Development Of Targeted Therapy For Tuberous Sclerosis Complex

Tuberous sclerosis complex（TSC）is an autosomal dominant inheritance muti-system benign tumor syndrome.It is caused by either TSC1 or TSC2 mutation.Loss of either TSC1 or TSC2 activates m TOR which leads to uncontrolled cell proliferation.Tumor lesions can lead to seizures,developmental delay,mental impairment and autism.Kidney tumor rupture and hemorrhage,renal failure,respiratory failure may endanger patient’s life.Although m TOR inhibition is the standard targeted therapy for TSC,the efficacy is limited due to its cell static nature.Novel therapeutics is thus warrented.Since m TOR activation induces aerobic glycolysis and glutamate dependent metabolic abnormalities,m TOR activated cells（TSC2-/-Pten-/-）are more sensitive to glycolysis inhibitor（3-Br PA）,glutaminase inhibitor（BPTES）,GSK3 beta inhibitors（Li Cl and Li2CO3）in vitro.Because systemic TSC1 or TSC2 gene knockout mice are embryonic lethal,we generated two brain specific TSC1 knockout mice: one in neuron(Synapsin-cre/TSC1^{Lox P/Lox P})and the other in glial cells(GFAP-cre/TSC1^{Lox P/Lox P}).3-Br PA and BPTES did not improve the survival of Synapsin-cre/TSC1^{Lox P/Lox P}mice.We will test the efficacy of aforementioned inhibitors in GFAP-cre/TSC1^{Lox P/Lox P}mice.Immune surveillance is the immune system that constantly monitors mutant cells in vivo,identifies and removes them.But at the same time,tumor cells escape the attack of immune system through a variety of mechanisms,so that they can generate and proliferate（immune escape）.The occurrence and prognosis of tumor depends on the interaction of immune surveillance and immune escape.T cells are an important component of anti-tumor immunity.It can be activated by combining of TCR and MHC-antigen complex on the surface of tumor cell（TCR pathway）.In addition to the TCR signal,the activation of T cell is regulated by co-stimulatory and co-suppression molecules,the latter is known as checkpoint molecules.After a breakthrough of the checkpoint molecule blockers（CTLA-4 fusion protein and monoclonal antibody）in the field of immunotherapy,immune checkpoint blockade therapy（especially PD1 / PD-L1 deprivation therapy）and the CAR-T therapy are becoming the two most important approaches of immunotherapy.Because of the diversity of tumor escape mechanisms,combination of immunotherapy and targeted therapies became a trend in cancer treatment.In this study,we designed and generated two Fc fusion proteins consisting of two immune checkpoint（LAG3 and PD1）extracellular domain: LAG3-PD1-Fc and PD1-LAG3-Fc fusion protein expression plasmids.We detected the successful expression of two Fc fusion proteins in HEK293 expression system,and analyzed the molecular size of both.Next,we will test these fusion proteins in vitro and in vivo for cancer immunotherapy.