Immunogenicity of a new gorilla adenovirus vaccine candidate for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic caused by the emergent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health, and there is an urgent need to develop safe and effective vaccines. Here, we report the generation and the preclinical evaluation of a novel replication-defective gorilla adenovirus-vectored vaccine encoding the pre-fusion stabilized Spike (S) protein of SARS-CoV-2. We show that our vaccine candidate, GRAd-COV2, is highly immunogenic both in mice and macaques, eliciting both functional antibodies that neutralize SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and a robust, T helper (Th)1-dominated cellular response. We show here that the pre-fusion stabilized Spike antigen is superior to the wild type in inducing ACE2-interfering, SARS-CoV-2-neutralizing antibodies. To face the unprecedented need for vaccine manufacturing at a massive scale, different GRAd genome deletions were compared to select the vector backbone showing the highest productivity in stirred tank bioreactors. This preliminary dataset identified GRAd-COV2 as a potential COVID-19 vaccine candidate, supporting the translation of the GRAd-COV2 vaccine in a currently ongoing phase I clinical trial (ClinicalTrials.gov: NCT04528641).

CD16 pre-ligation by defucosylated tumor-targeting mAb sensitizes human NK cells to γc cytokine stimulation via PI3K/mTOR axis.

Obinutuzumab is a glycoengineered tumor-targeting anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for the FcγRIIIA/CD16 receptor, which was recently approved for clinical use in CLL and follicular lymphoma. Here we extend our previous observation that, in human NK cells, the sustained CD16 ligation by obinutuzumab-opsonized targets leads to a markedly enhanced IFN-γ production upon a subsequent cytokine re-stimulation. The increased IFN-γ competence in response to IL-2 or IL-15 is attributable to post-transcriptional regulation, as it does not correlate with the upregulation of IFN-γ mRNA levels. Different from the reference molecule rituximab, we observe that the stimulation with obinutuzumab promotes the upregulation of microRNA (miR)-155 expression. A similar trend was also observed in NK cells from untreated CLL patients stimulated with obinutuzumab-opsonized autologous leukemia. miR-155 upregulation associates with reduced levels of SHIP-1 inositol phosphatase, which acts in constraining PI3K-dependent signals, by virtue of its ability to mediate phosphatidylinositol 3,4,5-trisphosphate (PIP3) de-phosphorylation. Downstream of PI3K, the phosphorylation status of mammalian target of rapamycin (mTOR) effector molecule, S6, results in amplified response to IL-2 or IL-15 stimulation in obinutuzumab-experienced cells. Importantly, NK cell treatment with the PI3K or mTOR inhibitors, idelalisib and rapamycin, respectively, prevents the enhanced cytokine responsiveness, thus, highlighting the relevance of the PI3K/mTOR axis in CD16-dependent priming. The enhanced IFN-γ competence may be envisaged to potentiate the immunoregulatory role of NK cells in a therapeutic setting.

Peripheral blood T cell alterations in newly diagnosed diffuse large B cell lymphoma patients and their long-term dynamics upon rituximab-based chemoimmunotherapy.

The importance of T cell-dependent immune responses in achieving long-term cure of chemoimmunotherapy-treated cancer patients is underscored by the recently described "vaccinal effect" exerted by therapeutic mAbs. In accordance, pre- and post-therapy peripheral blood lymphopenia represents a well-established negative prognostic factor in DLBCL. We analyzed the phenotypic and functional (IFNγ production, and Granzyme B (GrzB) cytotoxic granule marker expression) profile of peripheral blood T lymphocyte subsets ("conventional" CD4+ and CD8+, FOXP3+CD25bright Treg, and "innate-like" CD56+) in DLBCL patients at diagnosis, and assessed the long-term impact of R-CHOP chemoimmunotherapy, in a prospective study. At diagnosis, DLBCL patients showed lower lymphocyte counts, due to selective decrement of CD4+ T (including Treg) and B lymphocytes. While all T cell subsets transiently decreased during therapy, CD4+ T cell and Treg remained significantly lower than controls, up to 1 year after R-CHOP. Phenotypically skewed profile of CD4+ and CD8+ T cell subsets associated with higher frequencies of IFNγ+ and GrzB+ cells at diagnosis, that transiently decreased during therapy, and re-attained persistently elevated levels, till up to 1 year after therapy. Differently, the pre-therapy elevated levels of circulating monocytes, and of plasma IL-6 and IL-10 rapidly normalized upon R-CHOP. In sum, we describe a quantitatively and functionally altered status of the peripheral blood T cell compartment in DLBCL patients at diagnosis, that persists long-term after tumor eradication, and it is only transiently perturbed by R-CHOP chemoimmunotherapy. Moreover, data suggest the association of selected T cell functional features with DLBCL phenotype, and with therapy outcome.

T lymphocytes engineered to express a CD16-chimeric antigen receptor redirect T-cell immune responses against immunoglobulin G-opsonized target cells.

BACKGROUND AIMS: Chimeric antigen receptors (CARs) designed for adoptive immunotherapy need to achieve two functions: antigen recognition and triggering of the lytic machinery of reprogrammed effector cells. Cytotoxic T cells have been engineered with FcγRIII (CD16) chimeric molecules to be redirected against malignant cells by monoclonal antibodies (mAbs). These cells have been proven to mediate granule-dependent cellular cytotoxicity, but it is not clear whether they can also kill malignant cells by a granule-independent mechanism of cell cytotoxicity. METHODS: We engineered a CD16A-CAR equipped with the extracellular CD16A, the hinge spacer and the transmembrane region of CD8, and the ζ-chain of the T-cell receptor/CD3 complex in tandem with the CD28 co-stimulatory signal transducer module. The CD16A-CAR was expressed and functionally tested in the MD45 cell line, a murine T-cell hybridoma with a defective granular exocytosis pathway but capable of killing target cells by a Fas ligand-mediated lysis. RESULTS: Our results indicate that in vitro cross-linking of CD16A-CAR on MD45 cells by the Fc fragment of mAb opsonized tumor cells induced interleukin-2 release and granule-independent cellular cytotoxicity. CONCLUSIONS: We conclude that strategies aimed to implement the therapeutic functions of mAbs used in the clinic with T-dependent immune responses driven by engineered T cells expressing FcγR-CAR can boost the antitumor efficacy of mAbs used in the clinic.

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition.

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

Improved memory CD8 T cell response to delayed vaccine boost is associated with a distinct molecular signature.

Effective secondary response to antigen is a hallmark of immunological memory. However, the extent of memory CD8 T cell response to secondary boost varies at different times after a primary response. Considering the central role of memory CD8 T cells in long-lived protection against viral infections and tumors, a better understanding of the molecular mechanisms underlying the changing responsiveness of these cells to antigenic challenge would be beneficial. We examined here primed CD8 T cell response to boost in a BALB/c mouse model of intramuscular vaccination by priming with HIV-1 gag-encoding Chimpanzee adenovector, and boosting with HIV-1 gag-encoding Modified Vaccinia virus Ankara. We found that boost was more effective at day(d)100 than at d30 post-prime, as evaluated at d45 post-boost by multi-lymphoid organ assessment of gag-specific CD8 T cell frequency, CD62L-expression (as a guide to memory status) and in vivo killing. RNA-sequencing of splenic gag-primed CD8 T cells at d100 revealed a quiescent, but highly responsive signature, that trended toward a central memory (CD62L+) phenotype. Interestingly, gag-specific CD8 T cell frequency selectively diminished in the blood at d100, relative to the spleen, lymph nodes and bone marrow. These results open the possibility to modify prime/boost intervals to achieve an improved memory CD8 T cell secondary response.

Corrigendum: Improved memory CD8 T cell response to delayed vaccine boost is associated with a distinct molecular signature.

[This corrects the article DOI: 10.3389/fimmu.2023.1043631.].

GRAd-COV2 vaccine provides potent and durable humoral and cellular immunity to SARS-CoV-2 in randomized placebo-controlled phase 2 trial.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and heterologous immunization approaches implemented worldwide for booster doses call for diversified vaccine portfolios. GRAd-COV2 is a gorilla adenovirus-based COVID-19 vaccine candidate encoding prefusion-stabilized spike. The safety and immunogenicity of GRAd-COV2 is evaluated in a dose- and regimen-finding phase 2 trial (COVITAR study, ClinicalTrials.gov: NCT04791423) whereby 917 eligible participants are randomized to receive a single intramuscular GRAd-COV2 administration followed by placebo, or two vaccine injections, or two doses of placebo, spaced over 3 weeks. Here, we report that GRAd-COV2 is well tolerated and induces robust immune responses after a single immunization; a second administration increases binding and neutralizing antibody titers. Potent, variant of concern (VOC) cross-reactive spike-specific T cell response peaks after the first dose and is characterized by high frequencies of CD8s. T cells maintain immediate effector functions and high proliferative potential over time. Thus, GRAd vector is a valuable platform for genetic vaccine development, especially when robust CD8 response is needed.

(Auto)Antibody Responses Shape Memory NK Cell Pool Size and Composition.

In vivo establishment and long-term persistence of a heterogeneous memory or an adaptive NK cell pool represents a functional adaptation to human cytomegalovirus (HCMV) infection in humans. Memory NK cells are commonly identified by lack of the FcεRIγ signalling chain, variably associated to the preferential but not completely overlapping expression of the HLA-E receptor NKG2C and CD57 maturation marker. Although characterized by selective hyperresponsiveness to IgG stimulation, the impact of the CD16/antibody interaction in regulating the establishment/maintenance and size, and in determining the relative abundance of this population, is still under investigation. Memory NK cell subset ex vivo profile and in vitro responsiveness to CD16 stimulation was evaluated in HCMV+ healthy donors and in patients affected by immune thrombocytopenia (ITP), an antibody-mediated autoimmune disease. We identified the FcεRIγ- NKG2C+CD57+ memory NK cell subset, whose abundance is uniquely associated with anti-HCMV antibody levels in healthy seropositive donors, and which is significantly expanded in ITP patients. This fully mature memory subset robustly and selectively expands in vitro in response to mAb-opsonized targets or ITP-derived platelets and displays superior CD16-dependent IFNγ production. Our work identifies opsonizing antibodies as a host-dependent factor that shapes HCMV-driven memory NK cell compartment. We first demonstrate that chronic exposure to auto-antibodies contributes to the establishment/expansion of a highly specialized and unique memory NK cell subset with distinct CD16-dependent functional capabilities. We also identify the specific contribution of the lack of FcεRIγ chain in conferring to NKG2C+CD57+ memory cells a higher responsivity to CD16 engagement.

GRAd-COV2, a gorilla adenovirus-based candidate vaccine against COVID-19, is safe and immunogenic in younger and older adults.

Safe and effective vaccines against coronavirus disease 2019 (COVID-19) are essential for ending the ongoing pandemic. Although impressive progress has been made with several COVID-19 vaccines already approved, it is clear that those developed so far cannot meet the global vaccine demand alone. We describe a COVID-19 vaccine based on a replication-defective gorilla adenovirus expressing the stabilized prefusion severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein named GRAd-COV2. We assessed the safety and immunogenicity of a single-dose regimen of this vaccine in healthy younger and older adults to select the appropriate dose for each age group. For this purpose, a phase 1, dose-escalation, open-labeled trial was conducted including 90 healthy participants (45 aged 18 to 55 years old and 45 aged 65 to 85 years old) who received a single intramuscular administration of GRAd-COV2 at three escalating doses. Local and systemic adverse reactions were mostly mild or moderate and of short duration, and no serious adverse events were reported. Four weeks after vaccination, seroconversion to spike protein and receptor binding domain was achieved in 43 of 44 young volunteers and in 45 of 45 older participants. Consistently, neutralizing antibodies were detected in 42 of 44 younger-age and 45 of 45 older-age volunteers. In addition, GRAd-COV2 induced a robust and T helper 1 cell (TH1)­skewed T cell response against the spike protein in 89 of 90 participants from both age groups. Overall, the safety and immunogenicity data from the phase 1 trial support the further development of this vaccine.

Safety and immune response kinetics of GRAd-COV2 vaccine: phase 1 clinical trial results.

Despite the successful deployment of efficacious vaccines and therapeutics, the development of novel vaccines for SARS-CoV-2 remains a major goal to increase vaccine doses availability and accessibility for lower income setting. We report here on the kinetics of Spike-specific humoral and T-cell response in young and old volunteers over 6 months follow-up after a single intramuscular administration of GRAd-COV2, a gorilla adenoviral vector-based vaccine candidate currently in phase-2 of clinical development. At all three tested vaccine dosages, Spike binding and neutralizing antibodies were induced and substantially maintained up to 3 months, to then contract at 6 months. Potent T-cell responses were readily induced and sustained throughout the study period, with only minor decline. No major differences in immune response to GRAd-COV2 vaccination were observed in the two age cohorts. In light of its favorable safety and immunogenicity, GRAd-COV2 is a valuable candidate for further clinical development and potential addition to the COVID-19 vaccine toolbox to help fighting SARS-CoV-2 pandemic.

Harnessing CD16-Mediated NK Cell Functions to Enhance Therapeutic Efficacy of Tumor-Targeting mAbs.

Natural killer (NK) cells hold a pivotal role in tumor-targeting monoclonal antibody (mAb)-based activity due to the expression of CD16, the low-affinity receptor for IgG. Indeed, beyond exerting cytotoxic function, activated NK cells also produce an array of cytokines and chemokines, through which they interface with and potentiate adaptive immune responses. Thus, CD16-activated NK cells can concur to mAb-dependent "vaccinal effect", i.e., the development of antigen-specific responses, which may be highly relevant in maintaining long-term protection of treated patients. On this basis, the review will focus on strategies aimed at potentiating NK cell-mediated antitumor functions in tumor-targeting mAb-based regimens, represented by (a) mAb manipulation strategies, aimed at augmenting recruitment and efficacy of NK cells, such as Fc-engineering, and the design of bi- or trispecific NK cell engagers and (b) the possible exploitation of memory NK cells, whose distinctive characteristics (enhanced responsiveness to CD16 engagement, longevity, and intrinsic resistance to the immunosuppressive microenvironment) may maximize therapeutic mAb antitumor efficacy.

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.

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+FcεRIγ- 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 characterized 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 expansions. 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.

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 program.

Natural killer (NK) cells and anti-tumor therapeutic mAb: unexplored interactions.

Tumor-targeting mAb are widely used in the treatment of a variety of solid and hematopoietic tumors and represent the first immunotherapeutic approach successfully arrived to the clinic. Nevertheless, the role of distinct immune mechanisms in contributing to their therapeutic efficacy is not completely understood and may vary depending on tumor- or antigen/antibody-dependent characteristics. Availability of next-generation, engineered, tumor-targeting mAb, optimized in their capability to recruit selected immune effectors, re-enforces the need for a deeper understanding of the mechanisms underlying anti-tumor mAb functionality. NK cells participate with a major role to innate anti-tumor responses, by exerting cytotoxic activity and producing a vast array of cytokines. As the CD16 (low-affinity FcγRIIIA)-activating receptor is expressed on the majority of NK cells, its effector functions can be ideally recruited against therapeutic mAb-opsonized tumor cells. The exact role of NK cells in determining therapeutic efficacy of tumor-targeting mAb is still unclear and much sought after. This knowledge will be instrumental to design innovative combination schemes with newly validated immunomodulatory agents. We will summarize what is known about the role of NK cells in therapeutic anti-tumor mAb therapy, with particular emphasis on RTX chimeric anti-CD20 mAb, the first one used in clinical practice for treating B cell malignancies.

Regulation and trafficking of the HLA-E molecules during monocyte-macrophage differentiation.

HLA-E is a nonclassical HLA-class I molecule whose best known role is to protect from the natural killer cells. More recently, an additional function more similar to that of classical HLA-class I molecules, i.e., antigen presentation to T cells, is emerging. However, much remains to be explored about the intracellular trafficking of the HLA-E molecules. With the use of 3 different cellular contexts, 2 monocytic cell lines, U937 and THP1, and peripheral blood monocytes, we show here a remarkable increase of HLA-E during monocyte-macrophage differentiation. This goes independently from the classical HLA-class I, the main source of HLA-E-specific peptides, which is found strongly up-regulated upon differentiation of peripheral blood monocytes but not at all in the case of U937 and THP1 cell lines. Although in all cases, there was a moderate increase of HLA-E expressed in the cell surface, lysis by natural killer cells is comparably restored by an anti-NKG2A antibody in untreated as well as in PMA-differentiated U937 cells. Instead, the great majority of the HLA-E is retained in the vesicles of the autophagy-lysosome network, where they colocalize with the microtubule-associated protein light chain 3, as well as with the lysosomal-associated membrane protein 1. We conclude that differently from the classical HLA-class I molecules, the primary destination of the newly synthesized HLA-E molecules in macrophages is, rather than the cell membrane, the intracellular autophagy-lysosomal vesicles where they are stored and where they can encounter the exogenous antigens.

Tumor-associated and immunochemotherapy-dependent long-term alterations of the peripheral blood NK cell compartment in DLBCL patients.

Natural Killer (NK) cells are a key component of tumor immunosurveillance and thus play an important role in rituximab-dependent killing of lymphoma cells via an antibody-dependent cellular cytotoxicity (ADCC) mechanism. We evaluated the phenotypic and functional assets of peripheral blood NK cell subsets in 32 newly-diagnosed diffuse large B-cell lymphoma (DLBCL) patients and in 27 healthy controls. We further monitored long-term modifications of patient NK cells for up to 12 months after rituximab-based immunochemotherapy. At diagnosis, patients showed a higher percentage of CD56dim and CD16+ NK cells, and a higher frequency of GrzB+ cells in CD56dim, CD56bright, and CD16+ NK cell subsets than healthy controls. Conversely, DLBCL NK cell killing and interferon γ (IFNγ) production capability were comparable to those derived from healthy subjects. Notably, NK cells from refractory/relapsed patients exhibited a lower "natural" cytotoxicity. A marked and prolonged therapy-induced reduction of both "natural" and CD16-dependent NK cytotoxic activities was accompanied by the down-modulation of CD16 and NKG2D activating receptors, particularly in the CD56dim subset. However, reduced NK cell killing was not associated with defective lytic granule content or IFNγ production capability. This study firstly describes tumor-associated and therapy-induced alterations of the systemic NK cell compartment in DLBCL patients. As these alterations may negatively impact rituximab-based therapy efficacy, our work may provide useful information for improving immunochemotherapeutic strategies.

Effect of surgery on pancreatic tumor-dependent lymphocyte asset: modulation of natural killer cell frequency and cytotoxic function.

OBJECTIVES: Tumor burden and invasiveness establish a microenvironment that surgery could alter. This study shows a comprehensive analysis of size, dynamics, and function of peripheral lymphocyte subsets in pancreatic cancer patients before and at different times after duodenopancreatectomy. METHODS: Lymphocyte frequency and natural cytotoxicity were evaluated by flow cytometry and in vitro assay on peripheral blood from initial and advanced-stage pancreatic cancer patients before (BS), at day 7 (PS7), and at day 30 (PS30) after surgery. RESULTS: An increase in natural killer (NK) cells and the diminution of B-cells occurred at PS30, whereas cytotoxicity decreased at PS7. The positive correlation between NK frequency and cytotoxicity at BS and PS7 revealed an altered NK behavior. The elevation of NK cell frequency at PS30, an initial defect in CD56bright NK, and the aberrant correlation between NK frequency and cytotoxicity remained significant in advanced-stage patients, whereas the diminution of NK cytotoxicity only affected initial stage patients. CONCLUSIONS: The NK cell functional ability is altered in presurgery patients; duodenopancreatectomy is associated with short-term impairment of NK function and with a long-term NK cell augmentation and reversion of the aberrant NK behavior, which may impact on immunosurveillance against residual cancer.

Efficacy of aerosol therapy of lung cancer correlates with EGFR paralysis induced by AvidinOX-anchored biotinylated Cetuximab.

Lung cancer, as well as lung metastases from distal primary tumors, could benefit from aerosol treatment. Unfortunately, because of lung physiology, clearance of nebulized drugs is fast, paralleled by unwanted systemic exposure. Here we report that nebulized AvidinOX can act as an artificial receptor for biotinylated drugs. In nude and SCID mice with advanced human KRAS-mutated A549 metastatic lung cancer, pre-nebulization with AvidinOX enables biotinylated Cetuximab to control tumor growth at a dose lower than 1/25,000 the intravenous effective dose. This result correlates with a striking, specific and unpredictable effect of AvidinOX-anchored biotinylated Cetuximab, as well as Panitumumab, observed on a panel of tumor cell lines, leading to inhibition of dimerization and signalling, blockade of endocytosis, induction of massive lysosomal degradation and abrogation of nuclear translocation of EGFR. Excellent tolerability, together with availability of pharmaceutical-grade AvidinOX and antibodies, will allow rapid clinical translation of the proposed therapy.