Anti-B7H3 chimeric antigen receptor and application thereof
A technology of chimeric antigen receptor and antigen, applied in the field of biomedicine
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[0131] Example 1 Preparation of CAR-T cells
[0132] In this example, anti-B7H3 antibodies H26B6, H2B8, 26B6, 2B8, 23H1, 6F7, Enoblituzumab (Eno) and huM30 were selected as the antigen-binding domains to construct CAR molecules. Among them, 26B6 and its humanized H26B6, 2B8 and its humanized H2B8, 23H1, and 6F7 have significant binding ability to B7H3, not only can bind to free B7H3 protein, but also can bind to cell surface B7H3 protein; huM30 is a humanized B7H3 antibody (CN103687945B) from Daiichi Sankyo (Japan). ), is conducting a phase I clinical trial for the treatment of B7H3-positive solid tumors (NCT02192567); Enoblituzumab (MGA271) is a new monoclonal antibody that has been optimized by immune molecules, targeting the B7H3 target, by MacroGenics using exclusive Fc optimization technology Developed, with unique antibody advantages and therapeutic potential, no such drug has been approved in the world, Enoblituzumab represents the world's leading B7H3 antibody drug.
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[0149] Example 2 Expression efficiency of CAR by CAR-T cells
[0150] The expression rate of CAR in CAR-T cells was detected by flow cytometry.
[0151] like Figure 3A As shown, the expression rate of CAR in H26B6-CAR-T cells was 65.72%, and the expression rates of CAR in H2B8-CAR-T, L2B8-CAR-T, and L26B6-CAR-T cells were 31.73%, 38.15%, and 44.14%, respectively.
[0152] like Figure 3B As shown, the expression rate of CAR in Eno-CAR-T cells was 27.3%, and the expression rate of CAR in huM30-CAR-T cells was 45.2%.
[0153] In another experiment, as Figure 3C As shown, the expression rate of CAR in huM30-CAR-T cells was 23.09%, and the expression rate of CAR in 26B6-CAR-T cells was 7.67%;
[0154] In another experiment, as Figure 3D As shown, the expression rate of CAR in huM30-CAR-T cells was 33.12%, the expression rate of CAR in L2B8-CAR-T cells was 33.43%, and the expression rate of CAR in 2B8-CAR-T cells was 12.55%.
Example Embodiment
[0155] Example 3 Killing function of CAR-T cells
[0156] H26B6-CAR-T, H2B8-CAR-T, L2B8-CAR-T, L26B6-CAR-T were combined with human liver cancer cell HepG2, human pancreatic cancer cell PL45, and human cervical cancer cell SiHa in a ratio of 2:1, 1:1 , 1:4 effector-target ratio was incubated for 16h, and the killing efficiency of CAR-T was detected by RTCA technology.
[0157] Figure 4A , Figure 4B , Figure 4C The results showed that the four CAR-T cells had killing effects on the three tumor cells under different effector-target ratios, and the larger the effector-target ratio, the stronger the killing ability; when the effector-target ratio was 2:1, H26B6-CAR-T cells The killing efficiency of T on three tumor cells was better than that of H2B8-CAR-T, L2B8-CAR-T and L26B6-CAR-T.
[0158] Different CAR-T cells (huM30-CAR-T and 2B8-CAR-T) were prepared from PBMCs of healthy donors (donor 1 and donor 2), and the target cells were 3:1, 1:1, 1 The effector-target ratio of ...
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