Carolyn Cheney

Mcl-1 Is a Relevant Therapeutic Target in Acute and Chronic Lymphoid Malignancies: Down-Regulation Enhances Rituximab-Mediated Apoptosis and Complement-Dependent Cytotoxicity

Purpose: The antiapoptotic Bcl-2 family member protein Mcl-1 is dynamically regulated in transformed B-cells, has a short mRNA and protein half-life, and is rapidly processed during apoptosis. Multiple therapies cause down-regulation of Mcl-1 in chronic and acute lymphoid leukemia (CLL and ALL) cells. Mcl-1 has also been reported to mediate resistance to rituximab in CLL. We therefore investigated whether direct reduction of Mcl-1 was sufficient to induce apoptosis and increase sensitivity to rituximab. Experimental Design: We used Mcl-1–specific small interfering RNA in ALL cell lines and tumor cells from CLL patients to block transcription of Mcl-1. Results: We show that Mcl-1 down-regulation alone is sufficient to promote mitochondrial membrane depolarization and apoptosis in ALL and CLL cells. Given the importance of rituximab in B-cell malignancies, we next assessed the influence of Mcl-1 down-regulation on antibody-mediated killing. Mcl-1 down-regulation by small interfering RNA increased sensitivity to rituximab-mediated killing both by direct apoptosis and complement-dependent cytotoxicity, but did not enhance antibody-dependent cellular cytotoxicity. Conclusions: These results show that Mcl-1 is a relevant therapeutic target for ALL and CLL, and its down-regulation has the potential to enhance the therapeutic effect of rituximab in CD20-bearing lymphoid cells.

Ibrutinib antagonizes rituximab-dependent NK cell–mediated cytotoxicity

To the editor: Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (BTK) with promising activity in CD20+ B-cell malignancies including recent US Food and Drug Administration approval in mantle cell lymphoma.[1][1] Given the homology between BTK and interleukin-2 inducible tyrosine

Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism.

Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cell-membrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab+anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells.

CD19 targeting of chronic lymphocytic leukemia with a novel Fc-domain engineered monoclonal antibody

CD19 is a B cell-specific antigen expressed on chronic lymphocytic leukemia (CLL) cells but to date has not been effectively targeted with therapeutic monoclonal antibodies. XmAb5574 is a novel engineered anti-CD19 monoclonal antibody with a modified constant fragment (Fc)-domain designed to enhance binding of FcgammaRIIIa. Herein, we demonstrate that XmAb5574 mediates potent antibody-dependent cellular cytotoxicity (ADCC), modest direct cytotoxicity, and antibody-dependent cellular phagocytosis but not complement-mediated cytotoxicity against CLL cells. Interestingly, XmAb5574 mediates significantly higher ADCC compared with both the humanized anti-CD19 nonengineered antibody it is derived from and also rituximab, a therapeutic antibody widely used in the treatment of CLL. The XmAb5574-dependent ADCC is mediated by natural killer (NK) cells through a granzyme B-dependent mechanism. The NK cell-mediated cytolytic and secretory function with XmAb5574 compared with the nonengineered antibody is associated with enhanced NK-cell activation, interferon production, extracellular signal-regulated kinase phosphorylation downstream of Fcgamma receptor, and no increased NK-cell apoptosis. Notably, enhanced NK cell-mediated ADCC with XmAb5574 was enhanced further by lenalidomide. These findings provide strong support for further clinical development of XmAb5574 as both a monotherapy and in combination with lenalidomide for the therapy of CLL and related CD19(+) B-cell malignancies.

IL-21 mediates apoptosis through up-regulation of the BH3 family member BIM and enhances both direct and antibody-dependent cellular cytotoxicity in primary chronic lymphocytic leukemia cells in vitro

Interleukin-21 (IL-21) is a recently identified gamma-chain receptor cytokine family member that promotes B-cell apoptosis as well as activation of innate immune system. Based on this, we hypothesized that IL-21 might enhance the apoptosis induced by fludarabine and rituximab and also play a role in augmenting immune-mediated clearance of the chronic lymphocytic leukemia (CLL) cells. Our studies demonstrate that the majority of CLL patients have surface IL-21 receptor-alpha, and its expression correlates with apoptosis, tyrosine phosphorylation of STAT1, and up-regulation of the proapoptotic BH3 domain protein BIM. IL-21-induced BIM up-regulation is critical for apoptosis because inhibition of BIM expression using small interfering RNA prevented IL-21-induced apoptosis. IL-21 treatment of CLL cells but not normal T cells with fludarabine or rituximab additively enhanced the direct cytotoxic effect of these therapies. In addition to its proapoptotic effect, IL-21 promoted STAT1 and STAT5 phosphorylation in natural killer cells with concurrent enhanced antibody-dependent cellular cytotoxicity against rituximab-coated CLL cells in vitro. These data provide justification for combination studies of IL-21 with fludarabine and rituximab in CLL and suggest that BIM up-regulation might serve as relevant pharmacodynamic end point to measure biologic effect of this cytokine in vivo.

Comparative Assessment of Clinically Utilized CD20-Directed Antibodies in Chronic Lymphocytic Leukemia Cells Reveals Divergent NK Cell, Monocyte, and Macrophage Properties

CD20 is a widely validated, B cell–specific target for therapy in B cell malignancies. Rituximab is an anti-CD20 Ab that prolongs survival of chronic lymphocytic leukemia (CLL) patients when combined with chemotherapy. Ofatumumab and GA101 (obinutuzumab) are CD20-directed Abs currently being developed as alternative agents to rituximab in CLL based upon different properties of enhanced direct cell death, NK cell-mediated Ab-dependent cellular cytotoxicity, or complement-dependent cytotoxicity. Despite widespread study, ofatumumab and GA101 have not been compared with each other, nor studied for their interactions with monocytes and macrophages which are critical for the efficacy of anti-CD20 Abs in murine models. In CLL cells, we show that direct cell death and complement-dependent cytotoxicity are greatest with GA101 and ofatumumab, respectively. GA101 promotes enhanced NK cell activation and Ab-dependent cellular cytotoxicity at high Ab concentrations. Ofatumumab elicits superior Ab-dependent cellular phagocytosis with monocyte-derived macrophages. GA101 demonstrated reduced activation of monocytes with diminished pERK, TNF-α release, and FcγRIIa recruitment to lipid rafts. These data demonstrate that GA101 and ofatumumab are both superior to rituximab against CLL cells via different mechanisms of potential tumor elimination. These findings bear relevance to potential combination strategies with each of these anti-CD20 Abs in the treatment of CLL.

Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes

Feline leukaemia viruses (FeLVs) are classified into subgroups A, B and C by their use of different host cell receptors on feline cells, a phenotype which is determined by the viral envelope. FeLV-A is the ubiquitous, highly infectious form of FeLV, and FeLV-C isolates are rare variants which are invariably isolated along with FeLV-A. The FeLV-C isolates share the capacity to induce acute non-regenerative anaemia and the prototype, FeLV-C/Sarma, has strongly age-restricted infectivity for cats. The FeLV-C/Sarma env sequence is closely related to that of common, weakly pathogenic FeLV-A isolates. We now show by construction of chimeric viruses that the receptor specificity of FeLV-A/Glasgow-1 virus can be converted to that of FeLV-C by exchange of a single env variable domain, Vr1, which differs by a three codon deletion and nine adjacent substitutions. Attempts to dissect this region further by directed mutagenesis resulted in disabled proviruses. Sequence analysis of independent natural FeLV-C isolates showed that they have unique Vr1 sequences which are distinct from the conserved FeLV-A pattern. The chimeric viruses which acquired the host range and subgroup properties of FeLV-C retained certain FeLV-A-like properties in that they were non-cytopathogenic in 3201B feline T cells and readily induced viraemia in weanling animals. They also induced a profound anaemia in neonates which had a more prolonged course than that induced by FeLV-C/Sarma and which was macrocytic rather than non-regenerative in nature. Although receptor specificity and a major determinant of pathogenicity segregate with Vr1, it appears that sequences elsewhere in the genome influence infectivity and pathogenicity independently of the subgroup phenotype.

Targeted nanoparticle delivery overcomes off-target immunostimulatory effects of oligonucleotides and improves therapeutic efficacy in chronic lymphocytic leukemia

Several RNA-targeted therapeutics, including antisense oligonucleotides (ONs), small interfering RNAs, and miRNAs, constitute immunostimulatory CpG motifs as an integral part of their design. The limited success with free antisense ONs in hematologic malignancies in recent clinical trials has been attributed to the CpG motif-mediated, TLR-induced prosurvival effects and inefficient target modulation in desired cells. In an attempt to diminish their off-target prosurvival and proinflammatory effects and specific delivery, as a proof of principle, in the present study, we developed an Ab-targeted liposomal delivery strategy using a clinically relevant CD20 Ab (rituximab)-conjugated lipopolyplex nanoparticle (RIT-INP)- and Bcl-2-targeted antisense G3139 as archetypical antisense therapeutics. The adverse immunostimulatory responses were abrogated by selective B cell-targeted delivery and early endosomal compartmentalization of G3139-encapsulated RIT-INPs, resulting in reduced NF-κB activation, robust Bcl-2 down-regulation, and enhanced sensitivity to fludarabine-induced cytotoxicity. Furthermore, significant in vivo therapeutic efficacy was noted after RIT-INP-G3139 administration in a disseminated xenograft leukemia model. The results of the present study demonstrate that CD20-targeted delivery overcomes the immunostimulatory properties of CpG-containing ON therapeutics and improves efficient gene silencing and in vivo therapeutic efficacy for B-cell malignancies. The broader implications of similar approaches in overcoming immunostimulatory properties of RNA-directed therapeutics in hematologic malignancies are also discussed.

The humanized CD40 antibody SGN-40 demonstrates pre-clinical activity that is enhanced by lenalidomide in chronic lymphocytic leukaemia.

Antibody‐based therapies, such as rituximab and alemtuzumab, have contributed significantly to the treatment of Chronic Lymphocytic leukaemia (CLL). The CD40 antigen is expressed predominantly on B‐cells and represents a potential target for immune‐based therapies. SGN‐40 is a humanized IgG1 monoclonal antibody currently in Phase I/II clinical trials for indolent lymphomas, diffuse large B cell lymphomas and Multiple Myeloma. Its biological effect on CLL cells has not been studied. The present study demonstrated that SGN‐40 mediated modest apoptosis in a subset of patients with secondary cross‐linking but did not mediate complement‐dependent cytotoxicity. SGN‐40 also mediated antibody‐dependent cellular cytotoxicity (ADCC) predominantly through natural killer (NK) cells. Previous studies by our group and others have demonstrated that lenalidomide upregulates CD40 expression on primary B CLL cells and activates NK‐cells. We therefore examined for the combinatorial effect of lenalidomide and SGN‐40 and demonstrated that both enhanced direct apoptosis and ADCC against primary CLL B cells. These data together provide justification for clinical trials of SGN‐40 and lenalidomide in combination for CLL therapy.

A feline large granular lymphoma and its derived cell line.

SummaryA lymphoma cell line (MCC) was derived from an abdominal mass from a 13-yr-old castrated male cat. The cells resemble natural killer precursor cells, have membrane-bound granules, and are positive for chloroacetate esterase, α-naphthyl butyrate esterase, and tartrate-resistant acid phosphatase activities. The MCC cells are negative for rearranged feline T-cell receptor genes, negative for feline T-cytotoxic antigen, Ia, and surface μ, τ, and lambda chains and do not form E-rosettes. The MCC cell line is negative for the feline leukemia virus (FeLV); e.g., negative for exogenous FeLV (exU3) sequences, negative for cytoplasmic and surface FeLV major core protein of 27 000 daltons (p27) by indirect, immunofluorescence assay, negative for helper FeLV by clone 81 assay, and negative for release of soluble FeLV p27 by enzyme-linked immunosorbent assay. Electron microscopy reveals budding type C retrovirus particles and MCC cells react with anti-RD-114 (anti-endogenous feline retrovirus) reference serum. After in vitro infection, MCC replicate FeLV readily, but replication is noncytopathic.