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

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.

Lenalidomide down-regulates the CD20 antigen and antagonizes direct and antibody-dependent cellular cytotoxicity of rituximab on primary chronic lymphocytic leukemia cells.

Lenalidomide, an immunomodulatory agent that enhances antibody-dependent cellular cytotoxicity (ADCC), is currently being investigated as a therapy for chronic lymphocytic leukemia (CLL). The anti-CD20 antibody rituximab is active in CLL and represents a rational agent to combine with lenalidomide. We therefore examined whether lenalidomide combined with rituximab enhances direct apoptosis and ADCC in CLL cells. In contrast to previous reports using CD20-positive lymphoma cell lines, lenalidomide down-regulated CD20 surface antigen expression in CLL patient cells via enhanced internalization, without influencing transcription. The CD20 surface antigen internalization enhanced delivery of an oligonucleotide incorporated into anti-CD20 immunoliposomes. In addition, CD20 surface antigen down-modulation by lenalidomide in CLL was accompanied by diminished rituximab-mediated apoptosis and ADCC. These observations suggest a need for alternative sequencing strategies to avoid antagonism between lenalidomide and rituximab therapy in CLL. In addition, they suggest that lenalidomide therapy might be useful to enhance targeted delivery of RNAi-based therapies using CD20 immunoliposomes in B-cell malignancies.

A novel Raji-Burkitt's lymphoma model for preclinical and mechanistic evaluation of CD52-targeted immunotherapeutic agents.

PURPOSE: To date, efforts to study CD52-targeted therapies, such as alemtuzumab, have been limited due to the lack of stable CD52 expressing transformed B-cell lines and animal models. We describe generation and utilization of cell lines that stably express CD52 both in vitro and in vivo. EXPERIMENTAL DESIGN: By limiting dilution, we have established several clones of Raji-Burkitt's lymphoma cell line that express surface CD52. Immunophenotype and cytogenetic characterization of these clones was done. In vivo usefulness of the CD52(high) cell line to evaluate the therapeutic efficacy of CD52-directed antibody was investigated using a SCID mouse xenograft model. RESULTS: Stable expression of CD52 was confirmed in cells cultured in vitro up to 52 weeks of continuous growth. The functional integrity of the expressed CD52 molecule was shown using alemtuzumab, which induced cytotoxic effects in vitro in the CD52(high) but not the CD52(low) clone. Compared with control antibody, alemtuzumab treatment in CD52(high) inoculated mice resulted in significantly increased median survival. Comparable levels of CD52-targeted direct cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cytotoxicity and anti-CD52 immunoliposome-mediated delivery of synthetic oligodeoxyribo nucleotides in CD52(high) clone and primary B-chronic lymphocytic leukemia cells implicated potential in vivo application of this model for evaluation of CD52-targeted antibody and immunoliposomes encapsulating therapeutic agents. CONCLUSIONS: These results show the in vitro utility of the cloned Raji cell lines that stably express high levels CD52. The disseminated leukemia-lymphoma mouse model described herein using these stable cell lines can serve as an excellent system for in vivo therapeutic and mechanistic evaluation of existing and novel antibodies directed against CD52 molecule.

Milatuzumab-conjugated liposomes as targeted dexamethasone carriers for therapeutic delivery in CD74+ B-cell malignancies.

PURPOSE: Corticosteroids are widely used for the treatment of B-cell malignancies, including non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia; however, this class of drug is associated with undesirable off-target effects. Herein, we developed novel milatuzumab-conjugated liposomes as a targeted dexamethasone carrier for therapeutic delivery in CD74(+) B-cell malignancies and explored its effect against the disease. EXPERIMENTAL DESIGN: The targeting efficiency of milatuzumab-targeted liposomes to CD74(+) cells was evaluated in vitro. The effect of CD74-targeted liposomal dexamethasone was compared with free dexamethasone in primary CLL cells and cell lines in vitro. The therapeutic efficacy of CD74-targeted liposomal dexamethasone was evaluated in a Raji-severe combined immunodeficient (SCID) xenograft model in vivo. RESULTS: Milatuzumab-targeted liposomes promoted selective incorporation of carrier molecules into transformed CD74-positive B cells as compared with CD74-negative T-cells. The CD74-dexamethasone-targeted liposomes (CD74-IL-DEX) promoted and increased killing in CD74-positive tumor cells and primary CLL cells. Furthermore, the targeted drug liposomes showed enhanced therapeutic efficacy against a CD74-positive B-cell model as compared with free, or non-targeted, liposomal dexamethasone in SCID mice engrafted with Raji cells in vivo. CONCLUSIONS: These studies provide evidence and support for a potential use of CD74-targeted liposomal dexamethasone as a new therapy for B-cell malignancies.

A novel ultrasensitive hybridization-based ELISA method for 2-methoxyphosphorothiolate microRNAs and its in vitro and in vivo application.

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that bind to target mRNAs and regulate their expression. Recent evidence has indicated the involvement of miRNAs in human malignancies. It has been suggested that aberrantly down-regulated or up-regulated miRNAs may be replaced with synthetic miRNAs or antagomiRNAs, respectively, and restore normal cell functions. As therapeutic development requires analytical support, we developed and validated an ultrasensitive and selective assay for quantification of synthetic 2'-methoxyphosphorothiolate-miRNA in mouse plasma and cell lysate for the first time. The method is based on a hybridization-ligation fluorescence enzyme-linked immunosorbent assay and has provided a linear dynamic range of 10-1,000,000 pM for three synthetic miRNAs both singly and in a mixture. The intra- and inter-day coefficients of variation were <20% and the accuracy values nearly 100%. Using this assay, we performed pharmacokinetic studies of three synthetic miRNAs in mice treated with a single i.v. bolus dose of 7.5 mg kg⁻¹. The 2-methoxyphosphorothiolate-miRNAs reached peak concentrations in the µM and nM ranges in plasma and bone marrow, respectively, and remained measurable at 24 h. These concentrations are in a range that shows biological activities. We conclude that this method provides a general and valuable tool for the pharmacologic study and clinical development of synthetic miRNAs.