Biology subjects

Scheeren, F. A.

Publications and source records attributed to Scheeren, F. A..

5 recordsLinked to original sources

bioRxiv2021

Malaria parasite evades mosquito immunity by glutaminyl cyclase mediated protein modification

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that Plasmodium sporozoites of QC-null mutants are recognized by the mosquito immune system and melanized when they reach the hemocoel. Sporozoite numbers in salivary glands are also reduced in mosquitoes infected with QC-null or QC catalytically-dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito hemocytes or melanization immune responses. Mutation of a single QC-target glutamine of the major sporozoite surface protein (CSP) also results in immune recognition of sporozoites. These findings reveal QC-mediated post-translational modification of surface proteins as a major mechanism of mosquito immune evasion by Plasmodium sporozoites.

microbiology↗

bioRxiv2020

Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

I.Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theragnostics, and antibody-drug conjugates (ADC). Antibody functionalization is classically achieved by coupling payloads onto lysine or cysteine residues. However, such stochastic strategies typically lead to heterogenous products, bearing a varying number of payloads. This affects bioconjugate efficacy and stability, as well as its in vivo biodistribution, and therapeutic index, while potentially obstructing the binding sites and leading to off-target toxicity. In addition, therapeutic and theragnostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labelling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogenous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab fragments bearing two distinct site-specific labelling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab molecule (DTFab), which can be easily isolated in high yields. To demonstrate feasibility, we functionalized the DTFab with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theragnostics, and next-generation ADCs.

biochemistry↗

bioRxiv2019

Single-cell transcriptional diversity is a hallmark of developmental potential

Single-cell RNA-sequencing (scRNA-seq) is a powerful approach for reconstructing cellular differentiation trajectories. However, inferring both the state and direction of differentiation without prior knowledge has remained challenging. Here we describe a simple yet robust determinant of developmental potential--the number of detectably expressed genes per cell-- and leverage this measure of transcriptional diversity to develop a new framework for predicting ordered differentiation states from scRNA-seq data. When evaluated on ~150,000 single-cell transcriptomes spanning 53 lineages and five species, our approach, called CytoTRACE, outperformed previous methods and ~19,000 molecular signatures for resolving experimentally-confirmed developmental trajectories. In addition, it enabled unbiased identification of tissue-resident stem cells, including cells with long-term regenerative potential. When used to analyze human breast tumors, we discovered candidate genes associated with less-differentiated luminal progenitor cells and validated GULP1 as a novel gene involved in tumorigenesis. Our study establishes a key RNA-based correlate of developmental potential and provides a new platform for robust delineation of cellular hierarchies (https://cytotrace.stanford.edu).

genomics↗

bioRxiv2019

Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas which secrete antibodies in the preferred format, species and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab fragments, isotype switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemo-enzymatic modification. We believe this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research. One Sentence SummaryWe demonstrate a universal CRISPR/HDR based platform for rapid genetic engineering of hybridomas to obtain functionally diverse antibody isotype panels in the species and format of choice.

bioengineering↗

bioRxiv2018

CDK19 is a Regulator of Triple-Negative Breast Cancer Growth

Triple-negative breast cancer (TNBC) is a poor prognosis disease with no clinically approved targeted therapies. Here, using in vitro and in vivo RNA interference (RNAi) screens in TNBC patient-derived xenografts (PDX), we identify cyclin dependent kinase 19 (CDK19) as a potential therapeutic target. Using in vitro and in vivo TNBC PDX models, we validated the inhibitory effect of CDK19 knockdown on tumor initiation, proliferation and metastases. Despite this, CDK19 knockdown did not affect the growth of non-transformed mammary epithelial cells. Using CD10 and EpCAM as novel tumor initiating cell (TIC) markers, we found the EpCAMmed/high/CD10-/low TIC sub-population to be enriched in CDK19 and a putative cellular target of CDK19 inhibition. Comparative gene expression analysis of CDK19 and CDK8 knockdowns revealed that CDK19 regulates a number of cancer-relevant pathways, uniquely through its own action and others in common with CDK8. Furthermore, although it is known that CDK19 can act at enhancers, our CHIP-Seq studies showed that CDK19 can also epigenetically modulate specific H3K27Ac enhancer signals which correlate with gene expression changes. Finally, to assess the potential therapeutic utility of CDK19, we showed that both CDK19 knockdown and chemical inhibition of CDK19 kinase activity impaired the growth of pre-established PDX tumors in vivo. Current strategies inhibiting transcriptional co-factors and targeting TICs have been limited by toxicity to normal cells. Because of CDK19s limited tissue distribution and the viability of CDK19 knockout mice, CDK19 represents a promising therapeutic target for TNBC.

cancer biology↗