T cell characteristics impact response and resistance to T cell-redirecting bispecific antibodies in multiple myeloma.

PURPOSE: Bispecific antibodies (BsAbs) directed against B-cell maturation antigen (BCMA; teclistamab) or the orphan G protein-coupled receptor GPRC5D (talquetamab) induce deep and durable responses in heavily pretreated MM patients. However, mechanisms underlying primary and acquired resistance remain poorly understood. EXPERIMENTAL DESIGN: The anti-MM activity of teclistamab and talquetamab was evaluated in bone marrow (BM) samples from MM patients. T-cell phenotype and function were assessed in BM/peripheral blood samples obtained from MM patients who were treated with these BsAbs. RESULTS: In ex vivo killing assays with 41 BM samples from BsAb-naïve MM patients, teclistamab- and talquetamab-mediated MM lysis were strongly correlated (r=0.73, P<0.0001). Both BsAbs exhibited poor activity in samples with high regulatory T-cell (Treg) numbers and a low T-cell/MM cell-ratio. Furthermore, comprehensive phenotyping of BM samples derived from patients treated with teclistamab or talquetamab, revealed that high frequencies of PD-1+ CD4+ T-cells, CTLA4+ CD4+ T-cells, and CD38+ CD4+ T-cells were associated with primary resistance. Although this lack of response was linked to modest increase in expression of inhibitory receptors, increasing T-cell/MM cell-ratios by adding extra T-cells enhanced sensitivity to BsAbs. Further, treatment with BsAbs resulted in an increased proportion of T-cells expressing exhaustion markers (PD-1, TIGIT, and TIM-3), which was accompanied by reduced T-cell proliferative potential and cytokine secretion, as well as impaired anti-tumor efficacy in ex vivo experiments. CONCLUSIONS: Primary resistance is characterized by a low T-cell/MM cell-ratio and Treg-driven immunosuppression, while reduced T-cell fitness due to continuous BsAb-mediated T-cell activation may contribute to development of acquired resistance.

Distinct peripheral T-cell and NK-cell profiles in HGBL-MYC/BCL2 vs patients with DLBCL NOS.

ABSTRACT: 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.

NK Cell Phenotype Is Associated With Response and Resistance to Daratumumab in Relapsed/Refractory Multiple Myeloma.

The CD38-targeting antibody daratumumab has marked activity in multiple myeloma (MM). Natural killer (NK) cells play an important role during daratumumab therapy by mediating antibody-dependent cellular cytotoxicity via their FcγRIII receptor (CD16), but they are also rapidly decreased following initiation of daratumumab treatment. We characterized the NK cell phenotype at baseline and during daratumumab monotherapy by flow cytometry and cytometry by time of flight to assess its impact on response and development of resistance (DARA-ATRA study; NCT02751255). At baseline, nonresponding patients had a significantly lower proportion of CD16+ and granzyme B+ NK cells, and higher frequency of TIM-3+ and HLA-DR+ NK cells, consistent with a more activated/exhausted phenotype. These NK cell characteristics were also predictive of inferior progression-free survival and overall survival. Upon initiation of daratumumab treatment, NK cells were rapidly depleted. Persisting NK cells exhibited an activated and exhausted phenotype with reduced expression of CD16 and granzyme B, and increased expression of TIM-3 and HLA-DR. We observed that addition of healthy donor-derived purified NK cells to BM samples from patients with either primary or acquired daratumumab-resistance improved daratumumab-mediated MM cell killing. In conclusion, NK cell dysfunction plays a role in primary and acquired daratumumab resistance. This study supports the clinical evaluation of daratumumab combined with adoptive transfer of NK cells.

Hypermethylated RASSF1A as Circulating Tumor DNA Marker for Disease Monitoring in Neuroblastoma.

PURPOSE: Circulating tumor DNA (ctDNA) has been used for disease monitoring in several types of cancer. The aim of our study was to investigate whether ctDNA can be used for response monitoring in neuroblastoma. METHODS: One hundred forty-nine plasma samples from 56 patients were analyzed by quantitative polymerase chain reaction (qPCR) for total cell free DNA (cfDNA; albumin and ß-actin) and ctDNA (hypermethylated RASSF1A). ctDNA results were compared with mRNA-based minimal residual disease (qPCR) in bone marrow (BM) and blood and clinical patient characteristics. RESULTS: ctDNA was detected at diagnosis in all patients with high-risk and stage M neuroblastoma and in 3 of 7 patients with localized disease. The levels of ctDNA were highest at diagnosis, decreased during induction therapy, and not detected before or after autologous stem-cell transplantation. At relapse, the amount of ctDNA was comparable to levels at diagnosis. There was an association between ctDNA and blood or BM mRNA, with concordant results when tumor burden was high or no tumor was detected. The discrepancies indicated either low-level BM infiltration (ctDNA negative/mRNA positive) or primary tumor/soft tissue lesions with no BM involvement (ctDNA positive/mRNA negative). CONCLUSION: ctDNA can be used for monitoring disease in patients with neuroblastoma. In high-risk patients and all patients with stage M at diagnosis, ctDNA is present. Our data indicate that at low tumor load, testing of both ctDNA and mRNA increases the sensitivity of molecular disease monitoring. It is likely that ctDNA can originate from both primary tumor and metastases and may be of special interest for disease monitoring in patients who experience relapse in other organs than BM.

Targeted Therapy With Immunoconjugates for Multiple Myeloma.

The introduction of proteasome inhibitors (PI) and immunomodulatory drugs (IMiD) has markedly increased the survival of multiple myeloma (MM) patients. Also, the unconjugated monoclonal antibodies (mAb) daratumumab (anti-CD38) and elotuzumab (anti-SLAMF7) have revolutionized MM treatment given their clinical efficacy and safety, illustrating the potential of targeted immunotherapy as a powerful treatment strategy for MM. Nonetheless, most patients eventually develop PI-, IMiD-, and mAb-refractory disease because of the selection of resistant MM clones, which associates with a poor prognosis. Accordingly, these patients remain in urgent need of new therapies with novel mechanisms of action. In this respect, mAbs or mAb fragments can also be utilized as carriers of potent effector moieties to specifically target surface antigens on cells of interest. Such immunoconjugates have the potential to exert anti-MM activity in heavily pretreated patients due to their distinct and pleiotropic mechanisms of action. In addition, the fusion of highly cytotoxic compounds to mAbs decreases the off-target toxicity, thereby improving the therapeutic window. According to the effector moiety, immunoconjugates are classified into antibody-drug conjugates, immunotoxins, immunocytokines, or radioimmunoconjugates. This review will focus on the mechanisms of action, safety and efficacy of several promising immunoconjugates that are under investigation in preclinical and/or clinical MM studies. We will also include a discussion on combination therapy with immunoconjugates, resistance mechanisms, and future developments.