Bispecific Antibody Y150 Exhibited Potent Antitumor Effect On Multiple Myeloma

ObjectiveTo verify the ability of a novel anti-CD38/CD3 bispecific T-cell-engaging antibody Y150to bind CD38 and CD3.To explore the killing effects and the related molecular mechanisms of Y150 on multiple myeloma(MM)cells.Methods(1)In vitro and in vivo experiments were performed to identify the therapeutic potential of Y150 for MM.Firstly,the structure of Y150 was tested in vitro.At the cellular level,the Fab terminal of Y150 was tested by flow cytometry whether it could target CD38 and CD3simultaneously.DARZALEX,MOR202 analog and CD3 isotype antibody were used as the control antibodies to explore the immunity mechanisms of Y150.NK cells were extracted from the PBMCs,and subsequently the NK cells,MM cells and antibodies were co-cultured to detect the effect of antibody-dependent cellular cytotoxicity(ADCC).The complement was extracted from the blood of a healthy donor,and then the complement,MM cells and antibody were co-cultured to examined whether the antibody had complement-dependent cytotoxicity(CDC)phenomenon.Monocytes were extracted from PBMCs,and the monocytes were induced to macrophages by GM-CSF.Then,macrophages,MM cells and antibodies were co-cultured to verify the occurrence of antibody-dependent cellular phagocytosis(ADCP).MM cells,Y150 and cross-linking protein were co-cultured to analyze whether the antibody had programmed cell death(PCD).(2)Secondly,the function of Y150 was examined in vitro.The expression levels of CD38on humanized cell lines NCI-H929,Daudi,U266B1,RPMI-8226-Luc and HEK 293 were detected by flow cytometry.T cells were extracted from PBMCs and co-cultured with each cell line and antibody to assess the killing effect of Y150 on each cell lines with different CD38 expression levels by redirecting T cells.In the presence of MM cells,the effects of Y150 on the T cell activation and the release of cytokines such as IL-2,IL-4,IL-6,IL-10,TNF-αand IFN-γwere further analyzed.To explore the killing and proliferation effect of Y150 on immune cells in PBMCs,the expression levels of CD38 on T cells,NK cells,B cells and monocytes from two healthy donors were detected by flow cytometry.Then,Y150 was co-incubated with PBMCs to detect the killing effect of Y150 on immune cells by mediating T cells.Y150 was co-incubated with PBMCs and Daudi cells to detect the proliferation level of immune cells.(3)The antitumor effect of Y150 against MM was studied in immunocompromised NPG mice.The humanized immune system was reconstructed by intravenous injection of humanized PBMCs to mice.After 26 days,the humanized MM cells(NCI-H929)were inoculated subcutaneously.After 31 days,blood samples were taken from the mouse eyes to assess the proportion of h CD3~+m CD45~-cells(human-mouse).If the proportion was more than 1%,the humanized immune system in the mouse was reconstructed successfully.Then,the mice were divided into six groups,including saline,Y150 5mg/kg,Y150 1mg/kg,Y1500.2mg/kg,CD3 isotype 1mg/kg,and Y150 1mg/kg N/A PBMC(2 days interval for 5 times).The body weight and tumor volume of the mice were measured and documented every two days.The mice were executed after 41 days.The expression of CD3,CD4 and CD8 on tumor tissues were detected by immunohistochemistry.Part of tumor tissue was digested into single cell suspension,and subsequently,the activation,proliferation and cytokine secretion of human CD4~+T cells and human CD8~+T cells were detected respectively in the cell suspension and peripheral blood cells.Results(1)The Fab terminal of Y150 can target CD3 and CD38 simultaneously,while the modified Fc terminal cannot mediate the effects of ADCC,ADCP,CDC and PCD.However,DARZALEX and MOR202 analog could not target CD3 and CD38 at the same time,and had ADCC,ADCP,CDC and PCD effects,while CD3 isotype didin’t show the ability to induce the happening of ADCC,ADCP,CDC and PCD.(2)Daudi,NCI-H929 and RPMI-8226-Luc had high CD38 MFI and CD38-positive rate.CD38 was rarely expressed on U266B1 and hardly on HEK293.Y150 can effectively mediate human T cells against MM cells with high expression of CD38(p<0.001).When co-incubated with MM cells with high expression of CD38,Y150 induced significant T cell activation and increased the cytokine secretion such as IL-2,IL-4,IL-6,IL-10,TNF-αand IFN-γ.After co-incubation with PBMCs from two healthy donors,Y150 demonstrated strong cytotoxicity to monocytes,slight cytotoxicity to NK cells and B cells,and no cytotoxicity to T cells.After co-incubating with human PBMCs and Daudi for 3 days,Y150 stimulated T cell and B cell proliferation,whereas there was no significant change in NK cell number.After 6 days,Y150 still stimulated T cell proliferation,but not B cell or NK cell.DARZALEX and MOR202 analog stimulated NK cell proliferation.(3)NPG model showed that the tumor disappeared completely in Y150 5mg/kg group,significantly decreased in Y150 1mg/kg group,while the saline,CD3 isotype 1mg/kg and Y150 1mg/kg N/A PBMC group had no significant change.Furthermore,a huge amount of activated T cell was infiltrated in the peripheral blood and tumor microenvironment and cytokine secretion was significantly increased.ConclusionY150 can stimulate the T cell activation,proliferation and cytokine secretion,and mediate T cells to kill MM cells with high CD38 expression,and has no obvious effect on immune cells.Y150 might be promising in the treatment of MM and reduce tumor burden below the MRD detection range.