CD46-Targeted Theranostics for PET and 225Ac-Radiopharmaceutical Therapy of Multiple Myeloma

Abstract

Purpose: Multiple myeloma is a plasma cell malignancy with an unmet clinical need for improved imaging methods and therapeutics. Recently, we identified CD46 as an overexpressed therapeutic target in multiple myeloma and developed the antibody YS5, which targets a cancer-specific epitope on this protein. We further developed the CD46-targeting PET probe [89Zr]Zr-DFO-YS5 for imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of prostate cancer. These prior studies suggested the feasibility of the CD46 antigen as a theranostic target in multiple myeloma. Herein, we validate [89Zr]Zr-DFO-YS5 for immunoPET imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of multiple myeloma in murine models.

Experimental design: In vitro saturation binding was performed using the CD46 expressing MM.1S multiple myeloma cell line. ImmunoPET imaging using [89Zr]Zr-DFO-YS5 was performed in immunodeficient (NSG) mice bearing subcutaneous and systemic multiple myeloma xenografts. For radioligand therapy, [225Ac]Ac-DOTA-YS5 was prepared, and both dose escalation and fractionated dose treatment studies were performed in mice bearing MM1.S-Luc systemic xenografts. Tumor burden was analyzed using BLI, and body weight and overall survival were recorded to assess antitumor effect and toxicity.

Results: [89Zr]Zr-DFO-YS5 demonstrated high affinity for CD46 expressing MM.1S multiple myeloma cells (Kd = 16.3 nmol/L). In vitro assays in multiple myeloma cell lines demonstrated high binding, and bioinformatics analysis of human multiple myeloma samples revealed high CD46 expression. [89Zr]Zr-DFO-YS5 PET/CT specifically detected multiple myeloma lesions in a variety of models, with low uptake in controls, including CD46 knockout (KO) mice or multiple myeloma mice using a nontargeted antibody. In the MM.1S systemic model, localization of uptake on PET imaging correlated well with the luciferase expression from tumor cells. A treatment study using [225Ac]Ac-DOTA-YS5 in the MM.1S systemic model demonstrated a clear tumor volume and survival benefit in the treated groups.

Conclusions: Our study showed that the CD46-targeted probe [89Zr]Zr-DFO-YS5 can successfully image CD46-expressing multiple myeloma xenografts in murine models, and [225Ac]Ac-DOTA-YS5 can effectively inhibit the growth of multiple myeloma. These results demonstrate that CD46 is a promising theranostic target for multiple myeloma, with the potential for clinical translation.

Figure 1.

[⁸⁹Zr]Zr-DFO-YS5 can detect CD46 expression in various multiple myeloma cell lines. A,K_d measurement of [⁸⁹Zr]Zr-DFO-YS5 on the MM.1S cell line (n = 3), determined by a saturation binding assay (K_d = 16.31 ± 2.6 nmol/L) with a receptor density of 1.49 × 10⁵ per cell. B, Cell binding assay to measure the percent cell-associated activity of [⁸⁹Zr]Zr-DFO-YS5 using different multiple myeloma cell lines (n = 3). C, Flow cytometry analysis of CD46 cell surface expression in various multiple myeloma cell lines (n = 3).

Figure 2.

[⁸⁹Zr]Zr-DFO-YS5 PET/CT can detect subcutaneous MM.1S xenografts in vivo. MIP and CT and μPET/CT fusion images of (A) [⁸⁹Zr]Zr-DFO-YS5 (n = 4), (B) 50-fold YS5 + [⁸⁹Zr]Zr-DFO-YS5 (n = 3), and (C) [⁸⁹Zr]Zr-DFO-IgG in NSG mice (n = 4) bearing subcutaneous MM.1S xenografts at 6 days post-injection. D, MIP and CT and μPET/CT fusion images of [⁸⁹Zr]Zr-DFO-YS5 in NSG mice bearing (n = 5) subcutaneous CD46-KO MM.1S (CD46−/−) xenograft at 6 days post-injection. E, Biodistribution of [⁸⁹Zr]Zr-DFO-YS5, [⁸⁹Zr]Zr-DFO-YS5 + 50-fold YS5, [⁸⁹Zr]Zr-DFO-IgG in NSG mice bearing subcutaneous MM.1S xenograft, and biodistribution of [⁸⁹Zr]Zr-DFO-YS5 in NSG mice bearing subcutaneous CD46-KO MM.1S (CD46−/−) xenograft at 6 days after injection. Two-way ANOVA P values are indicated as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 3.

[⁸⁹Zr]Zr-DFO-YS5 imaging and biodistribution in RPMI8226 and ANBL6 subcutaneous xenograft models. MIP and CT and μPET/CT fusion images of (A) [⁸⁹Zr]Zr-DFO-YS5 (n = 4) and (B) [⁸⁹Zr]Zr-DFO-IgG (n = 5) in NSG mice bearing subcutaneous RPMI8226 xenografts at 6 days after injection. C, Biodistribution of [⁸⁹Zr]Zr-DFO-YS5 and [⁸⁹Zr]Zr-DFO-IgG in NSG mice bearing subcutaneous RPMI8226 xenografts at 6 days after injection. MIP and CT and μPET/CT fusion images of (D) [⁸⁹Zr]Zr-DFO-YS5 (n = 4) and (E) [⁸⁹Zr]Zr-DFO-IgG (n = 5) in NSG mice bearing subcutaneous ANBL6 xenografts at 6 days after injection. F, Biodistribution of [⁸⁹Zr]Zr-DFO-YS5 and [⁸⁹Zr]Zr-DFO-IgG in NSG mice bearing subcutaneous ANBL6 xenografts at 6 days after injection. Two-way ANOVA P values are indicated as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 4.

[¹⁸F]-FDG imaging and biodistribution in subcutaneous MM.1S, RPMI8226, and ANBL6 xenograft models. MIP and CT and μPET/CT fusion images of [¹⁸F]-FDG (coronal and transverse posture) in (A) MM.1S (n = 5), (B) RPMI8226 (n = 5), and (C) ANBL6 (n = 5) subcutaneous xenografts at 1 hour after injection. D, Biodistribution of [¹⁸F]-FDG in MM.1S, RPMI8226, and ANBL6 subcutaneous xenografts after μPET/CT imaging at 1 hour after injection.

Figure 5.

[⁸⁹Zr]Zr-DFO-YS5 demonstrates favorable PET imaging characteristics in the MM.1S systemic tumor model. A, BLI of NSG mice bearing MM.1S systemic model revealed tumor was located in the femoral bone marrow. Sites of disease involvement are indicated with an arrow. B, Maximum intensity projections (MIP) and μPET/CT fusion images of [⁸⁹Zr]Zr-DFO-YS5 in NSG mice (n = 5) bearing systemic MM.1S xenograft at 4 days after injection revealed high tumor uptake in femur bone marrow, indicated by arrows. C, Correlation of ex vivo [⁸⁹Zr]Zr-DFO-YS5 PET images with bioluminescence, demonstrating matching regions of osseous PET uptake with tumor BLI signal. D, BLI of NSG mice bearing MM.1S systemic model. Sites of disease involvement are indicated with an arrow. E, MIP and μPET/CT of [⁸⁹Zr]Zr-DFO-YS5 with 50-fold block of cold YS5 in NSG mice (n = 4) bearing MM.1S systemic xenograft at 4 days after injection, demonstrating no detectable tumor uptake above background at the documented sites of tumor involvement (arrows). F, Biodistribution of [⁸⁹Zr]Zr-DFO-YS5, [⁸⁹Zr]Zr-DFO-YS5 with 50-fold block of cold YS5 in NSG mice bearing systemic MM.1S xenograft at 4 days after injection. G, BLI of NSG mice bearing MM.1S systemic model. Sites of disease involvement are indicated with an arrow. H, MIP and μPET/CT fusion images of [¹⁸F]-FDG in NSG mice (n = 4) bearing MM.1S systemic xenograft at 1 hour after injection, demonstrating moderate tumor uptake. I, Biodistribution of [¹⁸F]-FDG in NSG mice bearing systemic MM.1S xenograft.

Figure 6.

[²²⁵Ac]Ac-DOTA-YS5 is an effective treatment for multiple myeloma in the MM.1S metastatic model. A, Schematic for therapeutic study. B, Serial BLI imaging indicates reduced tumor burden in the treatment groups (ventral view; n = 8). C, Body weight measurements in control and treatment groups. D, Kaplan–Meier curve demonstrates dose-dependent improvement in overall survival in the treatment arms. E, CD138 expressing cells in femurs in mice from the control group (n = 1) treated with saline after 15 days (F) decrease in CD138 expressing cells in femurs in mice treated with 0.125 μCi (n = 1) of [²²⁵Ac]Ac-DOTA-YS5 after 15 days, confirming tumor-targeted cell death and reduction in tumor burden.