CD7-CAR-NK-92MI Cell Targeted Therapy For T Cell Tumor And A Bispecific CAR-T Cell Targeted Therapy For B Cell Tumor

Chimeric antigen receptor(CAR)immunotherapy has recently shown promise in clinical trials for B-cell malignancies;however,designing CARs for T-cell based diseases remain a challenge.It is difficult to find a suitable target for T cell tumors Most target antigens are shared between normal and malignant cells,leading to CAR-T cell fratricide.CD7 is highly expressed in T-cell acute lymphoblastic leukemia(T-ALL),but it is not expressed in one small group of normal T lymphocytes.One study have shown CD7 appears not to be pivotal contribution to T-cell development or function,so CD7 may be a particularly appropriate target for treating T-ALL.But since CD7 molecules are also expressed on most normal T cells,CD7-CAR-T cells also cause suicide during preparation.CD7-CAR-T cells are difficult to prepare successfully.In addition,harvesting adequate numbers of autologous T cells from the patients with refractory disease,without contamination by malignant cells,is technically challenging It has been recently reported that T-ALL cells can be eliminated by allogeneic CD7-CAR-T cells that are simultaneously knock out of CD7 and T cell receptors alpha chain,however,due to the problem of gene editing efficiency in clinical applications.allogeneic CD7-CAR-T still has a risk of graft-versus-host disease(GVHD)Natural killer(NK)cells play an essential role in innate immune defenses against malignant cells.making them potentially effector cell or adoptive immunotherapy.The NK-92 cell line was derived from the peripheral blood mononuclear cells of a non-Hodgkin’s lymphoma patient and show potent cytotoxicity.Which is the only NK cell line that had been tested in clinical trials for immunotherapy of malignancies,NK-92MI cells were derived from the NK-92 cell line by stably transfecting an interleukin-2(IL-2)gene,making them IL-2-independent,Here,we constructed nonovalent CD7-CAR-NK-92MI and bivalent dCD7-CAR-NK-92MI cells using the CD7 nanobody sequences from our laboratory,to target the treatment of T-ALLPurpose:(1)To construct monovalent CD7-CAR-NK-92MI and bivalent dCD7-CAR-NK-92MI cells using the CD7 nanobody sequences identified in our laboratory;(2)To detect the cytotoxicity of C D7-C AR-N K-92MI and dCD7-CAR-NK-92MI cells on CD7-positive tumor cells;(3)The monovalent CD7-CAR-NK-92MI and bivalent dCD7-CAR-NK-92MI cell monoclonal cell lines were screened separately,and the cell viability of different monoclonal cell lines was compared,to obtain a monoclonal cell line with the best cell activity(mdCD7-CAR-NK-92MI);(4)To evaluate the antitumor activity of mdCD7-CAR-NK-92MI cells in vivo.Methods:we first designed,synthesized and constructed CD7-CAR and dCD7-CAR expression plasmids,then constructed CD7-CAR-NK-92MI and dCD7-CAR-NK-92MI cell lines by electroporation.CD92-CAR-NK-92MI and dCD7-CAR-NK-92MI cells were identified by cell flow and western blotting.The cytotoxicity of the CD7-CAR-NK-92M1 and dCD7-CAR-NK-92MI cells to T-ALL cells were examined by CSFE/7AAD cell killing assay.Monoclonal cell sorting was performed using a flow cytometer,and the cell viability of different monoclonal cell lines were compared by CSFE/7AAD cytotoxicity assay and CBA cytokine assay,to obtain the most active monoclonal cell line(mdCD7-CAR-NK-92MI).Finally,the anti-tumor effect of mdCD7-CAR-NK-92MI cell line was evaluated by a mouse model established by T-ALL primary tumor cells.Results:We successfully constructed monovalent CD7-CAR-NK-92MI and bivalent dCD7-CAR-NK-92MI cell lines.Both CD7-CAR-NK-92MI and dCD7-CAR-NK-92MI cells consistently showed specific and potent anti-tumor activity against T-cell leukemia cell lines and primary tumor cells.We screened eight monovalent CD7-CAR-NK-92MI and six bivalent dCD7-CAR-NK-92MI monoclonal cell lines,and compared the activities of the monoclonal cells.The mdCD7-CAR-NK-92MI monoclonal cell line was the most active cell line of all monoclonal cell lines.In PDX mouse models,mdCD7-CAR-NK-92MI cells robustly reduced tumor burdens,controlled tumor growth,and significantly prolonged survival when compared to those treated with NK-92MI cells as a control.Conclusion:We constructed CD7-CAR-NK-92MI and dCD7-CAR-NK-92MI cells based on CD7 nanobody sequences,and demonstrated anti-tumor effects on T-ALL tumor cells through a series of experiments.We not only confirmed the specific cytotoxicity of CD7-CAR-NK-92MI and dCD7-CAR-NK-92MI cells to T-ALL cell lines and primary tumor cells by in vitro experiments,but also demonstrated that mdCD7-CAR-NK-92MI cells significantly inhibited disease progression in xenograft mouse models of T-ALL primary tumor cells.In conclusion,CD7-CAR-NK-92MI cells can be used as a new method or a complementary therapy for treating T-cell acute lymphocytic leukemia.CD19-CAR-T therapy has made significant progress in the treatment of B-cell malignancies,especially B-cell acute lymphocytic leukemia(B-ALL),showing surprising clinical efficacy.However,the relapse rate after treatment with CD19-CAR-T is still very high.One mechanism of tumor relapse is that tumor cells can escape the killing of CD19-CAR-T cells by down-regulating the expression of CD 19 antigen on the surface of tumor cells.One strategy to tackle CD19-negative relapse is to use CAR-T cells that target other antigens.CD22,CD20,and CD 123 are also ideal targets for targeting B cell tumors.However,similar to CD19,targeting a single antigen may still result in tumor escape due to antigen loss.Therefore,the construction of CAR-T cells that can simultaneously target two antigens on cancer cells may be a new strategy to resolve tumor relapse due to single antigen loss.Purpose:(1)To develop humanized CD 19 and CD20 antibody sequences for construction of hCD19-hCD20-CAR vector;(2)To construct humanized bispecific hCD19-hCD20-CAR-T cells and hCD19-hCD20-CAR-Jurkat cells;(3)To detect whether hCD19-hCD20-CAR-Jurkat cells can be specifically activated by CD19-positive or CD20-positive target cells;(4)To evaluate the biological function of hCD19-hCD20-CAR-T cells against CD 19 overexpressing tumor cells or CD20 overexpressing tumor cells;(5)To evaluate the biological function of hCD19-hCD20-CAR-T cells against B cell tumor cell lines that express CD19 and CD20 antigens.Methods:To construct the bispecific hCD19-hCD20-CAR vector,we first developed the humanized CD19scFv and CD20scFv sequences,then designed.synthesized and constructed the hCD19-hCD20-CAR expression plasmid,and prepared hCD19-hCD20-CAR lentivirus.Then hCD19-hCD20-CAR-T cells and hCD 19-hCD20-CAR-Jurkat cell lines were constructed.We assessed whether hCD19-hCD20-CAR can be specifically activated by CD 19 or CD20 antigen by detecting the expression of CD69 on the surface of hCD19-hCD20-CAR-Jurkat cells after incubation with K562-CD19 or K562-CD20 cells.Then we tested the cvtotoxicity of hCD19-hCD20-CAR-T cells against K562-CD19,K562-CD20,K562-CD19-CD20,Raji,Nalm-6 cells by cell killing assay.Furthermore,it was further verified whether hCD19-hCD20-CAR-T can be specifically activated by target cells by detecting the secretion of cytokines and the expression of cell surface activation moleculesResults:We successfully developed humanized CD 19 antibody and CD20 antibody,obtained humanized CD19scFv and CD20scFv sequences,and constructed hCD19-hCD20-CAR-T cells and hCD19-hCD20-C AR-Jurkat cells.When hCD19-hCD20-CAR-Jurkat cells were incubated with K562-CD19 or K562-CD20 cells.the expression of CD69 was significantly increased,while after incubation with K562 cells,the expression of CD69 on hCD19-hCD20-CAR-Jurkat cells was not significantly elevated,indicating that hCD19-hCD20-CAR-Jurkat cells can be activated either by CD19-positive tumor cells or by CD20-positive tumor cells.The killing effect of hCD19-hCD20-CAR-T cells on K562-CD19 and K562-CD20 cells indicated that hCD19-hCD20-CAR-T has specific killing effect on CD19-positive or CD20-positive tumor cells,and can specifically kill CD 19-positive tumor cells or CD20 positive tumor cells.In addition,after incubation with K562-CD19 and K562-CD20 cells,the release of cytokines was significantly increased in the hCD19-hCD20-CAR-T cell group compared with the T cell group,and the expression of CD25 was also significantly increased,indicating that hCD19-hCD20-CAR-T cells can be specifically activated by K562-CD19 and K562-CD20 cells.The killing effect of hCD19-hCD20-CAR-T cells on Raji cells and Nalm-6 cells also showed that hCD19-hCD20-CAR-T cells also had significant specific killing of Raji and Nalm-6 cells,and hCD19-hCD20-CAR-T cells can also be specifically activated by Raji or Nalm-6 cellsConclusion:We successfully constructed hCD19-hCD20-CAR-T cells based on humanized CD19scFv and CD20scFv.and demonstrated that hCD19-hCD20-CAR-T cells have significant effects on CD19-positive tumor cells or CD20-positive tumor cells Therefore,the humanized hCD19-hCD20-CAR-T cells we constructed can be used as a novel treatment for targeted therapy of B cell tumors or as a remedy for tumor relapse after CD19-CAR-T treatment.