Milatuzumab immunoliposomes induce cell death in CLL by promoting accumulation of CD74 on the surface of B cells

Abstract

Chronic lymphocytic leukemia (CLL) is an incurable progressive disease for which new therapies are required. Therapy with monoclonal antibodies (mAbs) has improved the outcome of patients with CLL, making further investigation of novel antibodies directed against alternative and specific targets on B cells an important area of translational research. We now describe functional properties of an antagonistic humanized mAb to CD74, milatuzumab, showing that milatuzumab combined with a crosslinking antibody induces cytotoxicity in vitro in CLL cells in a caspase- and stromal-independent manner associated with aggregation of CD74 on the cell surface. Furthermore, incorporation of milatuzumab into an immunoliposome induces even more of a cytotoxic response than in vitro crosslinking, representing a novel therapeutic formulation for this mAb. Based on these data, future development of the milatuzumab-immunoliposome formulation as a therapeutic agent for CLL is warranted.

Figures

Figure 1

Milatuzumab mediates direct cytotoxicity in CLL cells. (A) Viability of chronic lymphocytic leukemia (CLL) patient cells at 4, 24, and 48 hours by propidium iodide (PI) staining. Cells were untreated, treated with goat anti–human Fc crosslinker alone, or treated with 5 μg/mL milatuzumab (Mila) or 10 μg/mL rituximab (Ritux) in the presence or absence of Fc crosslinker (in 5 × excess of binding antibody; N = 26; P < .0001 for anti-Fc vs mila + anti-Fc). Y-axis indicates the percent of PI positive cells and the active metabolite of fludarabine (2-F-ara A; Flud) was used as a positive control for cell death. (B) Viability of CLL patient cells at 24 hours by PI staining (N = 26 from panel A; each line represents individual patient sample). (C) Immunoblots for caspases-2, -3, -8, -6, -9, and PARP in whole cell lysates isolated from CLL cells treated with anti-Fc, 5 μg/mL mila + anti-Fc, or 5μM fludarabine in the presence or absence of 20μM caspase inhibitor, Q-VD-OPH, for 24 hours. Jurkat cells treated with ultraviolet (UV) or staurosporine were used as controls for caspase cleavage. Pro-caspase (P) and cleaved (C) forms are indicated. (D) Viability of CLL patient cells by PI staining treated with anti-Fc, 5 μg/mL mila + anti-Fc, or 5μM fludarabine in the presence or absence of 20μM caspase inhibitor, Q-VD-OPH, for 24 hours (N = 5; P = .03). (Ei) Viability of CLL patient cells treated with anti-Fc alone or 5 μg/mL mila + anti-Fc in the presence or absence of stroma coculture. After 6 hours in drug, cells were washed and cultured in fresh media alone or in the presence of HS-5 stromal cells. Viability was determined by PI staining after 48 hours of coculture (N = 11; P = .10). (ii) Viability of CLL patient cells treated with vehicle or 1μM 2-F-ara A in the presence or absence of stroma coculture. After 6 hours in drug cells were washed and cultured in fresh media alone or in the presence of HS-5 cells. Viability by PI staining was determined after 48 hours of coculture (N = 6). (iii) Viability of CLL patient cells treated with anti-Fc alone or 5 μg/mL mila + anti-Fc in the presence or absence of 500 ng/mL soluble CD40L. Viability by PI staining was determined at 48 hours (N = 17; P = .11).

Figure 2

Milatuzumab immunoliposome increases CD74 on the surface of CLL cells and induces cell death. (A) Mean fluorescent intensity (MFI) of surface CD74 in CLL patient cells either untreated, treated with 25 μg/mL anti-Fc, or 5 μg/mL milatuzumab with or without 25 μg/mL anti-Fc for 1 hour. Histogram shown is representative of 14 patients. (B left) Mean fluorescent intensity of CD74 in CLL patient cells either untreated, treated with 25 μg/mL anti-Fc, or 5 μg/mL milatuzumab with or without 25 μg/mL anti-Fc for 1 hour (N = 14; P = .0003). (Right) MFI of CD20 in CLL patient cells either untreated, or treated with 5 μg/mL milatuzumab with or without 25 μg/mL anti-Fc for 1 hour (N = 14; P = .14). (C) MFI of CD74 in CLL patient cells either untreated, or treated with empty liposome or milatuzumab-immunoliposome (mila-IL) for 1 hour (N = 4; P = .0003). (D) Viability by PI staining in CLL patient cells either untreated, or treated with empty liposome, IgG-immunoliposome (IgG-IL), mila-IL, 25 μg/mL anti-Fc, or 5 μg/mL milatuzumab with 25 μg/mL anti-Fc for 24 hours (N = 11; P = .0003).