抗Cbk1单克隆抗体人源化改造及其抗耐药真菌活性与机制研究

Most patients suffered from invasive fungal infection have immunodeficiency.But the existing antifungal drugs have limited types, narrow spectrums, and several side effects.While antibody drugs targeted at fungal cell wall protein have high selectivity, low toxicity, they will fundamentally improve immunologic function in immunocompromised patients against pathogenic fungi with high specificity.Cbk1 protein presents in both the cell bud and hyphal tip.Cbk1p is essential for budding and hyphae formation, which is indispensable for virulence.We have previously constructed and obtained one specific anti-Cbk1 monoclonal antibody with known specific epitope.The anti-Cbk1 antibody binds to the specific antigen on the cell surfaces of both yeast and hyphae in C.albicans by immunological fluorescence assay, and combines with the specific cell wall protein by Western blot.Particularly, the anti-Cbk1 monoclonal antibody significantly improve the survival rate of mice infected with invasive candidiasis to 80%, prolong the survival time of more than 30 days, and reduce the amount of fungal loading in liver and kidney.Such protection is similar to that of anti-Hsp90 antibody which is made by researchers in USA. In this study, we will develop humanized anti-Cbk1 antibody to reduce the immunogenicity and improve the safety.To construct the chimeric antibody light and heavy chain expression vectors, the cloned anti-Cbk1 antibody light and heavy chain variable region cDNA will be fused to the human antibody light and heavy chain constant region genes.The chimeric light and heavy chain will be inserted into the expression vectors.Then appropriate light and heavy chain expression vector will be co-transferred into CHO cells and the clones producing the highest amout of chimeric antibody will be selected and purified. To further reduce the immunogenicity of chimeric antibody, we will develop a humanized anti-Cbk1 antibody. The complementarity-determining regions from anti-Cbk1 light chain or heavy chain will be grafted into human antibody light chain or heavy chain frameworks to generate humanized antibody genes.Important residues that may have influences on binding activity in the human FRs will be analyzed by computer-assisted designing and back mutation assay will be carried out.A number of light and heavy chain versions will be produced to evaluate the contribution of FR residues to antigen binding activity.The humanized antibody will be expressed in CHO cells and be purified. We will then examine whether the humanized anti-Cbk1 antibody has functions of neutralization, opsonophagocytosis, and complement activation in removing fungal cells.Meanwhile,we will investigate in vivo anti-fungal activity of the humanized anti-Cbk1 antibody against drug-resistant and-sensitive fungi in mice with normal or deficient immunity.We will examine the synergistic anti-fungal activity of the combination of the humanized anti-Cbk1 and fluconazole or amphotericin B.

多数深部真菌感染患者免疫力缺陷，现有抗真菌化药种类有限，抗菌谱窄，毒副作用大，而特异结合真菌细胞壁蛋白的抗体药物选择性高、低毒，能改善免疫缺陷患者对致病真菌的抵御作用。Cbk1蛋白位于白念珠菌酵母态细胞出芽颈环和菌丝尖端，是出芽增殖、菌丝形成必需蛋白，还能提高菌株致病力，本项目前期已获得明确抗原表位的鼠源抗Cbk1单抗，能与白念珠菌酵母态及菌丝态活细胞表面抗原特异结合，能使侵袭性白念珠菌感染小鼠存活率提高至80%，并延长存活时间达30天以上，减轻肝肾载菌量，作用与国外抗Hsp90抗体相当。本项目拟将抗Cbk1可变区基因与人源抗体恒定区连接，表达获得嵌合抗体；利用计算机辅助设计，将可变区CDR移植到人源抗体模板FR区，对可能影响亲和力FR区重要残基进行回复突变分析，构建并表达高亲和力及特异性抗原结合活性的人源化抗体。进而对其生物学功能进行鉴定，探讨Cbk1人源化抗体抗多种真菌活性及作用机制。

本项目前期研究表明抗Cbk1鼠杂交瘤单克隆抗体具有体内外抗白念珠菌作用，本项目拟构建抗Cbk1人源化抗体并对其生物学功能进行鉴定。.首先，杂交瘤抗体经轻重链N末端测序，以指导基因克隆。其次提取杂交瘤细胞总RNA，选用相应的可变区引物组合，通过反转录PCR的方法扩增获得可变区基因片段，克隆入T-载体测序分析可变区基因，然后亚克隆到带有人抗体恒定区基因的表达载体中进行重组表达和ELISA分析其与合成多肽的结合。Fortebio分析重组嵌合抗体以及鼠杂交瘤抗体与抗原的结合，确认获得了正确的重组嵌合抗体，体外实验也表明克隆得到的重组嵌合抗体与鼠杂交瘤抗体有类似的抗原结合活性。最后，通过将轻重链基因克隆到稳定表达载体转染CHO细胞，并筛选表达克隆，放大重组表达体系，收集上清通过亲和与离子交换层析的方法获得了纯度大于95%嵌合抗体。.ELISA结果表明，抗Cbk1嵌合抗体和鼠源抗Cbk1抗体对Cbk1抗原多肽及白念珠菌SC5314活菌均有结合特异性。流式细胞术和免疫荧光实验结果表明，抗Cbk1嵌合抗体以及鼠源抗Cbk1抗体与酵母态和菌丝态的白念珠菌均有较强的结合。但是，抗Cbk1嵌合抗体失去抗真菌活性，表现为对白念珠菌的菌丝及生物被膜形成能力没有抑制作用，不能提高系统性白念株菌感染小鼠的存活率，降低肝肾载菌量；而鼠源抗Cbk1抗体对白念珠菌的菌丝及生物被膜形成能力有抑制作用，能显著提高系统性白念株菌感染小鼠的存活率，降低肝肾载菌量。.结果表明，人鼠嵌合抗体保留了免疫原性，但失去体内外抗真菌生物活性。推测原因可能是最初的鼠源单克隆抗体细胞株中存在其他亚克隆细胞，是具有生物活性的。而通过N端测序获得序列进而推测获得的可变区序列是其中并无生物学功能的亚克隆细胞，所以由此序列进一步构建的嵌合抗体无生物活性。.针对这一问题，我们以Cbk1为靶点，重新设计并通过原核表达Cbk1蛋白抗原，免疫小鼠，取脾脏混合构建小鼠免疫源噬菌体展示抗体库，进而筛选抗体，以避免筛选亚克隆细胞不纯的问题。现已完成原核表达Cbk1蛋白。