Muramyl dipeptide CD10 monoclonal antibody immunoconjugates inhibited acute leukemia in nude mice

Abstract

Minimal residual disease (MRD) is one of the causes of leukemia recurrence. Previously, we developed anti-CD10 mAb conjugated to muramyl dipeptide immunoconjugate (MDP-Ab) for immune enhancement. The present study aimed to investigate anti-leukemia effect of MDP-Ab administered via different methods in leukemia ectopic graft nude mouse model. BALB/c nude mice were injected with Nalm-6 cells subcutaneously to establish leukemia xenografts in nude mice as a model. MDP-Ab or/and human lymphocytes (LYM) was injected into different sites of the nude mice. Immunohistochemistry staining of CDs in the bone marrow, liver and spleen was performed. IFN-γ was detected by ELISA. We detected the metastasis of leukemia cells to the liver, spleen and bone marrow in nude mouse leukemia model. MDP-Ab and LYM inhibited the growth of tumors, and simultaneous injection of MDP-Ab and LYM into the tumor inhibited the growth of tumors. IFN-γ levels in MDP-Ab (ca) + h-LYM (ca) group, MDP-Ab (ca) + h-LYM (ip) group, MDP-Ab (iv) + h-LYM (ip) group and PBS (ca) + h-LYM (ca) group were significantly higher than those in control group, while IFN-γ level in MDP-Ab (ca) + h-LYM (ca) group was the highest. Moreover, MDP-Ab and h-LYM promoted the expression of hCD4 and hCD8, with the highest expression in MDP-Ab (ca) + h-LYM (ca) group. In conclusion, MDP-Ab effectively promoted the production of IFN-γ, enhanced the antitumor immunity of T lymphocytes and inhibited leukemia.

Keywords: acute lymphoblastic leukemia; anti-CD10 mAb; immunotherapy; minimal residual disease; muramyl dipeptide.

Figure 1. The leukemia xenografts model in nude mice

(A) HE staining of peripheral blood (400×). (B) HE staining of bone marrow (1000×). (C) Positive staining of CD19 in bone marrow (400×). (D) Positive staining of CD10 in bone marrow (400×). (E) A white mass structure was observed on the surface of the liver. (F) HE staining of the liver. (G) Positive staining of CD19 in the liver. (H) White ring structure on the surface of the spleen. (I) HE staining of the spleen. (J) Positive staining of CD10 in the spleen.

Figure 2. MDP-Ab inhibited the growth of tumors in nude mice

(A) Compared with the first day, the weight changes of nude mice in different groups on the 15th day (n=6). (B) The subcutaneous xenograft tumors in different group on the 15th day. (C) The volume changes of tumors in different groups of nude mice and the changes on the 15th day compared with the first day (n=6). (D) Detection of IFN-γ levels in different groups of nude mice by ELISA. *P<0.05 vs. PBS (n=6).

Figure 3. The level of CD4- and CD8-positive T-cell infiltration in the tumor 15 days post-injection

Immunohistochemistry staining of CD4 (A) and CD8 (B) T-cell regions in nude mice from different groups with tumors. The results were shown as the percentage of tumor area (%) (staining area/ tumor area), the percentage of CD4-positive cells (C) in different groups and the percentage of CD8-positive cells (D) in different groups. *P<0.05 vs. PBS (n=6).

Figure 4. The infiltration levels of CD19-, CD14- and CD56-positive cells in tumors

Immunohistochemistry staining of CD19 (A), CD14 (B) and CD56 (C). Positive cell regions were detected in nude mice from different tumor groups. The infiltration levels of CD19- (D), CD14- (E) and CD56-positive cells (F) in different groups were expressed as the percentage of tumor area (%) (staining area/tumor area). *P<0.05 vs. PBS (n=6).