Patients with MYC overexpressing high grade B cell lymphoma (HGBL) face significant dismal prognosis after treatment with standard immunochemotherapy regimens. Recent preclinical studies indicate ...that MYC not only contributes to tumorigenesis by its effects on cell proliferation and differentiation, but also plays an important role in promoting escape from anti-tumor immune responses. This is of specific interest, since reversing tumor immune inhibition with immunotherapy has shown promising results in the treatment of both solid tumors and hematological malignancies. In this review, we outline the current understanding of impaired immune responses in B cell lymphoid malignancies with MYC overexpression, with a particular emphasis on diffuse large B cell lymphoma. We also discuss clinical consequences of MYC overexpression in the treatment of HGBL with novel immunotherapeutic agents and potential future treatment strategies.
Epcoritamab (DuoBody-CD3xCD20, GEN3013) is a novel bispecific IgG1 antibody redirecting T-cells toward CD20
tumor cells. Here, we assessed the preclinical efficacy of epcoritamab against primary ...tumor cells present in the lymph node biopsies from newly diagnosed (ND) and relapsed/refractory (RR) B-NHL patients. In the presence of T-cells from a healthy donor, epcoritamab demonstrated potent activity against primary tumor cells, irrespective of prior treatments, including CD20 mAbs. Median lysis of 65, 74, and 84% were achieved in diffuse large B-cell lymphoma (n = 16), follicular lymphoma (n = 15), and mantle cell lymphoma (n = 8), respectively. Furthermore, in this allogeneic setting, we discovered that the capacity of B-cell tumors to activate T-cells was heterogeneous and showed an inverse association with their surface expression levels of the immune checkpoint molecule Herpesvirus Entry Mediator (HVEM). In the autologous setting, when lymph node (LN)-residing T-cells were the only source of effector cells, the epcoritamab-dependent cytotoxicity strongly correlated with local effector cell-to-target cell ratios. Further analyses revealed that LN-residing-derived or peripheral blood-derived T-cells of B-NHL patients, as well as heathy donor T-cells equally mediated epcoritamab-dependent cytotoxicity. These results show the promise of epcoritamab for treatment of newly-diagnosed or relapsed/refractory B-NHL patients, including those who became refractory to previous CD20-directed therapies.
Cell surface expression levels of GPRC5D, an orphan G protein–coupled receptor, are significantly higher on multiple myeloma (MM) cells, compared with normal plasma cells or other immune cells, which ...renders it a promising target for immunotherapeutic strategies. The novel GPRC5D-targeting T-cell redirecting bispecific antibody, talquetamab, effectively kills GPRC5D+ MM cell lines in the presence of T cells from both healthy donors or heavily pretreated MM patients. In addition, talquetamab has potent anti-MM activity in bone marrow (BM) samples from 45 patients, including those with high-risk cytogenetic aberrations. There was no difference in talquetamab-mediated killing of MM cells from newly diagnosed, daratumumab-naïve relapsed/refractory (median of 3 prior therapies), and daratumumab-refractory (median of 6 prior therapies) MM patients. Tumor cell lysis was accompanied by T-cell activation and degranulation, as well as production of pro-inflammatory cytokines. High levels of GPRC5D and high effector:target ratio were associated with improved talquetamab-mediated lysis of MM cells, whereas an increased proportion of T cells expressing PD-1 or HLA-DR, and elevated regulatory T-cell (Treg) counts were associated with suboptimal killing. In cell line experiments, addition of Tregs to effector cells decreased MM cell lysis. Direct contact with bone marrow stromal cells also impaired the efficacy of talquetamab. Combination therapy with daratumumab or pomalidomide enhanced talquetamab-mediated lysis of primary MM cells in an additive fashion. In conclusion, we show that the GPRC5D-targeting T-cell redirecting bispecific antibody talquetamab is a promising novel antimyeloma agent. These results provide the preclinical rationale for ongoing studies with talquetamab in relapsed/refractory MM.
•Talquetamab, the new GPRC5DxCD3 bispecific antibody, actively kills GPRC5D+ MM cell lines and primary MM cells in vitro.•Tumor and immune characteristics (eg, GPRC5D expression level, effector:target ratio, Treg count) are determinants of ex vivo response.
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Patients with MYC rearranged (MYC-R) diffuse large B-cell lymphoma (DLBCL) have a poor prognosis. Previously, we demonstrated in a single-arm phase II trial (HOVON-130) that addition of lenalidomide ...to R-CHOP (R2CHOP) is well-tolerated and yields similar complete metabolic remission rates as more intensive chemotherapy regimens in literature. In parallel with this single-arm interventional trial, a prospective observational screening cohort (HOVON-900) was open in which we identified all newly diagnosed MYC-R DLBCL patients in the Netherlands. Eligible patients from the observational cohort that were not included in the interventional trial served as control group in the present risk-adjusted comparison. R2CHOP treated patients from the interventional trial (n = 77) were younger than patients in the R-CHOP control cohort (n = 56) (median age 63 versus 70 years, p = 0.018) and they were more likely to have a lower WHO performance score (p = 0.013). We adjusted for differences at baseline using 1:1 matching, multivariable analysis, and weighting using the propensity score to reduce treatment-selection bias. These analyses consistently showed improved outcome after R2CHOP with HRs of 0.53, 0.51, and 0.59, respectively, for OS, and 0.53, 0.59, and 0.60 for PFS. Thus, this non-randomized risk-adjusted comparison supports R2CHOP as an additional treatment option for MYC-R DLBCL patients.
•Distinct peripheral T-cell and NK-cell phenotypes exist in patients with HGBL-MYC/BCL2 vs patients with DLBCL NOS without MYC rearrangements.•Patients with HGBL-MYC/BCL2 encompass exhausted PD-1+ T ...cells without other immune effector cell impairments.
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Patients with high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBL-MYC/BCL2) respond poorly to immunochemotherapy compared with patients with diffuse large B-cell lymphoma not otherwise specified (DLBCL NOS) without a MYC rearrangement. This suggests a negative impact of lymphoma-intrinsic MYC on the immune system. To investigate this, we compared circulating T cells and natural killer (NK) cells of patients with HGBL-MYC/BCL2 (n = 66), patients with DLBCL NOS (n = 53), and age-matched healthy donors (HDs; n = 16) by flow cytometry and performed proliferation, cytokine production, and cytotoxicity assays. Compared with HDs, both lymphoma subtypes displayed similar frequencies of CD8+ T cells but decreased CD4+ T cells. Regulatory T-cell (Treg) frequencies were reduced only in patients with DLBCL NOS. Activated (HLA-DR+/CD38+) T cells, PD-1+CD4+ T cells, and PD-1+Tregs were increased in both lymphoma subtypes, but PD-1+CD8+ T cells were increased only in HGBL-MYC/BCL2. Patients with DLBCL NOS, but not patients with HGBL-MYC/BCL2, exhibited higher frequencies of senescent T cells than HDs. Functional assays showed no overt differences between both lymphoma groups and HDs. Deeper analyses revealed that PD-1+ T cells of patients with HGBL-MYC/BCL2 were exhausted with impaired cytokine production and degranulation. Patients with DLBCL NOS, but not patients with HGBL-MYC/BCL2, exhibited higher frequencies of NK cells expressing inhibiting receptor NKG2A. Both lymphoma subtypes exhibited lower TIM-3+– and DNAM-1+–expressing NK cells. Although NK cells of patients with HGBL-MYC/BCL2 showed less degranulation, they were not defective in cytotoxicity. In conclusion, our results demonstrate an increased exhaustion in circulating T cells of patients with HGBL-MYC/BCL2. Nonetheless, the overall intact peripheral T-cell and NK-cell functions in these patients emphasize the importance of investigating potential immune evasion in the microenvironment of MYC-rearranged lymphomas.
Introduction: Daratumumab-based quadruplets are the current standard of care for newly diagnosed transplant-eligible multiple myeloma (MM) patients, because of improved depth of response and ...survival, compared with triplet regimens without daratumumab. Since one of the primary mechanisms of action of daratumumab is the induction of antibody-dependent cellular cytotoxicity (ADCC) via the Fc-gamma receptor CD16 on natural killer (NK) cells, it is crucial to understand the role of NK cells in combination therapy. We therefore set out to comprehensively profile NK cells by flow cytometry in newly diagnosed MM (NDMM) patients treated with either daratumumab, bortezomib, thalidomide, and dexamethasone (D-VTD) or VTD. Methods: A subset of NDMM patients (n = 152) who were treated in the Cassiopeia trial (EudraCT: 2014-004781-15) was included in this immunomonitoring analysis. In this trial, patients were first randomized to receive either four induction cycles and two consolidation cycles of D-VTD or VTD in conjunction with autologous stem cell transplantation (ASCT). In the second part of this trial, patients who achieved partial response or better at day 100 post-ASCT were randomized to either receive daratumumab maintenance or observation for the duration of two years. Bone marrow (BM) and peripheral blood (PB) samples were collected at study entry (baseline), and for PB at additional time points: cycle 1 day 28, cycle 4 day 28, day 100 (post-ASCT) and week 52 (maintenance). All baseline BM and PB samples and a selection of longitudinal PB samples were profiled by flow cytometry, with a focus on NK cell phenotype (CD56, CD16, CD57, DNAM-1, TIGIT, KLRG1, PD1, TIM3, NKG2A, NKG2C, NKG2D). To identify NK cell profiles associated with minimal residual disease (MRD) negativity, baseline PB and BM data were compared between MM patients based on MRD status after consolidation. Additionally, longitudinal data of PB samples collected during treatment were assessed to understand the impact of D-VTD and VTD on NK cell profiles. Results: In this cohort, 49 patients out of 77 (64%) randomized to the D-VTD arm achieved MRD negativity post-consolidation, while this was the case in 28 out of 75 patients (37%) in the VTD arm (fisher exact-test, p = 0.008). Patients with a higher proportion of BM-residing NK cells expressing the ADCC receptor CD16 (median 86 vs 72%, p = 0.007), the activating receptor DNAM-1 (median 94 vs 89%, p = 0.029), or the maturation marker CD57 (median 58 vs 48 %, p = 0.049) at baseline were more likely to achieve MRD-negativity post-consolidation in the D-VTD arm (Figure 1). Notably, this association was not present in the VTD arm. Although not significant, we found a similar trend for CD16 in PB in the D-VTD arm. During D-VTD treatment, we observed an increase in the proportion of CD56 bright and NKG2A + in PB NK-cells, and a corresponding decline in cells expressing CD16, CD57 and DNAM-1 (linear mixed effect model (LME) p <0.001). During VTD treatment we also observed a decrease in CD16 + NK-cells and an increase in CD56 bright and NKG2A + NK-cells (LME p <0.05), although these changes were more pronounced in the D-VTD arm. In patients who were assigned D-VTD at the first randomization and were subsequently randomized to the observation arm, we observed a restoration of the proportion of CD16 +, CD57 +, and CD56 bright NK cells to baseline values at week 52 after ASCT (Figure 2). As expected, the daratumumab associated NK cell changes persisted in the group that was randomized to the daratumumab maintenance arm. Conclusion: This study highlights the importance of NK cell immunophenotypic profiles in bone marrow as predictive markers for MRD negativity in NDMM patients treated with D-VTD in in conjunction with ASCT. Additionally, our findings illustrate that NK cell changes associated with daratumumab return to baseline levels after treatment cessation, indicating the potential for sequential NK cell-based therapies. In conclusion, this study not only provides valuable insights into the role of NK cells in the treatment of NDMM patients with D-VTD and ASCT, but also indicates that further research to optimize NK cell function in the context of combination therapies is warranted.
Background: Patients with diffuse large B-cell lymphoma/high-grade B-cell lymphoma with MYC and BCL2 rearrangements (DLBCL/HGBL- MYC/BCL2, hereafter HGBL- MYC/BCL2) respond poorly to standard ...immunochemotherapy treatment compared with DLBCL not otherwise specified (LBCL-NOS) patients without a MYC rearrangement. This suggests a negative interaction between MYC and the immune system. As novel T-cell and NK-cell based immunotherapeutic approaches emerge as potential treatment options for aggressive lymphomas, we conducted comprehensive immune profiling to compare peripheral blood T-cells and NK-cells of HGBL- MYC/BCL2 with LBCL NOS patients. Methods: Peripheral blood mononuclear cells (PBMCs) were prospectively collected from HGBL- MYC/BCL2 patients (registered in the HOVON-152 trial, NCT03620578), LBCL-NOS (registered in the HOVON-902 cohort, NCT04139252) and age-matched healthy donors (HDs). Flow-cytometry-based immune profiling of T-cells and NK-cells was performed on baseline samples available from n=66 HGBL- MYC/BCL2 patients (median age 62 years), n=67 LBCL NOS patients (median age 69 years) and n=17 HDs (median age 64 years). The flow cytometry data were computationally analyzed using FlowSOM and UMAP. T-cells were also functionally assessed for intracellular cytokine secretion (ICS) after stimulation with PMA/ionomycin (4h) and proliferation after stimulation with CD3/CD28 beads (5 days) using CellTrace Violet. NK-cells were assessed for cytotoxicity against K562 and degranulation (CD107a/b upregulation) after 4h. Mann-Whitney's U tests were used to compare HGBL-MYC/BCL2 patients with LBCL-NOS patients (primary comparison), and for pairwise comparisons of the lymphoma groups with HDs. To correct for baseline characteristics (age), linear regression analysis was performed. Results: CD4 + helper T-cells did not significantly between the lymphoma subgroups, but were decreased in LBCL-NOS patients as compared to HDs. Frequencies of cytotoxic CD8 + and CD4 +CD8 + T-cells did not significantly between the lymphoma subgroups and were similar as in HDs. Regulatory T-cell (Treg) frequencies of HGBL- MYC/BCL2 patients were similar to that of HDs, but were decreased in LBCL NOS patients. The frequency of CD4 -CD8 - T-cells was increased in LBCL NOS patients. Previously activated (HLA-DR +) CD4 + and CD8 + T-cells, as well as recently activated CD25 +, CD38 + or CD127 + CD4 + T-cells (Figure 1A) or activated/exhausted (PD-1 +) CD4 + T-cells and PD1 + Tregs were increased in both lymphoma subtypes (Figure 1B), but only HGBL- MYC/BCL2 displayed an increase in PD1 + CD8 + T-cells (Figure 1B). Conversely, recently activated CD8 + T-cells were decreased exclusively in LBCL NOS. HGBL- MYC/BCL2 patients and HDs had similar percentages of T-cells with a senescent phenotype (CD28 -CD57 +KLRG1 +), but the frequency of T-cells with this senescent phenotype was increased in LBCL NOS patients (Figure 1A), also after adjustment for age. Despite all these phenotypic differences, functional in vitro assays detected no differences in proliferation or cytokine secretion of peripheral T-cells from HGBL- MYC/BCL2 patients, LBCL NOS patients and HDs. In the NK-cell compartment, both lymphoma subtypes contained lower frequencies of CD56 brightTim3 +, CD56 bright/dimDNAM-1 + NK-cells, but LBCL NOS patients expressed a higher amount of NK-cells expressing inhibitory receptor NKG2A compared with HDs. NK-cells of HGBL- MYC/BCL2 patients, LBCL NOS patients and HDs displayed similar cytotoxic activity, suggesting no apparent functional impairment. Conclusions: HGBL- MYC/BCL2 patients have a distinct peripheral T-cell and NK-cell phenotype from LBCL NOS patients without a MYC rearrangement, but show no apparent functional deficiencies in terms of proliferation, cytokine secretion or cytotoxic activity. These results are not contradicting the application of T-cell and NK-cell based immunotherapeutic approaches for patients with aggressive B-cell lymphoma. Simultaneously, it emphasize the need to investigate the potential immunomodulatory impact of lymphoma intrinsic MYC in the tumor microenvironment.
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