Francisco J. Hernandez-Ilizaliturri

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.

Higher response to lenalidomide in relapsed/refractory diffuse large B-cell lymphoma in nongerminal center B-cell-like than in germinal center B-cell-like phenotype.

There is a need to develop novel therapies for relapsed/refractory diffuse large B‐cell lymphoma (DLBCL) and to identify biomarkers predictive for therapeutic response. Lenalidomide was previously shown to induce an overall response rate (ORR) of 28% in patients with relapsed/refractory DLBCL. It is currently unknown if response rates differ between patients with different DLBCL subtypes.

Properties and structure-function relationships of veltuzumab (hA20), a humanized anti-CD20 monoclonal antibody

Veltuzumab is a humanized anti-CD20 monoclonal antibody with complementarity-determining regions (CDRs) identical to rituximab, except for one residue at the 101st position (Kabat numbering) in CDR3 of the variable heavy chain (V(H)), having aspartic acid (Asp) instead of asparagine (Asn), with framework regions of epratuzumab, a humanized anti-CD22 antibody. When compared with rituximab, veltuzumab has significantly reduced off-rates in 3 human lymphoma cell lines tested, as well as increased complement-dependent cytotoxicity in 1 of 3 cell lines, but no other in vitro differences. Mutation studies confirmed that the differentiation of the off-rate between veltuzumab and rituximab is related to the single amino acid change in CDR3-V(H). Studies of intraperitoneal and subcutaneous doses in mouse models of human lymphoma and in normal cynomolgus monkeys disclosed that low doses of veltuzumab control tumor growth or deplete circulating or sessile B cells. Low- and high-dose veltuzumab were significantly more effective in vivo than rituximab in 3 lymphoma models. These findings are consistent with activity in patients with non-Hodgkin lymphoma given low intravenous or subcutaneous doses of veltuzumab. Thus, changing Asn(101) to Asp(101) in CDR3-V(H) of rituximab is responsible for veltuzumabs lower off-rate and apparent improved potency in preclinical models that could translate into advantages in patients.

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.

Immunomodulatory Drug CC-5013 or CC-4047 and Rituximab Enhance Antitumor Activity in a Severe Combined Immunodeficient Mouse Lymphoma Model

New thalidomide derivatives CC-5013 and CC-4047 (immunomodulatory drugs, IMiD) are up to 10,000 times more potent than Thalidomide. The biological effects of IMiDs are presumed to be mediated by (a) activation of some components of the innate [natural killer (NK) cells] or adoptive immune system (T cells), (b) modification of cytokine microenvironment in the tumor bed, or by (c) inhibition of angiogenesis. In this article, we tested an innovative combination strategy involving rituximab and IMiDs in aggressive lymphoma cell lines and human lymphoma xenografts. Treatment of non-Hodgkins lymphoma cells with CC-5013 resulted in a 40% to 70% growth inhibition when compared with controls (P < 0.05). Exposure of lymphoma cells to CC-4047 resulted in a lesser degree of growth inhibition. Induction of apoptosis was shown in 10% to 26% of lymphoma cells 24 hours following exposure to either IMiD. In vivo studies in severe combined immunodeficient mice showed synergistic activity between CC-4047 (and to a lesser degree, CC-5013) plus rituximab. Animals treated with the CC-4047/rituximab combination had a median survival of 74 days (P = 0.0012) compared with 58 days (P = 0.167) in CC-5013/rituximab-treated animals compared with 45 days in rituximab monotherapy–treated animals. The synergistic effect between IMiDs and rituximab in our mouse model was attributed to NK cell expansion. The enhancement of rituximab activity by IMiDs was abrogated by in vivo depletion of NK cells. Augmenting NK cell function by CC-4047 or CC-5013 exposure may increase the antitumor effects of rituximab against B-cell lymphomas and warrants further exploration in the context of a clinical trial.

Impact of induction regimen and stem cell transplantation on outcomes in double-hit lymphoma: A multicenter retrospective analysis

Patients with double-hit lymphoma (DHL), which is characterized by rearrangements of MYC and either BCL2 or BCL6, face poor prognoses. We conducted a retrospective multicenter study of the impact of baseline clinical factors, induction therapy, and stem cell transplant (SCT) on the outcomes of 311 patients with previously untreated DHL. At median follow-up of 23 months, the median progression-free survival (PFS) and overall survival (OS) rates among all patients were 10.9 and 21.9 months, respectively. Forty percent of patients remain disease-free and 49% remain alive at 2 years. Intensive induction was associated with improved PFS, but not OS, and SCT was not associated with improved OS among patients achieving first complete remission (P = .14). By multivariate analysis, advanced stage, central nervous system involvement, leukocytosis, and LDH >3 times the upper limit of normal were associated with higher risk of death. Correcting for these, intensive induction was associated with improved OS. We developed a novel risk score for DHL, which divides patients into high-, intermediate-, and low-risk groups. In conclusion, a subset of DHL patients may be cured, and some patients may benefit from intensive induction. Further investigations into the roles of SCT and novel agents are needed.

Chemoimmunotherapy with O-FC in previously untreated patients with chronic lymphocytic leukemia.

We conducted an international phase 2 trial to evaluate 2 dose levels of ofatumumab, a human CD20 mAb, combined with fludarabine and cyclophosphamide (O-FC) as frontline therapy for chronic lymphocytic leukemia (CLL). Patients with active CLL were randomized to ofatumumab 500 mg (n = 31) or 1000 mg (n = 30) day 1, with fludarabine 25 mg/m(2) and cyclophosphamide 250 mg/m(2) days 2-4, course 1; days 1-3, courses 2-6; every 4 weeks for 6 courses. The first ofatumumab dose was 300 mg for both cohorts. The median age was 56 years; 13% of patients had a 17p deletion; 64% had β2-microglobulin > 3.5 mg/L. Based on the 1996 National Cancer Institute Working Group (NCI-WG) guidelines, the complete response (CR) rate as assessed by an independent review committee was 32% for the 500-mg and 50% for the 1000-mg cohort; the overall response (OR) rate was 77% and 73%, respectively. Based on univariable regression analyses, β2-microglobulin and the number of O-FC courses were significantly correlated (P < .05) with CR and OR rates and progression-free survival (PFS). The most frequent Common Terminology Criteria (CTC) grade 3-4 investigator-reported adverse events were neutropenia (48%), thrombocytopenia (15%), anemia (13%), and infection (8%). O-FC is active and safe in treatment-naive patients with CLL, including high-risk patients. This trial was registered at www.clinicaltrials.gov as NCT00410163.

Efficacy of lenalidomide in patients with chronic lymphocytic leukemia with high-risk cytogenetics.

Patients with chronic lymphocytic lymphoma (CLL) with high-risk cytogenetics [del(11q)(q22.3) or del(17p)(p13.1)] have limited therapeutic options and their prognosis remains poor. This analysis was conducted to determine the clinical activity of lenalidomide in patients with high-risk disease. Relapsed/refractory patients with CLL enrolled in a phase II clinical trial who had del(11q)(q22.3) or del(17p)(p13.1) were included in this analysis. Patients received single agent lenalidomide for 21 days of the 4 week treatment cycle. The overall response rate among patients with high-risk cytogenetics was 38%, with 19% of patients achieving a complete response. Median progression-free survival was 12.1 months, which is higher than demonstrated with other agents in comparable patient populations. In addition, the estimated 2-year survival probability was 58%, demonstrating that the responses achieved with lenalidomide are durable, even in patients with CLL with high-risk disease with poor risk cytogenetics.

Potent antitumor activity of the anti-CD19 auristatin antibody-drug conjugate hBU12-vcMMAE against rituximab sensitive and resistant lymphomas

Despite major advances in the treatment of non-Hodgkin lymphoma (NHL), including the use of chemotherapeutic agents and the anti-CD20 antibody rituximab, the majority of patients eventually relapse, and salvage treatments with non-cross-resistant compounds are needed to further improve patient survival. Here, we evaluated the antitumor effects of the microtubule destabilizing agent monomethyl auristatin E (MMAE) conjugated to the humanized anti-CD19 antibody hBU12 via a protease-sensitive valine-citrulline (vc) dipeptide linker. hBU12-vcMMAE induced potent tumor cell killing against rituximab-sensitive and -resistant NHL cell lines. CD19 can form heterodimers with CD21, and high levels of CD21 were reported to interfere negatively with the activity of CD19-targeted therapeutics. However, we observed comparable internalization, intracellular trafficking, and drug release in CD21(low) and CD21(high), rituximab-sensitive and -refractory lymphomas treated with hBU12-vcMMAE. Furthermore, high rates of durable regressions in mice implanted with these tumors were observed, suggesting that both rituximab resistance and CD21 expression levels do not impact on the activity of hBU12-vcMMAE. Combined, our data suggest that hBU12-vcMMAE may represent a promising addition to the treatment options for rituximab refractory NHL and other hematologic malignancies, including acute lymphoblastic leukemia.

Acquired resistance to rituximab is associated with chemotherapy resistance resulting from decreased Bax and Bak expression.

Purpose: Targeting malignant B cells using rituximab (anti-CD20) has improved the efficacy of chemotherapy regimens used to treat patients with non-Hodgkins lymphoma. Despite the promising clinical results obtained using rituximab, many patients relapse with therapy-resistant disease following rituximab-based treatments. We have created a cell line model of rituximab resistance using three B-cell non-Hodgkins lymphoma–derived cell lines (Raji, RL, and SUDHL-4). In an attempt to define strategies to overcome rituximab resistance, we sought to determine the chemotherapy sensitivity of our rituximab-resistant cell lines (RRCL). Experimental Design: Parental, rituximab-sensitive cell lines (RSCL) Raji, RL, and SUDHL-4, along with RRCLs derived from them, were exposed to several chemotherapeutic agents with different mechanisms of action and the ability of these agents to induce apoptotic cell death was measured. Expression of multidomain Bcl-2 family proteins was studied as potential mediators of chemotherapy/rituximab resistance. Results: We found that RRCLs are resistant to multiple chemotherapeutic agents and have significantly decreased expression of the Bcl-2 family proteins Bax, Bak, and Bcl-2. RRCLs do not undergo rituximab- or chemotherapy-induced apoptosis but die in a caspase-dependent manner when either wild-type Bax or Bak is exogenously expressed. Furthermore, forced expression of Bak sensitized RRCL to chemotherapy-induced apoptosis. Conclusions: Whereas a single or limited exposure of lymphoma cells to rituximab may lead to a favorable ratio of proapoptotic to antiapoptotic Bcl-2 family proteins, repeated exposure to rituximab is associated with a therapy-resistant phenotype via modulation of Bax and Bak expression.