| Objective To prepare and identify the immunoreactivity of a novel immunomodulator composed of muramyl dipeptide and anti-CD 10 monoclonal antibody.Methods The immunomodulator (MDP-Ab) was constructed by coupling anti-CD 10 monoclonal antibody (MAb) and muramyl dipeptide (MDP) using heterobifunctional reagent SPDP. Electrospray ionization mass spectrometry (ESI-MS) and Ultraviolet spectrophotometer were used to identify the molecular weight and the molecular ratio of MDP and MAb. The activity of anti-CD 10 antibody on CD 10 antigen in the MDP-Ab immunoconjugate was assayed by indirect flow cytometry (FCM). Peripheral blood mononuclear cells (PBMC) were separated from children with acute lymphoblastic leukemia by use of Ficoll-Hypaque method and were adhered for 3h. The adherent cells were induced into the DC, which were cultured with 10% fetal calf serum (FCS) (volume fraction) of RPMI-1640 and Granulocyte-macrophage colony-stimulating factor (GM-CSF),interleukin-4 (IL-4) in the control group. Besides GM-CSF and IL-4, the test groups were divided into five subgroups, adding unconjugated anti-CD 10 alone, unconjugated MDP alone, MDP-Ab alone, LPS alone and MDP-Ab+LPS, respectively. The mitomycin C-treated DC were cultured with autologous lymphocytes at the ratio of 1:10 for 72h.The growth of DC was observed under an invert microscope everyday. The immunophenotypes and the endocytosis of DC were detected by direct and indirect FCM on the 8th day. The stimulation index of autologous lymphocytes was assayed by CFSE-staining method. The supernatants of DC and autologous lymphocytes were used to detect the level of cytokines (IL-12 and IFN-y).Results 1.Purification of MDP-Ab:The molecular weight of every complex, The molecular weight (MW) of every complex, including PDP-arm-Boc, PDP-arm and PDP-MDP, which was expressed in the form of MW+[H], was 372.1,272.1,746.4. The absorption of IT-Ab complex (concentration 0.1g/ml) was 0.30. The number of thiol group in IT-Ab complex was 4.5. The molecular weight (MW+[H]) of purified final conjugate (MDP-Ab) was 101KD. The molecular weight was about 101KD and the molecular ratio of MDP and MAb was about 2:1.2. Identification of antibody activity: the activity of anti-CD 10 antibody on CD 10 antigen in the MDP-Ab immunoconjugate by FCM was equal to unconjugated anti-CD 10 antibody, indicating that MDP-Ab immunoconjugate still effectively recognized and bound to CD 10 antigen.3. Identification of MDP activity:(1) DC morphological changes:On the 5th day, the morphology of immature DC showed round contours without evident dendrites. On the 8th day, DC triggered by the new immunoconjugate and LPS had morphological characteristics typical of mature DC, including loose adherence and multiple cytoplasmic projections, which indicated a promoted DC differentiation.(2) DC phenotype (HLA-DR, CD83, CD80, CD86):The expressions of HLA-DR, CD83 (a marker of mature DC) and co-stimulatory molecules (CD86 and CD80) were increased significantly upon DC triggered with MDP-Ab, LPS and combination of MDP-Ab with LPS, compared with the control group and unconjugated MAb group, unconjugated MDP group(p<0.05). All the surface markers expression of DC triggered by combination of MDP-Ab with LPS was the highest. (3) Endocytosis assay:The uptake of FITC-dextran of DC by FCM stimulated by unconjugated MAb, unconjugated MDP, MDP-Ab immunoconjugate, LPS or combination was (66.7±9.78)%, (62.5±6.72)%, (41.6±5.67)%, (33.9±3.42)%, (25.6±3.68)%, respectively, while that of immature DC in the control group was (81.3±10.1)%. (4) IL-12 secretion by DC:A significant increase in IL-12 level was detected in MDP-Ab, LPS, and combination of MDP-Ab with LPS, compared to that of unconjugated MAb, unconjugated MDP and control group (p<0.0.5). (5) Autologous T lymphocyte proliferation:MDP-Ab-treated DC, stimulated more proliferation of autologous T cells than the control group and unconjugated MAb group, unconjugated MDP group (p<0.05), while less than LPS and MDP-Ab+LPS group. The most significant T cell proliferation was observed in combination of MDP-Ab with LPS. (6) IFN-y secretion by T cells:MDP-Ab-treated DC, stimulated higher level of IFN-y than the control group and unconjugated MAb group, unconjugated MDP group (p<0.05), while lower than LPS and MDP-Ab+LPS group. The most significant was observed in combination of MDP-Ab with LPS.Conclusions1. A novel immunomodulator was synthesized successfully by coupling muramyl dipeptide with anti-CD 10 monoclonal antibody by use of SPDP method.2. The anti-CD10 monoclonal antibody in the novel immunomodulator (MDP-Ab) still keeps the specific combination with CD 10 antigen. 3. The immunomodulatory effect of MDP-Ab on DC exhibited up-regulated expression of HLA-DR, co-stimulatory marker (CD80 and CD86) and maturity marker (CD83), increased cytokine secretion, which are enhanced by combination with LPS.4. The MDP-Ab-treated DC could enhance autostimulatory activity, which prove that MDP-Ab immunoconjugate may be a suitable candidate for targeting trials and support the further development of vaccination with the new immunomodulator-triggered DC as a post-remission treatment to prevent relapse in ALL children. Objective To observe the effect of muramyl dipeptide conjugated anti-CD 10 immunoconjugate (MDP-Ab) on the function of lymphocytes and the activated lymphocytes on leukemia xenografts in nude mice in vivo.Methods PBMC were separated from children with acute lymphoblastic leukemia by use of Ficoll-Hypaque method and were adhered for 3h. The adherent cells and non-adherent cells were induced into the DC and cytotoxicity T cells, respectively. The Nalm-6 antigen-induced and mitomycin C-treated DC were cultured with autologous T cells at the ratio of 1:10 for 48h. The Nalm-6 cells were added and continued to foster for another 12h. The lactate dehydrogenase (LDH) release in each group was assayed to study T lymphocytes cytotoxic activity.36 BALB/C nude mice,4-week-old, male, were inoculated subcutaneously 1×10'Nalm-6 leukemia cells/0.2ml. After 7 to 10 days, leukemia xenografts in nude mice with formation of subcutaneous nodules were randomly divided into six groups (6 mice in each group), and the lymphocytes activated by different agents-treated DC was intratumorally administered into nude mice. The changes in tumor volume were observed, tumor growth curves were drawn, and then tumor weight inhibitory rate was calculated. The changes of the tumor, liver and spleen tissue were assessed by use of hematoxylin-eosin staining.Results 1.Cytotoxicity:The killing rate in MDP-Ab group was (35.66±5.81)% and significantly higher than the control group, unconjugated anti-CD10 antibody group and unconjugated MDP group, which was (7.29±2.42)%, (13.83±2.90)% and (15.16±3.10)%, respectively. The rate in MDP-Ab group was lower than the LPS group ((51.33±6.41)%) and MDP-Ab+LPS group ((63.33±6.92)%), among which MDP-Ab+LPS group was the strongest.2. Tumor formation rate of nude mice was 100% and all mice tumor volume was no significant difference. After administrating, the changes of tumor volume in MDP-Ab group was significantly smaller than the control group, unconjugated antibody group and unconjugated MDP group, but greater than the LPS group and MDP-Ab+LPS group, among which the MDP-Ab+LPS group was the smallest.3. On the 14th day after administrating with DC, all mice were sacrificed. The changes of tumor volume in each group showed that the rate of change in the control group, unconjugated antibody group and unconjugated MDP group was147.75,114.90,117.36, respectively, while the MDP-Ab group was 47.66, the LPS group was 32.31 and the MDP-Ab+LPS group was 12.43, which were significantly smaller than the control group, unconjugated antibody group and unconjugated MDP group.4. The early manifestation was subcutaneous tumor sections, which were round or oval, and at the late stage the surface was uneven integration with multiple nodules in the control group, in which three nude mice showed ulceration in the non-injection site, or even erosion. When peeling, the tumor showed clear boundaries, fewer adhesions, hard texture, and rich in blood vessels in the tumor profile. Under the optical microscope, tumor cells in the control group were mostly polygonal or round, disorganized, and nuclear size and shape with split-phase was common; while that in other groups showed different degrees of necrosis. Among them MDP-anti-CD10 conjugate group, LPS group and MDP-Ab+LPS group were of the most obvious, in three of which a large number of nuclear condensation, fragmentation and dissolution could be seen. Liver and spleen tissue of nude mice in all groups showed no infiltration of Nalm-6 cells.Conclusion1. MDP-Ab-induced DC can in vitro increase cytotoxic activity of autologous T lymphocytes, and the effect is more obvious when combining it with LPS.2. MDP-Ab-induced DC can stimulate autologous T lymphocytes to inhibit tumor growth in vivo, with a certain degree, and has a synergistic effect with LPS.3. Murine xenografts with Nalm-6 leukemia are established and there is no systemic metastasis.
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