Impact of anti-CD20 tumor-targeting therapeutic monoclonal antibodies on human Natural Killer cell responsiveness and plasticity: relevance of FcgammaRIIIA/CD16 affinity ligation conditions

My study is focused on understanding the mechanisms underlying the modulation of NK cell responsiveness and plasticity induced by tumor targeting therapeutic anti-CD20 monoclonal antibodies (mAbs) nowadays routinely used in the treatment of B-cell malignancies and autoimmune disorders. Anti-CD20 mAbs are grouped into type I and II subtypes. Type I mAbs induce CD20 redistribution into lipid rafts and display a remarkable ability to activate complement-dependent cytotoxicity (CDC). On the other hand, type II mAbs, which are not able to localize CD20 complexes into lipid rafts and induce weak or no CDC, evoke more homotypic adhesion and direct killing of target cells. Both type I and II mAbs demonstrate efficient phagocytosis and antibody-dependent cytotoxicity (ADCC). Natural Killer (NK) cell-mediated ADCC, based on the recognition of IgG-opsonized targets by the low affinity Fc receptor for IgG FcgammaRIIIA/CD16, represents one of the main mechanisms by which anti-CD20 mAbs mediate their anti-tumor effects. Besides ADCC, CD16 ligation also results in the production of cytokines such as IFN-gamma that plays a key role in the shaping of adaptive immune responses. Rituximab is a chimeric type I anti-CD20 mAb of 1st generation and is considered the reference molecule for the comparison with new generation anti-CD20 mAbs, designed to optimize clinical efficacy. Among them, obinutuzumab is a humanized Fc-glycoengineered type II anti-CD20 mAb of 3rd generation designed to increase the affinity for CD16 receptor and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood. Herein, I demonstrated that CD16 affinity ligation conditions may dictate both the amplitude of NK responsiveness (cytotoxicity and IFN-gamma production) as well as the ability to shift the NK functional program. Indeed, I observed that the interaction of NK cells with obinutuzumab-opsonized targets results in enhanced cytotoxicity and IFN-gamma production as compared with the parental non-glycoengineered mAb or the reference molecule rituximab, independently from the CD16-158V/F allotype. The affinity ligation conditions also strictly correlate with the ability to induce CD16 surface down-modulation and lysosomal targeting of receptor-coupled signaling elements. Indeed, a preferential degradation of FcepsilonRIgamma chain and Syk tyrosine kinase was observed upon obinutuzumab stimulation independently from the CD16-158V/F allotype. Notably, although the down-regulation of FcepsilonRIgamma/Syk module hesitates in the impairment of cytotoxic function induced by CD16, NKp46 and NKp30 activating receptors, obinutuzumab-experienced NK cells exhibit an increased ability to produce IFN-gamma in response to cytokines and target stimulation as well as to obinutuzumab-mediated CD16 re-stimulation. Relying on the observation that obinutuzumab-experienced NK cells, under molecular and functional profile, resemble the distinctive features of the long-lived and highly functional “memory” NK cells, a population recently identified in HCMV seropositive individuals, I assessed the capability of anti-CD20 mAbs to affect the expansion as well as the phenotypic and functional properties of the “memory” NK subset. My data show that the majority of the analysed healthy donors is HCMV seropositive and exhibits a detectable population of “memory” NK cells (CD3- CD56+ FcepsilonRIgamma- CD16+) accounting for 3 to 50% of peripheral blood NK cells. I observed that “memory” NK cells selectively undergo 2- to 12-fold expansion upon co-culturing with anti-CD20-opsonized targets; on the opposite, the proliferation of “conventional” NK cells (CD3- CD56+ FcepsilonRIgamma+ CD16+) is not affected by CD16 stimulation. I also noted that anti-CD20 mAb in vitro expanded “memory” NK cells show the molecular and functional hallmarks of their freshly isolated counterpart, including the increased expression of NKG2C receptor, the reduced expression of NKp46 receptor associated to an enhanced functional activity in response to CD16 re-stimulation, particularly in terms of IFN-gamma production

Memory NK cell features exploitable in anticancer immunotherapy

Besides their innate ability to rapidly produce effector cytokines and kill virus-infected or transformed cells, natural killer (NK) cells display a strong capability to adapt to environmental modifications and to differentiate into long-lived, hyperfunctional populations, dubbed memory or memory-like NK cells. Despite significant progress in the field of NK cell-based immunotherapies, some factors including their short life span and the occurrence of a tumor-dependent functional exhaustion have limited their clinical efficacy so that strategies aimed at overcoming these limitations represent one of the main current challenges in the field. In this scenario, the exploitation of NK cell memory may have a considerable potential. This article summarizes recent evidence in the literature on the peculiar features that render memory NK cells an attractive tool for antitumor immunotherapy, including their long-term survival and in vivo persistence, the resistance to tumor-dependent immunosuppressive microenvironment, the amplified functional responses to IgG-opsonized tumor cells, and in vitro expansion capability. Along with highlighting these issues, we speculate that memory NK cell-based adoptive immunotherapy settings would greatly take advantage from the combination with tumor-targeting therapeutic antibodies (mAbs), as a strategy to fully unleash their clinical efficacy

Anti-CD20 Therapy Acts via FcγRIIIA to Diminish Responsiveness of Human Natural Killer Cells

Natural killer (NK) immune cells mediate antibody-dependent cellular cytotoxicity (ADCC) by aggregating FcγRIIIA/CD16, contributing significantly to the therapeutic effect of CD20 monoclonal antibodies (mAb). In this study, we show that CD16 ligation on primary human NK cells by the anti-CD20 mAb rituximab or ofatumumab stably impairs the spontaneous cytotoxic response attributable to cross-tolerance of several unrelated NK-activating receptors (including NKG2D, DNAM-1, NKp46, and 2B4). Similar effects were obtained from NK cells isolated from patients with chronic lymphocytic leukemia in an autologous setting. NK cells rendered hyporesponsive in this manner were deficient in the ability of these cross-tolerized receptors to phosphorylate effector signaling molecules critical for NK cytotoxicity, including SLP-76, PLCγ2, and Vav1. These effects were associated with long-lasting recruitment of the tyrosine phosphatase SHP-1 to the CD16 receptor complex. Notably, pharmacologic inhibition of SHP-1 with sodium stibogluconate counteracted CD20 mAb-induced NK hyporesponsiveness, unveiling an unrecognized role for CD16 as a bifunctional receptor capable of engendering long-lasting NK cell inhibitory signals. Our work defines a novel mechanism of immune exhaustion induced by CD20 mAb in human NK cells, with potentially negative implications in CD20 mAb-treated patients where NK cells are partly responsible for clinical efficacy. Cancer Res; 75(19); 1-12. ©2015 AACR

Obinutuzumab-mediated high-affinity ligation of FcγRIIIA/CD16 primes NK cells for IFNγ production

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG FcγRIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFNγ is endowed with a well-recognized role in the shaping of adaptive immune responses. Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood. Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFNγ production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of FcεRIγ chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of FcεRIγ/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFNγ in response to different stimuli. These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional progra

Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming

Natural Killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human Cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C+Fc∝RI≥- long-lived “memory” NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterised yet. Here we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells, and we show the impact of donor' HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansion. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting, and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy

Phospho-proteomic analysis of mantle cell lymphoma cells suggests a pro-survival role of B-cell receptor signaling

BACKGROUND: Mantle cell lymphoma (MCL) is currently an incurable entity, and new therapeutic approaches are needed. We have applied a high-throughput phospho-proteomic technique to MCL cell lines to identify activated pathways and we have then validated our data in both cell lines and tumor tissues. METHODS: PhosphoScan analysis was performed on MCL cell lines. Results were validated by flow cytometry and western blotting. Functional validation was performed by blocking the most active pathway in MCL cell lines. RESULTS: PhosphoScan identified more than 300 tyrosine-phosporylated proteins, among which many protein kinases. The most abundant peptides belonged to proteins connected with B-cell receptor (BCR) signaling. Active BCR signaling was demonstrated by flow cytometry in MCL cells and by western blotting in MCL tumor tissues. Blocking BCR signaling by Syk inhibitor piceatannol induced dose/time-dependent apoptosis in MCL cell lines, as well as several modifications in the phosphorylation status of BCR pathway members and a collapse of cyclin D1 protein levels. CONCLUSION: Our data support a pro-survival role of BCR signaling in MCL and suggest that this pathway might be a candidate for therapy. Our findings also suggest that Syk activation patterns might be different in MCL compared to other lymphoma subtypes

Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells

Activation-induced cytidine deaminase (AID) is a B-cell specific enzyme that targets immunoglobulin (Ig) genes to initiate class switch recombination (CSR) and somatic hypermutation (SHM)(1). Through off-target activity, however, AID has a much broader impact on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in lymphoma development and progression(2). AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation(3). The phosphatidylinositol 3-kinase (PI3K) δ pathway plays a key role in AID regulation by suppressing its expression in B cells(4). Novel drugs for leukemia or lymphoma therapy such as idelalisib, duvelisib or ibrutinib block PI3Kδ activity directly or indirectly(5–8), potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation (SHM) and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both these effects were completely abrogated in AID deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumors in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IgH and AID off-target sites in human chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased SHM in AID off-targets. In summary, we show that PI3Kδ or BTK inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism, an effect that should be carefully considered as such inhibitors are administered for years to patients

BNT162B2 mRNA COVID-19 vaccine in heart and lung transplanted young adults: is an alternative SARS-CoV-2 immune response surveillance needed?

[no abstract available

DNAM-1-chimeric receptor-engineered NK cells, combined with Nutlin-3a, more effectively fight neuroblastoma cells in vitro: a proof-of-concept study

Adoptive transfer of engineered NK cells, one of clinical approaches to fight cancer, is gaining great interest in the last decade. However, the development of new strategies is needed to improve clinical efficacy and safety of NK cell-based immunotherapy. NK cell-mediated recognition and lysis of tumor cells are strictly dependent on the expression of ligands for NK cell-activating receptors NKG2D and DNAM-1 on tumor cells. Of note, the PVR/CD155 and Nectin-2/CD112 ligands for DNAM-1 are expressed primarily on solid tumor cells and poorly expressed in normal tissue cells. Here, we generated human NK cells expressing either the full length DNAM-1 receptor or three different DNAM-1-based chimeric receptor that provide the expression of DNAM-1 fused to a costimulatory molecule such as 2B4 and CD3ζ chain. Upon transfection into primary human NK cells isolated from healthy donors, we evaluated the surface expression of DNAM-1 and, as a functional readout, we assessed the extent of degranulation, cytotoxicity and the production of IFNγ and TNFα in response to human leukemic K562 cell line. In addition, we explored the effect of Nutlin-3a, a MDM2-targeting drug able of restoring p53 functions and known to have an immunomodulatory effect, on the degranulation of DNAM-1-engineered NK cells in response to human neuroblastoma (NB) LA-N-5 and SMS-KCNR cell lines. By comparing NK cells transfected with four different plasmid vectors and through blocking experiments, DNAM-1-CD3ζ-engineered NK cells showed the strongest response. Furthermore, both LA-N-5 and SMS-KCNR cells pretreated with Nutlin-3a were significantly more susceptible to DNAM-1-engineered NK cells than NK cells transfected with the empty vector. Our results provide a proof-of-concept suggesting that the combined use of DNAM-1-chimeric receptor-engineered NK cells and Nutlin-3a may represent a novel therapeutic approach for the treatment of solid tumors, such as NB, carrying dysfunctional p53