Syndecan-1 antigen, a promising new target for triple-negative breast cancer immuno-PET and radioimmunotherapy. A preclinical study on MDA-MB-468 xenograft tumors

Abstract

Background: Overexpression of syndecan-1 (CD138) in breast carcinoma correlates with a poor prognosis and an aggressive phenotype. The objective of this study was to evaluate the potential of targeting CD138 by immuno-PET imaging and radioimmunotherapy (RIT) using the antihuman syndecan-1 B-B4 mAb radiolabeled with either 124I or 131I in nude mice engrafted with the triple-negative MDA-MB-468 breast cancer cell line.

Method: The immunoreactivity of 125I-B-B4 (80%) was determined, and the affinity of 125I-B-B4 and the expression of CD138 on MDA-MB-468 was measured in vitro by Scatchard analysis. CD138 expression on established tumors was confirmed by immunohistochemistry. A biodistribution study was performed in mice with subcutaneous MDA-MB-468 and 125I-B-B4 at 4, 24, 48, 72, and 96 h after injection and compared with an isotype-matched control. Tumor uptake of B-B4 was evaluated in vivo by immuno-PET imaging using the 124I-B-B4 mAb. The maximum tolerated dose (MTD) was determined from mice treated with 131I-B-B4 and the RIT efficacy evaluated.

Results: 125I-B-B4 affinity was in the nanomolar range (Kd = 4.39 ± 1.10 nM). CD138 expression on MDA-MB-468 cells was quite low (Bmax = 1.19 × 104 ± 9.27 × 102 epitopes/cell) but all expressed CD138 in vivo as determined by immunohistochemistry. The tumor uptake of 125I-B-B4 peaked at 14% injected dose (ID) per gram at 24 h and was higher than that of the isotype-matched control mAb (5% ID per gram at 24 h). Immuno-PET performed with 124I-B-B4 on tumor-bearing mice confirmed the specificity of B-B4 uptake and its retention within the tumor. The MTD was reached at 22.2 MBq. All mice treated with RIT (n = 8) as a single treatment at the MTD experienced a partial (n = 3) or complete (n = 5) response, with three of them remaining tumor-free 95 days after treatment.

Conclusion: These results demonstrate that RIT with 131I-B-B4 could be considered for the treatment of metastatic triple-negative breast cancer that cannot benefit from hormone therapy or anti-Her2/neu immunotherapy. Immuno-PET for visualizing CD138-expressing tumors with 124I-B-B4 reinforces the interest of this mAb for diagnosis and quantitative imaging.

Figure 1

Characterization of the B-B4 mAb. (A) The affinity of B-B4 mAb and the number of antigenic sites on MDA-MB-468 breast cancer cells were evaluated by an equilibrium-binding assay with ¹²⁵I-labeled B-B4 mAb. The nonspecific binding of ¹²⁵I-labeled B-B4 mAb was evaluated with a tenfold excess of unlabeled B-B4 mAb at three concentrations of ¹²⁵I-labeled B-B4 mAb and subtracted from binding data. The mean results of a triplicate assay are represented. The Bmax corresponding to the number of antigenic sites on MDA-MB-468 cells, and the Kd of ¹²⁵I-labeled B-B4 were determined by nonlinear regression using a one-binding site equation. (B) The immunoreactivity of ¹²⁵I-labeled B-B4 mAb was estimated by measuring the binding of a constant concentration of ¹²⁵I-labeled B-B4 mAb to increasing numbers of U266 multiple myeloma cells that strongly expressed the CD138 antigen. This is a double-inverse plot of a triplicate assay. The fraction of immunoreactive ¹²⁵I-labeled B-B4 (R) was determined as the inverse of the ordinate at the origin of the abscissa according to the Lindmo method [23].

Figure 2

Immunohistochemistry of a MDA-MB-468 breast tumor xenograft. The expression of CD138 in MDA-MB-468 xenografts was assessed by immunohistochemistry. (A) Original magnification ×400 for hematoxylin-eosin staining. (B) Original magnification ×400 for CD138 immunostaining.

Figure 3

Biodistribution of ¹²⁵I-labeled B-B4 mAb. (A) The biodistribution of ¹²⁵I-labeled B-B4 mAb was estimated in nude mice engrafted with the MDA-MB-468 breast cancer cell line. The results are expressed as mean values ±95% confident intervals of the percentage of injected dose per gram (%ID/g) in the considered organ or tissue from three triplicate assays. (B) This panel shows the specific binding of the B-B4 antibody calculated by subtracting the %ID/g of the ¹²⁵I-labeled 7D4 isotype-matched control antibody to that of ¹²⁵I-labeled B-B4 measured in a triplicate assay under the same experimental conditions for both antibodies. (C) The %ID/g of ¹²⁵I-labeled B-B4 mAb obtained from biodistribution studies at different time points are plotted versus time in order to visualize the kinetics of activity uptake in different tissues. (D) The kinetics of 7D4 and B-B4 distribution in the blood and tumors are shown in the same graph to demonstrate the identical blood pharmacokinetics of both antibodies and the specific uptake of BB4 in the tumor. The difference between the uptake of the two antibodies in the tumor is not significant at 4 h but is clearly significant at 24, 48, and 72 h (p < 0.001 determined by a two-way ANOVA test).

Figure 4

Immuno-PET imaging with ^124I-labeled B-B4 mAb. PET image obtained 1 h after injection of 6 MBq ¹⁸F-FDG and at days 1, 3, and 8 after injection of 3.5 MBq of ¹²⁴I-labeled B-B4 mAb.

Figure 5

Figure 5. Hematological toxicity of ¹³¹I-labeled B-B4 mAb. Hematological toxicity was estimated by counting the leukocytes (A) and platelets (B) after treatment with increasing doses of ¹³¹I-labeled B-B4 mAb. All mice treated with 37 MBq died before day 14.

Figure 6

Radioimmuntherapy assay. (A) Tumor size variations in individual nude mice engrafted with the MDA-MB-468 cell line after treatment. Mice treated with 14.8 (n = 8) or 22.2 MBq (n = 8) of ¹³¹I-labeled B-B4 mAb can be compared with control mice (n = 9) that received unlabeled B-B4 mAb. The mean tumor volume of each group of mice is represented. The curves are very significantly different for the control versus 22.2 MBq (p < 0.0001) and the 14.8 MBq versus the 22.2 MBq groups (p < 0.0001) and significantly different for the control versus the 14.8 MBq groups (p = 0.028). (B) The effect of the unlabeled B-B4 mAb (n=5) and PBS (n=5) were tested in an independent experiment (left panel). The effect of RIT with 131I-labeled control isotype mAb (14.8, n=3 and 25.9 MBq, n=4) and 131I-B-B4 (14.8, n=4 and 22.2 MBq, n=4) and control PBS (n=4) were compared (right panel). Results are expressed as fold-increase in tumor volume compared to day 0. This figure summarizes the results of two independent assays performed under the same conditions. (C) The time required for tumors to double in size in groups given 14.8 and 22.2 MBq ¹³¹I-labeled B-B4 mAb compared with the control group..