Vaccine-boosted CAR T crosstalk with host immunity to reject tumors with antigen heterogeneity.

Chimeric antigen receptor (CAR) T cell therapy effectively treats human cancer, but the loss of the antigen recognized by the CAR poses a major obstacle. We found that in vivo vaccine boosting of CAR T cells triggers the engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited the priming of endogenous anti-tumor T cells. This process was accompanied by shifts in CAR T metabolism toward oxidative phosphorylation (OXPHOS) and was critically dependent on CAR-T-derived IFN-γ. Antigen spreading (AS) induced by vaccine-boosted CAR T enabled a proportion of complete responses even when the initial tumor was 50% CAR antigen negative, and heterogeneous tumor control was further enhanced by the genetic amplification of CAR T IFN-γ expression. Thus, CAR-T-cell-derived IFN-γ plays a critical role in promoting AS, and vaccine boosting provides a clinically translatable strategy to drive such responses against solid tumors.

Structural Alterations Driving Castration-Resistant Prostate Cancer Revealed by Linked-Read Genome Sequencing.

Nearly all prostate cancer deaths are from metastatic castration-resistant prostate cancer (mCRPC), but there have been few whole-genome sequencing (WGS) studies of this disease state. We performed linked-read WGS on 23 mCRPC biopsy specimens and analyzed cell-free DNA sequencing data from 86 patients with mCRPC. In addition to frequent rearrangements affecting known prostate cancer genes, we observed complex rearrangements of the AR locus in most cases. Unexpectedly, these rearrangements include highly recurrent tandem duplications involving an upstream enhancer of AR in 70%-87% of cases compared with <2% of primary prostate cancers. A subset of cases displayed AR or MYC enhancer duplication in the context of a genome-wide tandem duplicator phenotype associated with CDK12 inactivation. Our findings highlight the complex genomic structure of mCRPC, nominate alterations that may inform prostate cancer treatment, and suggest that additional recurrent events in the non-coding mCRPC genome remain to be discovered.

Tissue-specific abundance of interferon-gamma drives regulatory T cells to restrain DC1-mediated priming of cytotoxic T cells against lung cancer.

Local environmental factors influence CD8+ T cell priming in lymph nodes (LNs). Here, we sought to understand how factors unique to the tumor-draining mediastinal LN (mLN) impact CD8+ T cell responses toward lung cancer. Type 1 conventional dendritic cells (DC1s) showed a mLN-specific failure to induce robust cytotoxic T cells responses. Using regulatory T (Treg) cell depletion strategies, we found that Treg cells suppressed DC1s in a spatially coordinated manner within tissue-specific microniches within the mLN. Treg cell suppression required MHC II-dependent contact between DC1s and Treg cells. Elevated levels of IFN-γ drove differentiation Treg cells into Th1-like effector Treg cells in the mLN. In patients with cancer, Treg cell Th1 polarization, but not CD8+/Treg cell ratios, correlated with poor responses to checkpoint blockade immunotherapy. Thus, IFN-γ in the mLN skews Treg cells to be Th1-like effector Treg cells, driving their close interaction with DC1s and subsequent suppression of cytotoxic T cell responses.

TCR sequencing paired with massively parallel 3' RNA-seq reveals clonotypic T cell signatures.

High-throughput 3' single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3'-barcoded scRNA-seq samples. This approach is compatible with common 3' scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from patients with food allergy. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4+ T cell (TH2) states associated with food allergy. These results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.

Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies.

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

Single-cell technologies for monitoring immune systems.

The complex heterogeneity of cells, and their interconnectedness with each other, are major challenges to identifying clinically relevant measurements that reflect the state and capability of the immune system. Highly multiplexed, single-cell technologies may be critical for identifying correlates of disease or immunological interventions as well as for elucidating the underlying mechanisms of immunity. Here we review limitations of bulk measurements and explore advances in single-cell technologies that overcome these problems by expanding the depth and breadth of functional and phenotypic analysis in space and time. The geometric increases in complexity of data make formidable hurdles for exploring, analyzing and presenting results. We summarize recent approaches to making such computations tractable and discuss challenges for integrating heterogeneous data obtained using these single-cell technologies.

Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice.

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

WAT3R: recovery of T-cell receptor variable regions from 3' single-cell RNA-sequencing.

SUMMARY: Diversity of the T-cell receptor (TCR) repertoire is central to adaptive immunity. The TCR is composed of α and ß chains, encoded by the TRA and TRB genes, of which the variable regions determine antigen specificity. To generate novel biological insights into the complex functioning of immune cells, combined capture of variable regions and single-cell transcriptomes provides a compelling approach. Recent developments enable the enrichment of TRA and TRB variable regions from widely used technologies for 3'-based single-cell RNA-sequencing (scRNA-seq). However, a comprehensive computational pipeline to process TCR-enriched data from 3' scRNA-seq is not available. Here, we present an analysis pipeline to process TCR variable regions enriched from 3' scRNA-seq cDNA. The tool reports TRA and TRB nucleotide and amino acid sequences linked to cell barcodes, enabling the reconstruction of T-cell clonotypes with associated transcriptomes. We demonstrate the software using peripheral blood mononuclear cells from a healthy donor and detect TCR sequences in a high proportion of single T cells. Detection of TCR sequences is low in non-T-cell populations, demonstrating specificity. Finally, we show that TCR clones are larger in CD8 Memory T cells than in other T-cell types, indicating an association between T-cell clonotypes and differentiation states. AVAILABILITY AND IMPLEMENTATION: The Workflow for Association of T-cell receptors from 3' single-cell RNA-seq (WAT3R), including test data, is available on GitHub (https://github.com/mainciburu/WAT3R), Docker Hub (https://hub.docker.com/r/mainciburu/wat3r) and a workflow on the Terra platform (https://app.terra.bio). The test dataset is available on GEO (accession number GSE195956). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Minimal purification method enables developability assessment of recombinant proteins.

Analytical characterization of proteins is a critical task for developing therapeutics and subunit vaccine candidates. Assessing candidates with a battery of biophysical assays can inform the selection of one that exhibits properties consistent with a given target product profile (TPP). Such assessments, however, require several milligrams of purified protein, and ideal assessments of the physicochemical attributes of the proteins should not include unnatural modifications like peptide tags for purification. Here, we describe a fast two-stage minimal purification process for recombinant proteins secreted by the yeast host Komagataella phaffii from a 20 mL culture supernatant. This method comprises a buffer exchange and filtration with a Q-membrane filter and we demonstrate sufficient removal of key supernatant impurities including host-cell proteins (HCPs) and DNA with yields of 1-2 mg and >60% purity. This degree of purity enables characterizing the resulting proteins using affinity binding, mass spectrometry, and differential scanning calorimetry. We first evaluated this method to purify an engineered SARS-CoV-2 subunit protein antigen and compared the purified protein to a conventional two-step chromatographic process. We then applied this method to compare several SARS-CoV-2 RBD sequences. Finally, we show this simple process can be applied to a range of other proteins, including a single-domain antibody, a rotavirus protein subunit, and a human growth hormone. This simple and fast developability methodology obviates the need for genetic tagging or full chromatographic development when assessing and comparing early-stage protein therapeutics and vaccine candidates produced in K. phaffii.

Revealing the heterogeneity of CD4+ T cells through single-cell transcriptomics.

Single-cell RNA sequencing (scRNA-seq) offers the ability to resolve whole transcriptomes of single cells with substantial throughput, and it has revolutionized studies of gene expression. The transcriptional resolution available can uncover fine structures of biologic heterogeneity that are manifest among cell populations. Here, we review the applications of scRNA-seq to profile the phenotypes and clonotypes of CD4+ T cells. First, we describe challenges inherent to scRNA-seq that are important for analysis of CD4+ T cells, as well as the technical solutions that are emerging to address these challenges. We then consider major themes of the application of scRNA-seq to CD4+ T cells, including investigation of CD4+ T-cell heterogeneity in model systems, analysis of populations from the peripheral blood, and the profiling of tissue-resident populations. We place emphasis on capabilities unique to scRNA-seq, such as the ability to obtain paired T-cell receptor and transcriptome information from single T cells and the potential to elucidate interactions between CD4+ T cells and other cells in their environment. Finally, we conclude by considering future areas of technologic advancement and innovation through which scRNA-seq may further shape our understanding of the roles of CD4+ T cells in health and disease.

Modular development enables rapid design of media for alternative hosts.

Developing media to sustain cell growth and production is an essential and ongoing activity in bioprocess development. Modifications to media can often address host or product-specific challenges, such as low productivity or poor product quality. For other applications, systematic design of new media can facilitate the adoption of new industrially relevant alternative hosts. Despite manifold existing methods, common approaches for optimization often remain time and labor-intensive. We present here a novel approach to conventional media blending that leverages stable, simple, concentrated stock solutions to enable rapid improvement of measurable phenotypes of interest. We applied this modular methodology to generate high-performing media for two phenotypes of interest: biomass accumulation and heterologous protein production, using high-throughput, milliliter-scale batch fermentations of Pichia pastoris as a model system. In addition to these examples, we also created a flexible open-source package for modular blending automation on a low-cost liquid handling system to facilitate wide use of this method. Our modular blending method enables rapid, flexible media development, requiring minimal labor investment and prior knowledge of the host organism, and should enable developing improved media for other hosts and phenotypes of interest.

Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor-binding domain in engineered Komagataella phaffii.

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply ongoing demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor-binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5X by alleviating protein folding stress. Removal of methanol from the production process enabled to scale up to a 1200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

Divergent Functions of Tissue-Resident and Blood-Derived Macrophages in the Hemorrhagic Brain.

Background and Purpose: Brain tissue-resident microglia and monocyte-derived macrophages (MDMs) are innate immune cells that contribute to the inflammatory response, phagocytosis of debris, and tissue repair after injury. We have previously reported that both microglia and MDMs transition from proinflammatory to reparative phenotypes over days after an intracerebral hemorrhage (ICH). However, their individual functional properties in the brain remain largely unknown. Here we characterized the differences between microglia and MDMs and further elucidate their distinct activation states and functional contributions to the pathophysiology and recovery after ICH. Methods: Autologous blood injection was used to model ICH in mice. Longitudinal transcriptomic analyses on isolated microglia and MDMs from mice at days 1, 3, 7 and 10 after ICH and naive controls identified core transcriptional programs that distinguish these cells. Imaging flow cytometry and in vivo phagocytosis assays were used to study phagocytic ability of microglia and MDMs. Antigen presentation was evaluated by ovalbumin-OTII CD4 T-cell proliferation assays with bone marrow­derived macrophages and primary microglia cultures. Results: MDMs had higher phagocytic activity and higher erythrophagocytosis in the ICH brain. Differential gene expression revealed distinct transcriptional signatures in the MDMs and microglia after ICH. MDMs had higher expression of MHCII (major histocompatibility complex class II) genes than microglia at all time points and greater ability to induce antigen-specific T-cell proliferation. Conclusions: The different ontogeny of microglia and MDMs lead to divergent responses and functions in the inflamed brain as these 2 cell populations differ in phagocytic functions and antigen-presenting capabilities in the brain after ICH.

Diverse antiviral IgG effector activities are predicted by unique biophysical antibody features.

BACKGROUND: The critical role of antibody Fc-mediated effector functions in immune defense has been widely reported in various viral infections. These effector functions confer cellular responses through engagement with innate immune cells. The precise mechanism(s) by which immunoglobulin G (IgG) Fc domain and cognate receptors may afford protection are poorly understood, however, in the context of HIV/SHIV infections. Many different in vitro assays have been developed and utilized to measure effector functions, but the extent to which these assays capture distinct antibody activities has not been fully elucidated. RESULTS: In this study, six Fc-mediated effector function assays and two biophysical antibody profiling assays were performed on a common set of samples from HIV-1 infected and vaccinated subjects. Biophysical antibody profiles supported robust prediction of diverse IgG effector functions across distinct Fc-mediated effector function assays. While a number of assays showed correlated activities, supervised machine learning models indicated unique antibody features as primary contributing factors to the associated effector functions. Additional experiments established the mechanistic relevance of relationships discovered using this unbiased approach. CONCLUSIONS: In sum, this study provides better resolution on the diversity and complexity of effector function assays, offering a clearer perspective into this family of antibody mechanisms of action to inform future HIV-1 treatment and vaccination strategies.

Rapid optimization of processes for the integrated purification of biopharmaceuticals.

Straight-through chromatography, wherein the eluate from one column passes directly onto another column without adjustment, is one strategy to integrate and intensify manufacturing processes for biologics. Development and optimization of such straight-through chromatographic processes is a challenge, however. Conventional high-throughput screening methods optimize each chromatographic step independently, with limited consideration for the connectivity of steps. Here, we demonstrate a method for the development and optimization of fully integrated, multi-column processes for straight-through purification. Selection of resins was performed using an in silico tool for the prediction of processes for straight-through purification based on a one-time characterization of host-cell proteins combined with the chromatographic behavior of the product. A two-step optimization was then conducted to determine the buffer conditions that maximized yield while minimizing process- and product-related impurities. This optimization of buffer conditions included a series of range-finding experiments on each individual column, similar to conventional screening, followed by the development of a statistical model for the fully integrated, multi-column process using design of experiments. We used this methodology to develop and optimize integrated purification processes for a single-domain antibody and a cytokine, obtaining yields of 88% and 86%, respectively, with process- and product-related variants reduced to phase-appropriate levels for nonclinical material.

Modeling of copy number variability in Pichia pastoris.

Development of continuous biopharmaceutical manufacturing processes is an area of active research. This study considers the long-term transgene copy number stability of Pichia pastoris in continuous bioreactors. We propose a model of copy number loss that quantifies population heterogeneity. An analytical solution is derived and compared with existing experimental data. The model is then used to provide guidance for stable operating timescales. The model is extended to consider copy number dependent growth such as in the case of Zeocin supplementation. The model is also extended to analyze a continuous seeding strategy. This study is a critical step towards understanding the impact of continuous processing on the stability of Pichia pastoris and the resultant products.

Macroscopic modeling of bioreactors for recombinant protein producing Pichia pastoris in defined medium.

The methylotrophic yeast Pichia pastoris is widely used as a microbial host for recombinant protein production. Bioreactor models for P. pastoris can inform understanding of cellular metabolism and can be used to optimize bioreactor operation. This article constructs an extensive macroscopic bioreactor model for P. pastoris which describes substrates, biomass, total protein, other medium components, and off-gas components. Species and elemental balances are introduced to describe uptake and evolution rates for medium components and off-gas components. Additionally, a pH model is constructed using an overall charge balance, acid/base equilibria, and activity coefficients to describe production of recombinant protein and precipitation of medium components. The extent of run-to-run variability is modeled by distributions of a subset of the model parameters, which are estimated using the maximum likelihood method. Model prediction from the extensive macroscopic bioreactor model well describes experimental data with different operating conditions. The probability distributions of the model predictions quantified from the parameter distribution are quantifiably consistent with the run-to-run variability observed in the experimental data. The uncertainty description in this macroscopic bioreactor model identifies the model parameters that have large variability and provides guidance as to which aspects of cellular metabolism should be the focus of additional experimental studies. The model for medium components with pH and precipitation can be used for improving chemically defined medium by minimizing the amount of components needed while meeting cellular requirements.

Development of a platform process for the production and purification of single-domain antibodies.

Single-domain antibodies (sdAbs) offer the affinity and therapeutic value of conventional antibodies, with increased stability and solubility. Unlike conventional antibodies, however, sdAbs do not benefit from a platform manufacturing process. While successful production of a variety of sdAbs has been shown in numerous hosts, purification methods are often molecule specific or require affinity tags, which generally cannot be used in clinical manufacturing due to regulatory concerns. Here, we have developed a broadly applicable production and purification process for sdAbs in Komagataella phaffii (Pichia pastoris) and demonstrated the production of eight different sdAbs at a quality appropriate for nonclinical studies. We developed a two-step, integrated purification process without the use of affinity resins and showed that modification of a single process parameter, pH of the bridging buffer, was required for the successful purification of a variety of sdAbs. Further, we determined that this parameter can be predicted based only on the biophysical characteristics of the target molecule. Using these methods, we produced nonclinical quality sdAbs as few as 5 weeks after identifying the product sequence. Nonclinical studies of three different sdAbs showed that molecules produced using our platform process conferred protection against viral shedding of rotavirus or H1N1 influenza and were equivalent to similar molecules produced in Escherichia coli and purified using affinity tags.

Rho/SMAD/mTOR triple inhibition enables long-term expansion of human neonatal tracheal aspirate-derived airway basal cell-like cells.

BACKGROUND: Bronchopulmonary dysplasia remains one of the most common complications of prematurity, despite significant improvements in perinatal care. Functional modeling of human lung development and disease, like BPD, is limited by our ability to access the lung and to maintain relevant progenitor cell populations in culture. METHODS: We supplemented Rho/SMAD signaling inhibition with mTOR inhibition to generate epithelial basal cell-like cell lines from tracheal aspirates of neonates. RESULTS: Single-cell RNA-sequencing confirmed the presence of epithelial cells in tracheal aspirates obtained from intubated neonates. Using Rho/SMAD/mTOR triple signaling inhibition, neonatal tracheal aspirate-derived (nTAD) basal cell-like cells can be expanded long term and retain the ability to differentiate into pseudostratified airway epithelium. CONCLUSIONS: Our data demonstrate that neonatal tracheal aspirate-derived epithelial cells can provide a novel ex vivo human cellular model to study neonatal lung development and disease. IMPACT: Airway epithelial basal cell-like cell lines were derived from human neonatal tracheal aspirates. mTOR inhibition significantly extends in vitro proliferation of neonatal tracheal aspirate-derived basal cell-like cells (nTAD BCCs). nTAD BCCs can be differentiated into functional airway epithelium. nTAD BCCs provide a novel model to investigate perinatal lung development and diseases.