The Role Of Superkine Neo-2/15 In Anti-tumor Efficacy Of αMSLN-CAR-NK Cells For Pancreatic Cancer And Its Mechanisms

Pancreatic cancer is a leading cause of cancer death worldwide.It is estimated that 90%of pancreatic cancers originate from cells of the pancreatic ductal epithelium.Clinically,this cancer has a rapid disease progress and there are few treatment options resulting in poor prognosis.The morbidity and mortality rates of this cancer are almost similar(1:0.99),and it is thus called the " the king of cancer".The American Cancer Society reported that the total number of patients with pancreatic cancer in the United States is expected to reach64,050 in 2023,and the number of deaths is estimated to be 50,550.The overall 5-year survival rate of the cancer is only 12%.The latest statistics in China show that the incidence of pancreatic cancer has risen from the ninth position in 2016 to the seventh in 2022,and the mortality rate will be the sixth.Studies have shown that surgical resection of pancreatic cancer lesions can significantly improve the survival of patients,whereas only 5% to 22%of patients are suitable for tumor resection.Therefore,how to "downgrade" tumor lesions through chemotherapy,radiotherapy,and immunotherapy neoadjuvant methods in the treatment of pancreatic cancer in order to obtain surgery chance is a major scientific issue in the clinical research of pancreatic cancer.Although patients under neoadjuvant chemoradiotherapy could obtain more surgery opportunities and prolonged survival time,the current clinical benefits are still unsatisfactory.The progression of tumors is closely related to the function of the immune system.In healthy state,the immune system can sense the existence of tumors and eliminate tumor cells through various immune effector mechanisms.However,tumors have evolved escape mechanisms preventing immune system recognition and attack even induce immune suppression.Therefore,anti-tumor immune cells how to accurately identify tumors,overcome the immunosuppressive microenvironment and achieve precise killing are clinically important issues that urgent need to be solved.Adoptive cellular therapy(ACT)is a form of treatment that uses the cells of immune system to eliminate cancer by harvesting lymphocytes from patient,culturing and amplifying them in vitro and then infusing back to treat patients,also known as most prosperous immunotherapy.Among them,chimeric antigen receptor modified T-cell(CAR-T)has shown promising effects in the treatment of hematological malignancies,especially B-cell lymphoma.Multiple clinical trial data show that the complete tumor remission rate following CAR-T therapy is as high as 80% to 90%,with disease-free survival time of patient more than 5 years.However,there is no evidence to support the efficiency of CAR-T therapy in solid tumors,and some death cases have been reported in some clinical trials.With the advances in research,the limitations of CAR-T immunotherapy are gradually being revealed.First,CAR-T reinfusion will activate monocytes and macrophages,leading to the secretion and release of a large number of inflammatory cytokines such as interleukin-6(IL-6),IL-1βand tumor necrosis factor-α(TNF-α),triggering cytokine release syndrome(CRS)causing high fever,multiple organ failure and even death,which becomes one of the major challenge that limits the wider use of CAR-T cells to fight cancer.Secondly,T cells are activated in an MHC-dependent manner,and the use of allogeneic T cells can induce serious risks of graftversus-host disease(GVHD),as a result,the "Off-the-Shelf" cannot be realized.In addition,neurotoxicity,manufacturing time and costs also limit availability.Act as the first line of defense for immune surveillance and anti-tumor immunity,NK cells can exert strong target cells killing activity without antigen pre-sensitization and MHC-dependent recognition.Mature NK cells do not secrete IL-6 and have a short half-life.Compared with the "hug to die" killing mode of T cells,the "kiss to die" mode of NK cells has lower requirements for the affinity of the sc Fv on the target antigen,contributing to a wider range of antibody selection.The risk of life-threatening GVHD and CRS caused by CAR-NK is lower and CAR-NK cells would be able to efficiently eliminate tumors via both CAR-dependent and NK cells receptor-dependent mechanisms.Therefore,adoptive reinfusion of CAR-NK cells represents a promising approach for cell therapy-based treatment.However,the adoptive reinfusion of NK cells still faces many challenges in the treatment of solid tumors,which include the lack of effective strategies to improve the trafficking of CAR-NK cells into solid tumors,infiltrating into the tumor tissue and enhancement of the survival and proliferation in the immunosuppressive tumor microenvironment.In addition,it is also challenging to efficiently proliferate and activate CAR-NK cells in vitro for the treatment.At present,CAR-NK cells for ACT are expanded by IL-2 and IL-15 before reinfusion in order to obtain sufficient numbers of therapeutic cells with the desired genotype and phenotype.IL-2 could significantly increase the proportion of lymphokine-activated killer(LAK)in peripheral blood mononuclear cells(PBMC).IL-15 could escalate the killing effect of NK cells after adoptive reinfusion and up-regulate the expression of activating receptors NKG2 D and NKp46.However,short half-life in circulation,off-target effects and inherent pleiotropic functions of natural cytokines hinder their therapeutic use.On activated NK cells,IL-2 signals through a quaternary “high affinity” receptor complex consisting of IL-2,IL-2 receptor α(IL-2Rα,termed CD25),IL-2Rβ,and γc.NK cells express only a low density of IL-2Rβ and γc,and are therefore relatively insensitive to IL-2,but acquire sensitivity after CD25 expression which captures the cytokine and presents it to IL-2Rβ and γc.However,CD25 is mainly expressed in Treg cells,and the abundance of β and γ subunits in NK cell membrane is low.Therefore,even small doses of IL-2 may induce immune suppression.To avoid the serious side effects of IL-2 as well as bystander activation of other IL-2R positive cells like regulatory T cells(Treg)and enhance the stability of cytokines,“superkines” including H9,H9 T,N72D and Neo-2/15 were previously designed.Here,we first screened Neo-2/15 as NK cells agonist by comparing proliferation,expression of perforin(PFN)and granzyme B(Gr B)and anti-tumor activity of NK cell under different superkine stimulation respectively.Next,we constructed mesothelin(MSLN)targeting CAR-NK cells and Neo-2/15 expressing CAR-NK cells.Then we explored the anti-tumor effect of Neo-2/15 on anti-tumor efficiency using the pancreatic cancer model in vivo and in vitro.Neo-2/15 could improve the cytotoxic activities both in vivo and in vitro with an ability to restrict CAR-NK cells exhaustion and enhance tumor-bearing mice survival.The underlying mechanisms were further exploited.Part 1: Identification of the most suitable superkine for NK cells proliferation and activationWe first compared the expansion efficiency of natural cytokines IL-2,IL-15 and the superkines including H9,H9 T,N72D or Neo-2/15 on NK cells.It was uncovered that compared with natural cytokines and other superkines,NK-92 cells or primary NK cells from peripheral blood treated with Neo-2/15 had a superior expansion rate regardless of age,sex and immune status of donors.Neo-2/15 stimulation significantly increased the expression of phospho-STAT5,Ki67,perforin(PFN)and granzyme B(Gr B)in NK-92 cells and down-regulated expression of the inhibitory surface proteins molecules including LAG-3,TIGIT,TIM3,PD1 and NKG2 A.Moreover,compared with IL-2,IL-15,H9,and H9 T,the cytotoxicity of primary NK cells treated with Neo-2/15 was significantly enhanced in vitro.Collectively,these results suggested that Neo-2/15 could significantly promote expansion of NK-92 cells or primary NK cells from different donors,enhance NK cell activation rather than exhaustion resulting in the superior anti-tumor efficacy.Therefore,we selected the Neo-2/15 superkine as CAR-NK cells agonist to further investigate the role in anti-tumor effects and the underlying mechanisms of CAR-NK in pancreatic cancer.Part Ⅱ: The role of Neo-2/15 in the anti-tumor activity of αMSLNCAR-NK cellsCould Neo-2/15 enhance the anti-tumor efficacy of CAR-NK cells in pancreatic cancer model? We constructed the second-generation CAR targeting MSLN as well as Neo-2/15 sequence and packaged lentivirus respectively.The packaged virus infected NK-92 cells successively to generate αMSLN-CAR-NK(BBζ NK)cells and autocrine Neo-2/15αMSLN-CAR-NK(BBζ-Neo NK)cells.The cytotoxic activities of BBζ NK and BBζ-Neo NK cells were then compared.Cytotoxicity assay was performed in vitro and the results showed that BBζ-Neo NK cells exhibited stronger cytolytic activity in MSLN highexpression pancreatic cancer cells compared with BBζ-NK cells.BBζ-Neo NK cells specifically enhanced CD107 a,PFN and Gr B expression and cytokines secretion including TNF-α,IL-1β,IL-6 and IFN-γ.We established patient derived cancer organoid of pancreatic cancer and assessed BBζ-Neo NK cells-mediated specific cytolytic activity against organoid.We showed potentiated destruction of organoids by BBζ-Neo NK cells.Importantly,normal pancreatic tissue-derived organoids were not killed by BBζ-Neo NK cells,providing evidence of therapeutic safety.In both subcutaneous and orthotopic pancreatic cancer mouse models,BBζ-Neo NK could significantly enhance anti-tumor efficacy and prolong survival of tumor-bearing mice compared with other control groups.In addition,the anti-tumor efficacy of BBζ-Neo NK in vivo was further verified in human ovarian cancer mouse model.Compared to BBζ NK cells,BBζ-Neo NK cells decreased the expression of immune suppressive checkpoint molecules when co-cultured with As PC-1 cells.The proportion of BBζ-Neo NK cells in tumor tissue,peripheral blood and ascites was significantly increased after BBζ-Neo NK cells treatment.Collectively,these findings demonstrated that the adoptive reinfusion of BBζ-Neo NK cells decreased the expression of immune suppressive checkpoint molecules,increased the number of NK cells in intratumoral with enhanced antitumor efficacy.Part Ⅲ: The mechanism of Neo-2/15 in the anti-tumor activity ofαMSLN-CAR-NK cellsWhat is the mechanism by which Neo-2/15 enhances the tumor-killing efficacy ofαMSLN-CAR-NK cells? JAKs/STATs signaling,downstream of IL-2 and IL-15,are important for NK cell survival and activation.We found Neo-2/15 could induce stronger JAKs/STATs signal transduction,especially STAT5.After co-cultured with As PC-1 tumor cells,CAR-NK cells stimulated by Neo-2/15 could still up-regulate the expression of phosphor-STAT5.In addition,we found that Neo-2/15 also activated the Akt-mTOR signaling pathway.As the common effector,downstream of JAKs/STATs and Akt-mTOR signaling pathway,the c-Myc is a key molecule involved in the regulation of energy metabolism and cytotoxicity.In order to testify which is key factor for anti-tumor effect of BBζ NK cells under Neo-2/15 stimulation,we pretreated BBζ NK cells with Neo-2/15 and different inhibitor,target including STAT1,STAT3,STAT5,Akt,mTOR,c-Myc and STAT1/3/5 and then evaluated tumor killing potential of BBζ NK cells.Lysis effect was mostly maintained when STAT1 and STAT3 was respectively inhibited,whereas STAT5,AKT and mTOR inhibition significantly impaired tumor-killing function of BBζ NK cells.After inhibition of their downstream common molecule,c-Myc,the killing activity was almost relatively abolished,indicating that Neo-2/15 mainly maintains the cytotoxicity of BBζ NK cells through c-Myc.Transcriptome sequencing analysis showed that the metabolism profile of BBζ NK cells stimulated by Neo-2/15 was altered,and the associated metabolic pathways mainly included acetyl-Co A biosynthesis,pyruvate metabolism,glycolysis and tricarboxylic acid cycle(TCA cycle).In addition,the expression of key enzymes and mitochondrial respiratory chain complexes was up-regulated.Metabolome data revealed that differential metabolites were mainly enriched in glucose metabolism and TCA cycle.We further demonstrated that Neo-2/15 stimulation significantly increased the ATP production,glucose transporter1(Glut1)and amino acid transporter(ASCT2 / SNAT1)expression in BBζ NK cells even co-cultured with pancreatic cancer cells.Neo-2/15 stimulation significantly increased the oxidative phosphorylation(OXPHOS)of BBζ NK cells.Pretreatment with c-Myc inhibitor abolished these effects of Neo-2/15.These results indicated that Neo-2/15 might increased the ability of nutrient intake of BBζ NK cells by activating c-Myc to maintain their OXPHOS and improve energy production,leading to effective tumor-killing activity.After co-culture with tumor cells,Neo-2/15 can still maintain the energy production of BBζ NK cells.Mitochondria are the TCA cycle and the place where OXPHOS occurs,we speculated that Neo-2/15 may affect the mitochondrial homeostasis of BBζ NK cells after co-culture with tumor cells.When co-cultured BBζ NK cells and pancreatic cancer cells,the results showed that the mitochondrial activity and morphology of BBζ NK cells stimulated by Neo-2/15 remained stable,and the mitochondrial membrane potential did not decrease significantly compared with that before co-culture.Electron microscopy results also showed that the mitochondrial membranes of BBζ NK cells stimulated by Neo-2/15 remained intact without mitochondrial fragmentation.In addition,Neo-2/15 stimulation up-regulated the expression of anti-apoptotic molecules Bcl-2 and Bcl-xl in BBζ NK cells.Mitochondrial fragmentation was increased and tumor-killing activity was reduced in Neo-2/15-expanded BBζ NK cells when c-Myc was blocked.These results suggest that Neo-2/15 could maintain mitochondrial stability of BBζ NK cells through c-Myc in tumor environment,which may resist mitochondrial apoptosis.In summary,the biggest limitation to the success of adoptive immune cell therapy in solid tumors lies in the immunosuppressive tumor microenvironment,especially due to hypoxia,high lactic acid but few nutrients such as amino acids and glucose.CAR-NK cells cannot proliferate and activate normally as it lacks nutrients and energy.Being the most suitable agonist for NK cells screened in this study,Neo-2/15,functionally,could promote the survival and activation of adoptively reinfused BBζ NK cells in the tumor microenvironment,and enhance its anti-tumor activity in vivo and in vitro.Mechanismly,the expression of c-Myc in BBζ NK cells might promote its nutrients intake in tumor environment,increase OXPHOS and energy supply,which help to maintain the morphology and function of mitochondria in BBζ NK cells.As a result,Neo-2/15 induced higher targeted anti-tumor efficacy.All in all,the Neo-2/15-mediated anti-tumor mechanism was preliminarily explored from the perspective of metabolic adaptability in the tumor microenvironment to provide a new perspective for developing CAR-NK-based anti-solid tumor cell therapy.