| Background and Objective:B-cell non-Hodgkin’s lymphoma(B-NHL)represents a group of malignant tumor that derive from lymph nodes and other lymphatic tissues.Traditional treatment for B-NHL consists of chemotherapy,radiation therapy,or a combination of rituximab with various of chemotherapeutic agents.Although patients received enhanced complete remission rate after targeting treatments based on rituximab,over 50%of them became resistant to rituximab in clinic,which resulted in relapse or metastasis.With the discoveries of antigens on the surface of B-cell lymphoma and the development of technologies of molecular biology,targeting therapy for B-NHL receives more and more attentions.CD19,expressed by virtually all developmental stages of B-cell lineage with the important exception of stem cells,is emerging as a promising target antigen in B-NHL besides CD20.Bispecific antibodies(bsAbs)are genetically engineered antibodies which are comprised by domains of VH and VL from two different antibodies.BsAbs usually possess dual binding specificity,which could recruit effector cells to the tumor cells resulting in specific lysis.To date,the most promising for the therapeutic application is a bispecific antibody called blinatumomab which was approved by FDA for the treatment of B-precursor acute lymphoblastic leukemia(B-ALL).Although blinatumomab has impressive efficacy in the clinic,it still has some limitations.Pharmacokinetics from preclinical and early clinical studies on blinatumomab has shown a relatively short half-life due to its molecular weight(55 kDa),which is below the glomerular filtration threshold.Therefore,blinatumomab is administrated with continuous infusion for a long time,which results in inconvenience for patients and increased possibility of treatment-related adverse event.Mesenchymal stem cells(MSCs)are a population of adult stem cells that exist in various of organs and tissues with the capacity of highly self-renewal and multi-lineage potential.MSCs have been served as attractive carriers of agents for cancer therapy own to the characteristics of tumor tropism,low immunogenicity,and easily modification.In this study,we investigated a new dual-targeting therapeutic system based on the tumor migration of MSCs which were genetically engineered to secrete a tetravalent bispecific antibody named Tandab(CD3/CD19)targeting both CD3 and CD19.In addition,the antitumor effects of this targeting therapeutic system combining with D-1-methyl-tryptophan(D-1MT),an IDO pathway inhibitor,were also evaluated.Methods:The methods of PCR,overlap PCR,restriction enzyme cleavage and linkage were used to construct the lentiviral expression vectors pLentiR.SV40-Tandab(CD3/CD 19),pLentiR.SV40-fLuc,and pLentiR-EV(empty vector).The correctness of constructs was verified by DNA sequencing.The supernatants of 293T cells transiently transfected with the corresponding lentiviral expression vectors were collected,and the recombinant protein Tandab(CD3/CD19)was purified by affinity chromatography with His-tag.In addition,the purified or un-purified protein Tandab(CD3/CD19)was detected by Western blot analysis.Antigen binding activity of Tandab(CD3/CD19)to CD19-positive cells or CD3-positive cells was determined by flow cytometry analysis.Lactic dehydrogenase(LDH)release assay was employed to detect the cytotoxicity of T cells induced by Tandab(CD3/CD19).Lentiviral particles were packaged in 293T cells followed by infecting human umbilical cord-derived MSCs to stably express Tandab(CD3/CD19)(MSC-Tandab)or firefly luciferase(MSC-Luc).The influence of lentivirus on the proliferation of MSCs was detected by MTT assay.The expression of Tandab(CD3/CD19)in MSCs was determined by western blot analysis and the level of Tandab(CD3/CD 19)secreted in the supernatant from MSCs was also detected by ELISA.The migration ability of MSCs which were infected by lentivirus in vitro was determined by Transwell assay using cell culture inserts.To demonstrate the tumor tropism of MSCs in vivo,MSC-Luc were injected intravenously into nude mice which burdened well-established Raji xenograft tumor followed by bioluminescence imaging(BLI).To assess the bioactivity of Tandab(CD3/CD19)secreted by MSCs,a co-culture killing system was established.The specific lysis of CD19-positive Raji cells was determined by FACS analysis.The expression of activation of surface markers CD69 and CD25 of T cells was detected by FACS in the same conditions of co-culture system.And the supernatants in the inserts were collected for the assay of cytokines produced by T cells.Further more,D-1MT was added to this co-culture system for analysis of the cytotoxicity of T cells to Raji cells in the indicated time.Color-metric assays were employed to measure the kynurenine,a metabolite of tryptophan,in the co-culture system.And the T cell anergy-associated genes CD98 and Jumonji were detected by real-time PCR.To evaluate the therapeutic effect of MSC-Tandab + PBMC in combination with D-1MT in vivo,MSCs were injected intravenously into tumor-bearing mice followed by PBMCs via the vein two days later,every 7 days for 2 weeks.D-1MT was administrated in the drinking water(2 mg/ml).Growing tumors were measured every three days including tumor volumes and body weights.And the tumor tissues were weighed at the end of treatment to assess the therapeutic effects.Results:Lentiviral expression vectors pLentiR.S V40-Tandab(CD3/CD 19),pLentiR.SV40-fLuc,and pLentiR-EV were successfully constructed.Results from Western blot and FACS indicated that tetravalent bispecific antibody Tandab(CD3/CD19)could be successfully expressed in eukaryotic cells and could specifically bind to both CD19-positive cells(Raji,Daudi,and BJAB)and CD3-positive cells(Jurkat).However,Tandab(CD3/CD19)could not bind to K562 cells which were CD 19-negative and CD3-negative.Results from LDH release assays demonstrated that Tandab(CD3/CD19)secreted from 293T cells could induce specific cytotoxicity of T cells to CD19-positive cells.Lentiviral particles LentiR-Tandab(CD3/CD19),LentiR-EV,and LentiR-fLuc were successfully packaged in 293T cells.MSCs could express Tandab(CD3/CD19)efficiently after infected with LentiR-Tandab(CD3/CD19).And there were no differences on the capacity of proliferation and migration in vitro between MSC-Tandab and MSCs.The subcutaneous Raji cells xenograft model was successfully established in BALB/c nude mice.Results from the BLI indicated that MSC-Luc migrated to tumor site after 24 hours of intravenous injection.Results from co-culture killing experiments in vitro shown that the lysis of Raji cells killed by T cells induced by Tandab(CD3/CD19)released from MSC-Tandab arrived 60.6 ± 5.7%(P<0.001,compared with the control group).In additional,the expression of activation marker of T cell including CD69 and CD25 and cytokines(IL-2、IFN-γ、TNF-α)were obviously higher than that of control group(P<0.001).When combined with D-1MT,the lysis of Raji cells triggered by T cells was increased efficiently at 48 h and 72 h(P<0.05).However,there was no significant differences at 24 h(P>0.05).Although the level of Kyn in the supernatants shown no changes in the indicated time when combining with D-1MT,the expression of T cell anergy-associated genes CD98 and Jumonji decreased obviously at 72 h(P<0.001).Additionally,the proliferation of T cells in the co-culture system was also partly restored by D-1MT at 48 h and 72 h,respectively.Results from anti-tumor experiment indicated that the growth of xenograft tumor with Raji cells was inhibited significantly when mice were injected intravenously with MSC-Tandab and PBMCs combined with D-1MT in the drinking water.Conclusions:We have demonstrated in this study that human UC-MSCs can be acted as cell-based delivery vehicles for the treatment of B-NHL.Tandab(CD3/CD19)secreted from MSCs effectively redirected T cells to inhibit the growth of Raji lymphoma in mouse model in combination with D-1MT.Our findings indicate that the use of UC-MSCs as vehicles for engineered-antibodies combined with immunoregulatory agents represents a potential clinical application of gene therapy for cancers. |
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