Determinants of worse liver-related outcome according to HDV infection among HBsAg positive persons living with HIV: Data from the ICONA cohort.

OBJECTIVES: We aimed to study hepatitis D virus (HDV) prevalence and risk of progression to severe liver-related events (SLRE) in HBsAg positive people living with HIV (PLWH) in Italy; role of HDV-RNA copy levels, HCV coinfection and nadir CD4 counts were also investigated. METHODS: People living with HIV (PLWH) from Italian Foundation cohort Naïve antiretrovirals (ICONA) with available HBsAg and HDV Ab were enrolled. HBsAg, HDV Ab, HDV-RNA and HDV genotypes were tested. PRIMARY END-POINT: time from first HDV screening to Severe Liver Related Events (SLRE: decompensated cirrhosis, liver transplantation, HCC). Fine-grey regression models were used to evaluate the association of HDV Ab, HDV-RNA, HDV/HCV coinfection, CD4 nadir and outcome. Secondary end-points: time to SLRE or death; HDV Ab and HDV-RNA prevalence. RESULTS: A total of 152/809 (18.8%) HBsAg positive PLWH showed HDV Ab reactivity; 63/93 (67.7%) were HDV-RNA positive. Being male, persons who inject drugs (PWID), HCV Ab positive, with FIB-4 > 3.25 were independent factors of HDV Ab positivity. In a median follow-up of 5 years, 37 PLWH (4.1% at 5-year) developed SLRE and 97 (12.0%) reached the SLRE or death end-point. HDV-RNA positive (independently from HDV-RNA copy level) PLWH had a 4.6-fold (95%CI 2.0-10.5) higher risk of SLRE than HDV negatives. PLWH positive for both HCV Ab and HDV Ab showed the highest independent risk of SLRE (ASHR: 11.9, 95%CI: 4.6-30.9 vs. HCV neg/HDV neg). Nadir CD4 < 200/mL was associated with SLRE (ASHR: 3.9, 95% 1.0-14.5). CONCLUSIONS: One-fifth of the HBsAg positive PLWH harbour HDV infection, and are at high risk of progression to advanced liver disease. HCV contributes to worse outcomes. This population needs urgently effective treatments.

Targeted Phagocytosis Induction for Cancer Immunotherapy via Bispecific MerTK-Engaging Antibodies.

The Tyro, Axl, and MerTK receptors (TAMRs) play a significant role in the clearance of apoptotic cells. In this work, the spotlight was set on MerTK, as it is one of the prominent TAMRs expressed on the surface of macrophages and dendritic cells. MerTK-specific antibodies were previously isolated from a transgenic rat-derived immune library with suitable biophysical properties. Further characterisation resulted in an agonistic MerTK antibody that led to phospho AKT activation in a dose-dependent manner. In this proof-of-concept study, a MerTK-specific antibody, MerK28, was combined with tandem, biparatopic EGFR-binding VHH camelid antibody domains (7D9G) in different architectures to generate bispecific antibodies with the capacity to bind EGFR and MerTK simultaneously. The bispecific molecules exhibited appropriate binding properties with regard to both targets in their soluble forms as well as to cells, which resulted in the engagement of macrophage-like THP-1 cells with epidermoid carcinoma A431 cells. Furthermore, targeted phagocytosis in co-culture experiments was observed only with the bispecific variants and not the parental MerTK-binding antibody. This work paves the way for the generation of bispecific macrophage-engaging antibodies for targeted phagocytosis harnessing the immune-modulating roles of MerTK in immunotherapy.

Inflammation and microbial translocation measured prior to combination antiretroviral therapy (cART) and long-term probability of clinical progression in people living with HIV.

BACKGROUND: Despite the effectiveness of cART, people living with HIV still experience an increased risk of serious non-AIDS events, as compared to the HIV negative population. Whether pre-cART microbial translocation (MT) and systemic inflammation might predict morbidity/mortality during suppressive cART, independently of other known risk factors, is still unclear. Thus, we aimed to investigate the role of pre-cART inflammation and MT as predictors of clinical progression in HIV+ patients enrolled in the Icona Foundation Study Cohort. METHODS: We included Icona patients with ≥2 vials of plasma stored within 6 months before cART initiation and at least one CD4 count after therapy available. Circulating biomarker: LPS, sCD14, EndoCab, hs-CRP. Kaplan-Meier curves and Cox regression models were used. We defined the endpoint of clinical progression as the occurrence of a new AIDS-defining condition, severe non-AIDS condition (SNAEs) or death whichever occurred first. Follow-up accrued from the data of starting cART and was censored at the time of last available clinical visit. Biomarkers were evaluated as both binary (above/below median) and continuous variables (logescale). RESULTS: We studied 486 patients with 125 clinical events: 39 (31%) AIDS, 66 (53%) SNAEs and 20 (16%) deaths. Among the analyzed MT and pro-inflammatory markers, hs-CRP seemed to be the only biomarker retaining some association with the endpoint of clinical progression (i.e. AIDS/SNAEs/death) after adjustment for confounders, both when the study population was stratified according to the median of the distribution (1.51 mg/L) and when the study population was stratified according to the 33% percentiles of the distribution (low 0.0-1.1 mg/L; intermediate 1.2-5.3 mg/L; high > 5.3 mg/L). In particular, the higher the hs-CRP values, the higher the risk of clinical progression (p = 0.056 for median-based model; p = 0.002 for 33% percentile-based model). CONCLUSIONS: Our data carries evidence for an association between the risk of disease progression after cART initiation and circulating pre-cART hs-CRP levels but not with levels of MT. These results suggest that pre-therapy HIV-driven pro-inflammatory milieu might overweight MT and its downstream immune-activation.

Unexpected human cases of cutaneous anthrax in Latium region, Italy, August 2017: integrated human-animal investigation of epidemiological, clinical, microbiological and ecological factors.

On 31 August, a veterinarian and a farmworker were hospitalised for skin lesions. Both had been exposed to a dead cow on 19 August on a farm near Rome, where eight further cattle died of confirmed anthrax later the same month. At admission, the first case showed a black depressed eschar and another smaller lesion on one hand. The second case presented deep infection of the skin, with involvement of both arms. Anthrax diagnosis was confirmed by detection of B. anthracis DNA in eschar fragments from both patients. T-cell specific immunity was studied by flow cytometry and Elispot assay after stimulation with B. anthracis secretome in blood samples collected from Case 1. Immunoglobulin production was detected by complement fixation assay. In Case 1, specific CD4+ T-cell activation was detected, without antibody production. Specific antibodies were detected only in the second patient with severe cutaneous illness. Both patients recovered. The two human anthrax cases were epidemiologically linked, but anthrax was not suspected at admission in either case. The veterinarian had initially unrecognised professional exposure and the exposed farmworker did initially not report exposure to affected animals. A One Health strategy integrating human and animal investigations was essential to confirm the diagnosis.

Serological evaluation for Chagas disease in migrants from Latin American countries resident in Rome, Italy.

BACKGROUND: Chagas disease (CD) is a systemic parasitic infection caused by the protozoan Trypanosoma cruzi, whose chronic phase may lead to cardiac and intestinal disorders. Endemic in Latin America where it is transmitted mainly by vectors, large-scale migrations to other countries have turned CD into a global health problem because of its alternative transmission routes through blood transfusion, tissue transplantation, or congenital. Aim of this study was to compare the performance of two commercially available tests for serological diagnosis of CD in a group of Latin American migrants living in a non-endemic setting (Rome, Italy). The study was based on a cross-sectional analysis of seroprevalence in this group. Epidemiological risk factors associated to CD were also evaluated in this study population. METHODS: The present study was conducted on 368 subjects from the Latin American community resident in Rome. Following WHO guidelines, we employed a diagnostic strategy based on two tests to detect IgG antibodies against T. cruzi in the blood (a lysate antigen-based ELISA and a chemiluminescent microparticle CMIA composed of multiple recombinant antigens), followed by a third test (an immunochromatographic assay) on discordant samples. RESULTS: Our diagnostic approach produced 319/368 (86.7%) concordant negative and 30/368 (8.1%) concordant positive results after the first screening. Discrepancies were obtained for 19/368 (5.2%) samples that were tested using the third assay, obtaining 2 more positive and 17 negative results. The final count of positive samples was 32/368 (8.7% of the tested population). Increasing age, birth in Bolivia, and previous residence in a mud house were independent factors associated with T. cruzi positive serology. CONCLUSIONS: Serological diagnosis of CD is still challenging, because of the lack of a reference standard serological assay for diagnosis. Our results reaffirm the importance of performing CD screening in non-endemic countries; employing a fully automated and highly sensitive CMIA assay first could be a cost- and resource-effective strategy for mass screening of low-risk patients. However, our results also suggest that the WHO strategy of using two different serological assays, combined with epidemiological information, remains the best approach for patients coming from endemic countries.

3D topographies promote macrophage M2d-Subset differentiation.

In vitro cellular models denote a crucial part of drug discovery programs as they aid in identifying successful drug candidates based on their initial efficacy and potency. While tremendous headway has been achieved in improving 2D and 3D culture techniques, there is still a need for physiologically relevant systems that can mimic or alter cellular responses without the addition of external biochemical stimuli. A way forward to alter cellular responses is using physical cues, like 3D topographical inorganic substrates, to differentiate macrophage-like cells. Herein, protein secretion and gene expression markers for various macrophage subsets cultivated on a 3D topographical substrate are investigated. The results show that macrophages differentiate into anti-inflammatory M2-type macrophages, secreting increased IL-10 levels compared to the controls. Remarkably, these macrophage cells are differentiated into the M2d subset, making up the main component of tumour-associated macrophages (TAMs), as measured by upregulated Il-10 and Vegf mRNA. M2d subset differentiation is attributed to the topographical substrates with 3D fractal-like geometries arrayed over the surface, else primarily achieved by tumour-associated factors in vivo. From a broad perspective, this work paves the way for implementing 3D topographical inorganic surfaces for drug discovery programs, harnessing the advantages of in vitro assays without external stimulation and allowing the rapid characterisation of therapeutic modalities in physiologically relevant environments.

Bulk Reformatting of Antibody Fragments Displayed on the Surface of Yeast Cells to Final IgG Format for Mammalian Production.

While yeast surface display (YSD) has gained traction for antibody hit discovery efforts with the first therapeutic YSD-isolated antibody sintilimab approved in 2018, a major drawback that remains is the time-consuming reformatting of monoclonal antibody (mAb) candidates. By using a Golden Gate cloning (GGC)-dependent workflow, the bulk transfer of genetic information can be performed from antibody fragments displayed on yeast cells to a bidirectional mammalian expression vector. Herein, we describe in-depth protocols for the reformatting of mAbs, starting from the generation of Fab fragment libraries in YSD vectors and ending up with IgG molecules in bidirectional mammalian vectors in a consolidated two-pot, two-step procedure.

Generation of a symmetrical trispecific NK cell engager based on a two-in-one antibody.

To construct a trispecific IgG-like antibody at least three different binding moieties need to be combined, which results in a complex architecture and challenging production of these molecules. Here we report for the first time the construction of trispecific natural killer cell engagers based on a previously reported two-in-one antibody combined with a novel anti-CD16a common light chain module identified by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibodies simultaneously target epidermal growth factor receptor (EGFR), programmed death-ligand 1 (PD-L1) and CD16a with two Fab fragments, resulting in specific cellular binding properties on EGFR/PD-L1 double positive tumor cells and a potent ADCC effect. This study paves the way for further development of multispecific therapeutic antibodies derived from avian immunization with desired target combinations, valencies, molecular symmetries and architectures.

Parahydrogen-induced polarization allows 2000-fold signal enhancement in biologically active derivatives of the peptide-based drug octreotide.

Octreotide, a somatostatin analogue, has shown its efficacy for the diagnostics and treatment of various types of cancer, i.e., in octreotide scan, as radio-marker after labelling with a radiopharmaceutical. To avoid toxicity of radio-labeling, octreotide-based assays can be implemented into magnetic resonance techniques, such as MRI and NMR. Here we used a Parahydrogen-Induced Polarization (PHIP) approach as a cheap, fast and straightforward method. Introduction of L-propargyl tyrosine as a PHIP marker at different positions of octreotide by manual Solid-Phase Peptide Synthesis (SPPS) led to up to 2000-fold proton signal enhancement (SE). Cell binding studies confirmed that all octreotide variants retained strong binding affinity to the surface of human-derived cancer cells expressing somatostatin receptor 2. The hydrogenation reactions were successfully performed in methanol and under physiologically compatible mixtures of water with methanol or ethanol. The presented results open up new application areas of biochemical and pharmacological studies with octreotide.

TriTECM: A tetrafunctional T-cell engaging antibody with built-in risk mitigation of cytokine release syndrome.

Harnessing the innate power of T cells for therapeutic benefit has seen many shortcomings due to cytotoxicity in the past, but still remains a very attractive mechanism of action for immune-modulating biotherapeutics. With the intent of expanding the therapeutic window for T-cell targeting biotherapeutics, we present an attenuated trispecific T-cell engager (TCE) combined with an anti- interleukin 6 receptor (IL-6R) binding moiety in order to modulate cytokine activity (TriTECM). Overshooting cytokine release, culminating in cytokine release syndrome (CRS), is one of the severest adverse effects observed with T-cell immunotherapies, where the IL-6/IL-6R axis is known to play a pivotal role. By targeting two tumour-associated antigens, epidermal growth factor receptor (EGFR) and programmed death ligand 1 (PD-L1), simultaneously with a bispecific two-in-one antibody, high tumour selectivity together with checkpoint inhibition was achieved. We generated tetrafunctional molecules that contained additional CD3- and IL-6R-binding modules. Ligand competition for both PD-L1 and IL-6R as well as inhibition of both EGF- and IL-6-mediated signalling pathways was observed. Furthermore, TriTECM molecules were able to activate T cells and trigger T-cell-mediated cytotoxicity through CD3-binding in an attenuated fashion. A decrease in pro-inflammatory cytokine interferon γ (IFNγ) after T-cell activation was observed for the TriTECM molecules compared to their respective controls lacking IL-6R binding, hinting at a successful attenuation and potential modulation via IL-6R. As IL-6 is a key player in cytokine release syndrome as well as being implicated in enhancing tumour progression, such molecule designs could reduce side effects and cytotoxicity observed with previous TCEs and widen their therapeutic windows.

A Generic Strategy to Generate Bifunctional Two-in-One Antibodies by Chicken Immunization.

Various formats of bispecific antibodies exist, among them Two-in-One antibodies in which each Fab arm can bind to two different antigens. Their IgG-like architecture accounts for low immunogenicity and also circumvents laborious engineering and purification steps to facilitate correct chain pairing. Here we report for the first time the identification of a Two-in-One antibody by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibody simultaneously targets the epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PD-L1) at the same Fv fragment with two non-overlapping paratopes. The dual action Fab is capable of inhibiting EGFR signaling by binding to dimerization domain II as well as blocking the PD-1/PD-L1 interaction. Furthermore, the Two-in-One antibody demonstrates specific cellular binding properties on EGFR/PD-L1 double positive tumor cells. The presented strategy relies solely on screening of combinational immune-libraries and obviates the need for any additional CDR engineering as described in previous reports. Therefore, this study paves the way for further development of therapeutic antibodies derived from avian immunization with novel and tailor-made binding properties.

Antibody Library Screening Using Yeast Biopanning and Fluorescence-Activated Cell Sorting.

Yeast surface display (YSD) emerged as a prominent screening methodology for the isolation of monoclonal antibodies (mAbs) against various antigens. However, phage display remains the gold standard in cell panning-based screenings to isolate mAbs against difficult-to-screen targets, such as G-protein coupled receptors (GPCR) and ion channels. Herein we describe a step-by-step protocol to establish and perform the isolation of mAbs using YSD in a fluorescence-activated cell sorting (FACS)-assisted biopanning manner, yielding a variety of antibodies binding their antigen with high affinity in the natural environment of the cell. Upon mixing antibody-displaying yeast cells with antigen-displaying mammalian cells, complexes are specifically formed and isolated for enrichment of yeast cells encoding binders against the antigen. The utilization of mammalian cells expressing the respective target accounts for accessibility of the epitope and the correct conformation of the antigen. Furthermore, critical characterization methods mandatory for this kind of antibodies are illuminated.

Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells.

Yeast-surface display (YSD) is commonly applied to screen Fab immune or naïve libraries for binders of predefined target molecules. However, reformatting of isolated variants represents a time-intensive bottleneck. Herein, we present a novel approach to facilitate a lean transition from antibody screening using YSD Fab libraries to the production of full-length IgG antibodies in Expi293-F cells. In this study, utilizing Golden Gate Cloning (GGC) and a bidirectional promoter system, an exemplary Fab-displaying YSD library was generated based on immunised transgene rats. After subsequent screening for antigen-specific antibody candidates by fluorescence-activated cell sorting (FACS), the Fab-encoding genes were subcloned into a bidirectional mammalian expression vector, exhibiting CH2-CH3 encoding genes, in a GGC-mediated, PCR-free manner. This novel, straightforward and time-saving workflow allows the VH/VL pairing to be preserved. This study resulted in antibody variants exhibiting suitable biophysical properties and covered a broad VH diversity after two rounds of FACS screening, as revealed by NGS analysis. Ultimately, we demonstrate that the implication of such a gene transfer system streamlines antibody hit discovery efforts, allowing the faster characterisation of antibodies against a plethora of targets that may lead to new therapeutic agents.

Prevalence of Chagas disease and strongyloidiasis among HIV-infected Latin American immigrants in Italy - The CHILI study.

INTRODUCTION: Screening HIV-positive migrants for neglected tropical diseases having potential for life-threatening reactivation, such as Chagas disease and strongyloidiasis is not widely implemented. We evaluated the prevalence of these infections among a large cohort of HIV-infected migrants from Latin America living in Italy. METHOD: Cross-sectional study evaluating the prevalence of Trypanosoma cruzi and Strongyloides stercoralis infections in HIV-infected migrants from Latin America enrolled in the Italian Cohort of Antiretroviral-Naïve patients (ICONA) between 1997 and 2018, based on serology performed on sera stored in the ICONA Foundation biobank. Screening for Chagas disease was performed using two commercial ELISA complemented by commercial Immunoblot and CLIA if discordant. Strongyloidiasis was evaluated using a commercial ELISA. RESULTS: 389 patients were analysed. Fifteen (3.86%) had at least one positive Chagas ELISA test. Prevalence of Chagas disease was 0.5% or 1.29% depending on the confirmatory technique. Serology for strongyloidiasis was positive in 16 (4.11%) patients. Only Nadir CD4+ T cell count was associated with discordant serology for Chagas disease (p = 0.046). CONCLUSIONS: The accuracy of seroassays for Chagas disease and strongyloidiasis in HIV-positive patients is unclear. To avoid missing potentially life-threatening infections, we suggest implementing additional diagnostic strategies in at-risk patients with inconclusive serology results.

Recombinant Antibody Production Using a Dual-Promoter Single Plasmid System.

Monoclonal antibodies (mAbs) have demonstrated tremendous effects on the treatment of various disease indications and remain the fastest growing class of therapeutics. Production of recombinant antibodies is performed using mammalian expression systems to facilitate native antibody folding and post-translational modifications. Generally, mAb expression systems utilize co-transfection of heavy chain (hc) and light chain (lc) genes encoded on separate plasmids. In this study, we examine the production of two FDA-approved antibodies using a bidirectional (BiDi) vector encoding both hc and lc with mirrored promoter and enhancer elements on a single plasmid, by analysing the individual hc and lc mRNA expression levels and subsequent quantification of fully-folded IgGs on the protein level. From the assessment of different promoter combinations, we have developed a generic expression vector comprised of mirrored enhanced CMV (eCMV) promoters showing comparable mAb yields to a two-plasmid reference. This study paves the way to facilitate small-scale mAb production by transient cell transfection with a single vector in a cost- and time-efficient manner.

Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain Antibody Architecture.

Natural killer cell engagers gained enormous interest in recent years due to their potent anti-tumor activity and favorable safety profile. Simultaneously, chicken-derived antibodies entered clinical studies paving the way for avian-derived therapeutics. In this study, we describe the affinity maturation of a common light chain (cLC)-based, chicken-derived antibody targeting EGFR, followed by utilization of the same light chain for the isolation of CD16a- and PD-L1-specific monoclonal antibodies. The resulting binders target their respective antigen with single-digit nanomolar affinity while blocking the ligand binding of all three respective receptors. Following library-based humanization, bispecific and trispecific variants in a standard 1 + 1 or a 2 + 1 common light chain format were generated, simultaneously targeting EGFR, CD16a, and PD-L1. The trispecific antibody mediated an elevated antibody-dependent cellular cytotoxicity (ADCC) in comparison to the EGFR×CD16a bispecific variant by effectively bridging EGFR/PD-L1 double-positive cancer cells with CD16a-positive effector cells. These findings represent, to our knowledge, the first detailed report on the generation of a trispecific 2 + 1 antibodies exhibiting a common light chain and illustrate synergistic effects of trispecific antigen binding. Overall, this generic procedure paves the way for the engineering of tri- and oligospecific therapeutic antibodies derived from avian immunizations.

From cell line development to the formulated drug product: The art of manufacturing therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies and related products have steadily grown to become the dominant product class within the biopharmaceutical market. Production of antibodies requires special precautions to ensure safety and efficacy of the product. In particular, minimizing antibody product heterogeneity is crucial as drug substance variants may impair the activity, efficacy, safety, and pharmacokinetic properties of an antibody, consequently resulting in the failure of a product in pre-clinical and clinical development. This review will cover the manufacturing and formulation challenges and advances of therapeutic monoclonal antibodies, focusing on improved processes to minimize variants and ensure batch-to-batch consistency. Processes put in place by regulatory agencies, such as Quality-by-Design (QbD) and current Good Manufacturing Practices (cGMP), and how their implementation has aided drug development in pharmaceutical companies will be reviewed. Advances in formulation and considerations on the intended use of a therapeutic antibody, including the route of administration and patient compliance, will be discussed.

Milking the Cow: Cattle-Derived Chimeric Ultralong CDR-H3 Antibodies and Their Engineered CDR-H3-Only Knobbody Counterparts Targeting Epidermal Growth Factor Receptor Elicit Potent NK Cell-Mediated Cytotoxicity.

In this work, we have generated epidermal growth factor receptor (EGFR)-specific cattle-derived ultralong CDR-H3 antibodies by combining cattle immunization with yeast surface display. After immunization, ultralong CDR-H3 regions were specifically amplified and grafted onto an IGHV1-7 scaffold by homologous recombination to facilitate Fab display. Antigen-specific clones were readily obtained by fluorescence-activated cell sorting (FACS) and reformatted as chimeric antibodies. Binning experiments revealed epitope targeting of domains I, II, and IV of EGFR with none of the generated binders competing with Cetuximab, Matuzumab, or EGF for binding to EGFR. Cattle-derived chimeric antibodies were potent in inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against EGFR-overexpressing tumor cells with potencies (EC50 killing) in the picomolar range. Moreover, most of the antibodies were able to significantly inhibit EGFR-mediated downstream signaling. Furthermore, we demonstrate that a minor fraction of CDR-H3 knobs derived from generated antibodies was capable of independently functioning as a paratope facilitating EGFR binding when grafted onto the Fc part of human IgG1. Besides slightly to moderately diminished capacities, these engineered Knobbodies largely retained main properties of their parental antibodies such as cellular binding and triggering of ADCC. Hence, Knobbodies might emerge as promising tools for biotechnological applications upon further optimization.