Biology subjects

Arpaia, N.

Publications and source records attributed to Arpaia, N..

12 recordsLinked to original sources

bioRxiv2026

Engineered probiotics for tumor-targeted combination chemoimmunotherapy

Achieving tumor-specific delivery and sustained activation of both cytotoxic and immune-modulating agents remains a critical challenge in chemoimmunotherapy. Here, we present a bacterial platform engineered to combine enzyme/prodrug chemotherapy with immunotherapy, where tumor-homing E. coli Nissle 1917 expresses cytosine deaminase to convert the prodrug 5-fluorocytosine into the cytotoxic drug 5-fluorouracil within tumors. Concurrently, the engineered bacteria produce an IL-15 superagonist and a PD-L1 blocking nanobody to mitigate the immunosuppressive effects of tumor-localized chemotherapy. This platform demonstrated potent antitumor effects in the murine MC38 solid tumor model. Mechanistic studies showed that the combination therapy enhances activation of antigen-presenting cells, T cells and natural killer cells, while reducing immunosuppressive populations. In summary, our approach integrates enzyme/prodrug therapy and immunotherapy into a single bacterial delivery system, overcoming the limitations of conventional therapies and offering a scalable and precision-engineered strategy with an improved safety profile for synergistic cancer treatment. One Sentence SummaryA precision-engineered bacterial platform integrates enzyme/prodrug chemotherapy and immunotherapy to drive synergistic antitumor responses with enhanced safety, offering promise for clinical translation.

cancer biology↗

bioRxiv2025

Temperate phages enhance host fitness via RNA-guided flagellar remodeling

Bacterial flagella drive motility and chemotaxis while also playing critical roles in host-pathogen interactions, as their oligomeric subunit, flagellin, is specifically recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors known as TldR that regulate flagellin expression. However, the biological significance for this regulation, particularly in the context of host fitness, remained unknown. By focusing on a human clinical Enterobacter isolate that encodes a Flagellin Remodeling prophage (FR{varphi}), here we show that FR{varphi} exploits the combined action of TldR and its flagellin isoform to dramatically alter the flagellar composition and phenotypic properties of its host. This transformation has striking biological consequences, enhancing bacterial motility and mammalian immune evasion, and structural studies by cryo-EM of host- and prophage-encoded filaments reveal distinct architectures underlying these physiological changes. Moreover, we find that FR{varphi} improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodeling in a host-associated environment. Remarkably, flagellin-regulating TldR homologs emerged multiple times independently, further highlighting the strong selective pressures that drove evolution of RNA-guided flagellin control. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness.

microbiology↗

bioRxiv2024

Tumor-specific antibodies elicited by engineered bacteria promote bladder cancer immunotherapy

The intratumoral microbiome has recently emerged as a new hallmark of cancer, with implications for response or resistance to therapy. While bacteria can either promote or inhibit cancer growth, intratumoral bacteria can also be engineered using synthetic biology to remodel the tumor microenvironment. Here, we engineered the probiotic bacterium E. coli Nissle 1917 (EcN) to express the human chemokine CXCL13, a critical component of germinal center (GC) formation. The GC reaction is a fundamental aspect of adaptive immunity by which antibody affinity develops in secondary lymphoid organs for defense against pathogens. Using orthotopic models of bladder cancer, engineered CXCL13-expressing EcN colonized bladder tumors and elicited GC responses in bladder tumor-draining lymph nodes after intravesical delivery. Furthermore, when combined with PD-1 blockade, engineered EcN amplified the antitumor antibody response and promoted long-term survival and protective immunity upon tumor rechallenge. Thus, we demonstrate that synthetically engineered CXCL13-expressing EcN can enhance the efficacy of PD-1 checkpoint blockade immunotherapy by amplifying tumor-specific humoral immunity.

immunology↗

bioRxiv2024

An amphiregulin reporter mouse enables transcriptional and clonal expansion analysis of reparative lung Treg cells

Regulatory T (Treg) cells are known to play critical roles in tissue repair via provision of growth factors such as amphiregulin (Areg). Areg-producing Treg cells have previously been difficult to study because of an inability to isolate live Areg-producing cells. In this report, we created a novel reporter mouse to detect Areg expression in live cells (AregThy1.1). We employed influenza A and bleomycin models of lung damage to sort Areg-producing and -non-producing Treg cells for transcriptomic analyses. Single cell RNA-seq revealed distinct subpopulations of Treg cells and allowed transcriptomic comparisons of damage-induced populations. Single cell TCR sequencing showed that Treg cell clonal expansion is biased towards Areg-producing Treg cells, and largely occurs within damage-induced subgroups. Gene module analysis revealed functional divergence of Treg cells into immunosuppression-oriented and tissue repair-oriented groups, leading to identification of candidate receptors for induction of repair activity in Treg cells. We tested these using an ex vivo assay for Treg cell-mediated tissue repair, identifying 4-1BB agonism as a novel mechanism for reparative activity induction. Overall, we demonstrate that the AregThy1.1mouse is a promising tool for investigating tissue repair activity in leukocytes.

immunology↗

bioRxiv2024

Heparan sulfate regulates amphiregulin signaling towards reparative lung mesenchymal cells during influenza A infection

Amphiregulin (Areg), a growth factor produced by regulatory T (Treg) cells to facilitate tissue repair/regeneration, contains a heparan sulfate (HS) binding domain. How HS, a highly sulfated glycan subtype that alters growth factor signaling, influences Areg repair/regeneration functions is unclear. Here we report that inhibition of HS in various cell lines and primary lung mesenchymal cells (LMC) qualitatively alters downstream signaling and highlights the existence of HS-dependent vs. -independent Areg transcriptional signatures. Utilizing a panel of cell lines with targeted deletions in HS synthesis-related genes, we found that the presence of the glypican family of heparan sulfate proteoglycans is critical for Areg signaling and confirmed this dependency in primary LMC by siRNA-mediated knockdown. Furthermore, in the context of influenza A (IAV) infection in vivo, we found that an Areg-responsive subset of reparative LMC upregulate glypican-4 and HS. Conditional deletion of HS primarily within this LMC subset resulted in reduced blood oxygen saturation following infection with IAV, with no changes in viral load. Finally, we found that co-culture of HS-knockout LMC with IAV-induced Treg cells results in reduced LMC responses. Collectively, this study reveals the essentiality of HS on a specific lung mesenchymal population as a mediator of Treg cell-derived Areg reparative signaling during IAV infection.

immunology↗

bioRxiv2023

Probiotic neoantigen delivery vectors for precision cancer immunotherapy

Microbial systems have been synthetically engineered to deploy therapeutic payloads in vivo1-4. With emerging evidence that bacteria naturally home to tumors5-7 and modulate anti-tumor immunity8,9, one promising application is the development of bacterial vectors as precision cancer vaccines10-12. In this study, we engineered probiotic E. coli Nissle 1917 (EcN) as an anti-tumor vaccination platform optimized for enhanced production and cytosolic delivery of neoepitope-containing peptide arrays, with increased susceptibility to blood clearance and phagocytosis. These features enhance both safety and immunogenicity, achieving a system which drives potent and specific T cell-mediated anti-cancer immunity that effectively controls or eliminates tumor growth and extends survival in advanced murine primary and metastatic solid tumors. We demonstrate that the elicited anti-tumor immune response involves extensive priming and activation of neoantigen-specific CD4+ and CD8+ T cells, broader activation of both T and NK cells, and a reduction of tumor-infiltrating immunosuppressive myeloid and regulatory T and B cell populations. Taken together, this work leverages the advantages of living medicines to deliver arrays of tumor-specific neoantigen-derived epitopes within the optimal context to induce specific, effective, and durable systemic anti-tumor immunity.

synthetic biology↗

bioRxiv2023

Colorectal cancer detection and treatment with engineered probiotics

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate the phenomenon of selective, long-term colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition. We show that, after oral administration, adenomas can be monitored over time by recovering EcN from stool. We also demonstrate specific colonization of EcN to solitary neoplastic lesions in an orthotopic murine model of CRC. We then exploit this neoplasia-homing property of EcN to develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate, and demonstrate that oral delivery of this strain results in significantly increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. We also assess EcN engineered to locally release immunotherapeutics at the neoplastic site. Oral delivery to mice bearing adenomas, reduced adenoma burden by [~]50%, with notable differences in the spatial distribution of T cell populations within diseased and healthy intestinal tissue, suggesting local induction of robust anti-tumor immunity. Together, these results support the use of EcN as an orally-delivered platform to detect disease and treat CRC through its production of screening and therapeutic molecules.

bioengineering↗

bioRxiv2022

Engineered bacteria recruit and orchestrate anti-tumor immunity

Tumors employ multiple mechanisms to actively exclude or suppress adaptive immune cells involved in anti-tumor immunity. Strategies focused on overcoming these immunosuppressive or exclusion signals - through localized delivery of chemokines that directly recruit immune cells into the tumor microenvironment - remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor localized delivery, offering therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to produce and intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Intravenous or intratumoral delivery of bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16K42A) leads to the recruitment of activated T cells within tumors and offers therapeutic benefit in multiple mouse tumor models. Furthermore, we rationally target an additional step in the immune activation cascade - specifically, the presentation of tumor-derived antigens by dendritic cells - using a second engineered bacterial strain expressing CCL20. This combined targeting approach led to the recruitment of type 1 conventional dendritic cells and effectively synergized with hCXCL16K42A-induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to cooperatively recruit and activate both innate and adaptive anti-tumor immune responses, offering a new cancer immunotherapy strategy.

cancer biology↗

bioRxiv2021

Probiotic-guided CAR-T cells for universal solid tumor targeting

Synthetic biology enables the engineering of interactions between living medicines to overcome the specific limitations of any singular therapy. One major challenge of tumor-antigen targeting therapies like chimeric antigen receptor (CAR)-T cells is the identification of targetable antigens that are specifically and uniformly expressed on heterogenous solid tumors. In contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be readily engineered as antigen-independent platforms for therapeutic delivery. Bridging these approaches, we develop a platform of probiotic-guided CAR-T cells (ProCARs), in which T cells are engineered to sense synthetic antigens (SA) that are produced and released by tumor-colonizing probiotic bacteria. We demonstrate increased CAR-T cell activation and tumor-cell lysis when SAs anchor to components of the extracellular matrix. Moreover, we show that ProCARs are intratumorally activated by probiotically-delivered SAs, receive further stimulation from bacterial TLR agonists, and are safe and effective in multiple xenograft models. This approach repurposes tumor-colonizing bacteria as beacons that guide the activity of engineered T cells, and in turn builds the foundation for communities of living medicines.

bioengineering↗

bioRxiv2021

A programmable probiotic encapsulation system enhances therapeutic delivery in vivo

Recent advances in therapeutic modulation of human microbiota have driven new efforts to engineer living microbial medicines using synthetic biology. However, a long-standing challenge for live bacterial therapies is balancing the high dose required to achieve robust efficacy with the potential for sepsis. Here, we developed a genetically encoded microbial encapsulation system with tunable and dynamic expression of surface capsular polysaccharides to enhance therapeutic delivery. Following a synthetic small RNA knockdown screen of the capsular biosynthesis pathway, we constructed synthetic gene circuits that regulate bacterial encapsulation based on sensing the levels of environmental inducer, bacterial density, and blood pH. The induced encapsulation system enabled tunable immunogenicity and survivability of the probiotic Escherichia coli, resulting in increased maximum tolerated dose and enhanced efficacy in murine cancer models. Furthermore, triggering in situ encapsulation was found to increase microbial translocation between mouse tumors, leading to efficacy in distal tumors. The programmable encapsulation system demonstrates a new approach to control microbial therapeutic profiles in vivo using synthetic biology.

synthetic biology↗

bioRxiv2019

Programmable bacteria induce durable tumor regression and systemic antitumor immunity

SUMMARY PARAGRAPHSynthetic biology is driving a new era of medicine through the genetic programming of living cells1,2. This transformative approach allows for the creation of engineered systems that intelligently sense and respond to diverse environments, ultimately adding specificity and efficacy that extends beyond the capabilities of molecular-based therapeutics3-5. One particular focus area has been the engineering of bacteria as therapeutic delivery systems to selectively release therapeutic payloads in vivo6-8. Here, we engineered a non-pathogenic E. coli to specifically lyse within the tumor microenvironment and release an encoded nanobody antagonist of CD47 (CD47nb)9, an anti-phagocytic receptor commonly overexpressed in several human cancers10,11. We show that intratumoral delivery of CD47nb by tumor-colonizing bacteria increases activation of tumor-infiltrating T cells, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in a syngeneic tumor model. Moreover, we report that local injection of CD47nb bacteria stimulates systemic antitumor immune responses that reduce the growth of untreated tumors - providing, to the best of our knowledge, the first demonstration of an abscopal effect induced by a bacteria cancer therapy. Thus, engineered bacteria may be used for safe and local delivery of immunotherapeutic payloads leading to systemic antitumor immunity.

synthetic biology↗

bioRxiv2019

Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies

Immunotherapies such as checkpoint inhibitors have revolutionized cancer therapy yet lead to a multitude of immune-related adverse events, suggesting the need for more targeted delivery systems. Due to their preferential colonization of tumors and advances in engineering capabilities from synthetic biology, microbes are a natural platform for the local delivery of cancer therapeutics. Here, we present an engineered probiotic bacteria system for the controlled production and release of novel immune checkpoint targeting nanobodies from within tumors. Specifically, we engineered genetic lysis circuit variants to effectively release nanobodies and safely control bacteria populations. To maximize therapeutic efficacy of the system, we used computational modeling coupled with experimental validation of circuit dynamics and found that lower copy number variants provide optimal nanobody release. Thus, we subsequently integrated the lysis circuit operon into the genome of a probiotic E. coli Nissle 1917, and confirmed lysis dynamics in a syngeneic mouse model using in vivo bioluminescent imaging. Expressing a nanobody against PD-L1 in this strain demonstrated enhanced efficacy compared to a plasmid-based lysing variant, and similar efficacy to a clinically relevant monoclonal antibody against PD-L1. Expanding upon this therapeutic platform, we produced a nanobody against cytotoxic T-lymphocyte associated protein -4 (CTLA-4), which reduced growth rate or completely cleared tumors when combined with a probiotically-expressed PD-L1 nanobody in multiple syngeneic mouse models. Together, these results demonstrate that our engineered probiotic system combines innovations in synthetic biology and immunotherapy to improve upon the delivery of checkpoint inhibitors. SENTENCE SUMMARYWe designed a probiotic platform to locally deliver checkpoint blockade nanobodies to tumors using a controlled lysing mechanism for therapeutic release.

synthetic biology↗