The Anti-tumor Activity Of T Cells Expanded From PD-1~+ Peripheral Blood Lymphocytes

BackgroundTumor-infiltrating lymphocytes（TILs）therapy is a type of adoptive cell transfer（ACT）,and several clinical studies on advanced malignant melanoma have confirmed that the objective response rate of a combined therapy using TILs infusion and interleukin-2 was 49–72%and,impressively,22%of patients experienced a durable complete tumor response.In recent years,an increasing number of reports have shown that TIL reinfusion has definite efficacy in patients with malignancies other than melanoma,such as cholangiocarcinoma,cervical cancer,colon cancer,and breast cancer,and some patients have complete tumor remissions.In addition,a recently reported small sample clinical trial has shown a remarkable efficacy of TIL therapy in non-small cell lung cancer patients who had previously failed anti-programmed cell death protein 1（PD-1）antibody therapy,with 2 of the 13evaluable patients achieving complete responses lasting more than 18 months.However,the extensive application of TILs also faces two major problems:1.It is necessary to obtain enough samples for TIL extraction and culture by biopsy or surgery,which may exclude a large number of patients and 2.High technical requirements and costs.It is now clear that the root cause of excellent efficacy and mild adverse reactions of TIL therapy is that it is rich in tumor-specific T cells,which can accurately identify and kill tumor cells.In recent years,studies on some types of malignancies have demonstrated that tumor-specific T cells also exist in the peripheral blood,and further studies have found that PD-1⁺T lymphocytes in the peripheral blood are rich in tumor-specific T cells.Therefore,if the peripheral blood is used as a source to prepare cell products similar to TILs,the difficulty of obtaining samples by biopsy or surgery will be completely resolved.Therefore,the present study was designed and conducted.First,an efficient in vitro expansion protocol for PD-1⁺lymphocytes from the peripheral blood was established,and a preliminary phenotypic analysis of T cell products obtained by this protocol was performed.Furthermore,the anti-tumor activity of the cell products was preliminarily evaluated by in vitro experiments.T‐cell receptor（TCR）βsequencing was used to determine the overlap of TCR immunolibraries between the cell products and paired TILs.Finally,we conducted a small observational study to evaluate the safety and efficacy of the cell product in four patients with advanced cancer who had previously failed anti-PD-1 antibodies.PartⅠEstablishment of a Protocol for In Vitro Sorting and Expansion of PD-1⁺Lymphocytes in the Peripheral BloodObjectives1.To establish a protocol for the in vitro sorting and expansion of PD-1⁺lymphocytes in the peripheral blood.2.To perform an immunophenotypic analysis of PD-1⁺lymphocytes before and after expansion in vitro.3.To investigate the effect of anti-PD-1 antibody treatment on the proliferation of PD-1⁺lymphocytes in vivo and in vitro.Methods1.Peripheral blood mononuclear cells（PBMC）were extracted using Ficoll density gradient centrifugation.PD-1⁺lymphocytes that had been bound to anti-PD-1 antibodies were sorted using streptavidin-magnetic beads and biotin-conjugated anti-human immunoglobulin G4（Ig G4）antibodies（because all anti-PD-1antibodies currently on the market are Ig G4-subtype antibodies）.Subsequently,the sorted PD-1⁺lymphocytes were expanded in vitro using our in vitro expansion protocol(using X-Vivo ^TM15 serum-free medium,and Rereco Nectin,OKT-3,interleukin-2（IL-2）,and autologous plasma were added at specific time points).2.An immunophenotypic analysis of PD-1⁺lymphocytes before and after expansion in vitro was performed using flow cytometry.3.Flow cytometry was used to detect the proportion of PD-1⁺and Ki67⁺cells in the peripheral blood of patients before and 3 weeks after anti-PD-1 antibody treatment,and to compare the changes before and after treatment.Peripheral blood PD-1⁺lymphocytes were sorted and expanded in vitro before and after anti-PD-1antibody treatment to compare the differences in expansion ability in vitro.Results1.Starting with 50 ml peripheral blood,the sorted PD-1⁺lymphocytes could be expanded to（4.72±0.25）×10⁹in vitro within 2 weeks to harvest T cells expanded from PD-1⁺peripheral blood lymphocytes（FPD-1⁺T cells）.2.CD3⁺CD8⁺T cells and CD3⁺CD4⁺T cells were dominant in both PD-1⁺lymphocytes before expansion and FPD-1⁺T cells.The proportion of CD3⁺CD8⁺T cells in FPD-1⁺T cells was significantly higher than before expansion.3.After anti-PD-1 antibody treatment,the proportion of PD-1⁺lymphocytes and Ki67⁺PD-1⁺cells in the peripheral blood were both significantly increased,and the in vitro expansion rate of peripheral blood PD-1⁺lymphocytes was significantly increased.PartⅡAnti-tumor Activity of FPD-1⁺T cells In VitroObjectives1.To evaluate the anti-tumor activity of FPD-1⁺T cells in vitro.2.To investigate the relationship between the TCR immunolibrary of FTD-1⁺T cells and TCR immunolibrary of paired TIL cells.Methods1.Functional T-cell assay:T cell function was indirectly evaluated by detecting interferon（IFN）-γsecretion by FPD-1⁺T cells after co-incubation with autologous tumor cells（IFN-γsecretion by T cells suggested specific recognition and killing of tumor cells）,tumor-specific T cells（IFN-γ⁺T cells）were quantitatively analyzed by flow cytometry,and T cells expanded from PD-1-peripheral blood lymphocytes（PFD-1-T cells）were used as the control.2.Tetramer staining assay:the NY-ESO-1_157-165tetramer or Mart-1_26-35tetramer staining assay was used to quantitatively analyze the T cell-specific targeting tumor antigen NY-ESO or Mart-1 in PBMC,PD-1⁺lymphocytes and PD-1-cells before expansion,FPD-1⁺T cells and FPD-1^-T cells,respectively.3.TCR immunolibrary sequencing:Combined multiplex polymerase chain reaction amplification and high-throughput sequencing techniques to perform sequencing of the TCRβchain complementarity-determining region 3 in FPD-1⁺T cells,FPD-1^-T cells,and paired TIL cells,respectively were performed.We then analyzed the relationship of TCR immunolibrary among these three groups of cells.Results1.Functional T-cell assay:The proportion of IFN-γ⁺T cells in PFD-1⁺T cells was higher than that in PFD-1-T cells,specifically for CD8⁺T cell subsets（5.24±2.31%vs.0.65±0.20%,p=0.048）and CD4⁺T cell subsets（2.42±0.75%vs.0.38±0.17%,p=0.005）.2.Tetramer staining assay:Both PD-1⁺lymphocytes before expansion and FPD-1⁺T cells were enriched with tetramer-positive cells,and the proportion of tetramer positive cells in FPD-1⁺T cells was higher than that before expansion.3.TCR immunolibrary sequencing:Compared to FPD-1^-T cells,FPD-1⁺T cells had a higher clonal overlap with paired TIL cells.PartⅢPreliminary Evaluation of Safety and Efficacy of FPD-1⁺T cells in Clinical ApplicationObjectives1.To evaluate the safety of FPD-1⁺T cells in clinical application.2.To evaluate the anti-tumor activity of FPD-1⁺T cells in clinical application.Patients and MethodsAutologous FPD-1⁺T cell infusion combined with anti-PD-1 antibody therapy was applied to patients with advanced malignant tumors who did not response to multi-line therapy and were resistant to anti-PD-1 antibody therapy without standard treatment methods,and the safety and efficacy were preliminarily observed.Results1.Patient characteristics:Among the four patients,two female and two male,three had malignant melanoma and one had renal clear cell carcinoma,they all failed multi-line treatment（including anti-PD-1 antibody treatment failure）previously,with Eastern Cooperative Oncology Group（ECOG）performance status（PS）scores ranging from 0 to 2.2.FPD-1⁺T cells preparation and transfusion:The total amount of FPD-1⁺T cells ranged from 1.24×10⁹to 6.3×10⁹,and each patient completed four cycles of infusion.3.Safety:No serious adverse reactions related to cell infusion or serious immune-related adverse events were observed in the four patients.4.Efficacy:One patient achieved complete remission;his progression-free survival（PFS）was 22 months and was still in remission;partial remission was achieved in 2 patients,with PFS of 3.3 months and 11.2 months,respectively.One patient had stable disease,and the PFS was 4.9 months.Conclusions1.We established an efficient and convenient in vitro expansion protocol for PD-1⁺cells from peripheral blood,from which we could obtain（4.72±0.25）×10⁹PFD-1⁺T cells within 2 weeks starting from 50 ml peripheral blood of patients with malignancies.2.In vitro studies showed that FPD-1⁺T cells were enriched with tumor-specific T cells and that FPD-1⁺T cells had high clonal overlap with paired TILs.3.A small-sample observational clinical study showed that autologous FPD-1⁺T cell infusion combined with anti-PD-1 antibody therapy has good safety and efficacy in patients with advanced malignancies with previous ant-PD-1antibody resistance.