Berris van Kessel

Towards effective immunotherapy of myeloma: enhanced elimination of myeloma cells by combination of lenalidomide with the human CD38 monoclonal antibody daratumumab

Background In our efforts to develop novel effective treatment regimens for multiple myeloma we evaluated the potential benefits of combining the immunomodulatory drug lenalidomide with daratumumab. Daratumumab is a novel human CD38 monoclonal antibody which kills CD38+ multiple myeloma cells via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis. Design and Methods To explore the effect of lenalidomide combined with daratumumab, we first carried out standard antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity assays in which the CD38+ multiple myeloma cell line UM-9 and primary multiple myeloma cells isolated from patients were used as target cells. We also tested the effect of lenalidomide on daratumumab-dependent cell-mediated-cytotoxicity and complement-dependent cytotoxicity of multiple myeloma cells directly in the bone marrow mononuclear cells of multiple myeloma patients. Finally, we determined the daratumumab-dependent cell-mediated cytotoxicity using peripheral blood mononuclear cells of multiple myeloma patients receiving lenalidomide treatment. Results Daratumumab-dependent cell-mediated cytotoxicity of purified primary multiple myeloma cells, as well as of the UM-9 cell line, was significantly augmented by lenalidomide pre-treatment of the effector cells derived from peripheral blood mononuclear cells from healthy individuals. More importantly, we demonstrated a clear synergy between lenalidomide and daratumumab-induced antibody-dependent cell-mediated cytotoxicity directly in the bone marrow mononuclear cells of multiple myeloma patients, indicating that lenalidomide can also potentiate the daratumumab-dependent lysis of myeloma cells by activating the autologous effector cells within the natural environment of malignant cells. Finally, daratumumab-dependent cell-mediated cytotoxicity was significantly up-regulated in peripheral blood mononuclear cells derived from 3 multiple myeloma patients during lenalidomide treatment. Conclusions Our results indicate that powerful and complementary effects may be achieved by combining lenalidomide and daratumumab in the clinical management of multiple myeloma.

Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity

The replacement of bone marrow (BM) as a conventional source of stem cell (SC) by umbilical cord blood (UCB) and granulocyte‐colony stimulating factor‐mobilized peripheral blood SC (PBSC) has brought about clinical advantages. However, several studies have demonstrated that UCB CD34+ cells and PBSC significantly differ from BM CD34+ cells qualitatively and quantitatively. Here, we quantified the number of SC in purified BM, UCB CD34+ cells, and CD34+ PBSC using in vitro and in vivo assays for human hematopoietic SC (HSC) activity. A cobblestone area‐forming cell (CAFC) assay showed that UCB CD34+ cells contained the highest frequency of CAFCwk6 (3.6‐ to tenfold higher than BM CD34+ cells and PBSC, respectively), and the engraftment capacity in vivo by nonobese diabetic/severe combined immunodeficiency repopulation assay was also significantly greater than BM CD34+, with a higher proportion of CD45+ cells detected in the recipients at a lower cell dose. To understand the molecular characteristics underlying these functional differences, we performed several DNA microarray experiments using Affymetrix gene chips, containing 12,600 genes. Comparative analysis of gene‐expression profiles showed differential expression of 51 genes between BM and UCB CD34+ SC and 64 genes between BM CD34+ cells and PBSC. These genes are involved in proliferation, differentiation, apoptosis, and engraftment capacity of SC. Thus, the molecular expression profiles reported here confirmed functional differences observed among the SC sources. Moreover, this report provides new insights to describe the molecular phenotype of CD34+ HSC and leads to a better understanding of the discrepancy among the SC sources.

Preclinical evidence for the therapeutic potential of CD38-targeted immuno-chemotherapy in multiple myeloma patients refractory to lenalidomide and bortezomib

Purpose: Novel therapeutic agents have significantly improved the survival of patients with multiple myeloma. Nonetheless, the prognosis of patients with multiple myeloma who become refractory to the novel agents lenalidomide and bortezomib is very poor, indicating the urgent need for new therapeutic options for these patients. The human CD38 monoclonal antibody daratumumab is being evaluated as a novel therapy for multiple myeloma. Prompted with the encouraging results of ongoing clinical phase I/II trials, we now addressed the potential value of daratumumab alone or in combination with lenalidomide or bortezomib for the treatment of lenalidomide- and bortezomib-refractory patients. Experimental Design: In ex vivo assays, mainly evaluating antibody-dependent cell-mediated cytotoxicity, and in an in vivo xenograft mouse model, we evaluated daratumumab alone or in combination with lenalidomide or bortezomib as a potential therapy for lenalidomide- and bortezomib-refractory multiple myeloma patients. Results: Daratumumab induced significant lysis of lenalidomide/bortezomib-resistant multiple myeloma cell lines and of primary multiple myeloma cells in the bone marrow mononuclear cells derived from lenalidomide- and/or bortezomib-refractory patients. In these assays, lenalidomide but not bortezomib, synergistically enhanced daratumumab-mediated multiple myeloma lysis through activation of natural killer cells. Finally, in an in vivo xenograft model, only the combination of daratumumab with lenalidomide effectively reduced the tumorigenic growth of primary multiple myeloma cells from a lenalidomide- and bortezomib-refractory patient. Conclusions: Our results provide the first preclinical evidence for the benefit of daratumumab plus lenalidomide combination for lenalidomide- and bortezomib-refractory patients. Clin Cancer Res; 21(12); 2802–10. ©2014 AACR. See related commentary by Laubach and Richardson, p. 2660

Daratumumab-mediated lysis of primary multiple myeloma cells is enhanced in combination with the human anti-KIR antibody IPH2102 and lenalidomide

Despite recent treatment improvements, multiple myeloma remains an incurable disease. Since antibody-dependent cell-mediated cytotoxicity is an important effector mechanism of daratumumab, we explored the possibility of improving daratumumab-mediated cell-mediated cytotoxicity by blocking natural killer cell inhibitory receptors with the human monoclonal anti-KIR antibody IPH2102, next to activation of natural killer cells with the immune modulatory drug lenalidomide. In 4-hour antibody-dependent cell-mediated cytotoxicity assays, IPH2102 did not induce lysis of multiple myeloma cell lines, but it did significantly augment daratumumab-induced myeloma cell lysis. Also in an ex vivo setting, IPH2102 synergistically improved daratumumab-dependent lysis of primary myeloma cells in bone marrow mononuclear cells (n=21), especially in patients carrying the FcγRIIIa-158F allele or the FcγRIIa-131R allele, who bind IgG1 with lower affinity than patients carrying the FcγRIIIa-158V allele or the FcγRIIa-131H allele. Finally, a further synergistically improved myeloma cell lysis with the daratumumab-IPH2102 combination was observed by adding lenalidomide, which suggests that more effective treatment strategies can be designed for multiple myeloma by combining daratumumab with agents that independently modulate natural killer cell function.

Inhibition of the mevalonate pathway potentiates the effects of lenalidomide in myeloma.

The effects of the combination of simvastatin and lenalidomide were analyzed in myeloma. Myeloma cell lines and patient myeloma cells were incubated with different concentrations of lenalidomide, simvastatin, or the combination. Co exposure to simvastatin and lenalidomide resulted in a synergistic reduction of cell viability in myeloma cells. This effect was due to induction of apoptosis and inhibition of proliferation. The combination augmented induction of caspase-8 cleavage and enhanced down-regulation of pStat3. Mevalonate and GGOH abrogated the synergy between lenalidomide and simvastatin. These data provide a rationale for the clinical evaluation of lenalidomide and simvastatin in patients with myeloma.

Selective in vitro expansion and efficient retroviral transduction of human CD34+ CD38- haematopoietic stem cells.

Summary.  Ex vivo expansion of primitive human haematopoietic stem cells (HSC) is clinically relevant for stem cell transplantation and gene therapy. Here, we demonstrate the selective expansion of CD34+CD38– cells from purified CD34+ cells upon stimulation with Flt3‐ligand, stem cell factor and thrombopoietin. Over a 100‐fold (range 80 to 128‐fold) expansion of CD34+CD38– cells was observed with bone marrow and cord blood (CB). The expanded CD34+CD38– cells remained negative for lineage‐specific markers and could be induced to differentiate into granulocytes, monocytes, megakaryocytes, erythrocytes, and T and B‐lymphocytes in vitro. Lineage differentiation assays with single CD34+CD38– cells showed no loss of multilineage potential of expanded cells after ex vivo culture. We also demonstrated that the increase in frequency of CD34+CD38– cells was not as a result of the downregulation of CD38 expression during the culture. Quantitative analysis showed that the number of 6 week cobblestone area forming cells (CAFCwk6), a measure of proliferating HSC, in cytokine‐stimulated CD34+ cells were increased by 20‐fold. Expanded CD34+CD38– cells could be transduced efficiently with retroviruses encoding the low affinity nerve growth factor receptor (LNGFR) marker gene (17% to 44%, mean 27%), resulting in long‐lasting expression of retroviral‐encoded genes in progeny HSC and differentiated progenitors. We conclude that the combination Flt3‐ligand (FL), stem cell factor and thrombopoietin (TPO) induced strong ex vivo proliferation of CD34+CD38– cells and that the absolute number of expanded cells with stem cell activity increased substantially in this population.

Immunoglobulin gene analysis in polyneuropathy associated with IgM monoclonal gammopathy

Antineural antibody activity is the implicated pathogenic mechanism in polyneuropathy associated with monoclonal gammopathy. Recognition of antigen depends on immunoglobulin variable regions, encoded by V genes. We studied V(H)DJ(H) and V(L)J(L) gene use in monoclonal B cells by clonal analysis in 20 patients with polyneuropathy and IgM monoclonal gammopathy. V genes associated with bacterial responses appear over-represented and V(H)3-23 was preferentially used, without association with specific D, J(H) or V(L)J(L). V genes revealed somatic mutation and intraclonal variation was found in 9 of 20 patients. Polyneuropathy associated with monoclonal gammopathy may be caused by an immune response to bacterial antigens, which recruit somatically mutated autoreactive B cells.

Cytogenetic aberrations in neuropathy associated with IgM monoclonal gammopathy

The occurrence and nature of cytogenetic aberrations in polyneuropathy associated with IgM monoclonal gammopathy was determined. Therefore, interphase fluorescence in situ hybridization (FISH) was applied in 22 patients with polyneuropathy associated with IgM monoclonal gammopathy, multiplex ligation-dependent probe amplification (MLPA) assay in 18 of these patients and genome-wide-array-based comparative genomic hybridization (CGH) in eight of these 18 patients. Four patients had 10-20% and one patient had 30% B cells with IgH rearrangements; one patient had additional loss of 14qter; one patient had amplification of 6p and loss of 6q. Cytogenetic aberrations may be found in one third of the patients with neuropathy associated with IgM monoclonal gammopathy and are mainly associated with indolent Waldenstroms Macroglobulinemia.

Lenalidomide combined with low-dose cyclophosphamide and prednisone modulates Ikaros and Aiolos in lymphocytes, resulting in immunostimulatory effects in lenalidomide-refractory multiple myeloma patients

We recently showed that the outcome of multiple myeloma (MM) patients treated in the REPEAT study (evaluation of lenalidomide combined with low-dose cyclophosphamide and prednisone (REP) in lenalidomide-refractory MM) was markedly better than what has been described with cyclophosphamide-prednisone alone. The outcome with REP was not associated with plasma cell Cereblon expression levels, suggesting that the effect of REP treatment may involve mechanisms independent of plasma cell Cereblon-mediated direct anti-tumor activity. We therefore hypothesized that immunomodulatory effects contribute to the anti-MM activity of REP treatment, rather than plasma cell Cereblon-mediated effects. Consequently, we now characterized the effect of REP treatment on immune cell subsets in peripheral blood samples collected on day 1 and 14 of cycle 1, as well as on day 1 of cycle 2. We observed a significant mid-cycle decrease in the Cereblon substrate proteins Ikaros and Aiolos in diverse lymphocyte subsets, which was paralleled by an increase in T-cell activation. These effects were restored to baseline at day one of the second cycle, one week after lenalidomide interruption. In vitro, lenalidomide enhanced peripheral blood mononuclear cell-mediated killing of both lenalidomide-sensitive and lenalidomide-resistant MM cells in a co-culture system. These results indicate that the Cereblon-mediated immunomodulatory properties of lenalidomide are maintained in lenalidomide-refractory MM patients and may contribute to immune-mediated killing of MM cells. Therefore, combining lenalidomide with other drugs can have potent effects through immunomodulation, even in patients considered to be lenalidomide-refractory.

Abstract A12: Combination of the anti-CD38 monoclonal antibody daratumumab and all-trans retinoic acid.

Background: Daratumumab (DARA) is an anti-CD38 monoclonal antibody (mAb) with lytic activity on multiple myeloma (MM) cells, including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). In current clinical phase I/II trials, DARA induced anti-MM activity; however, the depth of the response varied between patients. Further improvement of DARA-treatment can be achieved by modulating the mechanisms hampering DARA responsiveness. Results: In a sub-analysis of 16 MM patients treated with DARA monotherapy (GEN501 trial), we observed that response to DARA was associated with baseline CD38 expression levels on the MM cells (R2= 0.40; P= 0.009). We also observed a significant decrease of CD38 expression on MM cells during DARA-treatment (median MFI decreased from 900 to 83). Consistent with this idea, in vitro experiments with isogenic MM cell lines expressing different levels of CD38 have revealed that the level of CD38 expression correlates with DARA-mediated ADCC and CDC. Similarly, in bone marrow samples from 125 and 56 MM patients, we observed a significant correlation between CD38 expression and DARA-mediated ADCC (Pearson R= 0.34; P= 0. Indeed, all-trans retinoic acid (ATRA) induced a ~5-fold increase in CD38 expression both in 4 MM cell lines and primary MM cells from 5 DARA-naive patients, which resulted in synergistic improvement of DARA-mediated ADCC and CDC. The synergy between ATRA and DARA was observed not only in DARA-naive patients, but also in 2 DARA-treated patients, whose residual MM cells had lower CD38 expression following therapy. Conclusion: Our results provide evidence that CD38 expression levels may predict response to DARA. Furthermore, we show that ATRA increases CD38 expression on MM cells, resulting in enhanced DARA-mediated lysis of MM cells. Our results provide the preclinical rationale for further evaluation of DARA combined with ATRA in MM patients. Citation Format: Inger S. Nijhof, Henk M. Lokhorst, Berris van Kessel, Parul Doshi, Kate Sasser, Tuna Mutis, Niels WCJ van de Donk. Combination of the anti-CD38 monoclonal antibody daratumumab and all-trans retinoic acid. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A12.