Antibody-dependent Cellular Cytotoxicity Research Articles

SHR-1806, a robust OX40 agonist to promote T cell-mediated antitumor immunity

ABSTRACT Anti-CTLA-4 and anti-PD-1/PD-L1 antibodies have significantly revolutionized cancer immunotherapy. However, the persistent challenge of low patient response rates necessitates novel approaches to overcome immune tolerance. Targeting immunostimulatory signaling may have a better chance of success for its ability to enhance effector T cell (Teff) function and expansion for antitumor immunity. Among various immunostimulatory pathways, the evidence underscores the potential of activating OX40-OX40L signaling to enhance CD8+ T cell generation and maintenance while suppressing regulatory T cells (Tregs) within the tumor microenvironment (TME). In this study, we introduce a potent agonistic anti-OX40 antibody, SHR-1806, designed to target OX40 receptors on activated T cells and amplify antitumor immune responses. SHR-1806 demonstrates a high affinity and specificity for human OX40 protein, eliciting FcγR-mediated agonistic effects, T cell activation, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities in vitro. In human OX40 knock-in mice bearing MC38 tumor, SHR-1806 shows a trend toward a higher potency than the reference anti-OX40 antibody produced in-house, GPX4, an analog of pogalizumab, the most advanced drug candidate developed by Roche. Furthermore, SHR-1806 displays promising anti-tumor activity alone or in combination with toll-like receptor 7 (TLR7) agonist or PD-L1 inhibitor in mouse models. Evaluation of SHR-1806 in rhesus monkeys indicates a favorable safety profile and typical pharmacokinetic characteristics. Thus, SHR-1806 emerges as a robust OX40 agonist with promising therapeutic potential.

In utero human cytomegalovirus infection expands NK-like FcγRIII+ CD8+ T cells that mediate Fc antibody functions.

Human cytomegalovirus (HCMV) profoundly impacts host T and natural killer (NK) cells across the lifespan, yet how this common congenital infection modulates developing fetal immune cell compartments remains underexplored. Using cord blood from neonates with and without congenital HCMV (cCMV) infection, we identify an expansion of Fcγ receptor III (FcγRIII)-expressing CD8+ T cells following HCMV exposure in utero. Most FcγRIII+ CD8+ T cells express the canonical αβ T cell receptor (TCR) but a proportion express non-canonical γδ TCR. FcγRIII+ CD8+ T cells are highly differentiated and have increased expression of NK cell markers and cytolytic molecules. Transcriptional analysis reveals FcγRIII+ CD8+ T cells upregulate T-bet and downregulate BCL11B, known transcription factors that govern T/NK cell fate. We show that FcγRIII+ CD8+ T cells mediate antibody-dependent IFNγ production and degranulation against IgG-opsonized target cells, similar to NK cell antibody-dependent cellular cytotoxicity (ADCC). FcγRIII+ CD8+ T cell Fc effector functions were further enhanced by interleukin-15 (IL-15), as has been observed in neonatal NK cells. Our study reveals that FcγRIII+ CD8+ T cells elicited in utero by HCMV infection can execute Fc-mediated effector functions bridging cellular and humoral immunity and may be a promising target for antibody-based therapeutics and vaccination in early life.

Dissecting Immunological Mechanisms Underlying Influenza Viral Nucleoprotein-induced Mucosal Immunity Against Diverse Viral Strains

The nucleoprotein (NP) of type A influenza virus (IAV) is highly conserved across all virus strains, making it an attractive candidate antigen for universal vaccines. While various studies have explored NP-induced mucosal immunity, here we interrogated the mechanistic differences between intramuscular (IM) and intranasal (IN) delivery of a recombinant adenovirus carrying NP fused with a bifunctional CD40 ligand. Despite being less effective than IM delivery in inducing systemic cellular immune responses and antibody-dependent cellular cytotoxicity (ADCC), IN immunization elicited superior antigen-specific recall humoral and cellular response in the nasal associated lymphoid tissue (NALT) of the upper respiratory tract, the initial site of immune recognition and elimination of inhaled pathogens. IN vaccination also induced significantly stronger pulmonary T cell responses in the lower respiratory tract than IM vaccination, in particular the CD8 T cells. Moreover, blocking lymphocyte circulation abrogated IM but not IN immunization induced protection, illustrating the critical role of local memory immune response upon viral infection. Notably, the CD40-targeted nasal delivery not only improved the magnitude but also the breadth of protection, including against lethal challenge with a newly isolated highly pathogenic avian H5N1 strain. These findings are informative for the design of universal mucosal vaccines, where the predominant mode of protection is independent of neutralizing antibodies.

Bendamustine–rituximab elicits dual tumoricidal and immunomodulatory responses via cGAS–STING activation in diffuse large B-cell lymphoma

BackgroundBendamustine–rituximab (BR) therapy stands out as a promising alternative for elderly patients with diffuse large B-cell lymphoma (DLBCL), demonstrating notable efficacy when conventional regimens pose challenges. Despite its clinical success,...

Nonclinical Similarity of the Biosimilar Candidate ABP 938 with Aflibercept Reference Product.

ABP 938 is being developed as a biosimilar to Eylea® (aflibercept reference product [RP]), an anti-vascular endothelial growth factor (VEGF) drug used in the management of retinal diseases. Previously, a comparative analytical similarity assessment demonstrated that ABP 938 and aflibercept RP have the same amino acid sequence and exhibit similar higher-order structure and biological activity. The nonclinical studies described here were designed to assess the in vitro pharmacology and the in vivo pharmacokinetics (PK), toxicokinetics (TK), and safety profiles of ABP 938 compared to aflibercept RP. In vitro target-binding kinetics and affinity for VEGF-A and placental growth factor (PIGF) isoforms were evaluated using surface plasmon resonance (SPR). Effector functions were assessed by cell-based assays. PK was evaluated in a nonterminal intravitreal (IVT) ocular distribution study in rabbits. Safety was assessed in a 1-month IVT study in cynomolgus monkeys. SPR results demonstrated that ABP 938 is similar to aflibercept RP in binding kinetics and affinity for VEGF-A111, VEGF-A121, VEGF-A165, VEGF-A189, PlGF-1, and PlGF-2 isoforms. No antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, or complement-dependent cytotoxicity was observed with ABP 938 and aflibercept RP. Results from thenonterminal ocular distribution study in rabbits indicated that there were no meaningful differences in the distribution kinetics between intravitreally injected ABP 938 and aflibercept RP. Additionally, there was no evidence of ocular or systemic toxicity associated with IVT administration of ABP 938 in a repeat-dose, 1-month toxicology study in cynomolgus monkeys; toxicokinetic and toxicology profiles were similar to aflibercept RP. This integrated assessment of results from the in vitro pharmacology assessment and in vivo PK and TK/toxicology profiles formed the nonclinical portion of the totality of evidence demonstrating ABP938 is a biosimilar to aflibercept RP.

A Cancer-Specific Anti-Podoplanin Monoclonal Antibody, PMab-117-mG2a Exerts Antitumor Activities in Human Tumor Xenograft Models

Podoplanin (PDPN) overexpression is associated with poor clinical outcomes in various tumors. PDPN is involved in malignant tumor progression by promoting invasiveness and metastasis. Therefore, PDPN is considered a promising target of monoclonal antibody (mAb)-based therapy. Because PDPN also plays an essential role in normal cells such as kidney podocytes, cancer specificity is required to reduce adverse effects on normal cells. We developed a cancer-specific mAb (CasMab) against PDPN, PMab-117 (rat IgM, kappa), by immunizing rats with PDPN-overexpressed glioblastoma cells. The recombinant mouse IgG2a-type PMab-117 (PMab-117-mG2a) reacted with the PDPN-positive tumor PC-10 and LN319 cells but not with PDPN-knockout LN319 cells in flow cytometry. PMab-117-mG2a did not react with normal kidney podocytes and normal epithelial cells from the lung bronchus, mammary gland, and corneal. In contrast, one of the non-CasMabs against PDPN, NZ-1, showed high reactivity to PDPN in both tumor and normal cells. Moreover, PMab-117-mG2a exerted antibody-dependent cellular cytotoxicity in the presence of effector splenocytes. In the human tumor xenograft models, PMab-117-mG2a exhibited potent antitumor effects. These results indicated that PMab-117-mG2a could be applied to antibody-based therapy against PDPN-expressing human tumors while reducing the adverse effects.

Enterovirus Antibodies: Friends and Foes.

Enteroviruses (EV) initiate replication by binding to their cellular receptors, leading to the uncoating and release of the viral genome into the cytosol of the host cell. Neutralising antibodies (NAbs) binding to epitopes on enteroviral capsid proteins can inhibit this infectious process through several mechanisms of neutralisation in vitro. Fc-mediated antibody effector functions such as antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis have also been described for some EV. However, antibody binding to virions does not always result in viral neutralisation. Non-neutralising antibodies, or sub-neutralising concentrations of antibodies, can enhance infection of viruses, leading to more severe pathologies. This phenomenon, known as antibody-dependent enhancement (ADE) of infection, has been described in vitro and/or in vivo for EV including poliovirus, coxsackievirus B and EV-A71. It has been shown that ADE of EV infection is mediated by FcγRs expressed by monocytes, macrophages, B lymphocytes and granulocytes. Antibodies play a crucial role in the diagnosis and monitoring of infections. They are valuable markers that have been used to establish a link between enteroviral infection and chronic diseases such as type 1 diabetes. Monoclonal and polyclonal antibodies targeting enteroviral proteins have been developed and shown to be effective to prevent or combat EV infections in vitro and in vivo. In addition, vaccines are under development, and clinical trials of vaccines are underway or have been completed, providing hope for the prevention of diseases due to EV. However, the ADE of the infection should be considered in the development of anti-EV antibodies or safe vaccines.

Autoimmune haemolytic anaemias.

Adult autoimmune haemolytic anaemias (AIHAs) include different subtypes of a rare autoimmune disease in which autoantibodies targeting autoantigens expressed on the membrane of autologous red blood cells (RBCs) are produced, leading to their accelerated destruction. In the presence of haemolytic anaemia, the direct antiglobulin test is the cornerstone of AIHA diagnosis. AIHAs are classified according to the isotype and the thermal optimum of the autoantibody into warm (wAIHAs), cold and mixed AIHAs. wAIHAs, the most frequent type of AIHAs, are associated with underlying conditions in ~50% of cases. In wAIHA, IgG autoantibody reacts with autologous RBCs at 37 °C, leading to antibody-dependent cell-mediated cytotoxicity and increased phagocytosis of RBCs in the spleen. Cold AIHAs include cold agglutinin disease (CAD) and cold agglutinin syndrome (CAS) when there is an underlying condition. CAD and cold agglutinin syndrome are IgM cold antibody-driven AIHAs characterized by classical complement pathway-mediated haemolysis. The management of wAIHAs has long been based around corticosteroids and splenectomy and on symptomatic measures and non-specific cytotoxic agents for CAD. Rituximab and the development of complement inhibitors, such as the anti-C1s antibody sutimlimab, have changed the therapeutic landscape of AIHAs, and new promising targeted therapies are under investigation.

Differences in IgG afucosylation between groups with and without carotid atherosclerosis

BackgroundA previous study demonstrated that N-glycosylation profiles of IgG are associated with subclinical atherosclerosis in a British population. However, the generalisability of this finding to other ethnic groups remains to be investigated, and it has yet to account for additional traditional atherosclerotic risk factors. The present study, thus, aims to explore IgG N-glycosylation profiles in Han Chinese with atherosclerosis, and their potential role in atherosclerosis, while controlling for traditional atherosclerotic risk factors.MethodsData of this case-control study were obtained from an established umbrella Health Examination Cohort Study (registration number: ChiCTR2100048740). The investigation was conducted at the Health Care Centre of the First Affiliated Hospital of Shantou University Medical College in China, from August 1, 2021, to July 31, 2022. A sample of 69 carotid atherosclerosis (CAS) cases was recruited from the umbrella cohort, along with 69 controls without carotid atherosclerosis, matched by traditional atherosclerosis-related risk factors, including gender, age, smoking, alcohol consumption, hypertension, diabetes, dyslipidemia and obesity. Subsequently, serum IgG N-glycosylation was profiled using Ultra-Performance Liquid Chromatography.ResultsAfter propensity score matching, the relative abundance of IgG fucosylation in CAS cases was significantly lower than that in controls [95.32 (92.96, 95.99) vs. 95.96 (94.70, 96.58), P = 0.022]. The traditional atherosclerosis-related risk factors showed no statistically significant difference between CAS cases and controls (P > 0.05).ConclusionsThe reduced fucosylation of IgG in CAS cases underscores the pivotal role of afucosylation in CAS. Enhancing the inflammatory capability of IgG via initiating antibody-dependent cell-mediated cytotoxicity could be the potential mechanism behind this, which should be further verified by functional studies.

Trastuzumab-Mediated Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Enhances Natural Killer Cell Cytotoxicity in HER2-Overexpressing Ovarian Cancer.

Ovarian cancer is the deadliest gynecologic cancer. Although human epidermal growth factor receptor-2 (HER2) overexpression, a poor prognostic molecular marker in ovarian cancer, is found in almost 30% of ovarian cancer cases, there are no established therapies for HER2-overexpressing ovarian cancer. In this study, we investigated the efficacy of combined samfenet, a biosimilar compound of trastuzumab, and natural killer (NK) cells in preclinical model of HER2-overexpressing ovarian cancer. Firstly, we screened the HER2 expression in three ovarian cancer cell lines and eight ovarian cancer patient-derived tumor xenograft (PDTX) samples. Then, immunohistochemistry and silver in situ hybridization (SISH) were performed following clinical criteria. HER2-overexpressing cells exhibited the highest sensitivity to samfenet compared with low-HER2-expressing cells. In addition, the combination of samfenet with natural killer (NK) cells resulted in significantly enhanced sensitivity to HER2-overexpressing cells and showed a significant antitumor effect on PDTX mice compared with monotherapy. It is known that anti-HER2-humanized IgG1 monoclonal antibodies, including trastuzumab, induce antibody-dependent cellular cytotoxicity (ADCC). Consequently, the combination of samfenet with NK cells demonstrated NK cell-mediated ADCC, as confirmed using an in vitro NK cytotoxicity assay and in vivo antitumor efficacy. A transferase dUTP nick end labeling (TUNEL) assay using xenografted tumors further supported the ADCC effects based on the increase in the number of apoptotic cells in the combination group. Furthermore, high HER2 expression was associated with shorter progression-free survival and overall survival based on public mRNA expression data. In this study, we demonstrated that the combination of samfenet and NK cell therapy could be a promising treatment strategy for patients with HER2-overexpressing ovarian cancer, through ADCC effects. Therefore, this study supports a rationale for further clinical studies of the combination of samfenet and NK cells as a therapy for patients with HER2-overexpressing ovarian cancer.

Microbiological aspects of cancer progression: A systematic review conducted according to the PRISMA 2020 guidelines and the Cochrane Handbook for Systematic Reviews of Interventions.

The complex interplay between the gut microbiota, cancer treatments and patient characteristics has emerged as a significant area of research. This study sought to examine these relationships in the context of colorectal cancer (CRC).A comprehensive search of relevant studies was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines and the Cochrane Handbook for Systematic Reviews of Interventions. The studies included a variety of treatment modalities and microbiological parameters. A data extraction form, designed specifically for this review, was used to assess a range of variables across all studies.The analysis revealed a multifaceted interaction between the gut microbiota, genetic factors and treatment outcomes. Elderly patients with CRC frequently received single-agent chemotherapy, with outcomes that were comparable to those of younger patients. The presence of tumorigenic bacteria, including Escherichia coli and Bacteroides fragilis, was associated with early colon neoplasia. Additionally, an abundance of Fusobacterium spp. was observed in colonic adenomas, contributing to a pro-inflammatory environment. Although the FcγRIIIa-158 V/V genotype was associated with higher cetuximab-mediated antibodydependent cellular cytotoxicity (ADCC), no direct influence of FcγR polymorphisms on treatment response was noted. Furthermore, the combination of programmed cell death protein-1 (PD-1), BRAF and MEK inhibition showed favorable response rates. The gut microbiome, especially the presence of Fusobacterium spp., had a notable influence on the therapeutic response in CRC.These findings underscore the role of the gut microbiota and genetic factors in cancer treatment outcomes, emphasizing the potential of a holistic approach to cancer management. Future research should exploit these findings in order to develop microbiota-modulating strategies and personalized medicine approaches for the purpose of improving the efficacy of cancer treatment.

EVA1-Antibody Drug Conjugate is a new therapeutic strategy for eliminating glioblastoma-initiating cells.

The discovery of glioblastoma (GBM)-initiating cells (GICs) has impacted GBM research. These cells are not only tumorigenic, but also exhibit resistance to radiotherapy and chemotherapy. Therefore, it is crucial to characterize GICs thoroughly and identify new therapeutic targets. In a previous study, we successfully identified Epithelial V-like antigen 1 (EVA1) as a novel functional factor specific to GICs. Hybridoma cells were generated by immunizing BALB/c mice with EVA1-Fc fusion protein. The reactivity of the supernatant from these hybridoma cells was examined using EVA1-overexpressing cells and GICs. Candidate antibodies were further selected using Biacore surface plasmon resonance analysis and two cytotoxicity assays, antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Among the antibodies, the cytotoxicity of the B2E5-antibody drug conjugate (B2E5-ADC) was evaluated by both adding it to cultured GICs and injecting it into GIC tumor-bearing brains. B2E5 demonstrated a high affinity for human EVA1 and effectively killed both EVA1-expressing cell lines and GICs in culture through ADCC and CDC. B2E5-ADC also exhibited strong cytotoxicity to GICs in culture and prevented their tumorigenesis in the brain when administered intracranially to the tumor-bearing brain. Our data indicate that B2E5-ADC is a new and promising therapeutic strategy for GBM.

Optimization of a Piperidine CD4-Mimetic Scaffold Sensitizing HIV-1 Infected Cells to Antibody-Dependent Cellular Cytotoxicity

Optimization of a Piperidine CD4-Mimetic Scaffold Sensitizing HIV-1 Infected Cells to Antibody-Dependent Cellular Cytotoxicity

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa/CD16a structure.

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa/CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues, we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step, with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating that the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa/CD16a structure

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa/CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues, we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step, with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating that the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.

Headless hemagglutinin-containing influenza viral particles direct immune responses toward more conserved epitopes.

Seasonal influenza vaccines provide mostly strain-specific protection due to the elicitation of antibody responses focused on evolutionarily plastic antigenic sites in the hemagglutinin head domain. To direct the humoral response toward more conserved epitopes, we generated an influenza virus particle where the full-length hemagglutinin protein was replaced with a membrane-anchored, "headless" variant while retaining the normal complement of other viral structural proteins such as the neuraminidase as well as viral RNAs. We found that a single administration of a headless virus particle-based vaccine elicited high titers of antibodies that recognized more conserved epitopes on the major viral glycoproteins. Furthermore, the vaccine could elicit these responses even in the presence of pre-existing, hemagglutinin (HA) head-focused influenza immunity. Importantly, these antibody responses mediated protective, but non-neutralizing functions such as neuraminidase inhibition and antibody-dependent cellular cytotoxicity. Additionally, we show the vaccine can provide protection from homologous and heterologous challenges in mouse models of severe influenza without any measurable HA head-directed antibody responses. Thus, headless hemagglutinin containing viral particles may represent a tool to drive the types of antibody responses predicted to increase influenza vaccine breadth and durability.IMPORTANCECurrent seasonal influenza vaccines provide incomplete protection from disease. This is partially the result of the antibody response being directed toward parts of the virus that are tolerant of mutations. Redirecting the immune response to more conserved regions of the virus has been a central strategy of next-generation vaccine designs and approaches. Here, we develop and test a vaccine based on a modified influenza virus particle that expresses a partially deleted hemagglutinin protein along with the other viral structural proteins. We demonstrate this vaccine elicits antibodies that recognize the more conserved viral epitopes of the hemagglutinin stalk and neuraminidase protein to facilitate protection against influenza viruses despite a lack of classical viral neutralization activity.

Expression of c-erb-B2 oncoprotein as a neoantigen strategy to repurpose anti-neu antibody therapy in a model of melanoma.

In this study, we tested a novel approach of "repurposing" a biomarker typically associated with breast cancer for use in melanoma. HER2/neu is a well characterized biomarker in breast cancer for which effective anti-HER2/neu therapies are readily available. We constructed a lentivirus encoding c-erb-B2, an animal (rat) homolog to HER2/neu. This was used to transfect B16 melanoma in vitro for use in an orthotopic preclinical mouse model, which resulted in expression of rat c-erb-B2 as a neoantigen target for anti-c-erb-B2 monoclonal antibody (7.16.4). The c-erb-B2-expressing melanoma was designated B16/neu. 7.16.4 produced statistically significant in vivo anti-tumor responses against B16/neu. This effect was mediated by NK-cell antibody-dependent cell-mediated cytotoxicity. To further model human melanoma (which expresses < 5% HER2/neu), our c-erb-B2 encoding lentivirus was used to inoculate naïve (wild-type) B16 tumors in vivo, resulting in successful c-erb-B2 expression. When combined with 7.16.4, anti-tumor responses were again demonstrated where approximately 40% of mice treated with c-erb-B2 lentivirus and 7.16.4 achieved complete clinical response and long-term survival. For the first time, we demonstrated a novel strategy to repurpose c-erb-B2 as a neoantigen target for melanoma. Our findings are particularly significant in the contemporary setting where newer anti-HER2/neu antibody-drug therapies have shown increased efficacy.

A recombinant fragment antigen-binding (Fab) of trastuzumab displays low cytotoxic profile in adult human cardiomyocytes: first evidence and the key implication of FcγRIIA receptor.

Fragment crystallizable gamma receptors (FcγRs) mediate various cellular responses with significant cardiovascular implications. They contribute to the anticancer activity of trastuzumab (TRZ), a recombinant humanized monoclonal antibody that interferes with human epidermal growth factor receptor 2 (HER2), thereby blocking its physiological function in cardiac cells. This is responsible for cardiac complications that hamper TRZ clinical application. In this study we investigated the involvement of FcγRs in the TRZ cardiotoxicity. We used a recombinant antigen-binding fragment (Fab) of TRZ (rFab-HER2) to examine whether the absence of the Fc region resulted in fewer cardiomyocyte toxicity while preserving TRZ's ability to inhibit HER2. When exposed to rFab-HER2, AC16 human adult ventricular cardiomyocytes were less vulnerable to damage and death, than to TRZ. Specifically, TRZ exhibited cytotoxicity at a lower concentration (150 µg/mL, corresponding to ~1 µM) compared to rFab-HER2 (250 µg/mL, corresponding to ~5 µM). Like TRZ, rFab-HER2 negatively modulated HER2 levels in cardiomyocyte (without inducing cytotoxic activity in BJ human fibroblast cells that either did not express or express very low levels of HER2) and inhibited the downstream ERK/AKT cascades. But rFab-HER2 did not alter cardiomyocyte mitochondrial dynamic balance, and affect apoptosis and inflammation, while it limited cytosolic and mitochondrial ROS indicators. On contrary, the Fc region (50-250 μg/mL) exerted direct cytotoxic action on cardiomyocytes (but not on human fibroblasts that lacked Fc receptors). TRZ (150 μg/mL) markedly upregulated the expression level of FcγRIIA (a FcγRs strongly involved in TRZ-induced antibody-dependent cellular toxicity) in cardiomyocytes, whereas the Fab fragment (150 μg/mL) had no effect. Our results demonstrate that Fc region plays an important pathogenic role in TRZ-induced cardiomyocyte toxicity. In addition, targeting FcγRIIA might contribute to the off-target effects of TRZ therapy.

Targeting the IL-5 pathway in eosinophilic asthma: A comparison of anti-IL-5 versus anti-IL-5 receptor agents.

Eosinophilic asthma is characterized by frequent exacerbations, poor symptom control and accelerated lung function decline. It is now recognized that the immune response underlying eosinophilic asthma involves a complex network of interconnected pathways from both the adaptive and innate immune systems. Within this response, interleukin-5 (IL-5) plays a central role in eosinophil differentiation, activation and survival and has emerged as a key target for therapies treating severe asthma. The monoclonal antibodies mepolizumab and reslizumab target the ligand IL-5, preventing its interaction with eosinophils; in contrast, benralizumab binds to the IL-5 receptor (IL-5R), preventing IL-5 from binding and leading to substantially greater eosinophil reduction by enhanced antibody-dependent cell-mediated cytotoxicity. Although no direct head-to-head clinical trials of asthma have been published to formally evaluate the clinical significance of these different therapeutic approaches, the potential benefits of partial versus complete eosinophil depletion continue to remain an important area of study and debate. Here, we review the existing real-world and clinical study data of anti-IL-5/anti-IL-5R therapies in severe eosinophilic asthma.

Anti-OAcGD2 antibody in combination with ceramide kinase inhibitor mediates potent antitumor cytotoxicity against breast cancer and diffuse intrinsic pontine glioma cells.

O-acetylated GD2 (OAcGD2) is a cancer-related antigen that is currently being explored for therapeutic use. Exploring the intricate mechanisms behind OAcGD2 synthesis in cancer cells has long been a challenge. Leveraging state-of-the-art high-throughput RNAi screening and confocal imaging technologies, our study delves into the genetic network orchestrating OAcGD2 synthesis in breast cancer cells. By conducting a comprehensive siRNA screen targeting the OAcGD2 phosphatome/kinome, we identified 43 genetic modulators, with 25 downregulating and 18 upregulating OAcGD2 synthesis. Among these, our study focused on CERK, the gene-encoding ceramide kinase, a pivotal player in glycosphingolipid metabolism. Through meticulous experimentation utilizing anti-CERK inhibitor and siRNAs, we made a significant discovery: CERK inhibition robustly upregulates OAcGD2 in both neuroblastoma and breast cancer cells, concurrently dampening cell migration. Furthermore, our findings highlight an exciting prospect: augmenting the antibody-dependent cell cytotoxicity of the chimeric human/mouse anti-OAcGD2 IgG1 monoclonal antibody (c8B6 mAb) against breast cancer and diffuse intrinsic pontine glioma cell lines in combination with specific CERK inhibitors. These results underscore the pivotal role of CERK inhibition in bolstering OAcGD2 synthesis, thus, presenting a promising strategy to increase the efficacy of anti-OAcGD2-based immunotherapy in patients with neuroectodermal tumors. By shedding light on this intricate interplay, our study paves the way for innovative therapeutic strategies poised to revolutionize the treatment landscape for these aggressive malignancies.