Tom van Meerten

The Biological Activity of Human CD20 Monoclonal Antibodies Is Linked to Unique Epitopes on CD20

We have previously defined a panel of fully human CD20 mAb. Most of these were unexpectedly efficient in their ability to recruit C1q to the surface of CD20-positive cells and mediate tumor lysis via activation of the classical pathway of complement. This complement-dependent cytotoxicity (CDC) potency appeared to relate to the unusually slow off-rate of these human Abs. However, we now present epitope-mapping data, which indicates that all human mAb bind a novel region of CD20 that may influence CDC potency. Epitope mapping, using both mutagenesis studies and overlapping 15-mer peptides of the extracellular loops of CD20, defined the amino acids required for binding by an extensive panel of mouse and human mAb. Binding by rituximab and mouse CD20 mAb, had an absolute requirement for alanine and proline at positions 170 and 172, respectively, within the large extracellular loop of CD20. Surprisingly, however, all of the human CD20 mAb recognize a completely novel epitope located N-terminally of this motif, also including the small extracellular loop of CD20. Thus, although off-rate may influence biological activity of mAb, another critical factor for determining CDC potency by CD20 mAb appears to be the region of the target molecule they recognize. We conclude that recognition of the novel epitope cooperates with slow off-rate in determining the activity of CD20 Ab in activation of complement and induction of tumor cell lysis.

Complement-induced cell death by rituximab depends on CD20 expression level and acts complementary to antibody-dependent cellular cytotoxicity

Purpose: The use of the CD20-specific antibody rituximab has greatly improved the response to treatment of CD20+ follicular lymphoma. Despite the success of rituximab, resistance has been reported and prognostic markers to predict individual response are lacking. The level of CD20 expression on tumors has been related to response, but results of several studies are contradictory and no clear relationship could be established. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are thought to be important effector mechanisms, but the exact mechanism of rituximab-mediated cell kill is still unknown. Importantly, no data have been reported on the combined contribution of CDC and ADCC. Experimental Design: We have developed a system of clonally related CEM-CD20 cells by retroviral transfer of the human CD20 cDNA (n = 90). This set of cells, with the CD20 molecule as the only variable, was used to study the importance of CD20 expression level on rituximab-mediated CDC, ADCC, and the combination. Results: We show a sigmoidal correlation of CD20 expression level and rituximab-mediated killing via CDC but not ADCC. On both high and low CD20-expressing cells, all CD20 molecules were translocated into lipid rafts after rituximab binding. Furthermore, CDC and ADCC act simultaneously and CDC-resistant cells are sensitive to ADCC and vice versa. Conclusions: These findings suggest that CDC depends on CD20 expression level and that both CDC and ADCC act complementary. These data give new insights into novel strategies to improve the efficacy of CD20-specific antibodies for the treatment of CD20+ tumors.

Target Antigen Density Governs the Efficacy of Anti–CD20-CD28-CD3 ζ Chimeric Antigen Receptor–Modified Effector CD8+ T Cells

The effectiveness of chimeric Ag receptor (CAR)–transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second- and later-generation CARs simultaneously transmit costimulatory signals with CD3ζ signals upon ligation, but may lead to severe adverse effects owing to the recognition of minimal Ag expression outside the target tumor. Currently, the threshold target Ag density for CAR-T cell lysis and further activation, including cytokine production, has not yet been investigated in detail. Therefore, we determined the threshold target Ag density required to induce CAR-T cell responses using novel anti-CD20 CAR-T cells with a CD28 intracellular domain and a CD20-transduced CEM cell model. The newly developed CD20CAR–T cells demonstrated Ag-specific lysis and cytokine secretion, which was a reasonable level as a second-generation CAR. For lytic activity, the threshold Ag density was determined to be ∼200 molecules per target cell, whereas the Ag density required for cytokine production of CAR-T cells was ∼10-fold higher, at a few thousand per target cell. CD20CAR–T cells responded efficiently to CD20-downregulated lymphoma and leukemia targets, including rituximab- or ofatumumab-refractory primary chronic lymphocytic leukemia cells. Despite the potential influence of the structure, localization, and binding affinity of the CAR/Ag, the threshold determined may be used for target Ag selection. An Ag density below the threshold may not result in adverse effects, whereas that above the threshold may be sufficient for practical effectiveness. CD20CAR–T cells also demonstrated significant lytic activity against CD20-downregulated tumor cells and may exhibit effectiveness for CD20-positive lymphoid malignancies.

Type I CD20 Antibodies Recruit the B Cell Receptor for Complement-Dependent Lysis of Malignant B Cells

Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20+ B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab′)2 fragments, as well as C1q-binding–deficient IgG mutants, retained an ability to induce CDC, albeit with lower efficiency than for whole or unmodified IgG. Experiments using human serum depleted of specific complement components demonstrated that the observed lytic activity, which we termed “accessory CDC,” remained to be dependent on C1 and the classical pathway. We hypothesized that CD20 Ab-induced clustering of the IgM or IgG BCR was involved in accessory CDC. Indeed, accessory CDC was consistently observed in B cell lines expressing an IgM BCR and in some cell lines expressing an IgG BCR, but it was absent in BCR− B cell lines. A direct relationship between BCR expression and accessory CDC was established by transfecting the BCR into CD20+ cells: OFA-F(ab′)2 fragments were able to induce CDC in the CD20+BCR+ cell population, but not in the CD20+BCR− population. Importantly, OFA-F(ab′)2 fragments were able to induce CDC ex vivo in malignant B cells isolated from patients with mantle cell lymphoma and Waldenström macroglobulinemia. In summary, accessory CDC represents a novel effector mechanism that is dependent on type I CD20 Ab–induced BCR clustering. Accessory CDC may contribute to the excellent capacity of type I CD20 Abs to induce CDC, and thereby to the antitumor activity of such Abs in the clinic.

Combined PD-1 and JAK1/2 inhibition in refractory primary mediastinal B-cell lymphoma

Dear Editor, Primarymediastinal large B-cell lymphoma (PMBL) represents 2–4% of all B-cell non-Hodgkin lymphoma [1]. It shares clinical and molecular features with classical Hodgkin lymphoma [2]. Amplification or gain of 9p24.1 is observed in more than half of PMBL cases, resulting in overexpression of the immune checkpoints programmed cell-death ligand 1 and 2 (PD-L1 and PD-L2) and the cytokine receptor signaling kinase Janus kinase 2 (JAK2) [3, 4]. Besides increased growth signaling through the JAK-STAT pathway, JAK2 overexpression enhances PD-L1 transcription and expression [3]. Although recent studies indicate a favorable outcome of PMBL patients treated with doseadjusted etoposide, doxorubicin, cyclophosphamide, vincristine, and prednisone plus rituximab in first line [5], outcome of relapsed large B-cell lymphoma remains poor. Herein, we describe a 39-year-old Caucasian female with a refractory PMBL who was treated with a novel combination of PD-1 and JAK2 inhibition. PMBL had progressed after RCHOP. Subsequent treatments consisted of dexamethasone, cytarabine, and cisplatinum (DHAP), brentuximab vedotin, and radiotherapy. Progression was histologically confirmed (Fig. 1a). Fluorescence in situ hybridization revealed 9p24.1 amplification (Fig. 1b) and polysomy (Fig. 1c). However, PDL1 or pSTAT3 (as a measure of increased JAK2 activity) expression could not be observed (Fig. 1d, e). PD-1 staining of Tcells (Fig. 1f) was limited. The patient had a life threatening vena cava superior syndrome (VCSS). In the absence of a clinical trial, with the patient’s consent, she was treated with off-label ruxolitinib at a dose of 10mg bid. Off-label pembrolizumabwas initiated 2 weeks later at 2 mg per kilogram in 3-week cycles (Fig. 2a). Four weeks after start of ruxolitinib the VCSS had resolved. Staging and response evaluation were performed with combined fluorodeoxyglucose positron emission tomography and computerized tomography scans according to the Lugano classification [6]. A complete metabolic remission was documented 10 weeks after start of ruxolitinib (Fig. 2b). The patient continued treatment until an allogeneic hematopoietic cell transplantation (HCT) after non-myeloablative conditioning with

Identification of relevant drugable targets in diffuse large B-cell lymphoma using a genome-wide unbiased CD20 guilt-by association approach

Forty percent of patients with diffuse large B-cell lymphoma (DLBCL) show resistant disease to standard chemotherapy (CHOP) in combination with the anti-CD20 monoclonal antibody rituximab (R). Although many new anti-cancer drugs were developed in the last years, it is unclear which of these drugs can be safely combined to improve standard therapy without antagonizing anti-CD20 efficacy. In this study, we aimed to identify rituximab compatible drug-target combinations for DLBCL. For this, we collected gene expression profiles of 1,804 DLBCL patient samples. Subsequently, we performed a guilt-by-association analysis with MS4A1 (CD20) and prioritized the 500 top-ranked CD20-associated gene probes for drug-target interactions. This analysis showed the well-known genes involved in DLBCL pathobiology, but also revealed several genes that are relatively unknown in DLBCL, such as WEE1 and PARP1. To demonstrate potential clinical relevance of these targets, we confirmed high protein expression of WEE1 and PARP1 in patient samples. Using clinically approved WEE1 and PARP1 inhibiting drugs in combination with rituximab, we demonstrated significantly improved DLBCL cell killing, also in rituximab-insensitive cell lines. In conclusion, as exemplified by WEE1 and PARP1, our CD20-based genome-wide analysis can be used as an approach to identify biological relevant drug-targets that are rituximab compatible and may be implemented in phase 1/2 clinical trials to improve DLBCL treatment.

Lactate dehydrogenase levels and F-18-FDG PET/CT metrics differentiate between mediastinal Hodgkin's lymphoma and primary mediastinal B-cell lymphoma

Purpose This study aims to investigate whether clinical, laboratory, and fluorine-18-fluorodeoxyglucose (18F-FDG) PET/CT findings can discriminate between mediastinal Hodgkin’s lymphoma and primary mediastinal B-cell lymphoma (PMBCL). Patients and methods This retrospective study included 56 patients (42 with mediastinal Hodgkin’s lymphoma and 14 with PBMCL). Differences in clinical, laboratory, and 18F-FDG PET/CT metrics were assessed between Hodgkin’s lymphoma and PMBCL. Results Lactate dehydrogenase (LDH) and 18F-FDG PET/CT-based maximum tumor diameter, lesion-to-liver ratio maximum standardized uptake value (SUVmax), and lesion-to-liver ratio peak standardized uptake value (SUVpeak) were all significantly higher (P<0.001) in PMBCL than in Hodgkin’s lymphoma, and PMBCL also significantly more frequently (P=0.001) exhibited necrosis on 18F-FDG PET/CT than Hodgkin’s lymphoma. LDH, maximum tumor diameter, lesion-to-liver ratio SUVmax, and lesion-to-liver ratio SUVpeak yielded areas under the receiver operating characteristic curve of 0.968 [95% confidence interval (CI): 0.923–1.000], 0.866 (95% CI: 0.765–0.968), 0.875 (95% CI: 0.776–0.975), and 0.874 (95% CI: 0.771–0.976), respectively. LDH (with cutoff of 236 U/l) achieved sensitivity and specificity of 81.6 and 100%, respectively; maximum tumor diameter (with cutoff of 9.98 cm) achieved sensitivity and specificity of 87.2 and 78.3%, respectively; lesion-to-liver ratio SUVmax (with cutoff of 7.12) achieved sensitivity and specificity of 94.9 and 64.3%, respectively; lesion-to-liver ratio SUVpeak (with cutoff of 11.45) achieved sensitivity and specificity of 97.4 and 64.3%, respectively; and the presence of necrosis achieved sensitivity and specificity of 78.6 and 74.4%, respectively, in discriminating PMBCL from Hodgkin’s lymphoma. Conclusion LDH levels and several 18F-FDG PET/CT findings (tumor size, presence of necrosis, and degree of 18F-FDG uptake) are helpful in discriminating mediastinal Hodgkin’s lymphoma from PMBCL.