The Natural Killer Cells Engineered With Anti-GPC3 Chimeric Antigen Receptors In Immunotherapy For Hepatocellular Carcinoma

Hepatocellular carcinoma?HCC?is the second leading cause of cancer deaths in the world.The incidence cases and number of deaths of HCC both account for more than 50%of the total all over the world respectively,while the mortality rate is in the third place.Although the HCC treatment protocols tend to be diversified and combined,it is difficult to achieve further improvement in the long-term survival of HCC patients in the last 10years.Therefore,it is urgent to find a new,effective and safe treatment protocol.In recent years,chimeric antigen receptor?CAR?-modified T cells is one of the tumor immunotherapies that attract the most attention.This technology allows T cells to specifically target tumor surface antigens by transfecting chimeric antigen receptors in T cells,while the intracellular domain provides the first and second signals to activate T cells,and it is a new generation of cell therapy with targeting ability,which overcomes the drawback of insufficient infiltrating cells and maintains the killing activity.This technique has shown excellent therapeutic effects in the treatment of various tumors,especially hematological tumors.At the same time,safety has become a major problem that affects the clinical applications of CAR-T therapy:cytokine storm,off-target effects,severe allergic reactions and neurotoxicity,and the potential safety hazards posed by viral vectors have severely limited further applications of CAR-T therapy.NK cells are important effector cells of the innate immune system.NK cells do not require pre-sensitization by antigen to perform extremely high cell killing activity and are not MHC restricted;mature NK cells do not secrete IL-6,and the physiological cycle is short and does not cause GVHD response.Studies in bone marrow transplantation have shown that hematological tumors can be effectively recognized and killed by allogeneic NK cells,which can significantly increase disease control and reduce recurrence rate.However,many tumor cells often acquire immune evasion ability by expressing non-classical HLA class I molecules,expressing immunosuppressive ligands,or secreting immunosuppressive factors,etc.,thus the clinical efficacy of NK cells adoptive therapy is poor.Therefore,CAR modified NK cells,which show target killing effect on tumor cells,has become an important strategy to improve the efficacy of NK cells adoptive therapy.On this basis,in this study we have studied the TCGA big data,identified the HCC-specific antigen GPC3,and obtained high-affinity and high-specificity antibodies of GPC3 by phage antibody display technology;and further optimized the CAR structure based on the activation signal of NK cells,and then the CAR structure was selected and used on NK cells.Finally,the key technologies in the clinical application of CAR-NK were explored,and a liposome system for increasing NK cell expansion and cytotoxic activity was established,and a PDX model based on circulating tumor cells of HCC patients was established.Part ?:Screening and Expression of HCC Tumor Antigens and Screening of AntibodiesFirst,we performed a difference analysis of HCC samples in the TCGA database.The results showed that several genes were significantly up-regulated in liver cancer,of which GPC3 was the most significant one.Combined with the data in the Human Protein Atlas database,GPC3 is hardly expressed in other normal tissues,suggesting that GPC3 is a good target for HCC treatment and hardly causes damage to other normal tissues during targeted therapy.Then we analyzed the physicochemical properties of GPC3,and found that the GPC3 carboxy-terminal subunits have more?-turns and random curls,which are suitable for immunizing animals as antigens.Therefore,we expressed and purified the GPC3 carboxy-terminal subunit?GPC3 in the CHO system.After the phage antibody display libraries were panned,we obtained three kinds of antibodies QX6.3F,QX1.11A and QX2.6C,in which antibody QX6.3F has the highest affinity and good specificity,and its K_d is approximately 20 nM.Subsequently,we examined the expression of GPC3 in HCC cell lines.After big data analysis and Western-Blot verification,we confirmed HepG2 and Hep3B as GPC3 positive cells,and SMMC-7721 and QYG-7703 as GPC3 negative cells.In addition,we established the corresponding GPC3 knockout cell line or overexpressing cell line and the corresponding stable cell line expressing Luciferase by FACS sorting and CRISPR/Cas9system,respectively.And their growth characteristics were studied.Part ?:Optimization of CAR Structure in Natural Killer CellsThe chimeric antigen receptor structure in CAR-NK currently used in clinical practice mainly follows the structure of second and third generation CAR-T cells,and is not optimized for the activation signal unique to NK cells.Therefore,we respectively optimized the intracellular and transmembrane domains of CAR in this section,and separately verified the biological effects of CARs with GC33,YP7 and QX6.3F antibodies as scFv-terminals.In the optimization of intracellular domain,we selected the active domains of CD137?4-1BB?,2B4,DNAM1,CD3?,DAP10,and DAP12 in tandem as the intracellular domain of CAR(CAR^Max).After establishment,its killing efficiency was compared with that of T-CAR.The results showed that CAR^Max significantly increased the cytotoxic activity of NK-92 against HepG2,Hep3B and SMMC-7721 and QYG-7703 after overexpression of GPC3.Then the above domains were deleted one by one,and the results showed that the CD137 domain had little effect on the activity of CAR and could be deleted.In the optimization of transmembrane domain,we selected the biologically active NKp44 and NKG2D transmembrane domain structures.The NKp44 transmembrane domain is rich in positively charged lysine and binds to the negatively charged DAP12 in the cytoplasmic domain,while NKG2D binds to the DAP10 molecule to deliver an activation signal.Different CARs were constructed to detect their cytotoxic activity against HepG2.The results showed that the NKp44 transmembrane domain could further enhance the killing activity of CAR-NK.The NKp44 transmembrane domain can mediate DAP12signal,so we explored whether DAP12 structure can be removed in the intracellular domain.The results indicated that there is no significant effect on biological function of CAR whether the intracellular domain contains DAP12 structure after replacement of NKp44 in the transmembrane domain.Finally,we simplified the CAR structure on the basis of the strategy of the Michel Sadelain team at the Memorial Sloan-Kettering Cancer Center.The results indicated that the ITAM motif close to the transmembrane domain can further increase the NK cells activation ratio and IFN-?secretion.In addition,the activation of three ITAM non-NK cells in CD3?is necessary,and the first ITAM retained is sufficient to maintain the physiological function of CAR.The best CAR structure in NK cells has thus been figured out:scFv-NKP44^TM-CD3?^?1-2B4-DNAM1-DAP10.We investigated the advantages of this structure compared to T-CAR in signal transduction.The results indicated that the phosphorylation level of kinase Fyn is significantly up-regulated in CAR^opti compared to that of T-CAR,which in turn mediated the activiation of PLC-?1 and PLC-?2;increased Syk phosphorylation level,mediated increased activation of Vav and ERK1/2;promoted the activation of IKK?/?,then activated I?B,thereby promoted phosphorylation of p65;slightly promoted the activation of PI-3K p85 subunit,which in turn mediated the increased activation of AKT.We verified the difference in killing activity of HepG2 by third-party GPC3 antibodies,antibody GC33,YP7 and self-developed antibody QX6.3F.The results showed that there was no significant difference in the killing activity of CAR-NK constructed on the basis of QX6.3F and GC33.In addition,in order to further improve safety,AP1903-induced suicide gene was transduced into the cells,which will induce significant apoptosis in the presence of AP1903 even at a concentration of pmol/L,and is safe even in high dose in mice?7.5×10⁷cell for one mouse?.Part ?:Exploring New Protocols for Improving NK Cells Proliferation and Killing Ability and Establishing PDX Model Based on Circulating Tumor CellsIncreasing the viability of reinfused cells and maintaining it in the body is a major problem in adoptive immunotherapy.After CAR-NK cells infused in the body,the surrounding environment lacks cytokines,and therefore their activity are difficult to maintain.The most effective way to maintain CAR-NK cells activity at low concentrations of cytokines is to use Feeder cells.However,the current common feeder cell K562 is a tumor cell that grows in suspension,and has a potential tumor-forming risk once infused in the human body.On this basis,we developed Feeder liposomes based on NK's K562Feeder cells.We incorporated ganglioside G_M2 into liposomes and attached to their surface using streptavidin-conjugated G_M2 single-chain antibodies to form mount centers.Feeder liposomes were then formed by co-incubation of biotin-conjugated IL-21,IL-15 and4-1BBL with liposomes.The liposome can increase NK cells proliferation and killing activity in the NK culture system,and does not require isolation before injection.Another major problem in the clinical application of CAR-NK cells is how to choose the right patient.Therefore,we have established a PDX model based on temperature-sensitive biogel suspension culture of HCC circulating tumor cells,and then implanted in vivo.Through this culture system,circulating tumor cells of HCC patients can be expanded in vitro and form polycell clone,which can be transplanted under the skin and into the liver in nude mice to form tumors,and establish a xenograft model.Combining the above three studies,we have established a complete CAR-NK system targeting GPC3 from tumor antigen screening and antibody development,and constructed a NK-specific CAR structure based on the biological characteristics of NK cells,and finally transfected suicide gene to guarantees clinical safety,constructed Feeder liposome to enhance NK cells activity in vivo,established HCC-PDX model for screening indication,and formed a complete CAR-NK cells therapy system.