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

Simultaneous management of renal carcinoma with caval vein thrombosis and double coronary artery disease

Introduction: Recent advances in surgical and anesthesiology techniques allow simultaneous thoracic and abdominal operations to be performed for severe heart disease and benignant or malignant abdominal diseases. Case report: The simultaneous surgical management in a 75-year-old patient suffering from severe double coronary artery disease and a renal cell carcinoma with extended intravascular growth into the inferior vena cava is reported. Conclusion: The postoperative course was uneventful. Simultaneous surgery proved to be beneficial and safe, showing optimal results in our patient

The clonal evolution of two distinct T315I-positive BCR-ABL1 subclones in a Philadelphia-positive acute lymphoblastic leukemia failing multiple lines of therapy: a case report

BACKGROUND: The treatment of Philadelphia chromosome-positive Acute Lymphoblastic Leukemia (Ph+ ALL) patients who harbor the T315I BCR-ABL1 mutation or who have two or more mutations in the same BCR-ABL1 molecule is particularly challenging since first and second-generation Tyrosine Kinase Inhibitors (TKIs) are ineffective. Ponatinib, blinatumomab, chemotherapy and transplant are the currently available options in these cases. CASE PRESENTATION: We here report the case of a young Ph+ ALL patient who relapsed on front-line dasatinib therapy because of two independent T315I-positive subclones, resulting from different nucleotide substitutions -one of whom never reported previously- and where additional mutant clones outgrew and persisted despite ponatinib, transplant, blinatumomab and conventional chemotherapy. Deep Sequencing (DS) was used to dissect the complexity of BCR-ABL1 kinase domain (KD) mutation status and follow the kinetics of different mutant clones across the sequential therapeutic approaches. CONCLUSIONS: This case presents several peculiar and remarkable aspects: i) distinct clones may acquire the same amino acid substitution via different nucleotide changes; ii) the T315I mutation may arise also from an 'act' to 'atc' codon change; iii) the strategy of temporarily replacing TKI therapy with chemo or immunotherapy, in order to remove the selective pressure and deselect aggressive mutant clones, cannot always be expected to be effective; iv) BCR-ABL1-mutated sub-clones may persist at very low levels (undetectable even by Deep Sequencing) for long time and then outcompete BCR-ABL1-unmutated ones becoming dominant even in the absence of any TKI selective pressure

Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity

<p>Abstract</p> <p>Background</p> <p>Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype.</p> <p>Methods</p> <p>The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14) by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored.</p> <p>Results</p> <p>We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs.</p> <p>Conclusion</p> <p>Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these effects can modulate the cell phenotype and can induce a higher sensitivity to tyrosine related anti-blastic drugs.</p

Surface functionalization of extracellular vesicle nanoparticles with antibodies: a first study on the protein corona "variable"

To be profitably exploited in medicine, nanosized systems must be endowed with biocompatibility, targeting capability, the ability to evade the immune system, and resistance to clearance. Currently, biogenic nanoparticles, such as extracellular vesicles (EVs), are intensively investigated as the platform that naturally recapitulates these highly needed characteristics. EV native targeting properties and pharmacokinetics can be further augmented by decorating the EV surface with specific target ligands as antibodies. However, to date, studies dealing with the functionalization of the EV surface with proteins have never considered the protein corona "variable", namely the fact that extrinsic proteins may spontaneously adsorb on the EV surface, contributing to determine the surface, and in turn the biological identity of the EV. In this work, we explore and compare the two edge cases of EVs modified with the antibody Cetuximab (CTX) by chemisorption of CTX (through covalent binding via biorthogonal click-chemistry) and by formation of a physisorbed CTX corona. The results indicate that (i) no differences exist between the two formulations in terms of binding affinity imparted by molecular recognition of CTX versus its natural binding partner (epidermal growth factor receptor, EGFR), but (ii) significant differences emerge at the cellular level, where CTX-EVs prepared by click chemistry display superior binding and uptake toward target cells, very likely due to the higher robustness of the CTX anchorage

Use of a high sensitive nanofluidic array for the detection of rare copies of BCR-ABL1 transcript in patients with Philadelphia-positiveacute lymphoblastic leukemia in complete responseIlaria

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Circulating functional T cells specific to human herpes virus 6 (HHV6) antigens in individuals with chromosomally integrated HHV6

Circulating functional T cells specific to human herpes virus 6 (HHV6) antigens in individuals with chromosomally integrated HHV

The baseline comorbidity burden affects survival in elderly patients with acute myeloid leukemia receiving hypomethylating agents: Results from a multicentric clinical study

Background: In older patients with acute myeloid leukemia (AML), the definition of fitness, prognosis, and risk of death represents an open question. Methods: In the present study, we tested the impact on survival of disease- and patient-related parameters in a large cohort of elderly AML patients homogeneously assigned to treatment with hypomethylating agents (HMAs). Results: In 131 patients with a median age of 76 years, we confirmed that early response (&lt;0.001) and biology-based risk classification (p&nbsp;=&nbsp;0.003) can select patients with better-predicted survival. However, a full disease-oriented model had limitations in stratifying our patients, prompting us to investigate the impact of baseline comorbidities on overall survival basing on a comorbidity score. The albumin level (p&nbsp;=&nbsp;0.001) and the presence of lung disease (p&nbsp;=&nbsp;0.013) had a single-variable impact on prognosis. The baseline comorbidity burden was a powerful predictor of patients' frailty, correlating with increased incidence of adverse events, especially infections, and predicted overall survival (p &lt; 0.001). Conclusion: The comorbidity burden may contribute to impact prognosis in addition to disease biology. While the therapeutic armamentarium of elderly AML is improving, a comprehensive approach that combines AML biology with tailored interventions to patients' frailty is likely to fully exploit the anti-leukemia potential of novel drugs