Decoration of Anti-CD38 on Nanoparticles Carrying a STAT3 Inhibitor Can Improve the Therapeutic Efficacy Against Myeloma

Abstract

STAT3 is an oncoprotein which has been shown to contribute to drug resistance in multiple myeloma (MM). Nonetheless, the clinical utility of STAT3 inhibitors in treating MM has been limited, partly related to some of their pharmacologic properties. To overcome these challenges, our group had previously packaged STAT3 inhibitors using a novel formulation of nanoparticles (NP) and found encouraging results. In this study, we aimed to further improve the pharmacologic properties of these NP by decorating them with monoclonal anti-CD38 antibodies. NP loaded with S3I-1757 (a STAT3 inhibitor), labeled as S3I-NP, were generated. S3I-NP decorated with anti-CD38 (labeled as CD38-S3I-NP) were found to have a similar nanoparticular size, drug encapsulation, and loading as S3I-NP. The release of S3I-1757 at 24 h was also similar between the two formulations. Using Cy5.5 labeling of the NP, we found that the decoration of anti-CD38 on these NP significantly increased the cellular uptake by two MM cell lines (p < 0.001). Accordingly, CD38-S3I-NP showed a significantly lower inhibitory concentration at 50% (IC50) compared to S3I-NP in two IL6-stimulated MM cell lines (p < 0.001). In a xenograft mouse model, CD38-S3I-NP significantly reduced the tumor size by 4-fold compared to S3I-NP on day 12 after drug administration (p = 0.006). The efficacy of CD38-S3I-NP in suppressing STAT3 phosphorylation in the xenografts was confirmed by using immunocytochemistry and Western blot analysis. In conclusion, our study suggests that the decoration of anti-CD38 on NP loaded with STAT3 inhibitors can further improve their therapeutic effects against MM.

Keywords: CD38; S3I-1757; STAT3; multiple myeloma; nanoparticle.

Figure 1

The synthesis of CD38-S3I-NP. (A) Chemical reactions of the anti-CD38 conjugation to PEO-b-PBCL, the building block of our nanoparticles (NP). The final product was mixed with S3I-NP to generate CD38-S3I-NP. (B) The release of S3I-1757 from S3I-NP or CD38-S3I-NP in vitro. The percentage of S3I-1757 released was calculated by the lost amount of S3I-1757 compared to the initial total amount of S3I-1757. The error bar represents the standard deviation from a triplicate experiment; * p < 0.05, via Student’s t-test.

Figure 2

Flow cytometry analysis of the Cy5.5-positive cell population 4 h after treatment of Cy5.5-NP or Cy5.5-CD38-NP. Anti-CD38-conjugated NP exhibits improved cellular uptake of NP by multiple myeloma (MM) cells. Cy5.5 was chemically conjugated to the core of NP. The gated area was defined using the cells without NP treatment. The representative dot plot from a triplicate experiment is shown. The error values represent the standard deviation from the triplicate experiment. A non-MM cell line, SupM2, was included for comparison. The fold change in cell uptake was calculated by dividing the percentage of Cy5.5-positive cells with Cy5.5-CD38-NP treatment by that with Cy5.5-NP treatment. The error bar represents standard deviation from a triplicate experiment; * p < 0.05, via Student’s t-test.

Figure 3

CD38-S3I-NP induces cytotoxicity and inhibits STAT3 activity in MM cells. (A) U266 and RPMI8226 cells were then treated with S3I-NP, CD38-S3I-NP, or S3I-NP with free CD38 antibody at a concentration which is equivalent to CD38-S3I-NP (1.4 mg/mL) with the presence of IL6 (2 ng/mL) for 24 and 48 h. Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay in triplicate; * p < 0.05, via Student’s t-test. (B) Western blot analysis of STAT3 and pSTAT3 levels in U266 and RPMI8226 cells treated with S3I-NP or CD38-S3I-NP with the presence of IL6 (2 ng/mL) for 24 h. β-actin was blotted as a loading control.

Figure 4

CD38-S3I-NP is more tumor suppressive than S3I-NP in MM xenograft. (A) SCID mice intravenously injected with PBS (black line, n = 2), 3 mg/kg S3I-NP (green line, n = 4) or CD38-S3I-NP (blue line, n = 4) every day for two days as indicated by arrows on the x-axis. Animal numbers other than the initial numbers at different time points are indicated. The tumor size was quantified by the bioluminescence intensity and normalized to the initial bioluminescence signal (i.e., 2 h post-injection). The representative bioluminescence images of animals treated with PBS, S3I-NP, or CD38-S3I-NP were shown. * p < 0.05, via Student’s t-test; EU—euthanized. (B) The pSTAT3 levels in bone marrow mononuclear cells extracted from the SCID mice in (A) at the endpoint. Non-tumorous brain tissue from a SCID mouse was used as a negative control. SupM2 cells were used as a positive control for pSTAT3. β-actin was blotted as a loading control. (C) Immunocytochemical staining of pSTAT3 and in bone marrow mononuclear cells from (B). Each image represents bone marrow cells from one animal. The images were taken at a magnification of 400×.