T cell immunoglobulin-3 (Tim-3) is one of the immune checkpoint molecules.It is expressed in cells that secrete IFN-γ, including Th1 cells, dendritic cells, monocytes, CD8+ T cells and other lymphocytes.2,3 The corresponding ligand of Tim-3 is Galectin-9. .Tumour cells or tumour antigen-presenting cells continuously express the antigen Galectin-9 and bind to the Tim-3 of lymphocytes, resulting in the consumption of T cells in the tumour microenvironment, thus resulting in a decline in immune tolerance. One of the immune checkpoint inhibitors, Tim-3 antibody, can effectively prevent the binding of Tim-3 and Galectin-9, promote the proliferation and activation of T cells, and improve the apoptotic effect of T cells on tumour cells.4,8 According to the expression of Tim-3 and the clinicopathological correlation of renal cancer, the polymorphism of Tim-3 is related to the prognosis of renal cancer.9 We therefore chose to determine the role of Tim-3 single-chain antibody in renal tumour immunotherapy.
Currently, the first-line treatment for advanced renal cancer is a combination of two immune checkpoint inhibitors, ipilimumab (CTLA-4 antibody) and nivolumab (PD-1 antibody), or immune checkpoint inhibitors combined with targeted drugs.10 However, the treatment costs of these immune checkpoint inhibitors or target drugs are very high, thus limiting their use. In this study, the transformed lactic acid bacteria were used to express Tim-3scFv, and immunotherapy of advanced renal tumours was carried out via the oral route.
The gastrointestinal tract is the simplest and the most economical bioreactor for the generation and transformation of biological drugs, and probiotics such as lactic acid bacteria are good carriers for use in oral administration of immunotherapeutic drugs. In recent years, research on recombinant protein expression, gene therapy, and oral vaccines with lactic acid bacteria and bifidobacteria as host bacteria has been highly valued by scientific researchers worldwide.11–14 A study has managed to transform the endostatin gene in bifidobacteria to produce an endostatin transgenic bifidobacteria that yielded better anti-tumour effects.15 Research by Japanese scholars has also shown that the immunologically active interleukin 6 can be expressed by transforming lactic acid bacteria.16 We used the food-grade lactic acid bacteria intestinal controllable expression vector pLAN to express the CTB-Tim3scFv fusion gene, Lactococcus lactis lacking the LacF gene as the host bacteria, the auxotrophic complementation method, and lactose as the sole carbon source to select the CTB -Tim3scFv fusion gene-transformed bacteria. Positive monoclonal bacteria were picked backward, the culture was expanded, the corresponding plasmid was extracted, separated using 1.2% agarose gel electrophoresis and observed under the gel imager, and gene expression was determined through PCR. Results showed that the plasmid size and PCR band size of pLAN-0, pLAN-Tim3scFv, and pLAN-CTB-Tim3scFv were consistent with the known sequences.
To identify whether the transformed bacteria constructed in the previous experiment could correctly express the CTB-Tim3scFv fusion protein, we used nisin to induce the transformed bacteria NZ-CTB-Tim3scFv and NZ-Tim3scFv in vitro, with the empty vector bacteria NZ-Vector as a control, and performed immunoblotting identification. Results showed that the induced expression of the fusion protein CTB-Tim3scFv had a molecular weight of 53 KD, similar to the protein molecular weight estimated by the known amino acid sequence. The transformed bacteria that did not undergo induction did not express the protein. Western blot analysis indicated that both RAG and CACO2 cells expressed Tim3. Using these two cells as antigens, the immunological activity of the secreted protein of transformed bacteria was detected by ELISA, and results showed that the Nisin-induced and secreted Tim-3scFv and CTB-Tim-3scFv of transformed bacteria had immunological activity. In vitro analysis of proteins expressed by the three transformed bacteria that caused RAG cell apoptosis detection showed that the transformed bacteria in the NZ-CTB-Tim3scFv group could significantly inhibit the proliferation of RAG cells.
We determined how macromolecular substances could enter the blood circulation through the intestinal mucosal barrier to exert systemic functions. We aimed to allow Tim3scFv and cholera toxin B subunit (CTB) to be fused and expressed in lactic acid bacteria, and to add a furin-specific enzyme cleavage sequence between the target functional gene and CTB and a Flag tag. With the help of receptor-mediated endocytosis, the target protein was absorbed through the intestinal epithelium, and the enzyme interpretation released free Tim3scFv into the blood circulation to exert a systemic therapeutic effect. In vivo analysis of the effect of Tim3 antibody-transformed lactic acid bacteria on the treatment of transplanted tumours in mice showed that the NZ-CTB-Tim3scFv group had a better effect than that did the NZ-Tim3scFv and NZ-Vector groups. This indicated that CTB plays an important role in the passage of macromolecular substances through the intestinal mucosal barrier.
Results of mouse spleen lymphocyte subtypes detection using flow cytometry showed that the splenic CD3+, CD4+ cells, CD3+, and CD8a+ cells in the NZ-CTB-Tim3scFv group increased significantly compared with those in the NZ-Tim3scFv and NZ-Vector groups, suggesting that the transformed bacteria can promote the proliferation of splenic lymphocytes and the generation of Th cells in the spleen to enhance Th-mediated cellular immunity The expression of CD69 is often used as an indicator of T-cell activation. The proportion of CD3+ and CD69+ cells in the NZ-CTB-Tim3scFv group was significantly higher than that in the NZ-Vector group, indicating that feeding CTB-Tim-3ScFv-transforming Lactococcus lactis promotes the activation of mouse spleen lymphocytes.
Tumour size measurements, immunofluorescence detection of Ki67 to analyse the proliferation of mouse tumour cells, and CD31 detection to analyse the micro-angiogenesis of mouse tumours indicated that the CTB-Tim3scFv transformed bacteria have obvious therapeutic effects and can inhibit the increase in tumour volume, tumour cell proliferation, and inhibits tumour angiogenesis.