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Purpose: Daratumumab treatment results in a marked reduction of CD38 expression on multiple myeloma cells. The aim of this study was to investigate the clinical implications and the underlying mechanisms of daratumumab-mediated CD38 reduction. Experimental Design: We evaluated the effect of daratumumab alone or in combination with lenalidomide-dexamethasone, on CD38 levels of multiple myeloma cells and nontumor immune cells in the GEN501 study (daratumumab monotherapy) and the GEN503 study (daratumumab combined with lenalidomide-dexamethasone). In vitro assays were also performed. Results: In both trials, daratumumab reduced CD38 expression on multiple myeloma cells within hours after starting the first infusion, regardless of depth and duration of the response. In addition, CD38 expression on nontumor immune cells, including natural killer cells, T cells, B cells, and monocytes, was also reduced irrespective of alterations in their absolute numbers during therapy. In-depth analyses revealed that CD38 levels of multiple myeloma cells were only reduced in the presence of complement or effector cells, suggesting that the rapid elimination of CD38 high multiple myeloma cells can contribute to CD38 reduction. In addition, we discovered that daratumumab-CD38 complexes and accompanying cell membrane were actively transferred from multiple myeloma cells to monocytes and granulocytes. This process of trogocytosis was also associated with reduced surface levels of some other membrane proteins, including CD49d, CD56, and CD138. Conclusions: Daratumumab rapidly reduced CD38 expression levels, at least in part, through trogocytosis. Importantly, all these effects also occurred in patients with deep and durable responses, thus excluding CD38 reduction alone as a mechanism of daratumumab resistance.The trials were registered at www.clinicaltrials.gov as NCT00574288 (GEN501) and NCT1615029 (GEN503). Clin Cancer Res; 23(24); 7498-511. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: The essential role of CD4(+) T cells as helpers of anticancer immunity is indisputable. Little is known, however, about their capacity to serve as effector cells in cancer treatment. Therefore, we explored the efficacy of immunotherapy with sole CD4(+) cytotoxic human T cells directed at a hematopoietic-restricted minor histocompatibility antigen (mHag)., Experimental Design: In macrophage-depleted Rag2(-/-)γc(-/-) mice, which were also devoid of T, B, and natural killer cells, mHag-specific native T cells or tetanus toxoid (TT)-specific T cells transduced with the mHag-specific T-cell receptor (TCR) were injected to treat full-blown mHag(+) human multiple myeloma tumors., Results: mHag-specific antitumor responses were achieved after injection of native or mHag-TCR-transduced T cells. Although the therapy completely eradicated the primary tumors in the bone marrow, it failed to control extramedullary relapses, even after repeated T-cell injections. Detailed analyses ruled out mHag or MHC downregulation as mechanisms of extramedullary tumor escape. Impaired T-cell survival in vivo or defective homing to the tumor site were also ruled out as mechanisms behind extramedullary relapses, because injections of TT-loaded antigen presenting cells could facilitate homing of long-term surviving T cells to s.c. tumor sites. Moreover, intratumoral treatment of extramedullary tumors with 3AB11 was also ineffective., Conclusions: Taken together, these results for the first time show the feasibility of immunotherapy of primary bone marrow tumors with sole CD4(+) human T cells directed to a tumor-associated mHag. Extramedullary relapses, probably due to microenvironment-dependent inhibitory mechanisms, remain a challenging issue towards effective cellular immunotherapy of hematologic malignancies., (©2010 AACR.)

Purpose: Identification of minor histocompatibility antigens (mHag) with classic methods often requires sophisticated technologies, determination, and patience. We here describe and validate a nonlaborious and convenient genetic approach, based on genome-wide correlations of mHag zygosities with HapMap single-nucleotide polymorphism genotypes, to identify clinical relevant mHags within a reasonable time frame., Experimental Design: Using this approach, we sought for the mHag recognized by a HLA-DRB1*1501-restricted T-cell clone, isolated from a multiple myeloma patient during a strong graft-versus-tumor effect associated with acute graft-versus-host disease grade 3., Results: In a period of 3 months, we determined the mHag phenotype of 54 HapMap individuals, deduced the zygosity of 20 individuals, defined the mHag locus by zygosity-genotype correlation analyses, tested the putative mHag peptides from this locus, and finally showed that the mHag is encoded by the arginine (R) allele of a nonsynonymous single-nucleotide polymorphism in the SLC19A1 gene., Conclusions: We conclude that this powerful and convenient strategy offers a broadly accessible platform toward rapid identification of mHags associated with graft-versus-tumor effect and graft-versus-host disease.

Purpose: Prenylation is essential for membrane localization and participation of proteins in various signaling pathways. This study examined the role of farnesylated and geranylgeranylated proteins in the regulation of myeloma cell proliferation., Experimental Design: Antiproliferative and apoptotic effects of various modulators of farnesylated and geranylgeranylated proteins were investigated in myeloma cells., Results: Depletion of geranylgeranylpyrophosphate inhibited myeloma cell proliferation through accumulation of cells in G(1) phase of the cell cycle and loss of cells in S phase. In contrast, depletion of farnesylpyrophosphate had no or only minor effects. Furthermore, inhibition of geranylgeranyl transferase I activity was more effective in reducing myeloma cell growth when compared with inhibition of farnesyl transferase activity. This indicates that protein geranylgeranylation is important for myeloma cell proliferation and cell cycle progression through G(1). Geranylgeranylated target proteins involved in the control of proliferation include GTPases, such as Rac-1, Cdc42, and RhoA. Inhibition of Rho, Rac, and Cdc42 GTPases by toxin B reduced proliferation, without affecting cell viability, whereas specific inhibition of Rho GTPases by C3 exoenzyme was without effect. This suggests a role for Rac and/or Cdc42 GTPases in myeloma cell growth. Rac-1 activity was found in all myeloma cell lines and was suppressed by the depletion of intracellular pools of geranylgeranylpyrophosphate, whereas interleukin-6 rapidly induced Rac-1 activation. Furthermore, dominant-negative Tat-Rac-1 reduced myeloma cell proliferation, whereas constitutively active Tat-Rac-1 enhanced proliferation., Conclusion: These results indicate that protein geranylgeranylation is essential for myeloma cell proliferation and suggest that Rac-1 is a regulator of myeloma cell growth.

Purpose: Prenylation is essential for membrane localization and participation of proteins in various signaling pathways. The following study was conducted to examine the importance of protein farnesylation and geranylgeranylation for the regulation of lymphoma cell survival and proliferation., Experimental Design: Lymphoma cells were treated with the beta-hydroxy-beta-methylglutaryl-CoA reductase inhibitor lovastatin, which inhibits protein farnesylation and geranylgeranylation by the depletion of intracellular pools of farnesylpyrophosphate and geranylgeranylpyrophosphate. In addition, farnesyl transferase and geranylgeranyl transferase activities were specifically inhibited by FTI-277 and GGTI-298, respectively., Results: Only inhibition of geranylgeranylation by lovastatin led to reduction of cell viability in lymphoma cell lines and purified tumor cells from lymphoma patients in a time- and dose-dependent way. Reduction in the number of viable cells was mediated by both induction of apoptosis and inhibition of proliferation. In addition, GGTI-298 was more effective in induction of apoptosis and inhibition of proliferation than FTI-277. Apoptosis induced by inhibition of protein geranylgeranylation was associated with a reduction of Mcl-1 protein levels, collapse of the mitochondrial transmembrane potential, and caspase-3 activation. Inhibition of proliferation resulted from the induction of G(1) arrest. Furthermore, lovastatin at low concentrations sensitized lymphoma cells to dexamethasone, including cells resistant to this drug., Conclusion: These results indicate that protein geranylgeranylation is critical for the regulation of lymphoma tumor cell survival and proliferation and that pharmacological agents such as lovastatin or geranylgeranyl transferase inhibitors, alone or in combination with other drugs, may be useful in the treatment of lymphoma.