Apolipoprotein E induces pathogenic senescent-like myeloid cells in prostate cancer.

Tumor cells promote the recruitment of immunosuppressive neutrophils, a subset of myeloid cells driving immune suppression, tumor proliferation, and treatment resistance. Physiologically, neutrophils are known to have a short half-life. Here, we report the identification of a subset of neutrophils that have upregulated expression of cellular senescence markers and persist in the tumor microenvironment. Senescent-like neutrophils express the triggering receptor expressed on myeloid cells 2 (TREM2) and are more immunosuppressive and tumor-promoting than canonical immunosuppressive neutrophils. Genetic and pharmacological elimination of senescent-like neutrophils decreases tumor progression in different mouse models of prostate cancer. Mechanistically, we have found that apolipoprotein E (APOE) secreted by prostate tumor cells binds TREM2 on neutrophils, promoting their senescence. APOE and TREM2 expression increases in prostate cancers and correlates with poor prognosis. Collectively, these results reveal an alternative mechanism of tumor immune evasion and support the development of immune senolytics targeting senescent-like neutrophils for cancer therapy.

Systematic Development of Peptide Inhibitors Targeting the CXCL12/HMGB1 Interaction.

During inflammatory reactions, the production and release of chemotactic factors guide the recruitment of selective leukocyte subpopulations. The alarmin HMGB1 and the chemokine CXCL12, both released in the microenvironment, can form a heterocomplex, which exclusively acts on the chemokine receptor CXCR4, enhancing cell migration, and in some pathological conditions such as rheumatoid arthritis exacerbates the immune response. An excessive cell influx at the inflammatory site can be diminished by disrupting the heterocomplex. Here, we report the computationally driven identification of the first peptide (HBP08) binding HMGB1 and selectively inhibiting the activity of the CXCL12/HMGB1 heterocomplex. Furthermore, HBP08 binds HMGB1 with the highest affinity reported so far (Kd of 0.8 ± 0.4 µM). The identification of this peptide represents an important step toward the development of innovative pharmacological tools for the treatment of severe chronic inflammatory conditions characterized by an uncontrolled immune response.

Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.

Label-Free Biosensor Detection of Endocrine Disrupting Compounds Using Engineered Estrogen Receptors.

Endocrine Disrupting Compounds (EDCs) are chemical substances shown to interfere with endogenous hormones affecting the endocrine, immune and nervous systems of mammals. EDCs are the causative agents of diseases including reproductive disorders and cancers. This highlights the urgency to develop fast and sensitive methods to detect EDCs, which are detrimental even at very low concentrations. In this work, we propose a label-free surface plasmon resonance (SPR) biosensor method to detect specific EDCs (17 ß-estradiol (E2), ethinyl-estradiol, 4-nonylphenol, tamoxifen) through their binding to estrogen receptor alpha (ERα). We show that the use of rationally designed ERα (as bio-recognition element) in combination with conformation-sensitive peptides (as amplification agent, resulting in increased responses) enables the detection of low parts per billion (ppb) levels of E2. As a proof of concept, this bioassay was used to detect E2 in (spiked) real water samples from fish farms, rivers and the sea at low ppb levels after concentration by solid phase extraction. In addition, the present SPR assay that combines a conformation-sensitive peptide with an array of ERα mutants is very promising for the assessment of the risk of potential estrogenic activity for chemical substances.

A Human Bi-specific Antibody against Zika Virus with High Therapeutic Potential.

Zika virus (ZIKV), a mosquito-borne flavivirus, causes devastating congenital birth defects. We isolated a human monoclonal antibody (mAb), ZKA190, that potently cross-neutralizes multi-lineage ZIKV strains. ZKA190 is highly effective in vivo in preventing morbidity and mortality of ZIKV-infected mice. NMR and cryo-electron microscopy show its binding to an exposed epitope on DIII of the E protein. ZKA190 Fab binds all 180 E protein copies, altering the virus quaternary arrangement and surface curvature. However, ZIKV escape mutants emerged in vitro and in vivo in the presence of ZKA190, as well as of other neutralizing mAbs. To counter this problem, we developed a bispecific antibody (FIT-1) comprising ZKA190 and a second mAb specific for DII of E protein. In addition to retaining high in vitro and in vivo potencies, FIT-1 robustly prevented viral escape, warranting its development as a ZIKV immunotherapy.

Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection.

Zika virus (ZIKV), a mosquito-borne flavivirus with homology to Dengue virus (DENV), has become a public health emergency. By characterizing memory lymphocytes from ZIKV-infected patients, we dissected ZIKV-specific and DENV-cross-reactive immune responses. Antibodies to nonstructural protein 1 (NS1) were largely ZIKV-specific and were used to develop a serological diagnostic tool. In contrast, antibodies against E protein domain I/II (EDI/II) were cross-reactive and, although poorly neutralizing, potently enhanced ZIKV and DENV infection in vitro and lethally enhanced DENV disease in mice. Memory T cells against NS1 or E proteins were poorly cross-reactive, even in donors preexposed to DENV. The most potent neutralizing antibodies were ZIKV-specific and targeted EDIII or quaternary epitopes on infectious virus. An EDIII-specific antibody protected mice from lethal ZIKV infection, illustrating the potential for antibody-based therapy.

Antibody Binding Modulates Conformational Exchange in Domain III of Dengue Virus E Protein.

UNLABELLED: Domain III of dengue virus E protein (DIII) participates in the recognition of cell receptors and in structural rearrangements required for membrane fusion and ultimately viral infection; furthermore, it contains epitopes for neutralizing antibodies and has been considered a potential vaccination agent. In this work, we addressed various structural aspects of DIII and their relevance for both the dengue virus infection mechanism and antibody recognition. We provided a dynamic description of DIII at physiological and endosomal pHs and in complex with the neutralizing human antibody DV32.6. We observed conformational exchange in the isolated DIII, in regions important for the packing of E protein dimers on the virus surface. This conformational diversity is likely to facilitate the partial detachment of DIII from the other E protein domains, which is required to achieve fusion to the host cellular membranes and to expose the epitopes of many anti-DIII antibodies. A comparison of DIII of two dengue virus serotypes revealed many common features but also some possibly unexpected differences. Antibody binding to DIII of dengue virus serotype 4 attenuated the conformational exchange in the epitope region but, surprisingly, generated exchange in other parts of DIII through allosteric effects. IMPORTANCE: Many studies have provided extensive structural information on the E protein and particularly on DIII, also in complex with antibodies. However, there is very scarce information regarding the molecular dynamics of DIII, and almost nothing is available on the dynamic effect of antibody binding, especially at the quantitative level. This work provides one of the very rare descriptions of the effect of antibody binding on antigen dynamics.

Prophylactic and postexposure efficacy of a potent human monoclonal antibody against MERS coronavirus.

Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a previously unidentified coronavirus (CoV), likely transmitted to humans by infected camels. There is no licensed vaccine or antiviral for MERS, therefore new prophylactic and therapeutic strategies to combat human infections are needed. In this study, we describe, for the first time, to our knowledge, the isolation of a potent MERS-CoV-neutralizing antibody from memory B cells of an infected individual. The antibody, named LCA60, binds to a novel site on the spike protein and potently neutralizes infection of multiple MERS-CoV isolates by interfering with the binding to the cellular receptor CD26. Importantly, using mice transduced with adenovirus expressing human CD26 and infected with MERS-CoV, we show that LCA60 can effectively protect in both prophylactic and postexposure settings. This antibody can be used for prophylaxis, for postexposure prophylaxis of individuals at risk, or for the treatment of human cases of MERS-CoV infection. The fact that it took only 4 mo from the initial screening of B cells derived from a convalescent patient for the development of a stable chinese hamster ovary (CHO) cell line producing neutralizing antibodies at more than 5 g/L provides an example of a rapid pathway toward the generation of effective antiviral therapies against emerging viruses.

Rationally modified estrogen receptor protein as a bio-recognition element for the detection of EDC pollutants: strategies and opportunities.

The estrogen receptor protein (ER) can bind a vast number of organic pollutants widely spread in the environment and collectively known as Endocrine Disrupting Chemicals, EDCs. Its broad selectivity makes it an ideal bio-recognition element for the detection of EDCs. Here we describe the strategy and rationale for the design of ER based biosensors and assays that generate a signal in the presence of EDCs. The opportunity to use either natural or rationally modified ER molecules is discussed. The latter approach was successfully applied in the EU-FP7 project RADAR, with the aim to develop a novel biosensor for the detection of organic pollutants both in the environment and in commercial water products.

Rational modification of estrogen receptor by combination of computational and experimental analysis.

In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally modifying the amino acid composition of its ligand binding domain. By combining sequence alignment and structural analysis of known estrogen receptor-ligand complexes with computational analysis, we were able to predict estrogen receptor mutants with altered binding properties. These predictions were experimentally confirmed by producing single point variants with up to an order of magnitude increased binding affinity towards some estrogen disrupting chemicals and reaching an half maximal inhibitory concentration (IC50) value of 2 nM for the 17α-ethinylestradiol ligand. Due to increased affinity and stability, utilizing such mutated estrogen receptor instead of the wild type as bio-recognition element would be beneficial in an assay or biosensor.

Receptor-based high-throughput screening and identification of estrogens in dietary supplements using bioaffinity liquid-chromatography ion mobility mass spectrometry.

A high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of recombinant human estrogen receptor α (ERα) ligands in dietary supplements. For screening, a semi-automated mass spectrometric ligand binding assay was developed applying (13)C2, (15) N-tamoxifen as non-radioactive label and fast ultra-high-performance-liquid chromatography-electrospray ionisation-triple-quadrupole-MS (UPLC-QqQ-MS), operated in the single reaction monitoring mode, as a readout system. Binding of the label to ERα-coated paramagnetic microbeads was inhibited by competing estrogens in the sample extract yielding decreased levels of the label in UPLC-QqQ-MS. The label showed high ionisation efficiency in positive electrospray ionisation (ESI) mode, so the developed BioMS approach is able to screen for estrogens in dietary supplements despite their poor ionisation efficiency in both positive and negative ESI modes. The assay was performed in a 96-well plate, and all these wells could be measured within 3 h. Estrogens in suspect extracts were identified by full-scan accurate mass and collision-cross section (CCS) values from a UPLC-ion mobility-Q-time-of-flight-MS (UPLC-IM-Q-ToF-MS) equipped with a novel atmospheric pressure ionisation source. Thanks to the novel ion source, this instrument provided picogram sensitivity for estrogens in the negative ion mode and an additional identification point (experimental CCS values) next to retention time, accurate mass and tandem mass spectrometry data. The developed combination of bioaffinity screening with UPLC-QqQ-MS and identification with UPLC-IM-Q-ToF-MS provides an extremely powerful analytical tool for early warning of ERα bioactive compounds in dietary supplements as demonstrated by analysis of selected dietary supplements in which different estrogens were identified.