Studies On Combination Of The LAG-3 Antibody And CGAMP Used For The Cancer Treatment

Immune checkpoint molecules are key molecules that regulate the homeostasis between activation and inhibition of immune responses.Under normal physiological conditions,suppressive immune checkpoints play an important role in autotolerance and autoimmune defense.Immune checkpoint therapy is a therapeutic method to kill tumor cells by regulating the activity of T cells through a series of pathways such as co-inhibition or co-stimulation signals.In recent years,immunotherapy targeting tumor microenvironment immunosuppression has achieved remarkable efficacy in the treatment of cancer,among which the most representative examples are the PD-1/PD-L1 immune checkpoint inhibitors.However,only 25%-30%of tumors use the PD-1/PD-L1 pathway to achieve immune escape.Following more discovery of the immune checkpoint proteins,LAG-3 has become a new generation of tumor immunotherapy target after PD-1 and has a great application prospect.Currently,studies on the c GAS-STING pathway mainly focus on tumor cells and natural immune cells,and it has been found that the c GAS-STING pathway plays an important role in tumor immunity,cell senescence,inflammatory disease and viral infection,etc.However,little is known about the changes of c GAS-STING pathway in T cells,especially in tumor microenvironment.As an important cytoplasmic DNA receptor,c GAS plays a monitoring role in the intake of heterologous DNA in cells such as bacteria,viruses and tumors,and catalyzes the generation of the second messenger c GAMP.As a natural ligand of STING,c GAMP activates STING to form the dimer,and induces IFN-βproduction by recruiting TBK1 to activate IRF3.Meanwhile,how the immune checkpoint LAG-3 is expressed and regulated during the activation of c GAS-STING innate immune pathway has not been reported.A number of immune-related clinical studies have selected combination therapy with LAG-3 target drugs,so it is particularly important to understand LAG-3 inhibition signaling pathway.However,so far,the specific mechanism of c GAS-STING pathway in the anti-tumor effect of LAG-3antibody remains unclear,and the combined effect of c GAMP and LAG-3 antibody remains unknown.In this study,a high purity human PD-1 antibody and a LAG-3 antibody were prepared using the Expi293F^TMeukaryotic expression system.The anti-tumor mechanism of LAG-3 antibody was studied by the combination of immune checkpoint LAG-3 antibody and immunomodulator c GAMP used to treat a mouse colon cancer model.The data are summarized as follows:（1）The Expi293F^TMeukaryotic expression system was used to successfully produce a high purity human PD-1 antibody and a LAG-3 antibody.In vivo experiments,it was found that the combination of PD-1 antibody and LAG-3 antibody could significantly inhibit the growth of tumor in mice by subcutaneous injection.（2）A prokaryotic system was used to express the c GAS protein which was used to synthesize c GAMP in vitro.The biological activity of c GAMP was verified by mass spectrometry and stimulation of BJ cells.Through the combined treatment of c GAMP and LAG-3 antibody in a mouse colon cancer model,we found that the combined treatment of c GAMP and the LAG-3 antibody has the better effect on mouse tumors compared to either c GAMP or the LAG3 antibody treated alone.These data suggest that,similar to the c GAS-STING innate immune pathway that plays a key role in anti-tumor immunity against PD-1,the activation of c GAS-STING pathway may also promote the anti-tumor effect of LAG-3 antibody.（3）Our data showed that activation of the c GAS-STING pathway can indeed increase the expression of LAG-3 in the cells stimulated by HT-DNA,Poly（I:C）,c GAMP and IFN-β,while the LAG-3 expression was decreased or not expressed in the corresponding knockout L929 and MC38 cells with c GAS^-/-,STING^-/-,or IRF3^-/-gene knockout genotype,indicating that activation of the c GAS-STING pathway is particularly important for tumor immunotherapy related to LAG-3 antibodies.This also explains why the combination of LAG-3 antibody and c GAMP has a better efficacy.The data of this study provides an important theoretical and experimental basis for the combined use of c GAMP and LAG-3 antibody in the treatment of solid tumors,and lay a cornerstone for its clinical application.It also provides a research basis for the regulation of LAG-3 immune signal pathway in the future.