Abstract
CD47 is a cell surface glycoprotein molecule, belonging to the immunoglobulin superfamily, binding to various proteins including integrins, thrombospondin-1, and signal regulatory protein α (SIRPα). CD47 is an important tumor antigen for the development and progression of various cancers. This study designed the chimeric antigen receptor T-cell (CAR-T) to bind to the CD47 to inhibit the expression of CD47. We used the complementarity-determining regions (CDRs) of the B6H12 mouse antibody grafted onto the IgG1 framework to create the humanized single-chain variable fragment (scFv) with linker (G4S)x3. scFv was used to design the chimeric antigen receptor with the structure CD8signal-CD47scFv-CD8a hinge-CD4TM-CD28-41BB-CD3ζ, which was then transformed into T lymphocytes by the lentivirus to create third generation of CAR-T. Results revealed that the new CAR-T cells efficiently killed A549 cancer cells. CAR-T inhibited the expression of genes involved in metastasis and invasion of cells A549 including beta actin, calreticulin, and cyclooxygenase 2 at mRNA levels.
Highlights
Current experimental evidence suggests an important role of immunoglobulins in the formation of cancerous tumors
Humanized anti-CD47 scFv was attached to phagemid pHEN2; the recombinant phagemid was transferred to E. coli TG1 strain, using helper phage to form M13 phage carrying CD47 scFv attached to the phage’s gIII
Our results showed that CD47 Chimeric Antigen Receptor (CAR)-T cells were toxic to the A549 cell line in the MTT assay
Summary
Current experimental evidence suggests an important role of immunoglobulins in the formation of cancerous tumors. In addition to functionally interacting with proteins such as integrins and thrombospondin-1, CD47 is considered a macrophage immune checkpoint when interacting with signal regulatory protein α (SIRPα) [7]. This activates SIRPα, phosphorylates the immunoreceptor tyrosine-based inhibition motif (ITIM), recruits SHP-1 phosphatases to the cell membrane, and limits accumulating myosin at the surface of the cell, which in turn prevent phagocytosis. The expression of CD47 is related to tumor growth and is proposed to be a critical clinical prognostic factor and mortality [6, 8]. The presence of CD47 has been found in patients with metastasis, immune evasion, or cell migration [8, 9]
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