本文關(guān)鍵詞：高親和力CD34人源化抗體的制備及鑒定　出處：《第二軍醫(yī)大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

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【摘要】： CD34是分子量約為110kDa的穿膜糖蛋白,是目前所知的最早期、最廣泛表達(dá)于人類HSC上的膜抗原。在臨床造血干細(xì)胞移植中,CD34單抗已被廣泛用于純化造血干細(xì)胞。但現(xiàn)有的抗人CD34單克隆抗體均為鼠源,進(jìn)入人體后可能誘發(fā)人抗鼠抗體免疫應(yīng)答(HAMA)。因此,為了提高造血干細(xì)胞移植的安全性,我們擬制備新型的抗CD34人源化抗體,以降低其免疫原性,提高其在臨床使用中的安全性。 我們首先采用傳統(tǒng)的雜交瘤技術(shù)制備了三株抗人CD34單克隆抗體(5812、4C8和2E10),利用試劑盒檢測(cè)其亞型分別為(IgG2a,κ)、(IgG1,κ)和(IgG3,κ);流式細(xì)胞術(shù)的結(jié)果表明,與商品化的CD34單抗581對(duì)照一樣,該三株抗體都能與表達(dá)CD34抗原的KG-1a細(xì)胞特異性結(jié)合;Western blotting分析進(jìn)一步證實(shí)它們特異性識(shí)別CD34分子;表位鑒定結(jié)果表明5B12、4C8和2E10分別識(shí)別CD34分子的Ⅰ、Ⅱ、Ⅲ類表位。我們采用5'RACE技術(shù)獲得了5812、4C8和2E10的可變區(qū)全長(zhǎng)cDNA,將可變區(qū)基因序列輸入計(jì)算機(jī)在GenBank數(shù)據(jù)庫(kù)中檢索,證明是新克隆的抗體基因。 在獲得5812、4C8和2E10這三株單抗的可變區(qū)基因之后,我們將它們分別與人源抗體的恒定區(qū)連接,然后將嵌合抗體輕、重鏈基因分別克隆到真核表達(dá)載體,最后將嵌合抗體輕、重鏈表達(dá)載體共轉(zhuǎn)染CHO-K1細(xì)胞,經(jīng)選擇培養(yǎng)基篩選后獲得穩(wěn)定表達(dá)抗體蛋白的CHO細(xì)胞克隆。獲得的CHO細(xì)胞培養(yǎng)上清經(jīng)Protein A親和分離純化,獲得純度在90%以上的CD34嵌合抗體c5812、c4C8、c2E10;抗原結(jié)合活性結(jié)果表明該三株嵌合抗體都能很好地與KG-1a細(xì)胞結(jié)合;競(jìng)爭(zhēng)抑制實(shí)驗(yàn)結(jié)果表明它們都能與各自對(duì)應(yīng)的鼠源抗體競(jìng)爭(zhēng),而且它們的IC_(50)值相似,說(shuō)明三株嵌合抗體都保留了與各自對(duì)應(yīng)的鼠源抗體相似的親和力和特異性。 為了進(jìn)一步減小嵌合抗體的免疫原性,我們利用計(jì)算機(jī)輔助設(shè)計(jì)對(duì)Ⅱ類CD34單抗4C8進(jìn)行人源化改造。在確定了4C8可變區(qū)中CDR區(qū)和FR區(qū)的范圍之后,我們將CDR區(qū)直接移植到人源抗體模板的FR中,構(gòu)建CDR移植抗體,并將獲得的人源化輕、重鏈抗體基因克隆到表達(dá)載體共轉(zhuǎn)染COS細(xì)胞,用流式細(xì)胞術(shù)測(cè)定其抗原結(jié)合活性,結(jié)果發(fā)現(xiàn)這個(gè)人源化抗體的親和力基本完全喪失。為了重建人源化抗體的親和力,我們利用計(jì)算機(jī)輔助設(shè)計(jì)對(duì)可能影響抗體親和力的FR區(qū)重要?dú)埢M(jìn)行回復(fù)突變分析,分別構(gòu)建了5條不同的人源化重鏈和5條不同的人源化輕鏈,經(jīng)過活性篩選,我們發(fā)現(xiàn)對(duì)于人源化重鏈來(lái)說(shuō),框架區(qū)殘基2,46,68,69,82,91的回復(fù)突變是必要的,將它們?nèi)客蛔優(yōu)槭笤礆埢蟮玫搅艘粋�(gè)活性超過嵌合抗體重鏈的人源化重鏈4C8VHb。我們采用同樣的方法對(duì)人源化輕鏈基因進(jìn)行改造,發(fā)現(xiàn)將輕鏈框架區(qū)殘基3,4,46全部回復(fù)突變獲得的人源化輕鏈4C8VLb與4C8VHb組成的人源化抗體h4C8的抗原結(jié)合活性超過了嵌合抗體c4C8,在篩選得到高親和力的人源化抗體h4C8之后,我們又建立了高效表達(dá)h4C8的CHO-K1細(xì)胞株,為今后的實(shí)驗(yàn)和臨床研究打下了基礎(chǔ)。競(jìng)爭(zhēng)抑制實(shí)驗(yàn)結(jié)果表明人源化抗體可競(jìng)爭(zhēng)抑制原鼠源抗體與CD34陽(yáng)性細(xì)胞的結(jié)合并具有與鼠源抗體相似的親和力,表明h4C8是一個(gè)改造成功的人源化抗體 綜上所述,本課題中構(gòu)建的抗CD34人源化抗體可能取代現(xiàn)有的鼠源CD34單抗用于臨床上造血干細(xì)胞的分選,從而提高臨床造血干細(xì)胞移植的安全性。
[Abstract]:CD34 is a molecular weight of approximately 110kDa transmembrane glycoprotein, is the earliest known at present, the most extensive membrane antigen expression in human HSC. In clinical hematopoietic stem cell transplantation, CD34 monoclonal antibody has been widely used for the purification of hematopoietic stem cells. But the existing anti human CD34 monoclonal antibodies are murine, enter the human body can induce human anti mouse antibody immune response (HAMA). Therefore, in order to improve the safety of hematopoietic stem cell transplantation, we prepared anti CD34 humanized antibody model, in order to reduce its immunogenicity, improve its safety in clinical use.
We used the traditional hybridoma technique was three strains of monoclonal antibodies against human CD34 were prepared (5812,4C8 and 2E10), to detect the subtype using kit respectively (IgG2a, K) (k, IgG1) and (IgG3, K); flow cytometry results showed that, with the commercial CD34 581 single anti control, the three antibodies can be combined with KG-1a cell specific expression of CD34 antigen; Western blotting analysis further confirmed their specific molecular recognition CD34; epitope identification results show that 5B12,4C8 and 2E10 respectively to identify CD34 molecules I, II, III epitopes. We obtained by 5'RACE 5812,4C8 2E10 and the variable region of the full-length cDNA gene sequences of variable regions of input computer retrieval in GenBank database, that is the new antibody gene cloning.
After obtaining the 5812,4C8 and 2E10 variable region genes of the three strains of monoclonal antibodies, we will connect respectively with human antibody constant region, then the chimeric antibody light and heavy chain genes were cloned into the eukaryotic expression vector of the chimeric antibody light and heavy chain expression vector were transfected into CHO-K1 cells, CHO cell clones selection of culture medium were screened with stable expression of antibody protein. The culture supernatant of CHO cells by Protein A affinity purification, the purity of CD34 chimeric antibody c5812, above 90% c4C8, c2E10; antigen binding activity results showed that the three strains of chimeric antibody can combine well with experimental results of competitive inhibition of KG-1a cells; that they can compete with their corresponding monoclonal antibody, and their IC_ (50) value similar to that of three strains of chimeric antibody have retained the mouse antibody with corresponding similar affinity and specificity.
In order to further reduce the immunogenicity of chimeric antibody, we use computer aided design of humanization of class II CD34 mAb 4C8. After determining the scope of the CDR and FR region 4C8 in variable regions, we will CDR transplanted directly to human antibody template FR, construct CDR transplantation and antibody. People will get the source of light, heavy chain antibody gene was cloned into the expression vector were transfected into COS cells. The antigen binding activity was determined by flow cytometry, the results showed that the humanized antibody affinity. In order to complete loss of basic affinity reconstruction of humanized antibody, we use the computer aided design of FR may influence area the important residues of the antibody affinity mutation analysis, we constructed 5 different human heavy chain and 5 different human light chain, through activity screening, we found that the humanized heavy chain, frame area The residue of 2,46,68,69,82,91 mutation is necessary, all of them are mutated into murine residues obtained after a heavy chain activity over the humanized chimeric antibody heavy chain 4C8VHb. we used the same method to transform the human light chain gene, found the residue light chain framework 3,4,46 reply to all human antibody h4C8 mutations of the human source light chain 4C8VLb and 4C8VHb composed of the antigen binding activity than the chimeric antibody c4C8, after screening the humanized antibody with high affinity for h4C8, we established the CHO-K1 cell line expressing h4C8, for future experimental and clinical research. This is the foundation of competitive inhibition the experimental results show that the humanized antibody can competitively inhibit the original mouse antibody and CD34 positive cells in combination with mouse antibody similar affinity, indicating that h4C8 is a successful transformation of the humanized antibody
To sum up, the anti CD34 humanized antibody constructed in this study may replace the existing murine CD34 monoclonal antibody for clinical hematopoietic stem cell sorting, thereby improving the safety of clinical hematopoietic stem cell transplantation.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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本文編號(hào)：1411994