首先,我们制备了大量的BCMA重组蛋白作为抗原免疫兔子,所有蛋白经SDS-PAGE和多种结合实验验证具有高纯度及高活性。我们从兔血清中分离出3~5 X108 B细胞,并快速冻存至液氮中,作为BCMA-B细胞种子库长期保存。从4mL兔全血中,我们筛选出70个ELISA阳性的B细胞克隆,其中,13个克隆呈现流式阳性。我们对这13个克隆进行单抗克隆和测序,通过系统发育分析和表位比较,鉴定出5个克隆。将这5个克隆人源化后应用于CAR-T研究,它们都显示出与蓝鸟公司的抗BCMA  huC11d5.3克隆具有相似的肿瘤细胞杀伤效果。

Figure 1: Workflow for BCMA DimAb® 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 DimAb®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. 

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.

Figure 3: Phylogenetic analysis of 13 different Anti-BCMA DimAb 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 DimAb 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.

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

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