研究案例:BCMA-先導抗體分子開發(fā)及CAR-T應用

1、簡介:

多發(fā)性骨髓瘤(MM,Multiple myeloma):僅次于非霍奇金淋巴瘤的第二大常見血液學惡性腫瘤,幾乎所有患者預后仍會復發(fā),被認為是無法治愈的疾病,新治療方案開發(fā)需求迫切;

BCMA是治療MM理想的抗原靶點:除了成熟B淋巴細胞及漿細胞表面,BCMA在其他組織細胞中幾乎不表達,但它在所有MM細胞中高表達;

市場開發(fā)賽道火熱:葛蘭素史克的Blenrep(2020上市);百時美施貴寶與藍鳥生物的Ide-cel(FDA優(yōu)先審評);南京傳奇和楊森的LCAR-B38M(三期臨床)……

2、BCMA開發(fā)流程:

首先,我們制備了大量的BCMA重組蛋白作為抗原免疫兔子,所有蛋白經SDS-PAGE和多種結合實驗驗證具有高純度及高活性。我們從兔血清中分離出3~5 X108 B細胞,并快速凍存至液氮中,作為BCMA-B細胞種子庫長期保存。從4mL兔全血中,我們篩選出70個ELISA陽性的B細胞克隆,其中,13個克隆呈現流式陽性。我們對這13個克隆進行單抗克隆和測序,通過系統發(fā)育分析和表位比較,鑒定出5個克隆。將這5個克隆人源化后應用于CAR-T研究,它們都顯示出與藍鳥公司的抗BCMA huC11d5.3克隆具有相似的腫瘤細胞殺傷效果。 

pages-776107b90ca653c
pages-17628dedd020d9b

Figure 1: Workflow for BCMA lead molecules project. From 4ml of rabbit whole blood, we identified 5 lead mAbs molecules for human BCMA targets with verified functional data and antibody sequences. We also created a B cell seed library for the human BCMA target which presumably contains 10 thousands Flow positive BCMA binders providing a good resource for additional screening.

2.1、BCMA功能性蛋白抗原開發(fā)

為了確保BCMA重組表達蛋白具有功能活性,我們對BCMA蛋白抗原進行了BCMA受體-配體互作驗證,同時也驗證了BCMA重組蛋白和huC11D5.3中和性抗體的體外結合能力(來源于藍鳥公司在臨床三期的CAR-T結構bb2121)。

Functional BCMA protein used as immunogen

Figure 2: Quality analysis of purified human BCMA protein, Fc-tagged. A. Human BCMA, Fc-tagged on SDS-PAGE under reducing condition. B. ELISA plate pre-coated by 2 μg/ml (100 μl/well) Human BAFF, hFc tagged protein can bind Human BCMA, Fc- tagged protein in a linear range of 0.03-15.625 ng/ml. C. ELISA plate pre-coated by 2 μg/ml (100 μl/well) Human BCMA, Fc-tagged protein can bind Anti-BCMA (huC11D5.3) (Its variable region was used to construct scFv portion of CAR-T Idecabtagene vicleucel (bb2121). ) in a linear range of 3.71-22.29 ng/ml.

2.2、BCMA先導分子發(fā)現和B細胞種子庫

利用重組單克隆抗體平臺,我們從4ml免疫后兔全血中篩選獲得了70株靶點結合陽性B細胞克隆,其中13株克隆進行了功能驗證。而這些只是BCMA B細胞種子庫中的一小部分,后期可以隨時擴大篩選。

pages-406107b921c4025

Figure 3: Phylogenetic analysis of 13 different Anti-BCMA clones A) heavy chain and B) Light chain. All these clones work for flow application. The boxed regions indicate heavy and light chains of the same clone come from the same lineage group.

Figure 4: Epitope comparison between different anti-BCMA clones and anti-BCMA huC11D5.3 clone (Bluebird bb2121). ELISA plate was coated with recombinant BCMA-hFc fusion protein, followed by pre-blocking with huC11D5.3 antibody (Grey bar) or rabbit control IgG (Black bar) and then different rabbit DimAbs antibodies were added to check the competitive inhibition of huC11D5.3. One clone exhibits the strongest inhibition (Red bar). This data indicated that one clone binds to the same epitope as bb2121.

2.3、BCMA抗體工程改造

根據重組單克隆抗體平臺提供的BCMA抗體基因序列信息,可以讓我們直接對篩選驗證的5株高親和力克隆株進行抗體人源化工程改造,同時構建了雙特異性BiTE分子和CAR分子,并進行體外功能驗證。(目前已有克隆株商業(yè)轉化)

pages-766107b93133dd2

BCMA雙特異性BiTE分子體外功能分析

pages-775fc73e583c57e

Figure 5. 人源化BCMA-BiTE分子(DM6克?。┝黾毎麣麢z測

pages-175fc73e655c3a5

BCMA CAR 分子體外功能分析

Figure 6: In-vivo testing of humanized anti-BCMA lead mAbs molecules. The preliminary tumor cell killing efficacy testing data is proprietary. It indicated that our 5 humanized CARs are comparable or better than BMK.

 

3、BCMA 開發(fā)周期:

項目總開發(fā)周期:約4.5個月

pages-136107b94399c95