Abstract Targeted cellular immunotherapies hold promise against pediatric brain tumors, evidenced by the preliminary efficacy of B7-H3- and GD2-targeting CAR T cells against DIPG. However, benefit was not universal, underscoring the need to further explore therapies capable of more durable treatment responses. While most cellular therapy advancements have centered on T cells, B cells are an attractive engineered option to design in vivo antibody production centers. In fact, B cells can be designed to secrete specific tumor-targeting antibodies and to initiate anti-tumor immune responses. Here, we engineered anti-GD2 antibody-producing B cells as a novel targeted immunotherapeutic approach against DIPG. Early data demonstrates functionality of the modified B cells, confirming cells were successfully engineered to produce anti-GD2 antibodies. In vitro affinity testing shows that anti-GD2 antibodies from engineered B cells binds to GD2 protein on DIPG cultures as well as commercially available anti-GD2 antibodies. In vivo, we have supporting data showing that the engineered human plasma B cells can be injected via ICV into the brains of NSG mice where they persist for over a week post-injection and, critically, secrete anti-GD2 antibodies. More importantly, immunostaining of treated mice indicates elevated immune cell recruitment to sites of anti-GD2 antibody secretion. These results represent the first foundational preclinical attempts to use engineered B cells secreting tumor-targeted antibodies against pediatric brain tumors and specifically validate the potential of anti-GD2 antibody secretion which is now in in vitro efficacy studies.

Cells secrete numerous proteins and other biomolecules into their surroundings to achieve critical functions—from communicating with other cells to blocking the activity of pathogens. Secretion of cytokines, growth factors, extracellular vesicles and even recombinant biologic drugs defines the therapeutic potency of many cell therapies. However, gene expression states that drive specific secretory phenotypes are largely unknown. We provide a protocol that enables the secretion amount of a target protein encoded (SEC) by oligonucleotide barcodes to be linked with transcriptional sequencing (seq) for thousands of single cells. SEC-seq leverages microscale hydrogel particles called Nanovials to isolate cells and capture their secretions in close proximity, oligonucleotide-labeled antibodies to tag secretions on Nanovials and flow cytometry and single-cell RNA-sequencing (scRNA-seq) platforms for readout. Cells on Nanovials can be sorted on the basis of viability, secretion amount or other surface markers without fixation or permeabilization, and cell- and secretion-containing Nanovials are directly introduced into microfluidic droplets-in-oil emulsions for single-cell barcoding of cell transcriptomes and secretions. We have used SEC-seq to link T cell receptor sequences to the relative amount of associated cytokine secretions, surface marker gene expression with a highly secreting and potential regenerative population of mesenchymal stromal cells and the transcriptome with high immunoglobulin secretion from plasma cells. Nanovial modification and cell loading takes <4 h, and once the desired incubation time is over, staining, cell sorting and emulsion generation for scRNA-seq can also be completed in <4 h. Compared to related techniques that link secretions to a cell’s surface, SEC-seq provides a general solution across any secretion target because of the ease with which biotinylated Nanovials can be modified. By linking gene expression and secretory strength, SEC-seq can expand our understanding of cell secretion, how it is regulated and how it can be engineered to make better therapies. SEC-seq is a single-cell method linking the secretion of a target protein to the cell’s transcriptome. Nanovials isolate cells and capture their secretions, and oligonucleotide-labeled antibodies tag them, followed by an scRNA-seq readout.

Millions of modular nanoliter-scale compartments that isolate functionally rich single-cell and cell-to-cell communication data can scale biological discovery for the age of AI. Here, we introduce capped nanovials-suspendable, sealable microscale compartments formed by the docking of hydrogel capping particles into bowl-shaped nanovials-as a versatile system for culturing, analyzing, and sorting single cells and small colonies. This two-particle architecture enables localized confinement of cells and secreted products while maintaining compatibility with standard laboratory workflows such as wash and reagent exchange steps, fluorescence microscopy, and flow cytometry. Crucially, these compartments are formed via simple pipetting and centrifugation steps, making the platform highly democratized. We demonstrate the ability of capped nanovials to compartmentalize single mammalian, bacterial, and yeast cells and support growth into colonies, enabling selection based on proliferation and bioproduction. We further show that capped nanovials enhance single-cell secretion assays by reducing molecular crosstalk and increasing signal-to-noise ratios. Importantly, we demonstrate functional co-culture assays by permitting stable confinement of cell pairs, enabling detection and enrichment of antibody-secreting cells based on the ability of their secreted antibodies to activate co-encapsulated reporter T cells, achieving a signal-to-noise ratio of >30 and up to 100% selection purity. By combining the simplicity of standard lab handling with the resolution and throughput of traditional microfluidic compartmentalization approaches, capped nanovials provide a new class of scalable, accessible test tubes for modern single-cell biology.

Antibody discovery can benefit from techniques to screen antibody-secreting cells (ASCs) at scale for the binding and functionality of a diverse set of secreted antibodies. Previously, we demonstrated the use of cavity-containing hydrogel microparticles (nanovials) coated with a single affinity agent, biotin, to capture and identify ASCs secreting antibodies against a recombinant antigen bound to the nanovial through biotin-streptavidin linkages. However, rapidly secreted antibodies from unbound cells or cells in adjacent nanovials can cause crosstalk leading to background signal. Earlier efforts address this by localizing capture sites to the nanovial cavity, emulsifying nanovials, or short secretion times to limit secreted antibodies from binding to neighboring nanovials. Here, we demonstrate a method to functionalize nanovials with moieties that impart orthogonal reactivity, enabling conjugation of cell capture antibodies and antigens at different times. We show that by using a strained alkyne moiety to attach cell-capture antibodies via click chemistry to nanovials, we can capture cells and subsequently quantify secretions via biotin-streptavidin linkages. By delaying the loading of antigens onto the nanovials until after cell capture, we were able to ensure high purity (>95%) isolation of hybridoma secreting an antigen-specific antibody in a background of other hybridoma. This approach allows tight temporal control of the secretion measurement, which is independent of the cell loading time and requires less convective transfer steps. Click chemistry-based coupling further improved cell loading into nanovials by 58% compared to biotin-streptavidin-biotin coupling and caused no reduction in cell viability. We demonstrate an implementation of this system to improve antigen-specific hybridoma screening, yielding an 8-fold improvement in hybridoma enrichment while maintaining similar workflow complexity. Hybridomas on nanovials sorted into well plates regrew into colonies following sorting using standard fluorescence-activated cell sorting and maintained secretion of antigen-specific antibodies with high purity (∼90%), as validated via standard enzyme-linked immunosorbent assays. This lab-on-a-particle approach can be applied more generally to decouple cell loading, treatment, or activation, from secretion measurements for single-cell functional assays.

Posttransplant lymphoproliferative disease (PTLD) is a major therapeutic challenge that has been difficult to study using human cells because of a lack of suitable models for mechanistic characterization. Here, we show that ex vivo-differentiated B cells isolated from a subset of healthy donors can elicit pathologies similar to PTLD when transferred into immunodeficient mice. The primary driver of PTLD-like pathologies were IgM-producing plasmablasts with Epstein-Barr virus (EBV) genomes that expressed genes commonly associated with EBV latency. We show that a small subset of EBV+ peripheral blood-derived B cells expressing self-reactive, nonmutated B cell receptors (BCRs) expand rapidly in culture in the absence of BCR stimulation. Furthermore, we found that in vitro and in vivo expansion of EBV+ plasmablasts required BCR signaling. Last, treatment of immunodeficient mice with the BCR pathway inhibitor, ibrutinib, delays onset of PTLD-like pathologies in vivo. These data have implications for the diagnosis and care of transplant recipients who are at risk of developing PTLD.

Colicin (Col) plasmid contains colicin encoding genes arranged in an operon controlled by an SOS inducible promoter. Therefore, any external stresses to the host cell can induce the expression of the downstream genes in the Col operon, including a lysis gene. The lysis protein is involved in the extracellular release of colicin through lysis of the producer cells, which causes a decline in culture turbidity. However, it is not yet known that E. coli cells with the native pColE9-J plasmid hold the same level of cell death at the population level following a set of induced conditions. In this study, using a mitomycin C sensitivity assay along with a live dead staining method of detection, we showed that the native pColE9-J plasmid, which unusually carries an extended Col operon (ColE9) containing two lysis genes, did not confer a rapid decline in the culture turbidity following induction with mitomycin C. Interestingly a subset of the cells suffered perturbation of their outer membrane, which was not observed from single lysis mutant (∆celE or ∆celI) cells. This observed heterogeneity in the colicin E9 release leading to differential outer membrane perforation may bring a competitive advantage to these cells in a mixed population.

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.

Pathogenic long-lived plasma cells (LLPCs) secrete autoreactive antibodies, exacerbating autoimmune diseases and complicating solid organ transplantation. Targeted elimination of the autoreactive B cell pool represents a promising therapeutic strategy, yet current treatment modalities fall short in depleting mature PCs. Here, we demonstrate that chimeric antigen receptor (CAR) T cells, targeting B cell maturation antigen (BCMA) utilizing a split-receptor design, offer a controlled and effective therapeutic strategy against LLPCs. Dimerizing agent-regulated immune-receptor complex (DARIC) T cells demonstrated robust rapamycin-dependent targeting of tumor and PCs. Notably, in humanized mouse models, DARIC T cells regulated peripheral human immunoglobulin levels through specific elimination of human LLPCs from the bone marrow. Furthermore, DARIC constructs were efficiently integrated into the T cell receptor α constant (TRAC) locus while maintaining potent antigen-specific cytotoxicity. These findings underscore the potential of split-receptor CAR T cells in autoimmune and transplant medicine, highlighting their versatility in applications beyond oncology.

Cells secrete numerous proteins and other biomolecules into their surroundings to achieve critical functions - from communicating with other cells to blocking the activity of pathogens. Secretion of cytokines, growth factors, extracellular vesicles, and even recombinant biologic drugs defines the therapeutic potency of many cell therapies. However, gene expression states that drive specific secretory phenotypes are largely unknown. We provide a protocol that enables linking the Secretion amount of a target protein EnCoded (SEC) by thousands of single cells with transcriptional sequencing (seq). SEC-seq leverages microscale hydrogel particles called Nanovials to isolate cells and capture their secretions in close proximity, oligonucleotide-labeled antibodies to tag secretions on Nanovials, and flow cytometry and single-cell RNA-sequencing platforms for readout. Cells on Nanovials can be sorted based on viability, secretion amount, or other surface markers without fixation or permeabilization, and cell and secretion-containing Nanovials are directly introduced into microfluidic droplets-in-oil emulsions for single-cell barcoding of cell transcriptomes and secretions. We have used SEC-seq to link T-cell receptor sequences to the relative amount of associated cytokine secretions, surface marker gene expression with a highly secreting and potential regenerative population of mesenchymal stromal cells, and the transcriptome with high immunoglobulin secretion from plasma cells. Nanovial modification and cell loading takes under 4 hours, and once the desired incubation time is over, staining, cell sorting, and emulsion generation for scRNA-seq can also be completed in under 4 hours. By linking gene expression and secretory strength, SEC-seq can expand our understanding of cell secretion, how it is regulated, and how it can be engineered to make better therapies. ### Competing Interest Statement D.D. and the Regents of the University of California have financial interests in Partillion Bioscience which sells Nanovials.

Antibody inhibitors pose an ongoing challenge to the treatment of subjects with inherited protein deficiency disorders, limiting the efficacy of both protein replacement therapy and corrective gene therapy. Beyond their central role as producers of serum antibody, B cells also exhibit many unique properties that could be exploited in cell therapy applications, notably including antigen-specific recognition and the linked capacity for antigen presentation. Here we employed CRISPR/Cas9 to demonstrate that ex vivo antigen-primed Blimp1-knockout “decoy” B cells, incapable of differentiation into plasma cells, participated in and downregulated host antigen-specific humoral responses after adoptive transfer. Following ex vivo antigen pulse, adoptively transferred high affinity antigen-specific decoy B cells were diverted into germinal centers en masse, thereby reducing participation by endogenous antigen-specific B cells in T-dependent humoral responses and suppressing both cognate and linked antigen-specific IgG following immunization with conjugated antigen. This effect was dose-dependent and, importantly, did not impact concurrent unrelated antibody responses. We demonstrated the therapeutic potential of this approach by treating factor VIII (FVIII)-knockout mice with antigen-pulsed decoy B cells prior to immunization with a FVIII conjugate protein, thereby blunting the production of serum FVIII-specific IgG by an order of magnitude as well as reducing the proportion of animals exhibiting functional FVIII inhibition by 6-fold.

Dysregulated expansion and/or activation of mast cells have detrimental consequences in allergic disease. In humans, homozygous or compound heterozygous deletions of DOCK8 cause a combined immunodeficiency characterized by allergic disorders. Based on this evidence, we hypothesized that DOCK8 may be a negative regulator of mast cell function. To address this hypothesis, we used mice with a loss-of-function mutation in Dock8 (primuris, Dock8 pri/primice) and conditional mice in which DOCK8 is ablated in connective tissue mast cells (Mcpt5-Cre +; Dock8 fl/flmice). Dock8 pri/primice exhibited increased plasma levels of mast cell protease-1 (MCPT1) in homeostatic conditions. Furthermore, Dock8 pri/primice and Mcpt5-Cre +; Dock8 fl/flmice experienced a more severe IgE-dependent passive cutaneous anaphylactic reaction (PCA) than control mice. This data suggests that DOCK8 deficiency is linked to increased mast cell activation in homeostatic conditions and upon stimulation by IgE-dependent mechanisms. Fetal skin derived cultured mast cells (FSMCs) generated from Dock8 pri/primice released increased amounts of β-hexosaminidase upon IgE-dependent activation. Consistent with this finding, naïve DOCK8-deficient FSMCs exhibited alterations in cytoskeletal dynamics that may facilitate mast cell degranulation. Finally, we observed an increased number of tryptase positive cells in skin biopsies obtained from three pediatric patients diagnosed with hyper IgE syndrome (HIES) due to DOCK8 mutations, suggesting that DOCK8 immunodeficiency may contribute to skin mast cell expansion in humans. Our findings provide strong evidence that DOCK8 can contribute to negative regulation of skin mast cell activation and expansion. R01 AI140626-05

The secreted products of cells drive many functions in vivo; however, methods to link this functional information to surface markers and transcriptomes have been lacking. By accumulating secretions close to secreting cells held within cavity-containing hydrogel nanovials, we demonstrate workflows to analyze the amount of IgG secreted from single human B cells and link this information to surface markers and transcriptomes from the same cells. Measurements using flow cytometry and imaging flow cytometry corroborate the association between IgG secretion and CD38/CD138. By using oligonucleotide-labeled antibodies we find that upregulation of pathways for protein localization to the endoplasmic reticulum and mitochondrial oxidative phosphorylation are most associated with high IgG secretion, and uncover surrogate plasma cell surface markers (e.g., CD59) defined by the ability to secrete IgG. Altogether, this method links quantity of secretion with single-cell sequencing (SEC-seq) and enables researchers to fully explore the links between genome and function, laying the foundation for discoveries in immunology, stem cell biology, and beyond.

Background Bispecific T Cell Engagers (BiTEs), consisting of an anti-CD3 scFv fused to an anti-tumor antigen scFv, are highly effective in the treatment of relapsed/refractory Acute Lymphoblastic Leukemia (ALL). However, the short half-life of BiTEs necessitates continuous intravenous administration at high doses for four-week increments. To overcome these pharmacokinetic shortcomings, we developed a method to engineer plasma cell precursors to continuously secrete transgenic biologics. Plasma cells were chosen for their high antibody production capacity (thousands of Ig molecules/cell/sec) and long-term survival (persisting for decades), making them a highly attractive cell-based platform for continuous biologic delivery.1 Methods To demonstrate proof-of-concept, we integrated a transgene coding for a bispecific non-Ig anti-CD3:CD19 scFv into the CCR5 safe-harbor locus of primary human B cells with CRISPR/Cas9, then initiated differentiation into plasma cells using a modified feeder-free culture system. Cells were characterized by flow cytometry, indel frequency, and droplet digital PCR. Edited cell supernatant was tested for therapeutic protein production with a timed ELISA and in vitro function with a cytolytic activity assay incorporating co-cultured T effector and CD19 expressing tumor cells. We also assessed in vivo anti-tumor activity of anti-CD3:CD19 scFv engineered B cell medicines (BeCMs) in NSG mice harboring a patient-derived xenograft (PDx). To avoid targeting of the BeCM by CD3+ cells, CD19 was knocked-out via a multiplexed CRISPR/Cas9 editing protocol. Mice were then inoculated with a luciferized B-ALL PDx line. Autologous T cells were delivered 24 hours and 72 hours following tumor transfer, and IVIS imaging was performed over the course of 17 days. A control GFP-engineered BeCM arm and PBS-dosed cohort were monitored in parallel, with n=6 mice per group. Results Flow cytometric analysis confirmed robust differentiation of BeCMs along the plasma cell lineage. We observed >85% cutting efficiency and 40–50% targeted integration, which translated to secretion rates (0.6–0.8 μg BiTE/106 cells/day) that were sufficient for in vitro functional assay tumor cytolytic activity. Significant reduction in tumor burden (bioluminescent flux, area under the curve) was observed in vivo in the anti-CD3:CD19 scFv cohort compared to the controls, which was in concordance with heightened in vivo T cell activation. The ~1000 pg/mL BiTE detected in mouse plasma demonstrates that BeCM-derived biologics can meet or even exceed the steady-state plasma concentrations achieved with clinically relevant doses.2 Conclusions These findings underscore the clinical potential of BeCMs as an emerging platform for sustained delivery of anti-tumor biologics. References Hung KL, Meitlis I, Hale M, Chen C, Singh S, Jackson SW, Miao CH, Khan IF, Rawlings DJ, James RG. Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells. Mol. Ther. 2018; 26:456–467. Franquiz MJ and Short NJ. Blinatumomab for the Treatment of Adult B-Cell Acute Lymphoblastic Leukemia: Toward a New Era of Targeted Immunotherapy. Biologics. 2020; 14: 23–34. Ethics Approval Deidentified human PBMCs were acquired under informed consent from the Fred Hutch Specimen Processing and Research Cell Bank (protocol #3942).

Macroautophagy/autophagy proteins have been linked with the development of immune-mediated diseases including lupus, but the mechanisms for this are unclear due to the complex roles of these proteins in multiple immune cell types. We have previously shown that a form of noncanonical autophagy induced by ITGAV/alpha(v) integrins regulates B cell activation by viral and self-antigens, in mice. Here, we investigate the involvement of this pathway in B cells from human tissues. Our data reveal that autophagy is specifically induced in the germinal center and memory B cell subpopulations of human tonsils and spleens. Transcriptomic analysis show that the induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of ITGAV/alpha(v) integrin-dependent autophagy in human B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells, we found that ITGAV/alpha(v)-dependent autophagy limits activation of specific pathways related to B cell responses, while promoting others. These data provide new mechanistic links for autophagy and B-cell-mediated immune dysregulation in diseases such as lupus.

Barriers to effective gene therapy for many diseases include the number of modified target cells required to achieve therapeutic outcomes and host immune responses to expressed therapeutic proteins. As long-lived cells specialized for protein secretion, antibody-secreting B cells are an attractive target for foreign protein expression in blood and tissue. To neutralize HIV-1, we developed a lentiviral vector (LV) gene therapy platform for delivery of the anti-HIV-1 immunoadhesin, eCD4-Ig, to B cells. The EμB29 enhancer/promoter in the LV limited gene expression in non-B cell lineages. By engineering a knob-in-hole-reversed (KiHR) modification in the CH3-Fc eCD4-Ig domain, we reduced interactions between eCD4-Ig and endogenous B cell immunoglobulin G proteins, which improved HIV-1 neutralization potency. Unlike previous approaches in non-lymphoid cells, eCD4-Ig-KiHR produced in B cells promoted HIV-1 neutralizing protection without requiring exogenous TPST2, a tyrosine sulfation enzyme required for eCD4-Ig-KiHR function. This finding indicated that B cell machinery is well suited to produce therapeutic proteins. Lastly, to overcome the inefficient transduction efficiency associated with VSV-G LV delivery to primary B cells, an optimized measles pseudotyped LV packaging methodology achieved up to 75% transduction efficiency. Overall, our findings support the utility of B cell gene therapy platforms for therapeutic protein delivery.

Plasma cells are specialized immune cells that can survive in the bone marrow for decades and continually secrete large amounts of antibodies, thus providing long-term, often lifelong protection from repeated exposures to a pathogen. The long-term survival and the capability to secrete high amounts of protein make plasma cells a potential candidate for stable delivery of therapeutic proteins and antibodies. However, engineering plasma cells or B cells has been challenging due to low lentiviral transduction efficiency, incompatibility with plasmid transfection and loss of episomal DNA expression over time. We have previously shown that human plasma cells can be engineered to secrete antibodies and proteins in in vitro culture systems via gene editing. In the current study, we have generated engineered murine plasma cells that can secrete the clotting factor IX (FIX). We hypothesize that the FIX-secreting plasma cells will engraft long-term in FIX-deficient immunocompetent mice and secrete therapeutic levels of FIX leading to protection from Hemophilia B. The standard of care for Hemophilia B is repeated intravenous infusion of recombinant FIX or AAV-based gene therapy. However, 30-60% humans have pre-existing immunity to AAV-based gene therapy and gene therapy can induce an immune response in people lacking pre-existing antibodies. Moreover, gene therapy is unsuitable for pediatric patients due to their rapid growth and proportional loss of AAV. Plasma cell-based therapy is lowly immunogenic and can be used for redosing in pediatric patients that require more product with age. Here, we will describe proof-of-concept studies showing that engineered murine plasma cells can engraft into immunocompetent mice without the need for conditioning and produce stable levels of protein for >10 weeks. We developed a three-step in vitro co-culture system that can be used to engineer and differentiate naïve B cells into plasma cells. These in vitro generated murine plasma cells had a comparable gene expression profile and phenotype to natural plasma cells and can engraft stably, in the absence of conditioning, into immunocompetent mice whereupon they secreted antibodies (Figure 1). Moreover, we show that plasma cells can be re-dosed to increase antibody levels without triggering a secondary immune response (Figure 1). For delivery of therapeutic proteins, we have developed adeno-associated virus-based homology directed repair reagents to edit murine B cells at several loci, including beta-2 microglobulin, Rosa-26, and the antibody heavy chain. By co-delivery of cis-linked fluorescent markers, we were able to sort and expand only edited cells, enabling enrichment of cells expressing a therapeutic protein. In proof-of-concept studies, we have used this approach to generate a human FIX-secreting plasma cell product with close to 90% edited cells at the end of the B cell culture. The enriched plasma cells generated 0.28 pg FIX/cell/day compared to 0.19 pg FIX/cell/day generated by the bulk edited cells (figure 2). We estimate that engraftment of 1 million edited plasma cells is sufficient to generate therapeutic levels of FIX upon (figure 2). We have developed reagents for a novel cell-based therapeutic tool with protein secretion capabilities and far-reaching applications. Out studies will provide insights into the feasibility and efficacy of a B cell based therapeutic product.