The Establishment Of CHO-hIL-15 Cell Line And Its Continuous Secretion On Proliferation And Killing Activity Of CAR-T Cells In Vitro

Background and aimsAcute lymphoblastic leukemia?ALL?is a hematological malignant tumor characterized by the clonal proliferation of a large number of immature lymphocytes.ALL is the most common malignancy in childhood,accounting for about 25%of cancer diagnoses in children under 15 years of age.With the advancement of leukemia cell genomics and epigenomics and the development of modern treatment programs,the overall survival?OS?of children with ALL has now exceeded 80%,but 15-35%of children will still relapse.The prognosis of multiple relapses is extremely poor,and treatment of these cases will become increasingly challenging.In the past decade,the activation of patients'autoimmune responses has become the fourth treatment option for cancer treatment.Chimeric antigen receptor?CAR?modified T?CAR-T?cells are typical of immunotherapy.representative.The CAR gene is an unnatural chimeric gene based on a single-chain antibody?Sc Fv?that recognizes target antigens on the surface of tumor cells and transmembrane regions,costimulatory factors such as CD137?4-1BB?,CD28,and the CD3?motif.Packaging and infecting activated immune-active cells such as T cells,NK cells,etc.,redirect the patient's own T or NK cells and exert the specific targeted killing and clearance functions of tumor cells.CARs programmed for tumor-associated antigens can be replicated quickly and uniformly.CAR-modified T cells with superphysiological activity can specifically recognize tumor-associated antigens and rapidly expand,becoming a magical“live”drug that exerts anti-Immediate and long-term effects of tumors.In particular,adoptive transfer of CAR to T cells in cancer patients has achieved significant remission of leukemia,in which the anti-CD19 CAR-T cell product CTL019?tisagenlecleucel,Kymriah,Novartis?is used for pediatric relapse/refractory B cell acute Lymphocytic leukemia?B-ALL?is the first genetically modified cell therapy approved by the US Food and Drug Administration?FDA?.Interleukin-15?IL-15?,with a molecular weight of 17.8KDa,is a cytokine that regulates the activation and proliferation of T and natural killer cells.It has many similar biology with interleukin-2?IL-2?.active.At present,some laboratories have adopted the method of combining IL-15 cytokines with CAR-T to construct fourth-generation CAR-T cells for clinical trials.Studies have shown that CD8+CD45RA+CCR7?Cxc Chemokine Receptor 7,CCR7?+subpopulations are related to the in vivo expansion of CAR-T cells injected into patients with B-cell malignancies,and IL-15 retains CAR-T cells expanded in vitro.CD8+CD45RA+CCR7+and other cells are better than IL-2,and compared with IL-2,IL-15 can make CAR-T cells exposed to a series of antigens have a higher survival rate,IL-15 expands The increased expression of CCR7 in CAR-T cells helps to efficiently homing it to secondary lymphoid organs,and CAR-T cells exposed to IL-15 can effectively increase its durability and antitumor activity in vivo.Studies have shown that compared with CARs without IL-15 structure,CARs with IL-15 structure can increase the persistence of CAR-T cells and increase the number of central memory T cells,which can enhance the ability to kill target cells.The advantages of reducing recurrence,but there is the disadvantage of not knowing the exact concentration of IL-15,everyone is in the exploration stage,and this is related to the current lack of standard guidelines for reference.We hypothesized that continuous secretion of IL-15 and CAR-T cells in vitro may increase the expansion and maintenance capacity of CAR-T cells,and thus increase the anti-tumor activity of CAR-T cells.There have been no reports of IL-15 secreted by feeder cells co-cultured with CAR-T.3A4CAR is a successful CD45 subclass chimeric antigen receptor constructed in our laboratory in the early stage.It has a novel chimeric antigen receptor targeting leukemia stem cells?LSCs?.This study intends to construct a Chinese hamster ovary(Chinese hamster cells?CHO?cell line?CHO-h IL-15?that stably expresses IL-15,and to identify the biological activity of IL-15 secreted by CHO-h IL-15 To feed cells,they were co-cultured with CAR-T cells,and the effects of continuously secreted IL-15 on the proliferation and killing activity of 3A4-CAR were observed to lay the foundation for further improving its immunotherapy effect.Methods1 Construction of p Lenti-IL-15 lentivirus expression vector1.1 Design of IL-15:search the literature to determine the genetic structure of IL-15;Search the national center for biotechnology information?NCBI?to verify the gene sequence of IL-15;Send company whole gene synthesis target fragment.1.2 Amplification of the target gene fragment:all the returned plasmids were cloned by PCR and TA to obtain a large number of IL-15 target fragments.1.3 Build IL-15 eukaryotic expression vector:use Bam H?single enzyme p Lenti-RFP-Blasticidin empty carrier,after purification,together with IL-15 purpose fragment,T4 connection enzyme reaction 16?for 30 min.After smears,cloning,shaking bacteria,sequencing,the construction pLenti IL-15 expression vector.1.4 Instant transfer of Chinese hamster cells?CHO?:transfection grade plasmid IL-15 was extracted.Transfected CHO cells with liposome method were observed by fluorescence after 48h.The concentration of IL-15 was determined by enzyme linked immunosorbent assay?ELISA?.2 IL-15 was successfully expressed in CHO cells2.1 IL-15 stable transfer CHO cell:human embryonic kidney cell?293T?was used as the packaging cell,and was co-transfected with the packaging plasmid and expression plasmid to obtain lentivirus particles carrying IL-15 gene.RT-PCR was used to determine the titer of lentivirus.Lentivirus infects CHO cells.2.2 Verify the expression of IL-15:The expression of IL-15 in CHO cells was verified by RT-PCR,ELISA,Western Blot and flow cytometry?FCM?.2.3 Culture of CHO-IL-15 monoclonal cell line:monoclonal cell lines were obtained by blasticidin-resistant pressurization and subcloning technology,and the IL-15fragment was verified by immunofluorescence.3 Effect of CHO-IL-15 cells on the activity of 3A4CAR-T cells3.1 Packaging and titer identification of chimeric antigen receptor 3A4CAR lentivirus.3.2 Lentiviral transfected T cells.3.3 Flow verification of 3A4CAR expression in T cells.3.4 The effect of CHO-IL-15 on the activity of T cells:Set the number ratio of T and cho-15 in different gradients,and culture IL-15 and T in the pure T control group and the recombinant human to see the proliferation level of T cells.3.5 The effect of CHO-IL-15 on the activity of 3A4-CAR-T:Set the number ratio of cells of 3A4CAR-T and CHO-IL-15 cell lines with different cell concentration gradients?2:1,5:1?,co-culture with simple 3A4CAR-T cells,3A4CAR-T and ordinary CHO cells,Co-culture with commercial recombinant human IL-15 and 3A4CAR-T as control groups to observe the proliferation and killing function of 3A4CAR-T cells.Results1 Construction of eukaryotic expression vector of IL-15 gene1.1 Determine the gene sequence of IL-15 by consulting the data.The target gene fragment is 489 bps in total.1.2 Amplification of target gene fragmentAccording to the structure of the gene fragment,design upstream and downstream primers containing the sequences at both ends of the reading frame.The returned plasmid was cloned by PCR and TA to obtain a large number of amplified IL-15 target gene fragments.There are bright bands at about 500bps,which correspond to the size of the designed gene.Further cut the gel and send it to a commercial company for sequencing and DNA-MAN software for gene sequence alignment.The results show that the sequence is completely consistent with the expected sequence.1.3 Use Bam H?single enzyme p Lenti-RFP-Blasticidin empty carrierpLenti-RFP-Blasticidin empty vector was digested with Bam H?,purified,and together with Bam H I digested IL-15 target gene fragment,in the presence of T4 ligase.The reaction was connected at 16?for 30 min.After coating,picking,cloning,shaking,and sequencing,the comparison results showed that the sequence and integration position of the IL-15 gene in the structure of the p Lenti IL-15 lentiviral expression vector was consistent with the expected design results,indicating that the vector was successfully constructed.1.4 Transfection grade plasmid p Lenti-IL-15 was transfected into CHO cells by liposome methodThe liposome method was used to infect the CHO cells with the transfection-grade plasmid p Lenti-IL-15,and 48 hours later,fluorescence was observed under the excitation of 576nm excitation light to find red fluorescence.The ELISA method was used to detect the expression of IL-15 in the culture supernatant.The results showed that the concentration of IL-15 was 43pg/ml,and 25.9%of the cells containing red fluorescence were detected by FCM.2 IL-15 was successfully expressed in CHO cells2.1 IL-15 stabilized CHO cellsWhen the blasticidin concentration is 10?g/ml or more,all CHO cells die.It is determined that 10?g/ml is the best blasticidin screening concentration.2.2 Verify the expression of IL-15293T cells successfully packaged lentiviral particles expressing the IL-15 gene.By RT-PCR identification,the titer of the virus supernatant in the stock solution was in the order of 4x10⁶/?l;at a virus volume of 500?l,the ratio of the virus amount of 2x10⁹ to the number of 10⁶CHO,5%CO₂,and 37?culture conditions for lentivirus After 2 days of infection,the red fluorescent cells carried by the lentiviral plasmid were observed under a fluorescence microscope.The positive rate of FCM was 80%,which proved that the lentivirus successfully infected CHO cells.2.3 Construction of CHO-IL-15 monoclonal cell lineThe CHO cells successfully infected by the above lentivirus,namely CHO-IL-15,were individually seeded into 2ml culture dishes per well of a 6-well plate,106 CHO-IL was added to each well-15 cells,culture in a 5%CO₂,37?incubator for 1?2 and 3days,collect the culture supernatant,adherent cells are incubated with 0.25%trypsin-EDTA solution for 2 minutes,and the cells are collected in PBS?PH 7.4,0.1M?,500g x 5min,washed twice by centrifugation,and then extracted RNA.After RT-PCR amplification,the IL-15 gene fragment was amplified in CHO-IL-15 cells;then the concentration of IL-15 in the culture supernatant was detected by ELISA method to be288 pg/ml;the experiment can be observed under a fluorescence microscope The group had green fluorescence,but no green fluorescence was seen in the control cells;FCM determined that red fluorescent cells could be as high as 85%.2.4 Establishment of CHO-IL-15 monoclonal cell lineThe above-mentioned CHO-IL-15 cells were cloned and cultured in a 96-well plate by limiting dilution method,and the intracellular fluorescence of the cloned growth was observed with a fluorescence microscope.After cloning and culture,CHO-IL-15monoclonal cell line stably secreting IL-15 was obtained;CHO-IL-15 cells were immunofluorescently stained with goat anti-rabbit Ig G FITC secondary antibody,observed under a fluorescent microscope,and the experiment was found There was bright green fluorescent protein expression in the GREEN field of the group,but no green fluorescence in the no-load group and the blank control group;red fluorescence protein expression in the red fluorescence field of the experimental group and the no-load group,and no fluorescence in the blank control group.Appeared,indicating that the infected CHO-IL-15 monoclonal cell line can successfully express the target protein of IL-15.3 Effect of CHO-IL-15 cells on the activity of 3A4CAR-T cells3.1 Packaging and titer identification of chimeric antigen receptor 3A4CAR lentivirusAccording to the previous research results of this laboratory,3A4CAR lentiviral particles were successfully packaged,and the lentiviral solution was concentrated 20times by ultracentrifugation.The titer was 9.25x10⁵copes/?l by quantitative PCR.3.2 Lentiviral transfected T cellsAfter ethical review,2 ml of peripheral blood of children with normal physical examination were obtained,and heparin was anticoagulated.Intermediate layer mononuclear cells?MNC?were isolated with Ficoll lymphocyte separation solution at1500 rpm x 15 min 10⁵T cells were infected with 100?l of lentivirus,cultured for 24hours under 5%CO₂,37?culture conditions,and T lymphocytes were cultured,T lymphocytes were stably transfected by lentivirus.3.3 Flow verification of 3A4CAR expression in T cellsAdd GAM antibody and streptomycin PE for indirect labeling,and then add CD5APC and CD45 Percp for labeling to see if 3A4CAR successfully transfects T cells.3.4 The effect of CHO-IL-15 on the activity of T cellsIt was found that when T:CHO-IL-15=10:2,the T cell proliferation was the largest,which was greater than the pure T control group and recombinant human IL-15With T culture group.3.5 The effect of CHO-IL-15 on the activity of 3A4-CAR-T It was found that when CAR-T:CHO-IL-15=2:1,the proliferation of CAR-T cells was the largest,which was greater than that of the simple CAR-T control group,the co-culture group of CAR-T and ordinary CHO,and recombinant human IL-15 with CAR-T culture group.Conclusion?1?Plenti-IL-15 lentiviral expression vector was successfully constructed.?2?The human IL-15 gene was successfully transferred into CHO cells for expression by lentiviral vector p Lenti IL-15 infection,and a monoclonal cell line CHO-IL-15capable of continuously and stably secreting human IL-15 was successfully established.?3?Successfully established a culture system using CHO-IL-15 as feeder cells.When co-cultured with normal children's peripheral blood T cells,it can significantly increase T cell proliferation compared to the control group co-cultured with exogenous IL-15Extend survival.?4?CHO-IL-15 can continuously secrete IL-15,promote the proliferation of CAR-T cells,increase the expansion factor and enhance the killing ability,however,there is no significant extend the survival time of CAR-T cells,.