Joy Knight

Immunomodulatory drugs stimulate natural killer‐cell function, alter cytokine production by dendritic cells, and inhibit angiogenesis enhancing the anti‐tumour activity of rituximab in vivo

The immunomodulatory drugs (IMiDs) lenalidomide and actimid (also known as CC‐4047) are thalidomide analogues which are more potent than their parental compound. In combination with rituximab, we have previously demonstrated that IMiDs have synergistic in vivo anti‐tumour activity in preclinical studies in a human lymphoma severe combined immunodeficiency mouse model. This report further explored the mechanisms by which IMiDs exert their anti‐lymphoma effects. Following exposure of subcutaneous lymphoma tumours in murine models to IMiDs, there was a significant increase in the recruitment of natural killer (NK) cells to tumour sites. This increase in NK cells was mediated via stimulation of dendritic cells and modification of the cytokine microenvironment associated with an increase in monocyte chemotactic protein‐1, tumour necrosis factor‐α and interferon‐γ and probably augmented rituximab‐associated antibody‐dependent cellular cytotoxicity. IMiDs also had significant anti‐angiogenic effects in our B‐cell lymphoma models. Thus, by modulation of the immune system mediated via dendritic cells and NK cells, changing the cytokine milieu, as well as by their anti‐angiogenic effects, IMiDs in combination with rituximab resulted in augmented in vivo anti‐tumour effects against B‐cell lymphoma. Our positive preclinical data adds additional support for the evaluation of IMiDs plus rituximab in patients with relapsed/refractory B‐cell lymphoma.

Acquirement of Rituximab Resistance in Lymphoma Cell Lines Is Associated with Both Global CD20 Gene and Protein Down-Regulation Regulated at the Pretranscriptional and Posttranscriptional Levels

Acquirement of resistance to rituximab has been observed in lymphoma patients. To define mechanisms associated with rituximab resistance, we developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 expression/structure, lipid raft domain (LRD) reorganization, calcium mobilization, antibody-dependent cellular cytotoxicity, and complement-mediated cytotoxicity (CMC) between parental and RRCL. Significant changes in surface CD20 antigen expression were shown in RRCL. Decreased calcium mobilization and redistribution of CD20 into LRD were found in RRCL. Western blotting identified a unique 35 kDa protein band in RRCL, which was not seen in parental cells and was secondary to an increase in surface and cytoplasmic expression of IgM light chains. CD20 gene expression was decreased in RRCL. In vitro exposure to PS341 increased CD20 expression in RRCL and minimally improved the sensitivity to rituximab-associated CMC. Our data strongly suggest that the acquisition of rituximab resistance is associated with global gene and protein down-regulation of the CD20 antigen affecting LRD organization and downstream signaling. CD20 expression seems to be regulated at the pretranscriptional and posttranscriptional levels. Proteasome inhibition partially reversed rituximab resistance, suggesting the existence of additional mediators of rituximab resistance. Future research is geared to identify drugs and/or biological agents that are effective against RRCL.

Augmented Antitumor Activity against B-Cell Lymphoma by a Combination of Monoclonal Antibodies Targeting TRAIL-R1 and CD20

Purpose: Mapatumumab and lexatumumab are fully humanized, high-affinity immunoglobulin G1λ monoclonal antibodies (mAb) that target/activate the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 1 (TRAIL-R1) and receptor 2 (TRAIL-R2), respectively, triggering the extrinsic apoptotic pathway. Theoretically, synergistic antitumor activity should be observed by combining TRAIL-R mAbs with agents (e.g., rituximab) that activate the intrinsic apoptotic pathway. Experimental Design: To this end, targeted antigen expression in a NHL-cell panel was evaluated by flow cytometry. NHL cells were exposed to mapatumumab or lexatumumab followed by rituximab, isotype, or RPMI. DNA synthesis was quantified by [3H]-thymidine incorporation assays. Induction of apoptosis was detected by flow-cytometric analysis. For antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) studies, standardized 51Cr-release assays were done. We inoculated severe combined immunodeficiency (SCID) mouse with Raji cells i.v. The animals then were treated with various combinations of rituximab, mapatumumab, lexatumumab, and isotype alone or in combination. Results:In vitro exposure to mapatumumab resulted in significant apoptosis (30-50%) and decreased DNA synthesis in sensitive lymphoma cells. Mapatumumab/rituximab combination resulted in a significant inhibition of cell proliferation (90% reduction) when compared with mapatumumab (60% reduction) or rituximab (5% reduction). In vivo, the median survival time of animals treated with mapatumumab and rituximab was longer (not reached) than those treated with rituximab monotherapy [33 days (95% confidence interval, 29-37), log-rank test, P = 0.05]. Conclusions: Mapatumumab induces apoptosis, cell growth arrest, ADCC, and CMC. The combination of mapatumumab plus rituximab is more effective in controlling lymphoma growth in vivo than either antibody. Rituximab and mapatumumab warrant further evaluation against B-cell lymphoma.

Distinct cellular and therapeutic effects of obatoclax in rituximab-sensitive and -resistant lymphomas

Bcl‐2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3‐mimetic, has been designed to specifically target and counteract anti‐apoptotic Bcl‐2 proteins. We evaluated the biological effects of obatoclax on the anti‐tumour activity of rituximab and chemotherapy agents. Obatoclax induced cell death of rituximab/chemotherapy‐sensitive (RSCL), ‐resistant cell lines (RRCL) and primary tumour‐cells derived from patients with B‐cell lymphomas (N = 39). Obatoclax also enhanced the activity of rituximab and had synergistic activity when combined with chemotherapy agents. The ability of Obatoclax to induce PARP cleavage varied between patient samples and was not observed in some RRCL. Inhibition of caspase activity did not affect obatoclax activity, suggesting the existence of caspase‐independent death pathways. Autophagy was detected by LC3 conversion and/or electron microscopy in RRCL and in patient‐derived tumour cells. Moreover, obatoclax activity was inhibited by Beclin‐1 knockdown. In summary, obatoclax is an active Bcl‐2 inhibitor that potentiates the activity of chemotherapy agents and, to a lesser degree, rituximab. Defining the molecular events triggered by obatoclax is necessary to further its clinical development and identify potential biomarkers that are predictive of response.

CD52 over-expression affects rituximab-associated complement-mediated cytotoxicity but not antibody-dependent cellular cytotoxicity: Preclinical evidence that targeting CD52 with alemtuzumab may reverse acquired resistance to rituximab in non-Hodgkin lymphoma

In an attempt to define mechanisms by which B-cell non-Hodgkin lymphoma (NHL) may escape rituximab immunotherapy, we developed several rituximab-resistant cell lines (RRCL) generated from the rituximab-sensitive cell lines (RSCL) Raji and RL. Rituximab resistance was associated with CD20 downregulation and upregulation of CD52 and the complement inhibitory proteins (CIPs) CD55 and CD59. No significant alemtuzumab-associated complement-mediated cell lysis (CMC) or antibody-dependent cellular cytotoxicity (ADCC) was demonstrated in RSCL. In contrast, in vitro exposure of RRCL to alemtuzumab resulted in a significant degree of CMC and ADCC. Of note, in vitro blocking of CD52 with anti-CD52 F(ab’)2 fractions in RRCL improved rituximab-associated CMC as compared to unblocked RRCL. Our current data provides a basis for further evaluation of alemtuzumab-based clinical trials for patients with rituximab-resistant NHL.