Car t-cells targeting bcma and uses thereof
A cell and amino acid technology, applied in the field of immunotherapy and chimeric antigen receptors, can solve problems such as persistence
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Embodiment 1
[0147] Example 1. Design of several BCMA CARs
[0148] In order to be able to compare the different binding parts against the improved BCMA CAR side-by-side, the same backbone construct was used, where only the binding part of the BCMA-CAR was changed between the different constructs to be tested. The backbone construct is as figure 1 As shown in A, it is based on a retroviral vector and further contains a marker. In addition, for use in an allogeneic environment, the same construct was prepared, which further contained shRNA against CD247 ( figure 1 A, bottom). When expressed in cells, this shRNA interferes with the function of the T cell receptor complex and is thus able to prevent the development of graft-versus-host disease (GvHD) in allogeneic ACT.
[0149] The design of the BCMA CAR is shown in more detail in figure 1 in B. Eleven different binding moieties were tested, but the hinge, transmembrane and CD3ζ signaling domains remained the same. Each construct was ...
Embodiment 2
[0150] Example 2. In vitro properties of different CARs
[0151] To test whether all CARs could be successfully expressed, PBMCs from three different healthy donors were transduced with the indicated BCMA CAR constructs or the Mock(tCD34) control vector. The cell expansion fold between the 4th day and the 10th day was compared ( figure 2 A). All constructs showed similar fold expansion. Survival at harvest was also comparable between cells transduced with different BCMA CAR constructs ( figure 2 B). Results from here were done using a 4-1BB based CAR.
[0152] Next, to assess the binding affinities of different scFvs to BCMA, cells transduced with different BCMA-CAR constructs were stained with BCMA-Fc fusion proteins. In a second step, cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies. Using the mean fluorescence intensity of tCD34 as a surrogate marker, the expression of the transgene on the cell surface was as follows image 3 ...
Embodiment 3
[0154] Example 3. Anti-tumor cell activity
[0155]To further evaluate the activity of different anti-BCMA CARs, the cytotoxicity and anticancer activity of different BCMA-CAR T cells were evaluated. The ability of different scFvs to elicit a functional response was explored by incubating CAR T cells at a 1:1 ratio for 24 h with different BCMA-expressing MM cancer cell lines (RPMI-8226, OPM-2, and U266). Lactate dehydrogenase (LDH) released into the culture medium was used as a biomarker of cytotoxicity and lysis. The result is as Figure 4 shown. Constructs #4, #6 and #11 showed the highest level of cytotoxic activity independent of cancer cell lines, while construct #3 showed very high cytotoxic activity against RPMI-8226 and OPM-2 tumor cells.
[0156] Likewise, interferon gamma secretion was used to measure anti-cancer cell activity. To this end, different BCMA-expressing cancer cell lines were co-cultured with Mock(tCD34) or BCMA-CAR-expressing T cells. After 24 ho...
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