Harnessing the fcμ receptor for potent and selective cytotoxic therapy of chronic lymphocytic leukemia

Abstract

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy in need of new, effective, and safe therapies. The recently identified IgM receptor FcμR is overexpressed on malignant B cells in CLL and mediates the rapid internalization and lysosomal shuttling of IgM via its Fc fragment (Fcμ). To exploit this internalization and trafficking pathway for targeted drug delivery, we engineered an IgM-derived protein scaffold (Fcμ) and linked it with the cytotoxic agent monomethylauristatin F. This Fcμ-drug conjugate was selectively toxic for FcμR-expressing cell lines in vitro and for CLL cells but not autologous normal T cells ex vivo. Notably, the cytotoxic activity of the Fcμ-drug conjugate was maintained in CLL cells carrying a 17p deletion, which predicts resistance to standard chemotherapy. Next, we tested the possible therapeutic application of the Fcμ-drug conjugate in immunodeficient NOD/SCID/IL-2Rγ(null) (NSG) mice engrafted with peripheral blood cells from patients with leukemia. Three intravenous injections of the Fcμ-drug conjugate over a 10-day period were well tolerated and selectively killed the human CLL cells but not the coengrafted autologous human T cells. In summary, we developed a novel strategy for targeted cytotoxic therapy of CLL based on the unique properties of FcμR. FcμR-targeted drug delivery showed potent and specific therapeutic activity in CLL, thus providing proof of concept for FcμR as a valuable therapeutic target in CLL and for IgM-based antibody-drug conjugates as a new targeting platform.

Figure 1

Fcμ-Sec engineering and structural formulas of auristatin F derivatives. Schematic of the Fcμ-Sec expression cassettes, which encode A, two or B, three C-terminal constant domains of the heavy chain of human IgM (gray). A C-terminal Sec (red) followed by a His₆ tag (black) was introduced by combining a TGA codon (red) with a 3’UTR that contains a SECIS element. The dominant product of these expression cassettes are pentameric and hexameric (shown) proteins with a single Sec-His₆-displaying C-terminus. (C) We synthesized a tertiary butyl ester of the tubulin polymerization inhibitor monomethyl auristatin F (compound 1a) and its maleimidocaproyl derivative (compound 1b) for site-specific conjugation to the unique selenol group in Fcμ-Sec.

Figure 2

Binding, internalization, and trafficking of Fcμ-Sec. HeLa cells stably transfected with human FcμR cDNA were incubated with 25 μg/mL Cμ3-Cμ4-Sec (Fcμ-Sec) for 30 min at 4°C. After washing, the cells were kept at 4°C or were incubated at 37°C for 30 and 120 min. The cells were then fixed, permeabilized, and co-stained with rabbit anti-human LAMP1 pAbs followed by donkey anti-rabbit IgG pAbs conjugated to Cy5 (red) and donkey anti-human Fcμ pAbs conjugated to DyLight 488 (green). Nuclei were stained with Hoechst 33258 (blue). Stained cells were visualized by confocal immunofluorescence microscopy. Note the co-localization (yellow) of Fcμ-Sec and lysosomal marker LAMP1 following internalization. Scale bars, 10 μm.

Figure 3

In vitro cytotoxicity of Fcμ-drug conjugate. A, FcμR is expressed in mantle cell lymphoma cell line Mino, but not HBL-2, as previously shown (15). Western blot of cell lysates probed with anti FcμR and p97 for loading control (inset box). Viability of Mino (red circles) and HBL-2 cells (purple squares) exposed to free MMAF compound 1a was determined using a colorimetric assay. B, The same experiment was carried out with MMAF compound 1b conjugated to Cμ2-Cμ3-Cμ4-Sec (Fcμ-Sec/drug).

Figure 4

Cell surface expression of FcμR on PBMC samples from CLL patients. PBMC samples from 30 untreated CLL patients were analyzed for cell surface expression of FcμR by flow cytometry. A, histograms comparing FcμR expression (red) by CD19+ malignant B cells (left) and autologous CD3+ T cells (right) of a representative sample. The percentages of FcμR+ cells are indicated in red. The isotype control is shown in blue. B, the specific expression of FcμR by CD3+ T cells and CD19+ malignant B cells is plotted for all 30 PBMC samples (for color-code and patient characteristics, please see Supplementary Table S1). A paired two-tailed t-test was used to calculate P.

Figure 5

Ex vivo cytotoxicity of Fcμ-drug conjugate. PBMC from 30 CLL patients were left untreated (control) or treated with (i) 100 nM Cμ2-Cμ3-Cμ4-Sec (Fcμ-Sec), (ii) 100 nM free MMAF compound 1a (free drug), or (iii) 100 nM MMAF compound 1b conjugated to Cμ2-Cμ3-Cμ4-Sec (Fcμ-Sec/drug). Cell viability was determined by flow cytometry using TO-PRO-3 staining. A, Fcμ-Sec/drug conjugate-mediated cytotoxicity of CD19+ malignant B cells (left panel) and autologous CD3+ T cells (right panel) in PBMC samples from CLL patients (Supplementary Table S1). Paired two-tailed t-tests were used to calculate P. B, Cytotoxicity of the Fcμ-Sec/drug conjugate and expression of FcμR are highly correlated (Spearman correlation: ρ = 0.8431, P < 0.0001). C, and D, the Fcμ-Sec/drug conjugate is equally effective against tumor cells with adverse prognostic features. C, comparison of cytotoxicity against cells with or without a deletion of chromosome 17p (del17p and no del17p, respectively). D, comparison of cytotoxicity against cells of the IGHV mutated (M-CLL) and of the IGHV unmutated CLL subtype (U-CLL).

Figure 6

In vivo cytotoxicity of the Fcμ-Sec/drug conjugate. A total of thirty NSG mice were injected i.v. on day 1 with 5 × 10⁷ PBMC from 4 different CLL patients and treated on days 4, 6, and 8 with 10 mg/kg Fcμ-Sec/drug conjugate in PBS or with PBS alone (control) by i.v. injection. On day 11, the absolute numbers of live (TO-PRO-3-negative) CD19+ malignant B cells and autologous CD3+ T-cells in blood and spleen were quantified by flow cytometry. Two-way ANOVA was used to compare cell numbers in treated and control mice. A, the leukemic cell count in the blood of the xenografted NSG mice is significantly reduced by Fcμ-Sec/drug, while there was no change in T cells numbers. B, live CD19+ malignant B cells relative to the total number of live nucleated cells in the spleen are also greatly reduced by Fcμ-Sec/drug. Again T-cells were unchanged. Note that while the human cells engraft in the spleen, the majority of nucleated cells are of murine origin. C, The column graph summarizes the effect of the Fcμ-Sec/drug conjugate on tumor burden (left panel) and T cells (right panel). Shown is the mean +/− SEM.