| BackgroundGastric cancer(GC)is the fifth most frequently diagnosed cancer and the third leading cause of death from cancer in the world,with a high estimated incidence and mortality rates of 50%in China.Many GC patients present with inoperable or metastatic disease during their first visit and have an inauspicious prognosis.Despite the development of multimodality therapies such as surgery,chemotherapy and radiation therapy,the mortality rates of metastatic GC remain dismal.Human epidermal growth factor receptor 2(HER2)is a well-recognized mediator of the cancerogenic process and is dysregulated in a wide range of solid tumors.The reported rates of HER2 overexpression in GC patients range from 9%to 23%.The ToGA study,a randomized,prospective,multicenter phase III trial,established trastuzumab as the first targeted therapeutic for patients with HER2-positive advanced or metastatic GC.Nevertheless,benefits from trastuzumab remain unsatisfactory in survival and some drawbacks are arising,such as cost,need for continuous treatment,increased risk of cardiac toxicity,what’s more,the development of resistance.Therefore,novel and powerful therapeutic strategies for treatment of GC are urgently needed,which prompted us to study active immunotherapeutic strategies targeting HER2.Among the immunotherapies that have been incorporated into cancer treatment,adoptive transfer of chimeric antigen receptor(CAR)modified T cells has been at the forefront for the last decade.Introduction of CAR into T cells enables these effector cells to recognize tumor associated antigen(TAA)via the single-chain variable fragment(scFv)and activate T cells through the cytoplasmic signaling domains,releasing perforin,granzyme and various cytokines to exert potent anti-tumor effect.Thus,CAR T cells function in non-MHC restricted manner,which cleverly combine the potent tumor-killing capacity of cytotoxic T cells and the specific antigen recognition of antibody together.The CAR T cells approach first showed compelling success in eradicating hematological tumors,and later also particular relevance and encouraging results for solid tumors in both preclinical studies and early phase clinical trials.Compared with monoclonal antibody therapy,CAR T cells approach is more effective in generating durable and also precise tumor response for solid tumors with lower risk of resistance.HER2 has been targeted by CAR T cells therapy in many tumor models,including breast cancer,ovarian cancer,biliary tract cancers and pancreatic cancer,medulloblastoma,glioblastoma,and osteosarcoma;some of the HER2 CAR T cells therapy have been evaluated in phase Ⅰ/Ⅱ clinical trials.However,this novel therapy for GC has not yet been broadly evaluated.AimsWe aim to construct a HER2 specific sencond-generation chA21 scFv-4-1BBz CAR and transduce human primary T cells;evaluate its anti-tumor effect towards HER2-overexxpressing gastric cancer in vitro and in vivo,attempting to providing a new immunotherapeutic strategies for advanced gastric cancer.Methods1.Detect the expression levels of HER2 on surface of human gastric cancer cell lines by flow cytometry.2.Construct the CD8a leader-chA21 scFv-CD8a hinge-4-1BB-CD3z plasmid by overlap extension PCR and then subcloned into pSin lentiviral backbone to create the pSin-GFP-chA21-4-1BBz plasmid;produce lentiviral particles by transfecting 293 T cells.3.Transduce activated human T lymphocytes by lentiviral vectors to generate chA21-4-1BBz CAR T cells.Analyze the transduction efficiency of CAR on T cells by flow cytometry.4.Detect multiple cytokines(IL-2、TNF-α、INF-γ、IL-4、IL-6、IL-10、IL-17A)release of chA21-4-1BBz CAR T cells when cocultured with tumor cells expressing different levels of HER2 by flow cytometry using the Multi-Analyte Flow Assay Kit.5.Set 1:1,3:1,10:1,30:1 different effector:target ratios for coculture and evaluate the killing effect of chA21-4-1BBz CAR T cells toward tumor cells expressing different levels of HER2 by LDH release assays.6.Establish the subcutaneous model of human gastric cancer by subcutaneously inoculating NOD-SICD female mice with NCI-N87 tumor cells on the left flank and MKN-28 tumor cells on the right flank at the same time;monitor the effect of chA21-4-1BBz CAR T cells administration for tumor growth by vernier calipers measurement.7.30 days after T cells administration,peripheral blood from mice was collected to detect the survival of human T cells in the peripheral mice circulation by flow cytometry.8.Collect tumor samples from each group and evaluate the infiltration and accumulation of human T cells in tumor sites by immunohistochemistry analysis.9.Establish the peritoneal metastasis model of human gastric cancer,by intraperitoneally injecting NOD-SICD female mice with NCI-N87 tumor cells;monitor the effect of chA21-4-1BBz CAR T cells administration for tumor progression and mice survival.Results1.Human gastric cancer cell line NCI-N87 expressed a high level of HER2,HGC-27,MKN-45 and BGC-823 expressed a moderate level of HER2,however a very low level of HER2 expression was detected on MKN-28 cells;the HER2 overexpressing ovarian cancer cell line SKOV3 was used as a known positive comparison.2.Successfully generated chA21-4-1BBz CAR T cells with CAR expression higher than 50%;both chA21-4-1BBz CAR T cells and untransduced T cells expand well in vitro.3.When stimulated with HER2 overexpressing tumor cell lines SKOV3 and NCI-N87,chA21-4-1BBz CART cells specifically secreted great amounts of IFN-y,IL-2 and TNF-a cytokines,while little to no IL-4,IL-6,IL-10,IL-17A,suggesting a preferential Thl-cytokine response.However,chA21-4-1BBz CAR T cells secreted much lower levels of cytokines when incubated with HGC-27,MKN-45,BGC-823 cells;and almost undetectable activity when in coculture with MKN-28 cells.The amount of cytokines secreted by chA21-4-1BBz CAR T cells positively correlated with the level of HER2 surface expression by target tumor cells.4.The chA21-4-1BBz CAR T cells exhibited direct and efficient lyse toward SKOV3 and NCI-N87 cells,decreased lyse toward MKN-45 cells,but no distinct lyse toward MKN-28 cells compared with the baseline killing activity of UT T cells.The killing efficiency was increased with higher effector:target cell ratio.5.Successfully established the subcutaneous model of human gastric cance.NCI-N87 xenograft treated with chA21-4-1BBz CAR T cells experienced rapid tumor regression which was significantly better than UT T cells(P<0.05).By contrast,MKN-28 tumor receiving whatever chA21-4-1BBz CAR or UT T cells administration both grew progressively(P>0.05).Mice bearing NCI-N87 tumor treated with UT T cells had heavier tumor burden compared with mice treated with chA21-4-1BBz CAR T cells(648 ± 18 mg vs 268 ± 50 mg,P<0.001).While the tumor weight in MKN-28 xenograft mice treated with CAR T cells or UT T cells was not significantly different(718 ± 79 mg vs 678 ± 72 mg,P>0.05).6.30 days after T cell administration,human CD3+ T cell were detected in blood from mice treated with chA21-4-1BBz CAR T cells but not in the UT T cells treated group;obvious accumulation of human T cells was detected in regressing NCI-N87 lesions while very few human T cells in the U T T cells treated NCI-N87 lesions and both groups of MKN-28 lesions.7.Successfully established the peritoneal metastasis model of human gastric cancer.All six mice in UT T cells treated group died and were found bloody ascites and multiple nodular peritoneal tumors under abdominal exploration;however,only two mice died in the chA21-4-1BBz CAR T cells treated group,very few tumor nodules with no bloody ascites were found in these two mice,while neither ascites nor tumor nodules in the other four mice.chA21-4-1BBz CAR T cells intraperitoneally injection prolonged tumor-related survival(P<0.001).Conclusions1.chA21-4-1BBz CAR T cells specifically recognize and produce great amounts of Th1 cytokines as well as exhibit direct and efficient cytotoxicity when stimulated with HER2 overexpressing tumor cell lines in vitro.2.chA21-4-1BBz CAR T cells can specifically home to,and accumulate in tumor sites and also persist in mice circulation,dramatically facilitated the regression of HER2 overexpressing tumor,whereas spared the progression of HER2 low-expressing tumor.3.chA21-4-1BBz CAR T cells i.p.administration dramatically suppressed the peritoneal carcinomatosis and progression and also prolonged the survival of tumor-bearing mice.4.These results provide the basis for the future clinical investigation of the humanized chA21 scFv based,4-1BB costimulated CAR T cells for the treatment of gastric cancer,and other HER2-expressing solid tumors.BackgroundTriple negative breast cancers(TNBC),an aggressive form of breast cancer that lacks significant expression of the human epidermal growth factor receptor 2(HER2),estrogen receptor(ER)and progesterone receptor(PR),accounts for approximately 15%-20%of invasive breast cancers.TNBC has unique clinical and pathological features with high tumor heterogeneity,high invasiveness,prone to local recurrence and distant metastasis,and a worse prognosis than other types of breast cancer.In the absence of obvious targets,patients with TNBC do not benefit from endocrine therapy or other available targeted agents.To date,the standard treatment still depends on surgery and adjuvant chemotherapy and radiotherapy and the clinical benefit is limited.Thus,powerful therapeutic strategies are now urgently needed for TNBC patients.The NK cell activating receptors NKG2D(natural-killer group 2,member D)have been reported to play an important role in the immune and tumor immunity of NK cells.The NKG2D ligands(NKG2DLs)mainly includes two members of the MIC(MHC class I-related chain)family and six members of the ULBP/RAET(UL 16-binding protein,or retinoic acid early transcript)family.Studies have demonstrated that NKG2D ligands are expressed on most epithelial-derived tumor cells,such as triple-negative breast cancer,ovarian cancer,colon cancer,leukemia,and multiple myeloma.The feasibility of targeting NKG2DLs utilizing chimeric antigen receptor(CAR)engineered T cell approach has been evaluated from animal experiments to clinical trials.Sentman et al.first constructed NKG2D based CAR in 2005,which contained the full-length NKG2D which fused to the cytoplasmic domain of CD3z with costimulation provided by Dap 10.They observed potent recognition and killing activity of NKG2D CAR T cells toward tumor cells expressing NKG2DLs.Later,our research group and other scientists tried to construct NKG2D CAR T cells incorporating 4-1BB or CD28 costimulatory signals,and identified their antitumor effect in animal experiments.The NKG2D CAR utilize human NKG2D receptors as the extracellular domain,whereas most CARs use scFvs derived from mouse monoclonal antibodies,so NKG2D CAR is under lower risk of host immune rejection.As studies report that NKG2DLs are frequently and broadly expressed in TNBC,we speculate that NKG2D CAR T cell therapy should be a promising targeted therapy for TNBC,which has not been reported so far.AimsThe purpose of this study is to construct NKG2D CAR T cells and to evaluate its recognition and killing effects torward NKG2DLs-expressing triple negative breast cancer cells in vitro,as well as its tumor-inhibiting effects in a subcutaneous xenograft model of TNBC.We also aim to study the roles of 4-1BB and CD27 costimulatory signaling domains in NKG2D CAR in generating potent T cells against TNBCs in vitro and in vivo,attempting to provide a new treatment strategy for TNBC.Methods1.The expression of NKG2DLs on the surface of human TNBC cell line was detected by flow cytometry.2.Utilizing the previous CAR backbone,NKG2D CAR was constructed by using Nhel and Sal1 double digestion.The NKG2D CAR construct are comprised of the extracellular portion of human NKG2D(aa 82-216)linked to a CD8a hinge and transmembrane region,followed by a CD3z signaling moiety alone(NKG2D-z)or in tandem with the 4-1BB or CD27 intracellular signaling motif.CAR sequences were preceded in frame by a green fluorescent protein(GFP)sequence followed by the 2A ribosomal skipping sequence.The constructed CAR DNA was inserted into the pELNS lentiviral backbone,and virus particles were made by liposome transfection in 293T cells.3.Transduce activated human T lymphocytes by lentiviral vectors to generate NKG2D-z,NKG2D-BBz,NKG2D-27z CAR T cells.Analyze the transduction efficiency of CAR on T cells by flow cytometry.4.NKG2D-z,NKG2D-BBz,NKG2D-27z CAR T cells were co-cultured with MDA-MB-231,MDA-MB-468,AE17 cell line.Detect the IFN-γsecretion by ELISA to evaluate the specific recognition of NKG2D CAR T cells toward tumor cells expressing NKG2DLs in vitro.5.Set 1:2,1:1,2:1 three different effector:target ratios for coculture and evaluate the killing effect of NKG2D CAR T cells for TNBC cell lines expressing NKG2DLs by bioluminescence cytotoxicity assay.6.Set two in vitro culture conditions with or without exogenous IL2(50 IU/ml).Continuously monitor NKG2D CAR T cells number and CAR expression levels by flow cytometry,to evaluate the effect of exogenous IL2 for NKG2D CAR T cells expansion and enrichment in vitro.7.Magnetically isolate CD25 expressing T cells and obtain two NKG2D CAR T cell populations with high or low CD25 expression.Continuously monitor NKG2D CAR T cells number and CAR expression levels by flow cytometry,to assess the effect of IL2-CD25 interaction in NKG2D-z CAR T cells enrichment.8.NSG female mice were subcutaneously injected with luciferase-labeled MDA-MB-231 cells to establish a human TNBC subcutaneous xenograft tumor model;40 and 45 days after tumor inoculation,mice in five group were administrated PBS,UT T cells,NKG2D-z,NKG2D-BBz or NKG2D-27z CAR T cell by tail vein.The tumor growth in each group was monitored by vernier calipers measurement and bioluminescence imaging system.9.10 days and 20 days after T cell therapy,peripheral blood from mice was collected twice.Detect the survival of human T cells in the peripheral blood circulation of mice and the proportion of CD4+ and CD8+ T cells by flow cytometry,as well as analyze CAR expression level of human T cell populations.Results1.NKG2DLs are widely expressed in human TNBC cell lines.MICA/B was highly expressed in BT549 cells but expressed lower in MDA-MB-436 cells.The expression of ULBPs in TNBC cell lines varied:only MDA-MB-468 cells expressed ULBP1 at low level,while ULBP-2/5/6 showed higher expression in all TNBC cell lines except BT549;ULBP-3 and ULBP-4 were only found on MDA-453 and MDA-MB-231 cells.2.Successfully constructed NKG2D-z,NKG2D-BBz,NKG2D-27z CAR.Lentiviral vectors were transfected into human T lymphocytes and the expression efficiency of the three CARs were around 90%.3.NKG2D CAR T cells effectively recognize NKG2DLs expressing TNBC cell lines and secrete high levels of IFN-y in vitro;when the E:T ratio was as low as 1:2,the killing efficiency of NKG2D CAR T cells was more than 60%and increased as the E:T ratio increased.NKG2D CAR T cells did not response to the NKG2DLs-negative AE17 cells.Compared to the 1st generation of NKG2D-z CAR T cells,the second generation of NKG2D-27z and NKG2D-BBz CAR T cells secrete higher levels of IFN-1 and exhibit stronger cytotoxicity.4.Flow analysis identified the expression of NKG2DLs(～10%)on activated T cells.In the presence of IL-2,NKG2D-z,NKG2D-BBz and NKG2D-27z CAR T cells expanded more than 300-fold and were highly enriched for CAR+ cells,only-30%of T cells were positive for GFP on day 5,but were preferentially enriched to more than 95%after additional two weeks of culture.In the absence of IL-2,NKG2D CAR T cells did not expand well and only NKG2D-BBz and NKG2D-27z CAR T cells were still highly enriched for CAR+ cells,while NKG2D-z CAR was stable at-30%over the time.5.In vitro cultures with exogenous IL-2,the CD25 high-expressing populations of NKG2D-z,NKG2D-BBz,and NKG2D-27z CAR T cells all have CAR enrichment to approximately 75%to 90%;however in the CD25 low-expressing populations of NKG2D CAR T cells,the second-generation NKG2D-BBz and NKG2D-27z CAR T cells achieved 80%to 90%CAR enrichment,while the first generation NKG2D-z CAR T cells only have about 30%CAR enrichment.6.Successfully established the subcutaneous xenograft model of human TNBC in NSG mice with luciferase-labeled MDA-MB-231 cells.The MDA-MB-231 tumors treated with PBS or UT T cells grew progressively,NKG2D-z T cells modestly delayed tumor growth compared with control group but NKG2D-BBz or NKG2D-27z CAR T cells showed significantly stronger inhibition of tumor growth than NKG2D-z CAR T cells(P<0.001).7.Ten days post first T cells injection,human CD4+ T cell counts were higher compared to CD8+ T cell counts in the peripheral circulation and both CD4+ and CD8+T cells in NKG2D-z,NKG2D-BBz and NKG2D-27z CAR cohorts were present in lower numbers in comparison to untransduced T cells;while the CAR expression by NKG2D-BBz and NKG2D-27z T cells(～20%)was higher than NKG2D-z CAR T cells(～10%).Twenty days after first T cell injection,human CD8+ T cells counts in the peripheral circulation was higher than CD4+ T cells counts,and the number of NKG2D-BBz or NKG2D-27z CAR T cells was significantly increased,whereas UT T and NKG2D-z CAR T cells counts were significantly less than the counts of 10 days ago;CAR in NKG2D-BBz and NKG2D-27z CAR T cell groups was highly enriched(75%-95%)and significantly higher than that of NKG2D-z CAR T cells(～20%).Conclusions1.NKG2DLs are widely expressed in human TNBC cell lines.NKG2D CAR T cells effectively recognized and killed TNBC cell lines expressing NKG2DLs in vitro,but exert no response to NKG2DLs-negative cell lines.Compared to the 1st generation of NKG2D-z CAR T cells,the 2nd generation of NKG2D-27z and NKG2D-BBz CAR T cells secrete higher levels of IFN-y and exhibit stronger cytotoxicity.2.Exogenous IL2 can promote the expansion and enrichment of NKG2D CAR T cells in vitro;NKG2D-BBz and NKG2D-27z CAR T cells can maintain good expansion and survival through the effect of co-stimulatory signals,while the enrichment of NKG2D-z CAR T cells is dependent on the IL2-CD25 interaction.3.The NKG2D CAR T cells intravenous administration effectively inhibited tumor growth of human TNBC xenograft;2nd generation of NKG2D-BBz or NKG2D-27z CAR T cells functioned significantly better than the 1st generation NKG2D-z CAR.T cells.4.4-1BB and CD27 co-stimulatory signals enhance the expansion and survival of CD8+ T cells in vivo.Second-generation NKG2D-BBz and NKG2D-27z CAR T cells can expand and persist well in vivo,with highly CAR enrichment;while 1st generations of NKG2D-z CAR T cells cannot expand and persist well in vivo and cannot maintain stable CAR enrichment.5.The study provide the basis for future clinical investigation of the 4-1BB or CD27 co-stimulated NKG2D CAR T cells for the treatment of triple-negative breast cancer. |
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