High-Throughput and Format-Agnostic Mispairing Assay for Multispecific Antibodies Using Intact Mass Spectrometry.

Multispecific antibodies have gained significant importance in a broad indication space due to their ability to engage multiple epitopes simultaneously and to thereby overcome therapeutic barriers. With growing therapeutic potential, however, the molecular complexity increases, thus intensifying the demand for innovative protein engineering and analytical strategies. A major challenge for multispecific antibodies is the correct assembly of light and heavy chains. Engineering strategies exist to stabilize the correct pairing, but typically individual engineering campaigns are required to arrive at the anticipated format. Mass spectrometry has proven to be a versatile tool to identify mispaired species. However, due to manual data analysis procedures, mass spectrometry is limited to lower throughputs. To keep pace with increasing sample numbers, we developed a high-throughput-capable mispairing workflow based on intact mass spectrometry with automated data analysis, peak detection, and relative quantification using Genedata Expressionist. This workflow is capable of detecting mispaired species of ∼1000 multispecific antibodies in three weeks and thus is applicable to complex screening campaigns. As a proof of concept, the assay was applied to engineering a trispecific antibody. Strikingly, the new setup has not only proved successful in mispairing analysis but has also revealed its potential to automatically annotate other product-related impurities. Furthermore, we could confirm the assay to be format-agnostic, as shown by analyzing several different multispecific formats in one run. With these comprehensive capabilities, the new automated intact mass workflow can be applied as a universal tool to detect and annotate peaks in a format-agnostic approach and in high-throughput, thus enabling complex discovery campaigns.

Honing-in antigen-specific cells during antibody discovery: a user-friendly process to mine a deeper repertoire.

Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Identifying these cells is akin to finding needle in a haystack. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire. We report on the bulk purification of antigen-specific B-cells and the benefits it offers to various antibody discovery platforms. Using five different antigens, we show hit rates of 51-88%, compared to about 5% with conventional methods. We also show that this purification is highly efficient with loss of only about 2% antigen specific cells. Furthermore, we compared clones in which cognate chains are preserved with those from display libraries in which chains either from total B cells (TBC) or antigen-specific B cells (AgSC) underwent combinatorial pairing. We found that cognate chain paired clones and combinatorial clones from AgSC library had higher frequency of functional clones and showed greater diversity in sequence and paratope compared to clones from the TBC library. This antigen-specific B-cell selection technique exemplifies a process improvement with reduced cycle time and cost, by removing undesired clones prior to screening and increasing the chance of capturing desirable and rare functional clones in the repertoire.

Surveillance of SARS-CoV-2 transmission in educational institutions, August to December 2020, Germany.

This study aims at providing estimates on the transmission risk of SARS-CoV-2 in schools and day-care centres. We calculated secondary attack rates (SARs) using individual-level data from state-wide mandatory notification of index cases in educational institutions, followed by contact tracing and PCR-testing of high-risk contacts. From August to December 2020, every sixth of overall 784 independent index cases was associated with secondary cases in educational institutions. Monitoring of 14 594 institutional high-risk contacts (89% PCR-tested) of 441 index cases during quarantine revealed 196 secondary cases (SAR 1.34%, 0.99-1.78). SARS-CoV-2 infection among high-risk contacts was more likely around teacher-indexes compared to student-/child-indexes (incidence rate ratio (IRR) 3.17, 1.79-5.59), and in day-care centres compared to secondary schools (IRR 3.23, 1.76-5.91), mainly due to clusters around teacher-indexes in day-care containing a higher mean number of secondary cases per index case (142/113 = 1.26) than clusters around student-indexes in schools (82/474 = 0.17). In 2020, SARS-CoV-2 transmission risk in educational settings was low overall, but varied strongly between setting and role of the index case, indicating the chance for targeted intervention. Surveillance of SARS-CoV-2 transmission in educational institutions can powerfully inform public health policy and improve educational justice during the pandemic.

Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease.

Parkinson's disease (PD) is a fatal neurodegenerative disorder that is primarily caused by the degeneration and loss of dopaminergic neurons of the substantia nigra in the ventral midbrain. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset PD identified to date, with G2019S being the most frequent LRRK2 mutation, which is responsible for up to 1-2% of sporadic PD and up to 6% of familial PD cases. As no treatment is available for this devastating disease, developing new therapeutic strategies is of foremost importance. Cellular models are commonly used for testing novel potential neuroprotective compounds. However, current cellular PD models either lack physiological relevance to dopaminergic neurons or are too complex and costly for scaling up the production process and for screening purposes. In order to combine biological relevance and throughput, we have developed a PD model in Lund human mesencephalic (LUHMES) cell-derived dopaminergic neurons by overexpressing wild-type (WT) and G2019S LRRK2 proteins. We show that these cells can differentiate into dopaminergic-like neurons and that expression of mutant LRRK2 causes a range of different phenotypes, including reduced nuclear eccentricity, altered mitochondrial and lysosomal morphologies, and increased dopaminergic cell death. This model could be used to elucidate G2019S LRRK2-mediated dopaminergic neural dysfunction and to identify novel molecular targets for disease intervention. In addition, our model could be applied to high-throughput and phenotypic screenings for the identification of novel PD therapeutics.

A platform-agnostic, function first-based antibody discovery strategy using plasmid-free mammalian expression of antibodies.

Hybridoma technology has been valuable in the development of therapeutic antibodies. More recently, antigen-specific B-cell selection and display technologies are also gaining importance. A major limitation of these approaches used for antibody discovery is the extensive process of cloning and expression involved in transitioning from antibody identification to validating the function, which compromises the throughput of antibody discovery. In this study, we describe a process to identify and rapidly re-format and express antibodies for functional characterization. We used two different approaches to isolate antibodies to five different targets: 1) flow cytometry to identify antigen-specific single B cells from the spleen of immunized human immunoglobulin transgenic mice; and 2) panning of phage libraries. PCR amplification allowed recovery of paired VH and VL sequences from 79% to 96% of antigen-specific B cells. All cognate VH and VL transcripts were formatted into transcription and translation compatible linear DNA expression cassettes (LEC) encoding whole IgG or Fab. Between 92% and 100% of paired VH and VL transcripts could be converted to LECs, and nearly 100% of them expressed as antibodies when transfected into Expi293F cells. The concentration of IgG in the cell culture supernatants ranged from 0.05 µg/ml to 145.8 µg/ml (mean = 18.4 µg/ml). Antigen-specific binding was displayed by 78-100% of antibodies. High throughput functional screening allowed the rapid identification of several functional antibodies. In summary, we describe a plasmid-free system for cloning and expressing antibodies isolated by different approaches, in any format of choice for deep functional screening that can be applied in any research setting during antibody discovery.

SAR439859, a Novel Selective Estrogen Receptor Degrader (SERD), Demonstrates Effective and Broad Antitumor Activity in Wild-Type and Mutant ER-Positive Breast Cancer Models.

Primary treatment for estrogen receptor-positive (ER+) breast cancer is endocrine therapy. However, substantial evidence indicates a continued role for ER signaling in tumor progression. Selective estrogen receptor degraders (SERD), such as fulvestrant, induce effective ER signaling inhibition, although clinical studies with fulvestrant report insufficient blockade of ER signaling, possibly due to suboptimal pharmaceutical properties. Furthermore, activating mutations in the ER have emerged as a resistance mechanism to current endocrine therapies. New oral SERDs with improved drug properties are under clinical investigation, but the biological profile that could translate to improved therapeutic benefit remains unclear. Here, we describe the discovery of SAR439859, a novel, orally bioavailable SERD with potent antagonist and degradation activities against both wild-type and mutant Y537S ER. Driven by its fluoropropyl pyrrolidinyl side chain, SAR439859 has demonstrated broader and superior ER antagonist and degrader activities across a large panel of ER+ cells, compared with other SERDs characterized by a cinnamic acid side chain, including improved inhibition of ER signaling and tumor cell growth. Similarly, in vivo treatment with SAR439859 demonstrated significant tumor regression in ER+ breast cancer models, including MCF7-ESR1 wild-type and mutant-Y537S mouse tumors, and HCI013, a patient-derived tamoxifen-resistant xenograft tumor. These findings indicate that SAR439859 may provide therapeutic benefit to patients with ER+ breast cancer, including those who have resistance to endocrine therapy with both wild-type and mutant ER.

Bringing the Heavy Chain to Light: Creating a Symmetric, Bivalent IgG-Like Bispecific.

Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic (DMPK) properties. However, IgG-like bispecifics do not possess target bivalency and current designs often require tedious engineering and purification to ensure appropriate chain pairing. Here, we present a near-native IgG antibody format, the 2xVH, which can create bivalency for each target or epitope and requires no engineering for cognate chain pairing. In this modality, two different variable heavy (VH) domains with distinct binding specificities are grafted onto the first constant heavy (CH1) and constant light (CL) domains, conferring the molecule with dual specificity. To determine the versatility of this format, we characterized the expression, binding, and stability of several previously identified soluble human VH domains. By grafting these domains onto an IgG scaffold, we generated several prototype 2xVH IgG and Fab molecules that display similar properties to mAbs. These molecules avoided the post-expression purification necessary for engineered bispecifics while maintaining a capacity for simultaneous dual binding. Hence, the 2xVH format represents a bivalent, bispecific design that addresses limitations of manufacturing IgG-like bispecifics while promoting biologically-relevant dual target engagement.

Quantitative Proteomic Profiling Reveals Novel Plasmodium falciparum Surface Antigens and Possible Vaccine Candidates.

Despite recent efforts toward control and elimination, malaria remains a major public health problem worldwide. Plasmodium falciparum resistance against artemisinin, used in front line combination drugs, is on the rise, and the only approved vaccine shows limited efficacy. Combinations of novel and tailored drug and vaccine interventions are required to maintain the momentum of the current malaria elimination program. Current evidence suggests that strain-transcendent protection against malaria infection can be achieved using whole organism vaccination or with a polyvalent vaccine covering multiple antigens or epitopes. These approaches have been successfully applied to the human-infective sporozoite stage. Both systemic and tissue-specific pathology during infection with the human malaria parasite P. falciparum is caused by asexual blood stages. Tissue tropism and vascular sequestration are the result of specific binding interactions between antigens on the parasite-infected red blood cell (pRBC) surface and endothelial receptors. The major surface antigen and parasite ligand binding to endothelial receptors, PfEMP1 is encoded by about 60 variants per genome and shows high sequence diversity across strains. Apart from PfEMP1 and three additional variant surface antigen families RIFIN, STEVOR, and SURFIN, systematic analysis of the infected red blood cell surface is lacking. Here we present the most comprehensive proteomic investigation of the parasitized red blood cell surface so far. Apart from the known variant surface antigens, we identified a set of putative single copy surface antigens with low sequence diversity, several of which are validated in a series of complementary experiments. Further functional and immunological investigation is underway to test these novel P. falciparum blood stage proteins as possible vaccine candidates.

Glutaminase is essential for the growth of triple-negative breast cancer cells with a deregulated glutamine metabolism pathway and its suppression synergizes with mTOR inhibition.

Tumor cells display fundamental changes in metabolism and nutrient uptake in order to utilize additional nutrient sources to meet their enhanced bioenergetic requirements. Glutamine (Gln) is one such nutrient that is rapidly taken up by tumor cells to fulfill this increased metabolic demand. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS). This study has identified GLS a potential therapeutic target in breast cancer, specifically in the basal subtype that exhibits a deregulated glutaminolysis pathway. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines with a deregulated glutaminolysis pathway led to profound tumor growth inhibition in vitro and in vivo. GLS knockdown had no effect on growth and metabolite levels in non-TNBC cell lines. We rescued the anti-tumor effect of GLS knockdown using shRNA resistant cDNAs encoding both GLS isoforms and by addition of an α-ketoglutarate (αKG) analog thus confirming the critical role of GLS in TNBC. Pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) activity and an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). Finally, we found that GLS inhibition synergizes with mTOR inhibition, which introduces the possibility of a novel therapeutic strategy for TNBC. Our study revealed that GLS is essential for the survival of TNBC with a deregulated glutaminolysis pathway. The synergistic activity of GLS and mTOR inhibitors in TNBC cell lines suggests therapeutic potential of this combination for the treatment of vulnerable subpopulations of TNBC.

Oncogenic dependency on ß-catenin in liver cancer cell lines correlates with pathway activation.

Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/ß-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (ß-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of ß-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on ß-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on ß-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) ß-catenin were significantly more sensitive to ß-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active ß-catenin. Finally, significant therapeutic benefit of ß-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. ß-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of ß-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant ß-catenin signaling in the maintenance of oncogenic phenotype in HCC.

Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells.

Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.

Selective inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule.

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

PIM inhibitors target CD25-positive AML cells through concomitant suppression of STAT5 activation and degradation of MYC oncogene.

Postchemotherapy relapse presents a major unmet medical need in acute myeloid leukemia (AML), where treatment options are limited. CD25 is a leukemic stem cell marker and a conspicuous prognostic marker for overall/relapse-free survival in AML. Rare occurrence of genetic alterations among PIM family members imposes a substantial hurdle in formulating a compelling patient stratification strategy for the clinical development of selective PIM inhibitors in cancer. Here we show that CD25, a bona fide STAT5 regulated gene, is a mechanistically relevant predictive biomarker for sensitivity to PIM kinase inhibitors. Alone or in combination with tyrosine kinase inhibitors, PIM inhibitors can suppress STAT5 activation and significantly shorten the half-life of MYC to achieve substantial growth inhibition of high CD25-expressing AML cells. Our results highlight the importance of STAT5 and MYC in rendering cancer cells sensitive to PIM inhibitors. Because the presence of a CD25-positive subpopulation in leukemic blasts correlates with poor overall or relapse-free survival, our data suggest that a combination of PIM inhibitors with chemotherapy and tyrosine kinase inhibitors could improve long-term therapeutic outcomes in CD25-positive AML.

Evaluation of cancer dependence and druggability of PRP4 kinase using cellular, biochemical, and structural approaches.

PRP4 kinase is known for its roles in regulating pre-mRNA splicing and beyond. Therefore, a wider spectrum of PRP4 kinase substrates could be expected. The role of PRP4 kinase in cancer is also yet to be fully elucidated. Attaining specific and potent PRP4 inhibitors would greatly facilitate the study of PRP4 biological function and its validation as a credible cancer target. In this report, we verified the requirement of enzymatic activity of PRP4 in regulating cancer cell growth and identified an array of potential novel substrates through orthogonal proteomics approaches. The ensuing effort in structural biology unveiled for the first time unique features of PRP4 kinase domain and its potential mode of interaction with a low molecular weight inhibitor. These results provide new and important information for further exploration of PRP4 kinase function in cancer.

A biomedically enriched collection of 7000 human ORF clones.

We report the production and availability of over 7000 fully sequence verified plasmid ORF clones representing over 3400 unique human genes. These ORF clones were derived using the human MGC collection as template and were produced in two formats: with and without stop codons. Thus, this collection supports the production of either native protein or proteins with fusion tags added to either or both ends. The template clones used to generate this collection were enriched in three ways. First, gene redundancy was removed. Second, clones were selected to represent the best available GenBank reference sequence. Finally, a literature-based software tool was used to evaluate the list of target genes to ensure that it broadly reflected biomedical research interests. The target gene list was compared with 4000 human diseases and over 8500 biological and chemical MeSH classes in approximately 15 Million publications recorded in PubMed at the time of analysis. The outcome of this analysis revealed that relative to the genome and the MGC collection, this collection is enriched for the presence of genes with published associations with a wide range of diseases and biomedical terms without displaying a particular bias towards any single disease or concept. Thus, this collection is likely to be a powerful resource for researchers who wish to study protein function in a set of genes with documented biomedical significance.

Sequence analysis and biochemical characterization of the nostopeptolide A biosynthetic gene cluster from Nostoc sp. GSV224.

The cloning, sequencing, annotation and biochemical analysis of the nostopeptolide (nos) biosynthetic gene cluster from the terrestrial cyanobacterium Nostoc sp. GSV224 is described. Nostopeptolides A1 and A2 are cyclic peptide-polyketide hybrid natural products possessing nine amino acid residues, a butyric acid group, and an internal acetate-derived unit that are linked by peptide and ester bonds. The nos gene cluster includes eight ORFs encompassing 40 kb and includes most of the genes predicted to be involved in the biosynthesis and transport of this group of nonapeptolides. The genetic architecture and domain organization of the nos synthetase, a mixed non-ribosomal peptide synthetase-polyketide synthase, is co-linear in arrangement with respect to the putative order of the biosynthetic assembly of the lipopeptolide. Biochemical analysis of the NosA1, NosC1 and NosD1 adenylation domains coupled with the recent characterization of the nosE and nosF gene products, which are involved in the biosynthesis of the rare non-proteinogenic amino acid residue L-4-methylproline from L-leucine, support the involvement of this gene cluster in nostopeptolide biosynthesis.

Occurrence and non-detectability of maytansinoids in individual plants of the genera Maytenus and Putterlickia.

Individual plants belonging to different species of the family Celastraceae collected from their natural habitats in South Africa (Putterlickia verrucosa (E. Meyer ex Sonder) Szyszyl., Putterlickia pyracantha (L.) Szyszyl., Putterlickia retrospinosa van Wyk and Mostert) and Brazil (Maytenus ilicifolia Mart. ex Reiss., Maytenus evonymoides Reiss., Maytenus aquifolia Mart.) were investigated for the presence of maytansinoids and of maytansine, an ansamycin of high cytotoxic activity. Maytansinoids were not detectable in plants grown in Brazil. Analysis of plants growing in South Africa, however, showed clearly that maytansinoids were present in some individual plants but were not detectable in others. Molecular biological analysis of a Putterlickia verrucosa cell culture gave no evidence for the presence of the aminohydroxybenzoate synthase gene which is unique to the biosynthesis of aminohydroxybenzoate, a precursor of the ansamycins including maytansinoids. Moreover, this gene was not detectable in DNA extracted from the aerial parts of Putterlickia plants. In contrast, observations indicate that this gene may be present in microbes of the rhizosphere of Putterlickia plants. Our observations are discussed with respect to the possibility that the roots of Putterlickia plants may be associated with microorganisms which are responsible for the biosynthesis of maytansine or maytansinoids.

Biosynthesis of 4-methylproline in cyanobacteria: cloning of nosE and nosF genes and biochemical characterization of the encoded dehydrogenase and reductase activities.

The biosynthesis of the unusual amino acid 4-methylproline in the Nostoc genus of cyanobacteria was investigated on the genetic and enzymatic level. Two genes involved in the biosynthesis were cloned and the corresponding enzymes, a zinc-dependent long-chain dehydrogenase and a Delta(1)-pyrroline-5-carboxylic acid (P5C) reductase homologue, were overexpressed in Escherichia coli and biochemically characterized. Putative substrates were synthesized to test enzyme substrate specificities, and deuterium labeling studies were carried out to reveal the stereospecificities of the enzymatic reactions with respect to the substrates as well as to the coenzymes.