究深入,发现肾癌特别是透明细胞癌多存在Von Hippel-Lindau(VHL)基因突变,乏氧诱导因子(hypoxia-inducible factor, HIF)及血管内皮生长因子(vascular endothelial growth factor, VEGF)表达增高,而且VEGF增高与肿瘤分期及预后密切相关。因此,抑制或阻断VHL-HIF-VEGF信号通路,特别是阻断VEGF或其受体VEGFR,就可能抑制肾癌的生长。
贝伐单抗(Bevacizumab)是VEGF特异性的经过人源化改造的鼠源抗体,能特异性与VEGF结合并阻断其生物活性。贝伐单抗联合IFN-α可以提高转移性肾癌的客观缓解率(ORR)和无疾病进展率(PFS)。2007年12月美国FDA批准贝伐单抗联合IFN-α作为靶向治疗转移性肾癌的一线治疗方案。
然而,通过杂交瘤技术获得的单抗需经过人源化改造,一方面不可能完全去除抗体的免疫原性,另一方面抗体空间构型可能发生改变,亲和力往往会降低,很难达到预期的作用效果。
为了尽可能减少药用抗体的免疫原性,研究者尝试利用哺乳动物细胞表面抗体展示技术用于人源抗体的筛选,但目前的这些哺乳动物细胞表面展示技术所构建抗体库容量多局限于104～106,筛选抗体多样性不足,不能满足筛选高亲和力抗体的要求。
为了克服哺乳动物细胞表面展示技术筛选全长人源抗体存在的上述不足,本课题组对现有的哺乳动物细胞展示技术作了技术改良。
人的VEGF是一种自身蛋白,以来自于自身免疫病患者的外周血淋巴细胞为材料构建抗体库筛选到抗VEGF抗体的几率更大。通过提取自身免疫病患者的外周血淋巴细胞的总RNA,进行RT-PCR扩增全套抗体基因,随后使用哺乳动物细胞表达载体pDGB-HC-TM,构建了三个全长人源抗体基因库,库容量达到1010以上。将抗体基因库通过四片段连接插入哺乳动物细胞表达载体pDGB4,然后将其稳定转染FCHO细胞。由于采用基因定点整合技术,每个细胞只表达一种抗体,从而成功建成能展示全长抗体的哺乳动物细胞库。
本课题通过全基因合成的方法合成了贝伐单抗的全长基因,并根据哺乳动物细胞对密码子的选择性对抗体基因的碱基序列进行表达优化,既作为筛选抗体的阳性对照,同时也用于抗体的链置换筛选技术,以提高筛选VEGF特异性抗体的成功率；与已经构建好的三个抗体库基因进行链置换,构建了全长人源抗VEGF抗体库,并成功将其展示于哺乳动物细胞表面。
一、全长人源初级抗体库的构建及展示
目的：
构建大容量的全长人源初级抗体基因库。
方法：
1.外周血淋巴细胞的分离及总RNA的提取
从供体中采集适量静脉血,置于肝素抗凝管中,采用密度梯度离心法分离外周血单核细胞,加入适量Buffer RLT,裂解细胞,—80℃冰箱保存备用或者直接用于提取总RNA。
2. cDNA第一链的合成
以总RNA为模板,应用根据V-BASE网站信息所设计合成的全套人抗体重链可变区引物和全套人抗体轻链恒定区引物,逆转录合成cDNA第一条链。
3.全套IgG1重链可变区基因和轻链K型全长基因的扩增
以合成的cDNA为模板,用相应的特异性引物扩增全套IgG1轻链K型全长基因及全套IgG1重链可变区基因(3套轻链引物,3套重链引物)。PCR条件为：94-C预变性5分钟,然后进行35个循环的PCR扩增(94℃变性30s,55℃退火30s,72℃延伸1min),随后在延长温度下反应7min,以确保全长目的片段的合成。
PCR扩增产物的分离纯化
用PCR扩增抗体全套Kappa轻链和重链可变区基因。PCR产物用1%的琼脂糖凝胶电泳分离纯化目的片段。然后将回收得到的3组重链基因PCR扩增产物,再次经1%琼脂糖凝胶电泳后回收目的片段备用。并用同样方法处理3组轻链PCR扩增产物。
重链抗体基因库及轻链基因库的构建及鉴定
以BsmBI对重链基因扩增产物及载体pDGB-HC-TM进行酶切,电泳分离纯化目的片段,将回收的载体片段和抗体基因片段用T4DNA连接酶按1：1比例混合连接,在16℃反应24h后导入化学转化感受态大肠杆菌TOPO-10,构建重链基因库。
轻链全长基因的扩增产物经SfiI酶切后回收,与同样经SfiI酶切回收的载体pDGB-HC-TM片段用T4DNA连接酶按1：1比例混合连接,在16℃反应24h后导入化学转化感受态大肠杆菌TOPO-10,构建轻链基因库。
分别从重链抗体库和轻链抗体库中各随机挑取10个单克隆,摇菌培养过夜后抽提质粒,测序分析抗体基因库的质量及多样性。
4.抗体库在哺乳动物细胞表面的展示
分别将中提所获得的重链库pDGB-HClib-TM与轻链库pDGB-LClib的质粒DNA共转染293T细胞,将上述转染后继续培养48-72h的293T细胞进行回收,然后行免疫荧光染色及流式细胞学分析。
结果与讨论：
分离获得淋巴细胞的数量总计为1.4×108个。取一定量的细胞裂解物,提取获得约3.5u g总RNA,其A260/A280的值为1.90。PCR扩增共获得约2μg的全套IgG1轻链K型全长基因和约2μg的全套重链可变区基因。
根据在带有氨苄青霉素抗性的LB平皿上长出的细菌克隆数,计算所构建的抗体重链基因库库容量为1.89×105,背景克隆的比例为3.60%；抗体轻链基因库库容量为6.54×104,背景克隆的比例为1.65%。用同样的方法一共构建了三个大容量的全长人源抗体基因库,库容量分别为1.19×1010、2.0×1010和5.27×1011。在其中一个抗体基因库里面的重链库和轻链库中各挑取10个单克隆进行测序分析,结果表明重链库的10个单克隆中有8个含有正确的VH编码序列,编码8个特异的氨基酸序列；轻链库的10个单克隆中有7个含有正确的LCκ编码序列,编码7个特异的氨基酸序列。将此重链库和轻链库共转染哺乳动物细胞后,理论上抗体库的多样性可以达到6.67×109[(1.89×105×80%)×(6.54x104x70%)],可以满足筛选高亲和力、高特异性抗体的要求。将抗体库瞬时转染293T细胞后,在63.40%的细胞表面可以检测到全长人源抗体的表达。
结论：
本研究采用哺乳动物细胞展示技术,成功用哺乳动物细胞表面展示载体pDGB-HC-TM构建了库容量大且多样性好的全长人源抗体基因库,可以满足筛选高亲和力、高特异性抗体的要求。
二、全长人源二级抗体库的构建及展示
目的：
将第一部分构建好的三个初级抗体基因库通过四片段连接插入哺乳动物细胞表达载体pDGB4,构建三个全长人源二级抗体库,并将此三个抗体基因库合并一起稳定转染哺乳动物细胞FCHO,获得能够稳定展示全长人源抗体的哺乳动物细胞库。
方法：
1.双表达载体pDGB4的制备
以BsmBI和SfiI对载体pDGB4进行双酶切,经1%琼脂糖凝胶电泳鉴定,并分离纯化5kb和3kb的两段目的DNA片段。
2.抗体基因的制备
以第一部分已经制备的抗体重链基因库(pDGB-HClib-TM)的细菌沉淀为来源,进行质粒中提获得抗体重链基因库的质粒DNA；用BsmBI进行酶切,用1%琼脂糖凝胶电泳分离纯化0.45kb的DNA片段。
以第一部分已经制备的抗体轻链基因库(pDGB-LClib)的细菌沉淀为来源,进行质粒中提获得抗体轻链基因库的质粒DNA；用SfiI进行酶切,用1%琼脂糖凝胶电泳分离纯化0.75kb的DNA片段。
3.转化感受态大肠杆菌TOPO-10及单克隆转染
将获得的抗体轻重链基因库片段插入到pDGB4载体的相应酶切位点之间,获得pDGB-Full-Length-lib。转化化学感受态大肠杆菌TOPO-10,构建全长人源二级抗体库。用同样的方法将第一部分所构建的另外2个初级抗体基因库构建成为四片段连接的全长人源二级抗体库。在转化菌落中随机挑取十个单克隆,进行质粒DNA的小提。
4.抗体库在哺乳动物细胞表面的展示
将二级抗体库的质粒DNA瞬时转染293T细胞,用PE标鼠抗人kappa链抗体进行细胞免疫荧光染色,用流式细胞仪进行检测分析。
5.构建稳定表达单克隆抗体的细胞库
将上述构建的三个四片段连接二级抗体库质粒中提后进行1：1：1合并,与pOG44的质粒DNA共转染常规培养至对数生长期的FCHO细胞,构建稳定表达抗体的细胞库。
结果与讨论：
根据在带有氨苄青霉素抗性LB平皿上长出的克隆数,计算所构建的全长抗体二级基因库库容量为2.476x106,背景克隆的比例为0.30%。用同样的方法将第一部分构建好的第二、第三个初级抗体库基因通过四片段连接插入哺乳动物细胞表达载体pDGB4,构建了第二、第三个四片段连接二级抗体库,库容量分别为9.22×105和1.0828×106,背景克隆的比例分别为0.06%和0.17%。从二级抗体库的建库菌平皿中随机挑取十个单克隆进行质粒小提后瞬时转染293T细胞,流式细胞仪检测有9个克隆能够表达出抗体。将中提获得二级抗体库的质粒DNA进行293T细胞的瞬时转染,有61.78%的细胞表面可成功展示可被检测到的全长抗体。将三个四片段连接二级抗体库基因进行质粒中提后稳定转染FCHO细胞,成功构建了稳定细胞库,库容量为3.76×106,用流式细胞仪检测有59.62%的细胞表面可成功展示可被检测的全长抗体。
结论：
成功将第一部分构建好的三个初级抗体库基因通过四片段连接插入哺乳动物细胞双表达载体pDGB4,构建了三个全长人源二级抗体库,并将此三个基因库合并一起稳定转染哺乳动物细胞FCHO,获得能够稳定展示全长人源抗体的哺乳动物细胞库,为特异性抗体的筛选奠定了基础。
三、全长人源抗VEGF抗体库的构建及展示
目的：
合成并优化贝伐单抗的全长基因,与前面构建好的三个抗体库进行链置换,构建全长人源抗VEGF抗体库。
方法：
1.贝伐单抗序列的合成与优化
在贝伐单抗的重链可变区碱基序列前面加上BstXI和BsmBI的酶切序列以及信号肽序列,将其序列进行人类密码子偏向性优化以及基因表达的优化,以BsmBI进行酶切,1%琼脂糖凝胶电泳分离纯化0.45kb大小的目的DNA片段。
在贝伐单抗的轻链可变区碱基序列前面加上SfiI的酶切序列以及信号肽序列,将其序列进行人类密码子偏向性优化以及基因表达的优化,采用重叠延伸PCR技术进行轻链全长的制备,再以SfiI进行酶切,用1%琼脂糖凝胶电泳分离纯化0.714kb大小的目的片段。
2.双表达载体pDGB4片段的制备
以BsmBI和SfiI对载体pDGB4进行双酶切,经1%琼脂糖凝胶电泳鉴定,并分离纯化5kb大小和3kb大小的两段目的DNA片段。
3.构建质粒pDGB4-avastin
将载体pDGB4片段和优化后合成、制备的贝伐单抗重链抗体片段、轻链抗体片段分别酶切并纯化后,抗体重链基因和抗体轻链基因同时插入到pDGB4载体的相应酶切位点之间,转化化学感受态大肠杆菌TOPO-10,随机挑取单克隆,使用两套引物分别对单克隆的贝伐单抗链可变区序列和轻链可变区序列进行PCR鉴定,根据PCR鉴定的结果,将单克隆菌液进行扩增培养、小提测序分析。
4.链置换法构建抗VEGF抗体库
以BsmBI对载体pDGB4进行单酶切,分离纯化8.65kb大小的目的片段。将该载体片段与经由BsmBI进行酶切并纯化后的贝伐单抗重链抗体片段进行连接获得新质粒pDGB4-avastin-vh,转化化学感受态大肠杆菌TOPO-10,用引物247和引物248来进行PCR鉴定后进行质粒中提,以SfiI进行酶切,分离纯化约8.3kb大小的目的片段,与同样经由SifI进行酶切全长人源轻链基因库(pDGB-LClib)手纯化出的轻链抗体库基因片段进行连接,获得新质粒pDGB4-avastin-vh-Hu-LCs,转化化学感受态大肠杆菌TOPO-10,构建含贝伐单抗重链的全长人源抗VEGF抗体库,计算抗体库的库容量。
同样用链置换方法,获得新质粒pDGB4-avastin-vk-Hu-HCs,转化化学感受态大肠杆菌TOPO-10,构建含贝伐单抗轻链的全长人源抗VEGF抗体库,计算抗体库的库容量。
结果与讨论：
分别在贝伐单抗的重链可变区序列前面加上了BstXI和BsmBI的酶切序列和信号肽序列、在贝伐单抗的轻链可变区序列前面加上SfiI的酶切序列和信号肽序列,并且进行了人类密码子偏向性优化以及基因表达的优化。经过优化,贝代单(?)重链可变区碱基序列的密码子适应指数(CAI)从0.80提高到0.87,最优密码子使用频率(FOP)也得到了优化,而GC含量变化不大；轻链可变区碱基序列的密码子适应指数(CAI)从0.80提高到0.85,最优密码子使用频率(FOP)也得到了优化,而GC含量变化不大。通过与前面构建好的三个抗体库进行链置换,构建了库容量分别为7.58x105和1.33×106,背景克隆的比例分别为2.51%和1.37%的全长人源抗VEGF抗体库。将抗体库瞬时转染293T细胞后,在细胞表面可以检测到全长人源抗VEGF抗体的表达；在抗VEGF抗体库1中,有22%的细胞表面可成功展示可被检测到的全长抗体,在抗VEGF抗体库2中,有28.05%的细胞表面可成功展示可被检测到的全长抗体。
结论：
成功在贝伐单抗的重链可变区序列和轻链可变区序列的前面加上了相应的酶切序列和信号肽序列并进行优化、全基因合成与制备,与前面构建好的三个抗体库基因进行链置换,构建了全长人源抗VEGF抗体基因库,将抗体库瞬时转染293T细胞后,在细胞表面可以检测到全长人源抗VEGF抗体的表达,为进一步利用贝伐单抗的抗VEGF导向选择作用,以便筛选具有与贝伐单抗相同的抗原亲和性和抗原结合表位的全长人源抗VEGF抗体做好了准备,也为开展全长人源抗VEGF单抗靶向治疗转移性肾癌奠定了实验基础。
Renal cell carcinoma (RCC) is the most common kidney malignant tumor, which is not sensitive to radiotherapy and chemotherapy. Due to its insidious onset,25to30percent of patients had metastasis in the time of diagnosis and nearly30%of localized renal cell carcinoma will relapse even after radical resection. People are looking forward to a new therapeutic approach to solve this problem.
With in-depth study, Von Hippel-Lindau (VHL) gene mutation is found in most renal cell carcinoma, especially clear cell carcinoma, with a high level of hypoxia-inducible factor and vascular endothelial growth factor. VEGF is closely related to tumor stage and prognosis. Therefore, inhibition or blocking of VHL-HIF-VEGF signaling pathway, in particular, blocking VEGF or its receptors VEGFR, may inhibit the growth of renal cell carcinoma.
Bevacizumab is a VEGF-specific murine antibody, which is humanized and due to its biological activity of blocking VEGF, is used for the treatment of patients with solid tumors and high level of VEGF. Bevacizumab combined with IFN-a can increase the Objective Response Rate (ORR) and Progression Free Survival (PFS) of metastatic renal cell carcinoma. Thus, U.S. FDA approved bevacizumab plus IFN-alpha as first-line treatment of the targeted therapy of metastatic renal cell carcinoma in December2007.
However, humanization of monoclonal antibodies obtained by hybridoma technology, on the one hand, can not completely remove antibodies' immunogenicity. On the other hand, the antibody space configuration may be changed, the affinity tends to reduce and it is verydifficult to achieve the desired effect.
In order to minimize the immunogenicity of pharmaceutical antibodies, the researchers are trying to use mammalian cell surface display technology for the screening of human antibodies. The diversity of the antibody library contructed by mammalian display technology was limited to104to106so far and can not meet the requirements of screening high affinity antibodies.
We had made technical improvements of the existing mammalian cell surface display technology in order to overcome these shortcomings of it.
Using total RNA isolated from peripheral blood lymphocytes of patients with autoimmune disease as starting material, whole set of antibody genes were amplified by RT-PCR and then the antibody genes were inserted into the mammalian cell expression vector pDGB-HC-TM, Three full-lenth fully human antibody gene libraries have been constructed with a diversity of1010. Using four-way ligation, the antibody heavy and light genes were simultaneously inserted into mammalian display vector pDGB4to construct the sub-libraries. After transfection of the libraries into FCHO cell, a cell library which could stably display full-length antibodies on cell surface was successfully constructed.
Both heavy and light chain genes of Bevacizumab were synthesized and the codons were optimized for high expression of the antibody in mammalian cells. It will be used as a positive control during antibody screen. And it has also been used in chain exchange screen strategy to increase the success rate of screen. Replacing the heavy chain or light chain libraries in vector pDGB4with light chain or heavy chain genes of Bevacizumab, the full-length fully human anti-VEGF antibody libraries have been successfully constructed.
Part Ⅰ:Construction of Full-length Fully Human primary Antibody Display Library
Objective:
To construct3full-length human primary antibody display libraries with large capacity.
Methods:
Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of selected donors by gradient centrifugation. The total RNA was isolated from the purified PBMC using RNA Easy kit. The concentration of the total RNA was measured.
The amplification of heavy chain variable domain and full length kappa chain was carried out by two-step RT-PCR using specific forward and reverse primers. The vector pDGB-HC-TM and RT-PCR amplified DNA fragments were digested by proper restriction enzymes. The ligation was performed at16℃for24hours. The transformation efficiency was calculated and antibody gene libraries were constructed. The transfection of libraries into293T cells was carried out in12-well plate. The antibody expression on cell surface was detected by FACS and the data were analazed using FCS Express V3software.
Results and discussion:
Total6PCR reactions were carried out to amplify antibody genes,3for heavy chain variable domain (VH), and3for full length kappa chain. The sizes of these fragments are about0.45kb for VH and0.75kb for kappa chain. The PCR fragments were separated by electrophoresis and the right size fragments were purified. After digestion of VH library by BsmBI, the VH fragments were inserted into vector pDGB-HC-TM between comparable BsmBI sites. The light chain fragments were insterted into the vector pDGB-HC-TM between SfiI to replace the HC-TM in the original vector. The heavy chain library constructed has a size of1.89×105and light chain library has a size of6.54×104. Totoal3pairs of heavy and light chain liraries were constructed using this method.10heavy chain clones and10light chain clones were randomly picked up for sequence and expression analysis. Results show that8heavy chain clones and7light chain clones contain right coding regions for unique amino acid sequences. The heavy chain and light chain gen libraries were co-transfected into the293T cells. The expression of full length antibodies on293T cell surface were analyzed by FACS. The results show that63.40%cells expressed detectable antibodies.
Conclusion:
Using vector pDGB-HC-TM, three full-length human mammalian display antibody libraries with a combinatory diversity of1.19×10、2.0×1010and5.27×1011were successfully constructed, which allows the display of full-length antibodies on mammalian cell surface.
Part Ⅱ:Construction of Full-length Fully Human secondary Antibody Display Library
Objective:
To construct three full-length human secondary antibody display libraries with large combinatory diversity.
Methods:
After digestion of VH library by BsmBI and light chain library with SfiI, both VH and light chain fragments were simultaneously inserted vector pDGB4by four-way ligation. Totoal3liraries were constructed using this method. After tranformation of libraries into competent bacteria cell, the transformation efficiency and library size were calculated. The stable transfection of libraries into293T cells was carried out in a12-well plate. The antibody expression on cell surface was detected by FACS and the data were analyzed using FCS Express V3software.
Results and discussion:
A four-way ligation library was constructed with a size of2.476x106and a background of0.30%. Another two libraries were constructed using this method, with sizes of9.22×105and1.0828×106, backgrounds of0.06%and0.17%. The transfection of one of these four-way ligation libraries to293T cells was carried out in a12-well plate with61.78%cells expressed detectable antibodies.
After the four way ligation library was stably transfected into FCHO cell, a cell library which could stably display full-length antibodies was successfully constructed, with a size of3.76×106. We could detect that there were59.62%cells in the cell library could display full-length library. This cell library would be used for screening of specific antibody.
Conclusion:
We have successfully constructed three secondary antibody display libraries with large combinatory diversity and a stable cell library with a size of3.76×106, which could stably display full-length antibodies for antibody screen.
Part Ⅲ:Construction of Anti-VEGF Antibody Display Library
Objective:
To construct a full-length human anti-VEGF antibody library by using chain exchange strategy.
Methods:
Both heavy and light chain genes of antibody avastin were synthesized and the codons were optimized for high expression of the antibody in mammalian cells. The appropriate restriction enzyme recognizing sequences and signal peptide sequences were added in front of the heavy chain and the light chain genes. Then both genes were simultaneously inserted into the vector pDGB4to construct the avastin expression vector. The heavy chain or light chain libraries in secondary Antibody Display Library constructed previously was replaced with avastin light chain or heavy chain. In that way, the heavy or light chain in the library was exchanged.
The appropriate restriction enzyme sequence and signal peptide sequence was added in front of the heavy chain variable variable region and the light chain variable region sequence, which was then optimiazed in order to improve gene expression and meet the human codon bias. After digestion of avastin-VH by BsmBl and digestion of avastin-Vk by SfiI, the fragments were inserted into the vector pDGB4between comparable BsmBI sites and SfiI sites; a new vetor pDGB4-avastin was constructed. And then another new vector pDGB4-avastin-vh was constructed. The light chain fragments were insterted into the new vector pDGB4-avastin-vh between SfiI sites in order to construct vector pDGB4-avastin-vh-Hu-LCs. Finally, a full-length human anti-VEGF antibody library was built by the transformation of the new vector pDGB4-avastin-vh-Hu-LCs.
Results and discussion:
Both heavy and light chain genes of antibody avastin were synthesized and the codons were optimized for high expression of the antibody in mammalian cells. After optimization, the CAI (Codon Adaptation Index) of heavy chain was improved from0.80to0.87and the CAI of light chain from0.80to0.85.
Three new expression vetors were constructed:vector pDGB4-avastin for avastin expression; vector pDGB4-avastin-vh containing avastin heavy chain and unrelated light chain; and vector pDGB4-avastin-LC containing avastin light chain and unrelated heavy chain. Finally, a full-length fully human anti-VEGF antibody library, pDGB4-avastin-vh-Hu-LCs, was constructed with a size of7.58×105and a background of2.51%by the replacement of the unrelated light chain in the vector pDGB4-avastin-vh by light chain library. Using the similar method, another full-length fully human anti-VEGF antibody library, pDGB4-avastin-vk-Hu-HCs, was constructed with a size of1.33×106and a background of1.37%by the replacement of the unrelated heavy chain in the vector pDGB4-avastin-vk by heavy chain library.
Conclusion:
We have successfully constructed a full-length fully human anti-VEGF antibody library by using chain exchange strategy.
引文
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