Generation of NK cells with chimeric-switch receptors to overcome PD1-mediated inhibition in cancer immunotherapy.

Multiple myeloma (MM) is an incurable hematological cancer, in which immune checkpoint inhibition (ICI) with monoclonal antibodies (mAbs) has failed due to uncontrollable immune responses in combination therapies and lack of efficacy in monotherapies. Although NK cell-specific checkpoint targets such as NKG2A and KIRs are currently being evaluated in clinical trials, the clinical impact of NK cells on the PD1 cascade is less well understood compared to T cells. Furthermore, while NK cells have effector activity within the TME, under continuous ligand exposure, NK cell dysfunctionality may occur due to interaction of PD1 and its ligand PD-L1. Due to above-mentioned factors, we designed novel NK cell specific PD1-based chimeric switch receptors (PD1-CSR) by employing signaling domains of DAP10, DAP12 and CD3ζ to revert NK cell inhibition and retarget ICI. PD1-CSR modified NK cells showed increased degranulation, cytokine secretion and cytotoxicity upon recognition of PD-L1+ target cells. Additionally, PD1-CSR+ NK cells infiltrated and killed tumor spheroids. While primary NK cells (pNK), expressing native PD1, showed decreased degranulation and cytokine production against PD-L1+ target cells by twofold, PD1-CSR+ pNK cells demonstrated increased activity upon PD-L1+ target cell recognition and enhanced antibody-dependent cellular cytotoxicity. PD1-CSR+ pNK cells from patients with MM increased degranulation and cytokine expression against autologous CD138+PD-L1+ malignant plasma cells. Taken together, the present results demonstrate that PD1-CSR+ NK cells enhance and sustain potent anti-tumor activity in a PD-L1+ microenvironment and thus represent a promising strategy to advance adoptive NK cell-based immunotherapies toward PD-L1+ cancers.

Challenges in αCD38-chimeric antigen receptor (CAR)-expressing natural killer (NK) cell-based immunotherapy in multiple myeloma: Harnessing the CD38dim phenotype of cytokine-stimulated NK cells as a strategy to prevent fratricide.

BACKGROUND AIMS: Adoptive cell therapy with chimeric antigen receptor (CAR)-expressing natural killer (NK) cells is an emerging approach that holds promise in multiple myeloma (MM). However, the generation of CAR-NK cells targeting CD38 is met with obstacles due to the expression of CD38 on NK cells. Knock-out of CD38 is currently explored as a strategy, although the consequences of the lack of CD38 expression with regards to engraftment and activity in the bone marrow microenvironment are not fully elucidated. Here, we present an alternative approach by harnessing the CD38dim phenotype occurring during long-term cytokine stimulation of primary NK cells. METHODS: Primary NK cells were expanded from peripheral blood mononuclear cells by long-term IL-2 stimulation. During expansion, the CD38 expression was monitored in order to identify a time point when introduction of a novel affinity-optimized αCD38-CAR confered optimal viability, i.e. prevented fratricide. CD38dim NK cells were trasduced with retroviral vectors encoding for the CAR trasngene and their functionality was assessed in in vitro activation and cytotoxicity assays. RESULTS: We verified the functionality of the αCD38-CAR-NK cells against CD38+ cell lines and primary MM cells. Importantly, we demonstrated that αCD38-CAR-NK cells derived from patients with MM have increased activity against autologous MM samples ex vivo. CONCLUSIONS: Overall, our results highlight that incorporation of a functional αCD38-CAR construct into a suitable NK-cell expansion and activation protocol results in a potent and feasible immunotherapeutic strategy for the treatment of patients with MM.

Phosphodiesterase 4A confers resistance to PGE2-mediated suppression in CD25+ /CD54+ NK cells.

Inadequate persistence of tumor-infiltrating natural killer (NK) cells is associated with poor prognosis in cancer patients. The solid tumor microenvironment is characterized by the presence of immunosuppressive factors, including prostaglandin E2 (PGE2), that limit NK cell persistence. Here, we investigate if the modulation of the cytokine environment in lung cancer with IL-2 or IL-15 renders NK cells resistant to suppression by PGE2. Analyzing Cancer Genome Atlas (TCGA) data, we found that high NK cell gene signatures correlate with significantly improved overall survival in patients with high levels of the prostaglandin E synthase (PTGES). In vitro, IL-15, in contrast to IL-2, enriches for CD25+ /CD54+ NK cells with superior mTOR activity and increased expression of the cAMP hydrolyzing enzyme phosphodiesterase 4A (PDE4A). Consequently, this distinct population of NK cells maintains their function in the presence of PGE2 and shows an increased ability to infiltrate lung adenocarcinoma tumors in vitro and in vivo. Thus, strategies to enrich CD25+ /CD54+ NK cells for adoptive cell therapy should be considered.

Assessment of T Cell Receptor Complex Expression Kinetics in Natural Killer Cells.

Among the polypeptides that comprise the T cell receptor (TCR), only CD3ζ is found in Natural Killer (NK) cells, where it transmits signals from activating receptors such as CD16 and NKp46. NK cells are potent immune cells that recognize target cells through germline-encoded activating and inhibitory receptors. Genetic engineering of NK cells enables tumor-specific antigen recognition and, thus, has a significant promise in adoptive cell therapy. Ectopic expression of engineered TCR components in T cells leads to mispairing with the endogenous components, making a knockout of the endogenous TCR necessary. To circumvent the mispairing of TCRs or the need for knockout technologies, TCR complex expression has been studied in NK cells. In the current study, we explored the cellular processing of the TCR complex in NK cells. We observed that in the absence of CD3 subunits, the TCR was not expressed on the surface of NK cells and vice versa. Moreover, a progressive increase in surface expression of TCR between day three and day seven was observed after transduction. Interestingly, the TCR complex expression in NK92 cells was enhanced with a proteasome inhibitor (bortezomib) but not a lysosomal inhibitor (chloroquine). Additionally, we observed that the TCR complex was functional in NK92 cells as measured by estimating CD107a as a degranulation marker, IFNγ cytokine production, and killing assays. NK92 cells strongly degranulated when CD3ε was engaged in the presence of TCR, but not when only CD3 was overexpressed. Therefore, our findings encourage further investigation to unravel the mechanisms that prevent the surface expression of the TCR complex.

NK cell frequencies, function and correlates to vaccine outcome in BNT162b2 mRNA anti-SARS-CoV-2 vaccinated healthy and immunocompromised individuals.

Adaptive immune responses have been studied extensively in the course of mRNA vaccination against COVID-19. Considerably fewer studies have assessed the effects on innate immune cells. Here, we characterized NK cells in healthy individuals and immunocompromised patients in the course of an anti-SARS-CoV-2 BNT162b2 mRNA prospective, open-label clinical vaccine trial. See trial registration description in notes. Results revealed preserved NK cell numbers, frequencies, subsets, phenotypes, and function as assessed through consecutive peripheral blood samplings at 0, 10, 21, and 35 days following vaccination. A positive correlation was observed between the frequency of NKG2C+ NK cells at baseline (Day 0) and anti-SARS-CoV-2 Ab titers following BNT162b2 mRNA vaccination at Day 35. The present results provide basic insights in regards to NK cells in the context of mRNA vaccination, and have relevance for future mRNA-based vaccinations against COVID-19, other viral infections, and cancer.Trial registration: The current study is based on clinical material from the COVAXID open-label, non-randomized prospective clinical trial registered at EudraCT and clinicaltrials.gov (no. 2021-000175-37). Description: https://clinicaltrials.gov/ct2/show/NCT04780659?term=2021-000175-37&draw=2&rank=1 .

Conditioning with melphalan 200 mg/m2 and subsequent ASCT improves progression-free and overall survival in elderly myeloma patients compared to standard of care.

OBJECTIVES: Despite the effectiveness of newer drugs for the treatment of multiple myeloma (MM), the outcomes are further improved by subsequent autologous stem cell transplantation (ASCT). Data on effectiveness in older patients are limited. We compared outcomes in patients aged 65-75 years depending on whether they were treated with ASCT or not and compared those to outcomes in patients <65 years. METHODS: This was a retrospective, single-center study. We compared progression-free survival (PFS) and overall survival (OS) for all MM patients below and above the age of 65 years treated ± ASCT at the Karolinska University Hospital between 2010 and 2020. PFS and OS were calculated by the Kaplan-Meier method. Variables affecting PFS and OS were evaluated using Cox regression model. RESULTS: Both PFS and OS were improved in the group 65-75 years treated +ASCT compared to those treated pharmacologically (p = 0.008 and p < 0.001, respectively). There were no significant differences between patients <65 years and those 65-75 years treated with ASCT. CONCLUSION: The findings indicate that even patients >65 years should be evaluated as candidates for ASCT. An individualized approach supported by a frailty/geriatric assessment score could assist clinicians to select the appropriate treatment for each patient.

Improved survival in multiple Myeloma patients undergoing autologous stem cell transplantation is entirely in the standard cytogenetic risk groups.

INTRODUCTION: Novel drugs and drug combinations have improved outcomes for multiple myeloma patients. However, subgroups of patients still have a poor progression-free survival (PFS) and overall survival (OS). In an attempt to identify how the novel drugs affect the outcome in standard-risk and high-risk patients, respectively, we have investigated 715 multiple myeloma (MM) patients who have undergone high dose treatment followed by autologous stem cell transplantation at our center during 1995 - 2020. Outcomes during three time periods, 1995-1999 (period I), 2000-2009 (period II), and 2010-2020 (period III), were compared separately for standard-risk and high-risk patients. Risk stratification was based on chromosome analysis for periods II and III. RESULTS: The whole cohort of patients showed significantly improved OS with time during the three periods being at a median of 5.8, 7.0, and 10.0 years, respectively. There is also a weak tendency for improved PFS, that is, a median of 2.4, 2.6, and 2.9 years, respectively, during the same periods. However, the separate analysis of standard-risk and high-risk patients showed that the overall improvement with time was due to improved standard-risk patients (median OS 8.4 years for the period I and not reached for period II and III). In contrast, no significant improvement was seen in high-risk patients. For patients with del17p, PFS was even worse during period III as compared to period II (median 1.6 vs 3.2 years respectively). CONCLUSION: Our results show that the dramatic improvement in outcome for MM patients during the last 20 years only applies for standard-risk patients, while high-risk MM patients still are doing poorly, indicating that the novel drugs developed during this time are preferentially effective in standard-risk patients. New treatment modalities like CAR-T cells, CAR-NK cells, and/or bispecific antibodies should be tried in clinical studies early in the course of the disease, especially in patients with high-risk cytogenetics.

Comparative evaluation of involved free light chain and monoclonal spike as markers for progression from monoclonal gammopathy of undetermined significance to multiple myeloma.

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant clonal plasma cell disorder, with a 1% yearly risk of progression to multiple myeloma (MM). Evolution of M-spike and serum free light chain (sFLC) during follow-up could identify patients at high risk of progression. In this region-wide study, including 4756 individuals, 987 patients with MGUS were identified, and baseline factors as well as evolving involved FLC (iFLC) were evaluated as potential markers for risk of progression from MGUS to MM. Furthermore, evolving iFLC and M-spike were assessed quarterly for a median of 5 years. At baseline, patients that progressed had significantly higher iFLC compared to non-progressors. The risk factors of M-spike >1.5 g/dL, age >65 years and iFLC >100 mg/L were all independently associated with increased risk of MGUS to MM progression. For patients that had any two or three risk factors, the 5-year cumulative probability of progression was significantly higher (31%) compared to no risk factors (2%). Evolving iFLC >100 mg/L during follow-up was consistently associated with increased risk of progression. Based on our observations, we propose to include iFLC as a monitoring tool for all MGUS patients. Furthermore, we recommend a quarterly monitoring in all high-risk patients. Finally, we suggest that the risk of MGUS progression should be stratified with age, M-spike, and iFLC at baseline.

Low dose venetoclax as a single agent treatment of plasma cell malignancies harboring t(11;14).

Approximately 20% of newly diagnosed multiple myeloma (NDMM) patients harbor t(11;14), a marker of inferior prognosis, resulting in up-regulation of CCND1. These patients respond to BCL2 inhibitor experimental drug venetoclax. Furthermore, t(11;14) is reported to be associated with increased BCL2/MCL1 ratio. We investigated the use of venetoclax (400 mg daily) in a cohort of 25 multiple myeloma (MM) and AL-amyloidosis patients harboring t(11;14) and assessed safety and efficacy. Efficacy was assessed by response rate (RR) and time on treatment. Furthermore, immunohistochemistry (IHC), for BCL2 family member expression was assessed at diagnosis and relapse in the venetoclax-treated group and analyzed for correlation with clinical RR. Additionally, patient material from venetoclax non-treated group including non-t(11;14) diagnosis (n = 27), t(11;14) diagnosis (n = 17), t(11;14) relapse (n = 7), hyperdiploidy (n = 6) and hyperdiploidy + t(11;14) (n = 6) was used for RNA sequencing (RNASeq) and validation by qPCR. Venetoclax treatment in t(11;14) patients demonstrated manageable safety and promising efficacy. Partial responses or better were observed in eleven patients (44%). Responding patients had significantly higher BCL2/MCL1 (p = 0.031) as well as BCL2/BCL-XL (p = 0.021) ratio, regardless of time of measurement before venetoclax treatment. Furthermore, an IRF5 motif was enriched (p < .001) in the downregulated genes in t(11;14) relapses vs diagnoses. The RR with single agent venetoclax was 71% in AL-amyloidosis and 33% in MM, and IHC proved useful in prediction of treatment outcome. We could also demonstrate possible resistance mechanisms of t(11;14), downregulation of IRF5 targeted genes, which can be exploited for therapeutic advantages.

Engineering antigen-specific NK cell lines against the melanoma-associated antigen tyrosinase via TCR gene transfer.

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL-TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off-target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/ß chains, along with CD3 subunits, enables the functional expression of an antigen-specific TCR complex on NK cell lines NK-92 and YTS, demonstrated by using a TCR against the HLA-A2-restricted tyrosinase-derived melanoma epitope, Tyr368-377 . Most importantly, the introduction of a TCR complex to NK cell lines enables MHC-restricted, antigen-specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of "mispairing" in TCR gene therapy.

Outcome of COVID-19 in multiple myeloma patients in relation to treatment.

COVID-19 has emerged as a global pandemic. Cancer patients have been reported to be at higher risk for adverse outcome of COVID-19. Studies are ongoing to decipher the risk factors and risk groups among cancer patients as well as strategies to refine treatment approaches. Here, we report eight patients with multiple myeloma that underwent immunomodulatory therapies with daratumumab or lenalidomide-based combination treatments and one patient with smoldering multiple myeloma, all of which presented with symptomatic COVID-19. We report that patients that succumbed to COVID-19 presented with either progressive tumor disease under daratumumab treatment or were in remission under lenalidomide-dexamethasone treatment.

Impact of performance status on overall survival in patients with relapsed and/or refractory multiple myeloma: Real-life outcomes of daratumumab treatment.

BACKGROUND: Little is reported on the real-life impact of daratumumab in relapsed and/or refractory multiple myeloma patients (RRMM). We analyzed a cohort of 156 patients who received daratumumab as a single agent concerning ECOG status, eGFR, cytogenetics, lines of prior treatment, and their impact on survival. RESULTS: Eighty-two (53%) patients were triple refractory, 54 (35%) patients were single or double refractory, and 20 (12%) patients were non-refractory. Following daratumumab treatment, the progression-free survival (PFS) in these groups was 7.2%, 11.4%, and 53% (P < .001), and overall survival (OS) was 34%, 73%, and 58% (P < .001) at 36 months, respectively. Poor ECOG, three lines of prior treatment, and triple refractoriness were all associated with inferior PFS and OS in a multivariate analysis including ECOG, high-risk chromosomal aberrations, refractoriness, number of treatment lines, and eGFR. CONCLUSION: Daratumumab remains an attractive treatment option, especially in patients with poor performance and increased frailty. Furthermore, our observations suggest that patients with ECOG 2 and 3 status require additional supportive and/or palliative therapies to compensate for a potentially effective but encompassing late-line therapy. In conclusion, further prospective studies are needed to elucidate the impact of ECOG 2 and 3 status in patients with RRMM.

Characterization of human natural killer cells for therapeutic use.

As a part of the innate immune system, natural killer (NK) cells are cytotoxic lymphocytes that can exert cytotoxic activity against infected or transformed cells. Furthermore, due to their expression of a functional Fc receptor, they have also been eluded as a major effector fraction in antibody-dependent cellular cytotoxicity. These characteristics have led to multiple efforts to use them for adoptive immunotherapy against various malignancies.  There are now at least 70 clinical trials testing the safety and efficacy of NK cell products around the world in early-phase clinical trials. NK cells are also being tested in the context of tumor retargeting via chimeric antigen receptors, other genetic modification strategies, as well as tumor-specific activation strategies such as bispecific engagers with or without cytokine stimulations. One advantage of the use of NK cells for adoptive immunotherapy is their potential to overcome HLA barriers. This has led to a plethora of sources, such as cord blood hematopoietic stem cells and induced pluripotent stem cells, which can generate comparatively high cytotoxic NK cells to peripheral blood counterparts. However, the variety of the sources has led to a heterogeneity in the characterization of the final infusion product. Therefore, in this review, we will discuss a comparative assessment strategy, from characterization of NK cells at collection to final product release by various phenotypic and functional assays, in an effort to predict potency of the cellular product.

Translocation (11;14) in newly diagnosed multiple myeloma, time to reclassify this standard risk chromosomal aberration?

OBJECTIVES: The most common translocation in multiple myeloma (MM) is t(11;14)(q13;q32), and its importance as prognostic factor has been controversial. The aim was to analyze its prognostic value. METHOD: In this retrospective study of 469 newly diagnosed myeloma patients, outcomes in patients with (11;14) and standard risk (t(11;14)SR) or high risk (t(11;14)HR) cytogenetics were compared to outcomes of patients without t(11;14) and SR (non-t(11;14)SR) or HR (non-t(11;14)HR), respectively. RESULTS: Overall progression-free survival (PFS) was shorter in t(11;14)SR than non-t(11;14)SR (median 28.9 vs 35.3 months); however, the difference was not significant (P = .2). Overall survival (OS) did not differ significantly between the groups. In the subgroup of patients that did not receive high-dose treatment, PFS was shorter for t(11;14)SR compared to non-t(11;14)SR, 10.6 vs 24.6 months (P = .01). Although OS were shorter for t(11,14)SR compared to non-t(11;14)SR (5-year OS 41.7% vs 63.8%), the difference was not significant (P = .1). In HDT patients, no significant difference was observed for OS or PFS between those with or without t(11;14). CONCLUSION: This study shows that t(11;14) is associated with poorer outcome in MM, particularly in non-high-dose-treated SR patients. It should be considered an intermediate or high-risk marker in these patients.

Upfront bortezomib, lenalidomide, and dexamethasone compared to bortezomib, cyclophosphamide, and dexamethasone in multiple myeloma.

OBJECTIVES: At our center, patients with multiple myeloma (MM) were treated upfront with bortezomib, cyclophosphamide, and dexamethasone (VCD) until cyclophosphamide was replaced with lenalidomide in the combination (VRD). These treatments have never been compared head-to-head in large real-life patient material. METHOD: A retrospective analysis of patients treated with VRD and VCD in the first line, both with and without subsequent high-dose treatment (HDT) and autologous stem cell transplantation. A total of 681 patients were included, 117 receiving VRD (71 with, 46 without HDT) and 564 receiving VCD (351 with, 213 without HDT). RESULTS: Overall response rate (≥partial response) was higher with VRD compared to VCD in the entire VRD group (98% vs 88%, P < 0.001) and in the non-HDT group (98% vs 79%, P < 0.001). Progression-free survival (PFS) at 18 months was longer with VRD compared to VCD in the entire VRD group, the non-HDT group and the HDT group (88% vs 63%, 82% vs 32% and 91% vs 73%, respectively). Overall survival at 18 months was better for VRD-treated patients in the entire VRD group (95% vs 89%, P = 0.048). CONCLUSION: Upfront VRD gives better responses and longer PFS compared to VCD in MM patients with or without subsequent HDT.

Case Report: Treatment of light-chain amyloidosis with daratumumab monotherapy in two patients.

Immunoglobulin light-chain amyloidosis (AL) affects multiple organs, most prominently the kidney and the heart. Renal and cardiac impairment are both associated with poor prognosis and most patients die as a consequence of renal or cardiac failure. Monoclonal antibodies such as daratumumab (human IgG1 anti-CD38) and elotuzumab (anti-SLAMF7) have shown promising efficacy for the treatment of relapsed and refractory multiple myeloma. In this case report we show 2 patients with severe AL, one with severe heart failure and one with heart and renal failure, undergoing treatment with daratumumab. Both patients showed a rapid decrease in FLC in response to daratumumab infusions, with few associated adverse events. Using therapeutic CD38 antibodies as a front-line treatment for AL could induce rapid responses while maintaining a tolerable safety profile in these ultra-fragile patients.

Proteasome inhibitors and IMiDs can overcome some high-risk cytogenetics in multiple myeloma but not gain 1q21.

Chromosomal aberrations have significant prognostic importance in multiple myeloma (MM). However, proteasome inhibitors (PI) and IMiDs may partly overcome the poor prognostic impact of some of them. In this study, we investigated a population-based consecutive cohort newly diagnosed patients with MM admitted during a defined time period to hospitals in Denmark, Norway, and Sweden. The impact of treatment modality on the prognostic importance of specific chromosomal aberration was investigated, with special reference to gain 1q21. The median follow-up of patients still alive at analysis was 40 months for the high-dose (HDT)-treated ones and 29 months for the whole population. Three hundred forty-seven patients with a known 1q21 status were included in this study. The 347 patients were divided into three groups, that is, 119 patients with the 1q21 gain, 105 patients with other aberrations (OA), that is, del(13q), del(17p), t(4,14), and/or (14;16), and 123 patients with no aberrations (NA). The groups were compared in terms of overall survival (OS), time to progression (TTP), and response. The 3-yr OS for patients with gain 1q21 was 60% compared to patients with OA 74% and NO 82% (gain 1q21 vs. NO P < 0.001; gain 1q21 vs. OA P = 0.095). If treated with PI or IMiDs, the 3-yr OS was 58% for patients with gain 1q21 compared to patients with OA 78% and NO 78%, respectively (P = 0.041, P = 0.140). In HDT patients, the 3-yr OS was 69% for patients with gain 1q21 compared to patients with OA 84% and NO 88%, respectively (P < 0.008, P = 0.600). Thus, neither HDT nor using PI or IMiDs could overcome the poor prognostic impact of gain 1q21, while these drugs and HDT seemed to improve OS in patients with OA, approaching the survival in NO. Further, gain 1q21 appears to be one of the most important poor prognostic chromosomal aberrations in multiple myeloma with current treatments. Trials using new drugs or allogeneic transplantation are warranted.