PTTRAP1 bispecific fusion protein and application thereof
A fusion protein, bispecific technology, applied in the field of PTTRAP1 bispecific fusion protein, can solve the problem of patient response, reduce the number, improve the effect of anti-tumor immunotherapy, and improve the tumor microenvironment.
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Embodiment 1
[0049] Embodiment 1, construction of objective recombinant vector
[0050] This example utilizes the existing AbVec2.0-IGHG1 vector (addgene, #80795, Tiller et al., JImmunol Methods. 2008 Jan 1; 329(1-2): 1; it is specific for secretory immunoglobulins in mammalian cells The protein heavy chain is expressed, and the nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing), the extracellular domain sequence of programmed death receptor 1 (PD-1) (named PD1, Pubmed, Gene ID: 817329 (programmed death receptor 1), PDCD1 programmed cell death 1 [Homo sapiens (human) Gene ID: 5133, updated on 20-Jan-2019, its nucleotide sequence is shown in SEQ ID NO: 2 in the sequence listing, The corresponding amino acid sequence is shown in SEQ ID NO: 3 in the sequence table), the extracellular domain sequence of human transforming growth factor (TGF-β) receptor II (named TGFBRII, Pubmed, Gene ID: 100329004 (human transforming growth factor (TGF-β) receptor II), its nucleotide sequen...
Embodiment 2
[0077] Embodiment 2, target plasmid expression, purification and verification
[0078] In this example, 293T cells were used to transiently transfect the four target plasmids obtained in Example 1 above, and AktaAvant 25-Protein A Column (GE-Instructions 71-7002-00 AR HiTrapTM Protein A HP) was used to purify the secreted protein after transfection of 293T cells. supernatant. The purified four fusion proteins TGFBRII-PD1-Fc, TGFBRII-Fc-PD1, TGFBRII-Fc, PD1-Fc were verified by Western Blotting and Coomassie Brilliant Blue assay. Specifically include the following steps:
[0079] 2.1. Transfection and purification of the target plasmid
[0080] 1) Transient transfection of the target plasmid into 293T cells
[0081] The 293T cells in good growth state were spread to 10 cm dishes (10 dishes) in advance, so that the cell density reached 80%-90% after 48 hours of culture. The 293T cells were transfected according to the instructions of the jet PRIME Polyplus transfection kit (p...
Embodiment 3
[0088] Embodiment 3, in vitro binding function detection
[0089] This example uses Elisa binding experiments, flow blocking, Fortebio detection, and Western Blotting experiments to verify that the PTTRAP1 bispecific fusion protein can effectively and specifically bind PD-L1 and TGF-β, thereby blocking PD-1 / PD -L1 and TGF-β-Smad canonical pathways. Specifically include the following steps:
[0090] 3.1, Elisa binding detection
[0091] 1), PD-L1-Fc-coated microtiter plate: Dilute PD-L1-Fc (purchased from Sangon Bioengineering Co., Ltd.) with 0.05 MPH9, carbonate coating buffer to 1-10 μg / ml, in enzyme Add 0.1ml to each well of the target plate, and coat overnight at 4°C; the next day, discard the solution in the well, and wash 3 times with washing buffer (10mM PBS buffer solution with pH 7.0-7.4), 3 minutes each time.
[0092] 2) Adding samples: Add 0.1ml serially diluted (0.05M PH9, carbonate coating buffer dilution) sample to be tested (TGFBRII-PD1-Fc or TGFBRII-Fc-PD1) t...
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