The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell-mediated antitumor response. Systemic ...administration of anti-4-1BB-agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity.
Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and
functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity
.
In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity
, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non-small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8
T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion
and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer.
These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.
Cladribine is a disease-modifying selective immune reconstitution oral therapy for adult patients with highly active relapsing multiple sclerosis (RMS). It was approved in the USA in 2019 and in ...Europe in 2017, thus there are still gaps in existing guidelines for using cladribine tablets in clinical practice. Nine experts with extensive experience in managing patients with multiple sclerosis in Spain identified some of the unanswered questions related to the real-life use of cladribine tablets. They reviewed the available clinical trial data and real-world evidence, including their own experiences of using cladribine, over the course of three virtual meetings held between November 2020 and January 2021. This article gathers their practical recommendations to aid treatment decision-making and optimise the use of cladribine tablets in patients with RMS. The consensus recommendations cover the following areas: candidate patient profiles, switching strategies (to and from cladribine), managing response to cladribine and safety considerations.
The capacity of
(
) to sense, respond and adapt to a variable and hostile environment within the host makes it one of the most successful human pathogens. During different stages of infection,
is ...surrounded by a plethora of lipid molecules and current evidence points out the relevance of fatty acids during the infectious process. In this study, we have compared the transcriptional response of
to hypoxia in cultures supplemented with a mix of even long-chain fatty acids or dextrose as main carbon sources. Using RNA sequencing, we have identified differential expressed genes in early and late hypoxia, defined according to the
Wayne and Hayes model, and compared the results with the exponential phase of growth in both carbon sources. We show that the number of genes over-expressed in the lipid medium was quite low in both, early and late hypoxia, relative to conditions including dextrose, with the exception of transcripts of stable and non-coding RNAs, which were more expressed in the fatty acid medium. We found that
and
were over-expressed in the early phase of hypoxia, confirming their pivotal role in early adaptation to low oxygen concentration independently of the carbon source. A drastic contrast was found with the transcriptional regulatory factors at early hypoxia. Only 2 transcriptional factors were over-expressed in early hypoxia in the lipid medium compared to 37 that were over-expressed in the dextrose medium. Instead of Rv0081, known to be the central regulator of hypoxia in dextrose, Rv2745c (ClgR), seems to play a main role in hypoxia in the fatty acid medium. The low level of genes associated to the stress-response during their adaptation to hypoxia in fatty acids, suggests that this lipid environment makes hypoxia a less stressful condition for the tubercle bacilli. Taken all together, these results indicate that the presence of lipid molecules shapes the metabolic response of
to an adaptive state for different stresses within the host, including hypoxia. This fact could explain the success of
to establish long-term survival during latent infection.
Recent studies demonstrated that the expression of coxsackievirus and adenovirus receptor (CAR) is implicated in the pathophysiology of myocarditis. The aim of the present study was to assess the ...association between active and borderline myocarditis and CAR expression in endomyocardial tissues, and analyze the association between CAR expression and treatment response. An analytic, cross‑sectional, retrospective study was performed in 26 patients with myocarditis and 10 control subjects without heart disease. Myocardial biopsies were obtained and CAR transcription was measured by reverse transcription‑quantitative polymerase chain reaction analysis. The association between CAR mRNA levels and the response to immunosuppressive or conventional therapy (treatment responders, n=17; non‑responders, n=9) or with the type of histological myocarditis (active myocarditis, n=16; borderline myocarditis, n=10) was analyzed. CAR transcription levels were significantly lower (P=0.012) in patients with myocarditis compared with controls, and a significant decrease was observed (P=0.023) in CAR mRNA levels among patients with borderline myocarditis compared with the no myocarditis group. Patients responding to therapy exhibited higher CAR mRNA levels (P=0.036) compared with patients not responding to treatment, as evaluated based on clinical and echocardiographic criteria (immunosuppressive therapy, n=8; conventional therapy, n=1). Myocarditis in non‑responders was associated with fewer clinical manifestations and lower CAR mRNA levels. A significant difference was only found regarding the use of oral steroids in patients with active myocarditis who responded to treatment (P=0.02), with no difference in borderline myocarditis. In conclusion, the transcriptional level of CAR is low in the endomyocardial tissue of patients with myocarditis, and it is lower in borderline myocarditis and in non‑responder patients. These findings may enable early identification of patients who may benefit from treatment and timely determination of prognosis.
Abstract Escherichia coli is a leading cause of invasive bacterial infections in humans. Capsule polysaccharide has an important role in bacterial pathogenesis, and the K1 capsule has been firmly ...established as one of the most potent capsule types in E. coli through its association with severe infections. However, little is known about its distribution, evolution and functions across the E. coli phylogeny, which is fundamental to elucidating its role in the expansion of successful lineages. Using systematic surveys of invasive E. coli isolates, we show that the K1- cps locus is present in a quarter of bloodstream infection isolates and has emerged in at least four different extraintestinal pathogenic E. coli (ExPEC) phylogroups independently in the last 500 years. Phenotypic assessment demonstrates that K1 capsule synthesis enhances E. coli survival in human serum independent of genetic background, and that therapeutic targeting of the K1 capsule re-sensitizes E. coli from distinct genetic backgrounds to human serum. Our study highlights that assessing the evolutionary and functional properties of bacterial virulence factors at population levels is important to better monitor and predict the emergence of virulent clones, and to also inform therapies and preventive medicine to effectively control bacterial infections whilst significantly lowering antibiotic usage.
Mycobacteria, like other microorganisms, survive under different environmental variations by expressing an efficient adaptive response, oriented by regulatory elements, such as transcriptional ...repressors of the TetR family. These repressors in mycobacteria also appear to be related to cholesterol metabolism. In this study, we have evaluated the effect of a fatty acid (oleic–palmitic–stearic)/cholesterol mixture on some phenotypic and genotypic characteristics of a tetR-mutant strain (
BCG_2177c
mutated gene) of
M. bovis
BCG, a homologous of
Rv2160A
of
M. tuberculosis
. In order to accomplish this, we have analyzed the global gene expression of this strain by RNA-seq and evaluated its neutral-lipid storage capacity and potential to infect macrophages. We have also determined the macrophage response by measuring some pro- and anti-inflammatory cytokine expressions. In comparison with wild-type microorganisms, we showed that the mutation in the
BCG_2177c
gene did not affect the growth of
M. bovis
BCG in the presence of lipids but it probably modified the structure/composition of its cell envelope. Compared to with dextrose, an overexpression of the transcriptome of the wild-type and mutant strains was observed when these mycobacteria were cultured in lipids, mainly at the exponential phase. Twelve putative intracellular redox balance maintenance genes and four others coding for putative transcriptional factors (including WhiB6 and three TetR-like) were the main elements repeatedly overexpressed when cultured in the presence of lipids. These genes belonged to the central part of what we called the “genetic lipid signature” for
M. bovis
BCG. We have also found that all these mycobacteria genotypic changes affected the outcome of BCG-infected macrophages, being the mutant strain most adapted to persist longer inside the host. This high persistence result was also confirmed when mutant-infected macrophages showed overexpression of the anti-inflammatory cytokine TGF-β versus pro-inflammatory cytokines. In summary, the lack of this TetR-like repressor expression, within a lipid environment, may help mycobacteria overcome intracellular redox stress and survive longer inside their host.
Biofilm formed in vitro by mycobacteria has been associated with increased antibiotic tolerance as compared with planktonic cells. Cellulose has been identified as a component of DTT-exposed biofilms ...formed by M. tuberculosis. The celA1 gene of M. tuberculosis encodes a cellulase, which could affect the formation of biofilm by slow-growing mycobacteria. In this work, the celA1 gene of M. tuberculosis was cloned into the integrative pMV361 plasmid and then transformed into M. bovis BCG Pasteur to produce BCG:celA1, to have celA1 expressed from the strong promoter hsp60. We compared planktonic and biofilm growth, possible presence of CelA1 in whole protein extracts, quantitated biofilm, presence of monosaccharides, and bacillary burden in lungs after aerosol infection in BALB/c mice. Differences in the appearance of the surface pellicle and of the biofilm attached to the substrate were observed. In biofilms, we observed a significant decrease of glucosamine in BCG:celA1 compared with BCG:pMV361. Finally, BCG:celA1 had lower viable bacteria than the BCG:pMV361 strain after 24 h and 3 weeks post-infection, but no difference was found at 9 weeks post-infection.
Focal and segmental glomerulosclerosis (FSGS) is a histological pattern that has several etiologies, including genetics. The autosomal dominant form of FSGS is a heterogenic disease caused by ...mutations within three known genes: α-actinin 4 (ACTN4), canonical transient receptor potential 6 (TRPC6), and the inverted formin 2 (INF2) gene. More recently, INF2 mutations have also been attributed to Charcot–Marie–Tooth neuropathy associated with FSGS. Here we performed direct sequencing, histological characterization, and functional studies in a cohort of families with autosomal dominant FSGS. We detected a novel mutation in exon 6 of the INF2 gene outside of the exon 2–4 candidate region used for rapid diagnosis of autosomal dominant FSGS. This new mutation is predicted to alter a highly conserved amino-acid residue within the 17th α-helix of the diaphanous inhibitory domain of the protein. A long-term follow-up of this family indicated that all patients were diagnosed in adulthood, as opposed to early childhood, and progression to end-stage renal disease was at different times without clinical or electrodiagnostic evidence of neuropathy. Thus, this novel mutation in INF2 linked to nonsyndromic FSGS indicates the necessity for full gene sequencing if no mutation is found in the current rapid-screen region of the gene.
Abstract Purpose: Chemotherapy can potentially enhance the activity of immune checkpoint inhibitors by promoting immune priming. The phase Ib/II JAVELIN Chemotherapy Medley trial (NCT03317496) ...evaluated first-line avelumab + concurrent chemotherapy in patients with advanced urothelial carcinoma or non–small cell lung cancer (NSCLC). Materials and Methods: Avelumab 800 or 1,200 mg was administered continuously every 3 weeks with standard doses of cisplatin + gemcitabine in patients with urothelial carcinoma, or carboplatin + pemetrexed in patients with nonsquamous NSCLC. Dual primary endpoints were dose-limiting toxicity (DLT; phase Ib) and confirmed objective response (phase Ib/II). Results: In phase Ib, urothelial carcinoma and NSCLC cohorts received avelumab 800 mg (n = 13 and n = 6, respectively) or 1,200 mg (n = 6 each) + chemotherapy. In evaluable patients with urothelial carcinoma treated with avelumab 800 or 1,200 mg + chemotherapy, DLT occurred in 1/12 (8.3%) and 1/6 (16.7%), respectively; no DLT occurred in the NSCLC cohort. In phase II, 35 additional patients with urothelial carcinoma received avelumab 1,200 mg + chemotherapy. Across all treated patients, safety profiles were similar irrespective of avelumab dose. Objective response rates (95% confidence internal) with avelumab 800 or 1,200 mg + chemotherapy, respectively, across phase Ib/II, were 53.8% (25.1–80.8) and 39.0% (24.2–55.5) in urothelial carcinoma, and 50.0% (11.8–88.2) and 33.3% (4.3–77.7) in NSCLC. Conclusions: Preliminary efficacy and safety findings with avelumab + chemotherapy in urothelial carcinoma and NSCLC were consistent with previous studies of similar combination regimens. Conclusions about clinical activity are limited by small patient numbers. Significance: This phase Ib/II trial evaluated avelumab (immune checkpoint inhibitor) administered concurrently with standard first-line chemotherapy in patients with advanced urothelial carcinoma or advanced nonsquamous NSCLC without actionable mutations. Efficacy and safety appeared consistent with previous studies of similar combinations, although patient numbers were small.
Enterohemorrhagic
Escherichia coli
(EHEC) is a significant cause of serious human gastrointestinal disease worldwide. EHEC strains contain a pathogenicity island called the locus of enterocyte ...effacement (LEE), which encodes virulence factors responsible for damaging the gut mucosa. The Cpx envelope stress response of
E. coli
is controlled by a two-component system (TCS) consisting of a sensor histidine kinase (CpxA) and a cytoplasmic response regulator (CpxR). In this study, we investigated the role of CpxRA in the expression of LEE-encoded virulence factors of EHEC. We found that a mutation in
cpxA
significantly affected adherence of EHEC to human epithelial cells. Analysis of this mutant revealed the presence of high levels of CpxR which repressed transcription of
grlA
and
ler
, the main positive virulence regulators of the LEE, and influenced negatively the production of the type 3 secretion system–associated EspABD translocator proteins. It is known that CpxR activates
rpoH
(Sigma factor 32), which in turns activates transcription of the
lon
protease gene. We found that transcription levels of
ler
and
grlA
were significantly increased in the
lon
and
cpxA lon
mutants suggesting that
lon
is involved in down-regulating LEE genes. In addition, the
Galleria mellonella
model of infection was used to analyze the effect of the loss of the
cpx
and
lon
genes in EHEC's ability to kill the larvae. We found that the
cpxA
mutant was significantly deficient at killing the larvae however, the
cpxA lon
mutant which overexpresses LEE genes
in vitro
, was unable to kill the larvae, suggesting that virulence in the
G. mellonella
model is T3SS independent and that CpxA modulates virulence through a yet unknown EHEC-specific factor. Our data provides new insights and broadens our scope into the complex regulatory network of the LEE in which the CpxA sensor kinase plays an important role in a cascade involving both global and virulence regulators.
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