Development of target antigen-displaying virus-like particles (VLPs) for the generation of antibodies using hybridoma technology

Formaldehyde-fixed, paraffin-embedded (FFPE) cell and tissue samples are of great importance for immunohistochemical studies of histological specimens. However, antibodies for FFPE samples pose a challenge to antibody discovery as current immunization strategies rely predominantly on soluble proteins that cannot adequately reflect the changes in target antigens during the FFPE process. Enveloped virus-like particles (VLPs) allow for the presentation of membrane-anchored target antigens on the VLP surface and elicit a strong target antigen-specific antibody response after immunization. This proof-of-concept study presents a novel FFPE-like fixation methodology for VLP preparation aiming at the generation of FFPE-compatible monoclonal antibodies (mAbs). Human 293-F-derived VLP-producing suspension cell pools were established to produce human immunodeficiency virus (HIV)-like particles decorated with the truncated human low affinity nerve growth factor receptor (trNGFR) as model antigen. The trNGFR antigen was efficiently incorporated into VLPs with an average of 284 } 24 trNGFR molecules per VLP. To develop a fixation protocol applicable to VLPs, trNGFR-expressing cells were subjected to a variety of fixation treatments. Changes in epitopes introduced by fixation were monitored using two mAbs recognizing either an epitope present in native NGFR or an epitope present in native and FFPE NGFR. The novel simplified fixation procedure consisted of only formaldehyde and 90 °C heat fixation (FF90). Transmission electron microscopic and dynamic light scattering analysis of FF90 VLPs revealed that the fixed VLPs withstood the FF90 treatment and showed no morphological changes, allowing for the FF90 trNGFR-VLPs to be used to immunize mice for hybridoma cell generation. Hybridoma clones were screened for mAbs specifically recognizing native, FF90 and FFPE trNGFR-expressing cells in a flow cytometric assay. The isolated hybridoma mAbs did not recognize native epitopes but were reactive with FF90 and FFPE epitopes. The use of FF90-trNGFR VLPs for immunization led to the discovery of nine FFPE-NGFR-specific mAbs. This proofof-concept study demonstrated that FF90-treated VLPs decorated with a membrane-anchored target antigen are suitable antigens to preferentially generate FFPE-compatible mAbs. The FF90-VLP platform should be useful for the future discovery of specific mAbs directed against a variety of FFPE cell surface antigens

Generation of Antibodies Selectively Recognizing Epitopes in a Formaldehyde-Fixed Cell-Surface Antigen Using Virus-like Particle Display and Hybridoma Technology

Efficient induction of target-specific antibodies can be elicited upon immunization with highly immunogenic virus-like particles (VLPs) decorated with desired membrane-anchored target antigens (Ags). However, for example, for diagnostic purposes, monoclonal antibodies (mAbs) are required to enable the histological examination of formaldehyde-fixed paraffin-embedded (FFPE) biopsy tissue samples. Aiming at the generation of FFPE-antigen-specific mAbs and as a proof of concept (POC), we first established a simplified protocol using only formaldehyde and 90 °C heat fixation (FF90) of cells expressing the target Ag nerve growth factor receptor (NGFR). The FF90 procedure was validated using flow cytometric analysis and two mAbs recognizing either the native and FFPE-Ag or exclusively the native Ag. C-terminally truncated NGFR (trNGFR)-displaying native and FF90-treated VLPs derived from HIV-1 did not reveal distinctive changes in particle morphology using transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. Mice were subsequently repetitively immunized with trNGFR-decorated FF90-VLPs and hybridoma technology was used to establish mAb-producing cell clones. In multiple screening rounds, nine cell clones were identified producing mAbs distinctively recognizing epitopes in FF90- and FFPE-NGFR. This POC of a new methodology should foster the future generation of mAbs selectively targeting FFPE-fixed cell-surface Ags

Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors

To date, the establishment of high-titer stable viral packaging cells (VPCs) at large scale for gene therapeutic applications is very time- and cost-intensive. Here we report the establishment of three human suspension 293-F-derived ecotropic MLV-based VPCs. The classic stable transfection of an EGFP-expressing transfer vector resulted in a polyclonal VPC pool that facilitated cultivation in shake flasks of 100 mL volumes and yielded high functional titers of more than 1 × 106 transducing units/mL (TU/mL). When the transfer vector was flanked by transposon terminal inverted repeats (TIRs) and upon co-transfection of a plasmid encoding for the transposase, productivities could be slightly elevated to more than 3 × 106 TU/mL. In contrast and using mRNA encoding for the transposase, as a proof of concept, productivities were drastically improved by more than ten-fold exceeding 5 × 107 TU/mL. In addition, these VPC pools were generated within only 3 weeks. The production volume was successfully scaled up to 500 mL employing a stirred-tank bioreactor (STR). We anticipate that the stable transposition of transfer vectors employing transposase transcripts will be of utility for the future establishment of high-yield VPCs producing pseudotype vector particles with a broader host tropism on a large scale

Integration of Tumor Mutation Burden and PD-L1 Testing in Routine Laboratory Diagnostics in Non-Small Cell Lung Cancer

In recent years, Non-small cell lung cancer (NSCLC) has evolved into a prime example for precision oncology with multiple FDA-approved "precision" drugs. For the majority of NSCLC lacking targetable genetic alterations, immune checkpoint inhibition (ICI) has become standard of care in first-line treatment or beyond. PD-L1 tumor expression represents the only approved predictive biomarker for PD-L1/PD-1 checkpoint inhibition by therapeutic antibodies. Since PD-L1-negative or low-expressing tumors may also respond to ICI, additional factors are likely to contribute in addition to PD-L1 expression. Tumor mutation burden (TMB) has emerged as a potential candidate; however, it is the most complex biomarker so far and might represent a challenge for routine diagnostics. We therefore established a hybrid capture (HC) next-generation sequencing (NGS) assay that covers all oncogenic driver alterations as well as TMB and validated TMB values by correlation with the assay (F1CDx) used for the CheckMate 227 study. Results of the first consecutive 417 patients analyzed in a routine clinical setting are presented. Data show that fast reliable comprehensive diagnostics including TMB and targetable alterations are obtained with a short turn-around time. Thus, even complex biomarkers can easily be implemented in routine practice to optimize treatment decisions for advanced NSCLC

Targeted thorium-227 conjugates as treatment options in oncology

Targeted alpha therapy (TAT) is a promising approach for addressing unmet needs in oncology. Inherent properties make α-emitting radionuclides well suited to cancer therapy, including high linear energy transfer (LET), penetration range of 2–10 cell layers, induction of complex double-stranded DNA breaks, and immune-stimulatory effects. Several alpha radionuclides, including radium-223 (223Ra), actinium-225 (225Ac), and thorium-227 (227Th), have been investigated. Conjugation of tumor targeting modalities, such as antibodies and small molecules, with a chelator moiety and subsequent radiolabeling with α-emitters enables specific delivery of cytotoxic payloads to different tumor types. 223Ra dichloride, approved for the treatment of patients with metastatic castration-resistant prostate cancer (mCRPC) with bone-metastatic disease and no visceral metastasis, is the only approved and commercialized alpha therapy. However, 223Ra dichloride cannot currently be complexed to targeting moieties. In contrast to 223Ra, 227Th may be readily chelated, which allows radiolabeling of tumor targeting moieties to produce targeted thorium conjugates (TTCs), facilitating delivery to a broad range of tumors. TTCs have shown promise in pre-clinical studies across a range of tumor-cell expressing antigens. A clinical study in hematological malignancy targeting CD22 has demonstrated early signs of activity. Furthermore, pre-clinical studies show additive or synergistic effects when TTCs are combined with established anti-cancer therapies, for example androgen receptor inhibitors (ARI), DNA damage response inhibitors such as poly (ADP)-ribose polymerase inhibitors or ataxia telangiectasia and Rad3-related kinase inhibitors, as well as immune checkpoint inhibitors

Nucleocytosolic depletion of the energy metabolite acetyl-coenzyme a stimulates autophagy and prolongs lifespan.

Healthy aging depends on removal of damaged cellular material that is in part mediated by autophagy. The nutritional status of cells affects both aging and autophagy through as-yet-elusive metabolic circuitries. Here, we show that nucleocytosolic acetyl-coenzyme A (AcCoA) production is a metabolic repressor of autophagy during aging in yeast. Blocking the mitochondrial route to AcCoA by deletion of the CoA-transferase ACH1 caused cytosolic accumulation of the AcCoA precursor acetate. This led to hyperactivation of nucleocytosolic AcCoA-synthetase Acs2p, triggering histone acetylation, repression of autophagy genes, and an age-dependent defect in autophagic flux, culminating in a reduced lifespan. Inhibition of nutrient signaling failed to restore, while simultaneous knockdown of ACS2 reinstated, autophagy and survival of ach1 mutant. Brain-specific knockdown of Drosophila AcCoA synthetase was sufficient to enhance autophagic protein clearance and prolong lifespan. Since AcCoA integrates various nutrition pathways, our findings may explain diet-dependent lifespan and autophagy regulation

Beyond spheres of influence: the myth of the state and Russia’s seductive power in Kyrgyzstan

This article questions the analytical value of “spheres of influence” for understanding power and the state in the post-Soviet region and beyond, based on a critical deconstruction of the ontological and epistemological assumptions inherent in the concept. It proposes an alternative reading of power and the state, drawing on the concept of “seductive power” at a distance and Timothy Mitchell’s “state effect.” Rather than the concept of a sphere of influence, a highly politicized concept that conveys an ontology that flattens and divides space, essentializes the state, and relies on an intentionalist account of power, we need an analytical framework that can help us make sense of the multiple, varied spatialities and historical legacies that produce the state and power. I demonstrate this through an extended discussion of Russian power in Kyrgyzstan, a country often described as a Russian client state. Mobilizing recent re-conceptualizations of state and power in anthropology and political geography, I present an analysis of Russia’s seductive power in Kyrgyzstan and the way it contributes to producing Kyrgyz state-ness. I also show how Russia’s Great Power myth is itself evolving and conclude that the differentiated, relational production of space and power in either Kyrgyz or Russian myths of the state is not captured by a the concept of a return to spheres of influence

Infectious RNA: Human Immunodeficiency Virus (HIV) Biology, Therapeutic Intervention, and the Quest for a Vaccine

Different mechanisms mediate the toxicity of RNA. Genomic retroviral mRNA hijacks infected host cell factors to enable virus replication. The viral genomic RNA of the human immunodeficiency virus (HIV) encompasses nine genes encoding in less than 10 kb all proteins needed for replication in susceptible host cells. To do so, the genomic RNA undergoes complex alternative splicing to facilitate the synthesis of the structural, accessory, and regulatory proteins. However, HIV strongly relies on the host cell machinery recruiting cellular factors to complete its replication cycle. Antiretroviral therapy (ART) targets different steps in the cycle, preventing disease progression to the acquired immunodeficiency syndrome (AIDS). The comprehension of the host immune system interaction with the virus has fostered the development of a variety of vaccine platforms. Despite encouraging provisional results in vaccine trials, no effective vaccine has been developed, yet. However, novel promising vaccine platforms are currently under investigation