| Objective:Lung cancer is the leading cause of cancer-related deaths worldwide.The majority of patients(approximately 75%)are already advanced at the time of diagnosis,with limited treatment options,and the 5-year survival rate for these patients is only 5-8%.Therefore,there is an urgent need for breakthroughs in treatment for this group of patients with advanced lung cancer.The immune system plays a key role in the recognition and clearance of tumor cells("immunosurveillance").In recent years,tremendous progress has been made in therapeutic strategies that utilize the specific immune system to eliminate tumors,and tumor immunotherapy is considered to be one of the most successful approaches in the field of oncology treatment.Tumor immunotherapy strategies commonly used today include immune checkpoint inhibitors(ICIs),tumor vaccine and adoptive cell transfer(ACT).Unlike traditional chemotherapy and radiotherapy,the essence of immunotherapy is to achieve the purpose of tumor treatment by activating the body’s immune system or relieving the body’s immune suppression.The core step requires the recognition of tumor antigens by T cells,so the tumor antigens targeted in immunotherapy are very important.During the development of tumor cells,there are a large number of genetic mutations,some of which occur within the coding regions of genes,resulting in changes in the sequences of the proteins they encode,thus forming immunogenic cellular proteins that are recognized by the body’s immune system as neoantigen.Therefore,tumor neoantigens are tumor-specific and do not form immune tolerance,making them ideal targets for tumor immunotherapy.A growing number of studies have also confirmed that neoantigen-specific T cells are key to the success of treatment with ICIs and ACT,and adoptive transfer autologous T cells targeting neoantigens has also been shown to mediate durable antitumor effects in a variety of solid tumors.Thus,immunotherapy targeting neoantigens is in a key position in tumor immunotherapy.With the development of genomics and bioinformatics,it has become possible to predict neoantigens produced by tumor.Currently,several research teams in China and abroad have validated the immune efficacy,clinical efficacy,safety and tolerability of RNA and peptide vaccines based on personalized neoantigens in preclinical and clinical studies.Dendritic cells(DCs)are considered to be the most efficient antigen-presenting cells(APCs)for initiating T-cell immunity.Direct loading of tumor antigens onto DCs can effectively increase the efficiency of antigen presentation by DCs and generate stronger antitumor immune responses,which is an important direction for personalized vaccine research.At present,DC vaccines based on personalized neoantigens have shown clinical efficacy in melanoma and other solid tumors,but no prospective clinical studies have been published for lung cancer.Lung cancer has a high tumor mutation burden(TMB)and a high level of tumor neoantigens.Therefore,neoantigen-based DC vaccine therapy may be a reasonable treatment option for these patients.To this end,in the first part of the study,we conducted a translational clinical study to explore the safety and efficacy of the DCs loaded with personalized neoantigens(hereinafter referred to as Neo-DCVac)in patients with advanced lung cancer,and explore whether Neo-DCVac induces neoantigen-specific T cell response after immunization.As mentioned earlier,neoantigen-specific T cells are the key effector cells for tumor killing.Theoretically,direct screening of T cells that targeting neoantigens for ACT is the most ideal immunotherapy modality.The current common approach is to identify neoantigen-specific T cells from tumor infiltrating lymphocytes(TILs),and then infuse them to patient after a large-sized expansion.However,this approach is difficult to perform universally,for most cancer patients,it is difficult to obtain enough tumor tissues to isolate TILs;in addition,it is very time-consuming to culture TILs,predict neoantigens,synthetic peptides or tandem minigene(TMG),and for cancer patients it is very important to buy time for treatment.The results of our clinical study and related literature reports have shown that neoantigen vaccines or tumor lysate vaccines can induce neoantigen-specific T cells response after immunization.Compared with neoantigen vaccines,tumor lysate vaccines are faster and easier to prepare,and theoretically,tumor lysates contain more immunogenic epitopes that help prevent tumor escape,making them an ideal antigen source for DC vaccines.Therefore,we speculate whether it is possible to obtain the peripheral blood from patients after immunization with tumor lysate vaccines,and then prepare neoantigen-reactive T cells(NRTs)for transfusion back to patients?If we adopt the above research assumption,when tumor tissues are obtained,we will prepare tumor lysate vaccines to immunize patient on the one hand,so that the patient can receive treatment as soon as possible;on the other hand,we will perform sequencing to screen neoantigens.In this way,patients do not simply wait in the process of screening for neoantigens,but receive treatment.After several cycles of immunization,peripheral blood mononuclear cells(PBMCs)are obtained from the patient and the neoantigen-reactive T cells are prepared for transfusion back to the patient.In this way,patients no need to isolate TILs,but can also receive treatment as early as possible.In addition,this is essentially a combination of vaccine and ACT immunotherapy,and patients are more likely to benefit from it.Based on the above assumptions,in the second part of the study,we carried out preclinical experiments on the basis of the previous research.By obtaining tumor tissues and blood samples of LL/2 tumor-bearing mice,the neoantigens were predicted(the research group has completed neoantigens prediction).The LL/2tumor-bearing mice were immunized with DCs loaded with LL/2 tumor lysate(hereinafter referred to as OCDC vaccine).After immunization with OCDC vaccine,immunogenic neoantigens were identified,and NRTs were prepared and reinfused to tumor-bearing mice.We hoped to explore a new and convenient method of preparing NRTs through this study,and further explore the efficacy and safety of OCDC vaccine combined with NRTs adoptive transfer,and provide new ideas and programs for current ACT treatment,so that more patients could receive ACT therapy and benefit from it.Materials and Methods:The first part of the study was a single-arm,multi-center,exploratory clinical study(Chi CTR-ONC-16009100,NCT02956551).Patients with advanced lung cancer who failed multiple lines of therapy were screened.The whole exome sequencing(WES)were performed on the tumor tissues and blood samples of patients to identify tumor mutations;tumor specimens were subjected to RNA sequencing(RNA seq)to determine the expression of mutations;then stepwise filtering by predefined conditions to predict the neoantigen.Synthesis of neoantigenic peptides based on predicted neoantigen information,then PBMCs from patients were obtained,DCs were cultured,and neoantigenic peptides were loaded onto the DCs to prepare neoantigen-loaded DC vaccines(Neo-DCVac).The patients were immunized with Neo-DCVac at week 1,2,4,6,and 8 respectively.All 5injections constituted a cycle of immunization.The patients needed to undergo imaging examination to evaluate the clinical efficacy.For patients with no disease progression,they could continue to receive the next cycle of Neo-DCVac treatment.If the patient had disease progression,study protocol was permanently ceased.And other palliative treatments including best support care(BSC)were provided.After immunization with Neo-DCVac,we obtained the patient’s PBMCs,and detected the release of cytokines by enzyme-linked immunospot assay(ELISpot),flow cytometry to evaluate whether Neo-DCVac could induce a neoantigen-specific T cell response.In the second part of the study,we first obtained LL/2 tumor cells,figured out the optimal hypochlorous acid(HOCL)concentration to induce necrosis of LL/2tumor cells,and then performed 6 cycles of repeated freezing and thawing to prepare LL/2 tumor lysate.Subsequently,we loaded the tumor lysate onto DCs to prepare a tumor lysate-loaded DC vaccine(OCDC).Next,we established the LL/2 orthotopic tumor model and the LL/2 subcutaneous tumor model,and explored the efficacy and safety of the OCDC vaccine by immunizing LL/2 tumor-bearing mice with the OCDC vaccine.Then,we obtained the spleen lymphocytes of mice immunized with OCDC vaccine.The release of interferon-γ(IFN-γ)and the expression of CD137 in spleen lymphocytes of mice immunized with OCDC vaccine were detected by ELISpot and flow cytometry to identify immunogenic neoantigens.Finally,we established LL/2 subcutaneous tumor model,loaded the immunogenic neoantigens onto DCs,and co-cultured them with the spleen lymphocytes of mice immunized with OCDC vaccine to prepare NRTs,and then these T cells were reinfused into LL/2 tumor-bearing mice immunized with OCDC vaccine to verify the anti-tumor effect and safety of OCDC vaccine combined with NRTs adoptive transfusion.Results:In the first part of the clinical study,from November 2017 to September 2019,we screened a total of 18 patients with advanced lung cancer who failed multi-line therapy.Finally,12 patients were included in the study.After extracting DNA from tumor tissues and blood samples of these 12 patients,and performing WES comparison,the median number of somatic non-synonymous mutations identified in each patient was 313.5(range 80-808),RNA sequencing(RNAseq)was performed on each tumor sample to confirm the expression status of these mutations,and then gradually filtered through the pre-set conditions,the median predicted neoantigens per patient were 16.5(range 12-30).After neoantigen peptides were synthesized,we loaded them onto DCs to prepare Neo-DCVac.In our study,a total of 85 doses of Neo-DCVac were produced.The median number of Neo-DCVac treatment was 5doses(range 3-14),and the median number of live cells in each dose of vaccine was1.60×10~7(range 0.65×10~7-2.4×10~7).The expression levels of CD11c,CD86,CD83and other molecular phenotypes in Neo-DCVac products were detected by flow cytometry.The results showed that the quality of Neo-DCVac products was qualified for infusion.In terms of safety,Neo-DCVac was well tolerated,all adverse events were grade 1 or 2,and there was no dose-limiting toxicity or leading to treatment delay or interruption.All patients experienced grade 1 injection site reactions.Patient 4developed a transient grade 1 neutropenia after receiving Neo-DCVac treatment.Patient 12 developed grade 2 pruritic rash on the trunk and limbs after receiving the vaccine.The adverse reactions gradually disappeared after the vaccination was stopped.In the 5 patients who were treated with ICIs during the vaccine immunization period,Neo-DCVac did not induce immune-related adverse events related to ICIs.In terms of efficacy,3 out of 12 patients(25%)achieved partial response(PR),6 cases(50%)were evaluated as stable disease(SD),and 6 cases(50%)had target lesions shrank,9 patients(75%)achieved disease control,and 3patients(25%)developed progressive disease(PD).The median follow-up time of12 patients treated with Neo-DCVac was 7.1 months(range 0.9-17.2 months),and the median progression-free survival(PFS)was 5.5 months(95%CI,1.9-9.2),the median overall survival(OS)was 7.9 months(95%CI,5.9-10.0).In addition,we divided the patients into patients who were treated with ICIs during Neo-DCVac treatment and patients who were not.We found that the combination therapy of ICIs and Neo-DCVac had longer PFS and a trend of better OS,indicating that these two treatment strategies might have potential synergistic therapeutic effects.Patient 1 was a 72-year-old male with advanced lung adenocarcinoma who participated in the clinical study after failing the third-line treatment.Computed tomography(CT)performed after one cycle of immunization with Neo-DCVac vaccine showed that the lung tumor lesions had shrunk,and the efficacy was evaluated as PR.ELISpot results showed that the PBMCs after vaccine immunization showed specific responses to the neoantigens MARCH6,CUX1 and B4GALNT1.Stimulation of PBMCs with MARCH6,CUX1 and B4GALNT1mutant peptides after Neo-DCVac vaccine immunization resulted in a significant increase in IFN-γ,tumor necrosis factor-α(TNF-α),interleukin-2(IL-2)and CD107a secretion in CD3+CD4+T cells,whereas the same results did not occur after stimulation with the corresponding wild peptides.PBMCs before and after Neo-DCVac vaccine immunization were stimulated with MARCH6,CUX1,B4GALNT1 mutant peptides and the corresponding WT peptides,and the secretion of IFN-γ,IL-2,and TNF-αin culture supernatants was measured by cytometric bead array(CBA).The results also showed that stimulation of PBMCs after vaccine immunization with the mutant peptides induced an increase in cytokine release,whereas this effect was not observed in PBMCs before vaccine immunization.These results showed that Neo-DCVac vaccine could induce neoantigen-specific T cell responses after immunization.In the second part of the preclinical study,we first set up different HOCL concentration gradients,and through trypan blue staining and flow cytometry to detect the proportion of necrotic cells,we found that when the HOCL concentration was 0.9μM,98.77%of the cells were PI and Annexin V double positive(necrotic cells),so the finally concentration of HOCL for oxidized LL/2 necrosis was 0.9μM.After 6 cycles of repeated freezing and thawing of oxidized necrotic LL/2 tumor cells,we prepared LL/2 tumor lysate,and then loaded them on DCs to prepare OCDC vaccine.The expression levels of CD11c,CD80 and CD86 in OCDC vaccine products were detected by flow cytometry,and the results showed that most of the cells in the vaccine production were mature DCs with antigen presentation function.Next,we established a LL/2 in situ lung cancer model and subcutaneously injected the OCDC vaccine to the mice.We found that the OCDC vaccine could significantly inhibit the growth of tumor.Hematoxylin-eosin(HE)staining was performed to evaluated the safety of the vaccine,and we found that there was no obvious toxicity in the main organs,suggesting that the OCDC vaccine had good efficacy and safety.Then,by establishing the LL/2 subcutaneous tumor model,we found that the OCDC vaccine could also significantly inhibit the growth of subcutaneous tumor.In addition,after OCDC vaccine immunization,the release of FN-γand the expression of CD137 in the spleen lymphocytes of mice immunized with OCDC vaccine were detected by ELISpot and flow cytometry.We identified five immunogenic neoantigens(Pank3,Tmem101,Rab3gap2,Neurl4,Supt6),which indicated that OCDC vaccine could induce neoantigen-specific immune response.Then,we loaded the immunogenic neoantigens onto DCs,co-cultured with the spleen lymphocytes of the mice immunized with the OCDC vaccine to prepare neoantigen-reactive T cells,and then transfer these T cells back to the LL/2tumor-bearing mice after immunization with OCDC vaccine.The results showed that OCDC vaccine combined with NRTs adoptive transfer could significantly inhibit the growth of LL/2 subcutaneous tumors,and the anti-tumor effect was better than that of OCDC vaccine alone.After the treatment,we used flow cytometry to detect the proportion of CD3+CD8+IFN-γ+T cells in the tumor microenvironment and found that after OCDC vaccine combined with NRTs treatment,the CD3+CD8+IFN-γ+T cell ratio in the tumor microenvironment increased significantly(P<0.0001).We speculated that after OCDC vaccine immunization,NRTs infusion could activate T cells in the tumor microenvironment to trigger an antigen-specific cytotoxic T lymphocyte(CTL)response to exert anti-tumor effects.Conclusion:In patients with advanced lung cancer,we conducted an attempt on a personalized neoantigen DC vaccine.Our results showed that it was feasible to prepare Neo-DCVac in patients with advanced lung cancer.Neo-DCVac was safe and effective for treatment of patients with advanced lung cancer,and Neo-DCVac could induce neoantigen-specific T cells response.However,screening of neoantigens,peptides synthesis,and preparation of Neo-DCVac was a complex and time-consuming process.For this reason,we turned our research attention to OCDC vaccine.Our preclinical research results showed that OCDC vaccine had good efficacy and safety.More importantly,OCDC vaccine could induce neoantigen-specific immunity after immunization,and OCDC vaccine combined with NRTs adoptive transfer could induce a stronger anti-tumor response.Our research provided new ideas and plans for personalized DC vaccine research,and explored a new breakthrough for"precision medicine". |
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