Compositions and methods for selective protein expression
A protein structure, protein technology, applied in the field of compositions and methods for selective protein expression
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Embodiment 1
[1136] Example 1: Materials and methods for Examples 1-13.
[1137] construct
[1138] The following constructs shown in Table 22 were used in Examples 1-13.
[1139] Note that the invention encompasses both orientations of heavy and light chains.
[1140] Table 22. Sequences of various constructs (signal peptide used in bold; furin cleavage site underlined)
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[1157] general method
[1158] The following general materials and methods were used for the assays described in Examples 1-13.
[1159] CAR construct generation
[1160]The scFv used in the CAR construct evaluated in Example 3 was synthesized independently based on the anti-CD19 scFv sequence described above. The scFv was cloned into a CD8 hinge region together with a 4-1BB molecule and a CD3δ molecule in the heavy chain to li...
Embodiment 2
[1173] Example 2: Furin is the most highly expressed proprotein convertase in primary human T cells
[1174] Literature sources show that furin (FUR; PACE; PCSK3; SPC1 ) has a broad expression profile (Seidah et al., Nature Reviews Drug Discovery 11, 367-383 (May 2012)). Expression of proprotein convertase family members was analyzed by qRT-PCR. RNA was harvested from normal donor T cells on days 0, 4, and 11 after stimulation with anti-CD3 / anti-CD28 activation beads. During the culture period, an additional group was supplemented with 100 U / mL IL-2 and RNA was harvested on day 11. These data show that after activation with anti-CD3 / anti-CD28 beads, furin mRNA is more highly expressed than other members of the proprotein convertase family ( figure 1 ).
Embodiment 3
[1175] Example 3: CD19 CAR Expression Controlled by Compound Addition
[1176] Furin degron domain (FKBP) fused to anti-CD19 scFv CAR construct in Jurkat T cells FD , ERα FD or DHFR FD ) transduction followed by treatment with the corresponding compound. FKBP were treated with 1 μM Shield1 FD transduced cells. ERα was treated with 1 μM bazedoxifene FD transduced cells. DHFR was treated with 1 mM TMP FD transduced cells. In the presence of the compound, the expression of anti-CD19 scFv was inducible ( figure 2 ). Black = UTD; gray = construct, no compound; white = construct, with compound. These data show that multiple degron domains lead to strict compound-dependent regulation of CAR 19 expression when combined with the furin cleavage domain.
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