Anti-tumor Effect Of Nanobody-specific Bispecific T Cell Engager CD3-FAP/nanoBiTE

BACKGROUND BiTE is a kind of bispecific T cell engager which uses T cells as the effector cells and connects with T cell and target cells.BiTE can acticate cytotoxic T cells to kill target cells,which is a novel type of antibody with great potential.BiTE has the advantages of high specificity and affinity,which make a good application prospect in tumor targeted therapy.While it also has many problems such as large molecular weight of scFv,poor tissue penetration,poor solubility,poor stability and low expression.Therefore,it is an urgent problem to make a more potent BiTE with stronger antibody and more specific target.OBJECTIVE To construct a novel bispecific T cell engager CD3-FAP/nanoBiTE based on nanobody using genetic engineering technology andidentify its biological function.To explore whether it can induce activated T cells to target tumor cells and exert anti-tumor effect,which provides a new strategy for BiTE tumor cell immune targeted therapy.METHODS In order to construct a nanobody-based novel bispecific T cell engager CD3-FAP/nanoBiTE,we selected affinity-matured anti-human FAP Nanobody sequences and anti-human CD3 Nanobody sequences via a flexible flexible Linker(-GGGGSGGGGSGGGGS-).After ligation and PCR amplification,the vector pET-30a(+)was ligated to transform the product into E.coli for optimal expression,and verified for identity by Western blot analysis.Flow cytometry was used to test the combination efficiency of target cell or T cells.The activated marker C69 and CD25 after modification of CD3-FAP/nanoBiTEwere detected by flow cytometry.Proliferation level of T cells was detected by flow cytometry.While the expression level of cytokine(IL-2?TNF?IFN-?)was detected by ELISA after CD3-FAP/nanoBiTE stimulated T cells activated by target cells.The killing-efficiency ofHepG2-FAPwas detected by flow cytometry when CD3-FAP/nanoBiTE T cells were activated by target cells.Xenograft liver carcinoma NOD/SCID mouse model was used to clarify the antitumor activity of CD3-FAP/nanoBiTE.RESULTS The anti-FAP nanobody and the anti-CD3 nanobody gene were fused together by PCR to obtain the novel bispecific T cell engager CD3-FAP/nanoBiTE gene based on nanobody.The linker peptide sequence was(Gly4Ser)3 and 6x his was introduced at the C-ter??us of the peptide chain to facilitate purification.The sequence of the novel CD3-FAP/nanoBiTE gene was verified by sequencing and then ligated into the expression vector pET-30a(+)to transform E.coli strains to induce expression.The supernatant was collected after ultrasonic cell disruption and separated by Ni affinity chromatography column.With a purity of more than 90%,it was detected that the CD3-FAP/nanoBiTE protein was expressed in the form of insoluble inclusion bodies,and a clear peak was observed in the elution curve between 50 and 100mM imidazole.Fractions were collected and the molecular weight of the expressed protein was identified by SDS-PAGE and Western blotting as 42 KD,which was in agreement with expectations.CD3-FAP/nanoBiTE specifically binds activated T cells and FAP-expressing target cells(HepG2-FAP).Under the stimulation of target cells,CD3-FAP/nanoTE showed significantly better stimulation of T cell activation than the control group.In the presence of target cell HepG2-FAP,the proliferation factor of activated T cells modified with CD3-FAP/nanoBiTE was higher than that of unmodified T cells,and the difference was statistically significant.The release of cytokines(IL-2,TNF,IFN-?)from CD3-FAP/nanoBiTE-modified T cells was higher than that of unmodified activated T cells,and the difference was statistically significant.Activation of CD3-FAP/nanoBiTE-modified T cells can target HepG2-FAP cells in vitro,and the higher the effect-target ratio,the better the killing effect,while the negative target cell HepG2 has no obvious killing advantage.And can effectively inhibit tumor growth in vivo and prolong the survival time of tumor-bearing mice.CONCLUSION The CD3-FAP/nanoBiTE,a novel bispecific T-cell engager based on nanobodies,demonstrated a good anti-tumor effect in vivo and in vitro.It provides a new strategy for tumor targeting therapy.