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.

Machine learning analyses of antibody somatic mutations predict immunoglobulin light chain toxicity.

In systemic light chain amyloidosis (AL), pathogenic monoclonal immunoglobulin light chains (LC) form toxic aggregates and amyloid fibrils in target organs. Prompt diagnosis is crucial to avoid permanent organ damage, but delayed diagnosis is common because symptoms usually appear only after strong organ involvement. Here we present LICTOR, a machine learning approach predicting LC toxicity in AL, based on the distribution of somatic mutations acquired during clonal selection. LICTOR achieves a specificity and a sensitivity of 0.82 and 0.76, respectively, with an area under the receiver operating characteristic curve (AUC) of 0.87. Tested on an independent set of 12 LCs sequences with known clinical phenotypes, LICTOR achieves a prediction accuracy of 83%. Furthermore, we are able to abolish the toxic phenotype of an LC by in silico reverting two germline-specific somatic mutations identified by LICTOR, and by experimentally assessing the loss of in vivo toxicity in a Caenorhabditis elegans model. Therefore, LICTOR represents a promising strategy for AL diagnosis and reducing high mortality rates in AL.

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.

Ligand-Receptor Interactions of Galectin-9 and VISTA Suppress Human T Lymphocyte Cytotoxic Activity.

Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.

The Tim-3-Galectin-9 Pathway and Its Regulatory Mechanisms in Human Breast Cancer.

Human cancer cells operate a variety of effective molecular and signaling mechanisms which allow them to escape host immune surveillance and thus progress the disease. We have recently reported that the immune receptor Tim-3 and its natural ligand galectin-9 are involved in the immune escape of human acute myeloid leukemia (AML) cells. These cells use the neuronal receptor latrophilin 1 (LPHN1) and its ligand fibronectin leucine rich transmembrane protein 3 (FLRT3, and possibly other ligands) to trigger the pathway. We hypothesized that the Tim-3-galectin-9 pathway may be involved in the immune escape of cancer cells of different origins. We found that studied breast tumors expressed significantly higher levels of both galectin-9 and Tim-3 compared to healthy breast tissues of the same patients and that these proteins were co-localized. Increased levels of LPHN2 and expressions of LPHN3 as well as FLRT3 were also detected in breast tumor cells. Activation of this pathway facilitated the translocation of galectin-9 onto the tumor cell surface, however no secretion of galectin-9 by tumor cells was observed. Surface-based galectin-9 was able to protect breast carcinoma cells against cytotoxic T cell-induced death. Furthermore, we found that cell lines from brain, colorectal, kidney, blood/mast cell, liver, prostate, lung, and skin cancers expressed detectable amounts of both Tim-3 and galectin-9 proteins. The majority of cell lines expressed one of the LPHN isoforms and FLRT3. We conclude that the Tim-3-galectin-9 pathway is operated by a wide range of human cancer cells and is possibly involved in prevention of anti-tumor immunity.

High mobility group box 1 (HMGB1) acts as an "alarmin" to promote acute myeloid leukaemia progression.

High mobility group box 1 (HMGB1) is a non-histone protein localised in the cell nucleus, where it interacts with DNA and promotes nuclear transcription events. HMGB1 levels are elevated during acute myeloid leukaemia (AML) progression followed by participation of this protein in triggering signalling events in target cells as a pro-inflammatory stimulus. This mechanism was hypothesised to be employed as a survival pathway by malignant blood cells and our aims were therefore to test this hypothesis experimentally. Here we report that HMGB1 triggers the release of tumour necrosis factor alpha (TNF-α) by primary human AML cells. TNF-α induces interleukin 1 beta (IL-1ß) production by healthy leukocytes, leading to IL-1ß-induced secretion of stem cell factor (SCF) by competent cells (for example endothelial cells). These results were verified in mouse bone marrow and primary human AML blood plasma samples. In addition, HMGB1 was found to induce secretion of angiogenic vascular endothelial growth factor (VEGF) and this process was dependent on the immune receptor Tim-3. We therefore conclude that HMGB1 is critical for AML progression as a ligand of Tim-3 and other immune receptors thus supporting survival/proliferation of AML cells and possibly the process of angiogenesis.

Highly specific targeting of human acute myeloid leukaemia cells using pharmacologically active nanoconjugates.

In this study we used 5 nm gold nanoparticles as delivery platforms to target cancer cells expressing the immune receptor Tim-3 using single chain antibodies. Gold surfaces were also covered with the cytotoxic drug rapamycin which was immobilised using a glutathione linker. These nanoconjugates allowed highly specific and efficient delivery of cytotoxic rapamycin into human malignant blood cells.

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.

The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a severe and often fatal systemic malignancy. Malignant cells are capable of escaping host immune surveillance by inactivating cytotoxic lymphoid cells. In this work we discovered a fundamental molecular pathway, which includes ligand-dependent activation of ectopically expressed latrophilin 1 and possibly other G-protein coupled receptors leading to increased translation and exocytosis of the immune receptor Tim-3 and its ligand galectin-9. This occurs in a protein kinase C and mTOR (mammalian target of rapamycin)-dependent manner. Tim-3 participates in galectin-9 secretion and is also released in a free soluble form. Galectin-9 impairs the anti-cancer activity of cytotoxic lymphoid cells including natural killer (NK) cells. Soluble Tim-3 prevents secretion of interleukin-2 (IL-2) required for the activation of cytotoxic lymphoid cells. These results were validated in ex vivo experiments using primary samples from AML patients. This pathway provides reliable targets for both highly specific diagnosis and immune therapy of AML.

Balance of Anti-CD123 Chimeric Antigen Receptor Binding Affinity and Density for the Targeting of Acute Myeloid Leukemia.

Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML.

The immune receptor Tim-3 acts as a trafficker in a Tim-3/galectin-9 autocrine loop in human myeloid leukemia cells.

The immune receptor Tim-3 is often highly expressed in human acute myeloid leukemia (AML) cells where it acts as a growth factor and inflammatory receptor. Recently, it has been demonstrated that Tim-3 forms an autocrine loop with its natural ligand galectin-9 in human AML cells. However, the pathophysiological functions of Tim-3 in human AML cells remain unclear. Here, we report for the first time that Tim-3 is required for galectin-9 secretion in human AML cells. However, this effect is cell-type specific and was found so far to be applicable only to myeloid (and not, for example, lymphoid) leukemia cells. We concluded that AML cells might use Tim-3 as a trafficker for the secretion of galectin-9 which can then be possibly used to impair the anticancer activities of cytotoxic T cells and natural killer (NK) cells.

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.

Interleukin-1 beta induces the expression and production of stem cell factor by epithelial cells: crucial involvement of the PI-3K/mTOR pathway and HIF-1 transcription complex.

Potential crosslinks between inflammation and leukaemia have been discussed for some time, but experimental evidence to support this dogma is scarce. In particular, it is important to understand the mechanisms responsible for potential upregulation of proto-oncogenic growth factor expressions by inflammatory mediators. Here, we investigated the ability of the highly inflammatory cytokine interleukin-1 beta (IL-1ß) to induce the production of stem cell factor (SCF), which is a major hematopoietic growth factor that controls the progression of acute myeloid leukaemia upon malignant transformation of haematopoietic myeloid cells. We found that human IL-1ß induced the expression/secretion of SCF in MCF-7 human epithelial breast cancer cells and that this process depended on the hypoxia-inducible factor 1 (HIF-1) transcription complex. We also demonstrated a crucial role of the phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway in IL-1ß-induced HIF-1α accumulation in MCF-7 cells. Importantly, mTOR was also found to play a role in IL-1ß-induced SCF production. Furthermore, a tendency for a positive correlation of IL-1ß and SCF levels in the plasma of healthy human donors was observed. Altogether, our results demonstrate that IL-1ß, which normally bridges innate and adaptive immunity, induces the production of the major haematopoietic/proleukaemic growth factor SCF through the PI-3K/mTOR pathway and the HIF-1 transcription complex. These findings strongly support a cross-talk between inflammation and acute myeloid leukaemia.

Differential expression and biochemical activity of the immune receptor Tim-3 in healthy and malignant human myeloid cells.

The T cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated receptor which is involved in a variety of biological responses in human immune cells. It is highly expressed in most acute myeloid leukaemia (AML) cells and therefore may serve as a possible target for AML therapy. However, its biochemical activities in primary human AML cells remain unclear. We therefore analysed the total expression and surface presence of the Tim-3 receptor in primary human AML blasts and healthy primary human leukocytes isolated from human blood. We found that Tim-3 expression was significantly higher in primary AML cells compared to primary healthy leukocytes. Tim-3 receptor molecules were distributed largely on the surface of primary AML cells, whereas in healthy leukocytes Tim-3 protein was mainly expressed intracellularly. In primary human AML blasts, both Tim-3 agonistic antibody and galectin-9 (a Tim-3 natural ligand) significantly upregulated mTOR pathway activity. This was in line with increased accumulation of hypoxia-inducible factor 1 alpha (HIF-1α) and secretion of VEGF and TNF-α. Similar results were obtained in primary human healthy leukocytes. Importantly, in both types of primary cells, Tim-3-mediated effects were compared with those induced by lipopolysaccharide (LPS) and stem cell factor (SCF). Tim-3 induced comparatively moderate responses in both AML cells and healthy leukocytes. However, Tim-3, like LPS, mediated the release of both TNF-α and VEGF, while SCF induced mostly VEGF secretion and did not upregulate TNF-α release.

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.

The immune receptor Tim-3 mediates activation of PI3 kinase/mTOR and HIF-1 pathways in human myeloid leukaemia cells.

The T-cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated protein that is highly expressed in human acute myeloid leukaemia cells. As an acute myeloid leukaemia antigen, it could therefore be considered as a potential target for immune therapy and highly-specific drug delivery. However, a conceptual understanding of its biological role is required before consideration of this protein for therapeutic settings. Here, we reveal the detailed mechanism of action underlying the biological responses mediated by the Tim-3 receptor in myeloid cells. Our studies demonstrate that Tim-3 triggers growth factor type responses in acute myeloid leukaemia cells by activating a phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway. In addition, the receptor activates hypoxic signalling pathways upregulating glycolysis and pro-angiogenic responses. These findings suggest that Tim-3 could be used as a potential therapeutic target for immune therapy and drug delivery in human acute myeloid leukaemia cells.