Biology subjects

Dougan, S. K.

Publications and source records attributed to Dougan, S. K..

9 recordsLinked to original sources

bioRxiv2026

De novo design of protease-activatable cytokine prodrugs

Cytokines can elicit potent antitumor immunity, but their clinical use is often limited by systemic immune activation and dose-limiting toxicity. Protease-responsive masking offers a way to improve selectivity by linking cytokine activity to disease-associated protease cleavage, but current approaches have faced several challenges. Here we describe a de novo protein design strategy for generating protease-activatable cytokine prodrugs that overcomes these challenges. We use this strategy to design and characterize cytokine prodrugs and well-behaved AND-gated split systems that require both target-dependent colocalization and proteolytic unmasking to generate active cytokines. In challenging syngeneic tumor models, our IL-21 prodrugs reduced treatment-associated toxicity while maintaining antitumor efficacy.

bioengineering↗

bioRxiv2026

Activation-dependent lentiviruses enable antigen-specific T cell expansion and transduction

Cancer immunotherapies rely on tumor-specific T cells, which arise endogenously in most patients with cancer, but can be low frequency and poorly functional. Methods to specifically identify, expand, and manipulate tumor-specific T cells at the rare frequencies found in peripheral blood would enable new immunotherapeutic strategies. Here, we demonstrate an approach to virally transduce polyclonal tumor-reactive T cells across any MHC haplotype and in the absence of knowing the cognate antigen. By generating lentiviral vectors that selectively transduce cells expressing 4-1BB (CD137), a marker of T cell activation, we can transduce antigen-specific T cells with user-defined genetic cargoes that can selectively expand and track individual clonotypes via single-cell sequencing. Anti-4-1BB lentiviruses (4-1BB LVs) encoding therapeutic cargoes can also enhance antigen-specific T cells to extend survival in a xenograft model of human melanoma and transduce tumor-infiltrating T cells from patients with ovarian cancer. Overall, the 4-1BB LV platform targets antigen-specific T cells in a manner agnostic to both the antigen and presenting MHC, with potential applications in adoptive cell therapy manufacturing and TCR identification. One Sentence SummaryEngineered lentiviral vectors targeting 4-1BB selectively activate, expand, and transduce antigen-specific T cells with immunomodulatory cargo.

immunology↗

bioRxiv2025

Scalable TCR synthesis and screening enables antigen reactivity mapping in vitiligo

T cell receptors (TCRs) mediate antigen recognition in adaptive immunity, yet large-scale mapping of TCR-antigen interactions remains a major challenge. Current approaches to synthesize and functionally screen TCRs remain technically complex and limited in throughput. We introduce a modular strategy, TCRAFT, to rapidly construct tens of thousands of TCRs for <$1 each while maintaining TCR-{beta} pairing with >99% accuracy. We integrate this approach with a high-throughput antigen discovery platform to enable library-on-library TCR-antigen screening. We reconstruct and screen 3,808 TCRs from vitiligo lesions, linking TCR specificity to transcriptional phenotypes for antigen-reactive T cells. To demonstrate scalability, we synthesize and screen 30,810 TCRs from donors with pancreatic ductal adenocarcinoma to capture antigen-specific TCRs. This workflow reduces the cost and complexity of large-scale TCR screening, enabling the expansion of the known landscape of antigen-specific TCRs in vitiligo with a method that can be readily extended to other immunological applications.

immunology↗

bioRxiv2024

Potent antitumor activity of a designed interleukin-21 mimic

Long-standing goals of cancer immunotherapy are to activate cytotoxic antitumor T cells across a broad range of affinities while dampening suppressive regulatory T (Treg) cell responses, but current approaches achieve these goals with limited success. Here, we report a de novo IL-21 mimic, 21h10, designed to have augmented stability and high signaling potency in both humans and mice. In multiple animal models and in ex vivo human melanoma patient derived organotypic tumor spheroids (PDOTS), 21h10 showed robust antitumor activity. 21h10 generates significantly prolonged STAT signaling in vivo compared with native IL-21, and has considerably stronger anti-tumor activity. Toxicities associated with systemic administration of 21h10 could be mitigated by TNF blockade without compromising antitumor efficacy. In the tumor microenvironment, 21h10 induced highly cytotoxic antitumor T cells from clonotypes with a range of affinities for endogenous tumor antigens, robustly expanding low-affinity cytotoxic T cells and driving high expression of interferon- (IFN-) and granzyme B compared to native IL-21, while increasing the frequency of IFN-+ Th1 cells and reducing that of Foxp3+ Tregs. As 21h10 has full human/mouse cross-reactivity, high stability and potency, and potentiates low-affinity antitumor responses, it has considerable translational potential.

biochemistry↗

bioRxiv2024

SMAC mimetics induce human macrophages to phagocytose live cancer cells

Macrophages engulf apoptotic bodies and cellular debris as part of homeostasis, but they can also phagocytose live cells such as aged red blood cells. Pharmacologic reprogramming with the SMAC mimetic LCL161 in combination with T cell-derived cytokines can induce macrophages to phagocytose live cancer cells in mouse models. Here we extend these findings to encompass a wide range of monovalent and bivalent SMAC mimetic compounds, demonstrating that live cell phagocytosis is a class effect of these agents. We demonstrate robust phagocytosis of live pancreatic and breast cancer cells by primary human macrophages across a range of healthy donors. Unlike mouse macrophages where combination of SMAC mimetics with lymphotoxin enhanced phagocytosis, human macrophages were more efficiently polarized to phagocytose live cells by the combination of SMAC mimetics and IFN{psi}. We profiled phagocytic macrophages by transcriptional and proteomic methodologies, uncovering a positive feedback loop of autocrine TNF production.

immunology↗

bioRxiv2024

Peptide-MHC-targeted retroviruses enable in vivo expansion and gene delivery to tumor-specific T cells

Tumor-infiltrating-lymphocyte (TIL) therapy has demonstrated that endogenous T cells can be harnessed to initiate an effective anti-tumor response. Despite clinical promise, current TIL production protocols involve weeks-long ex vivo expansions which can affect treatment efficacy. Therefore, additional tools are needed to engineer endogenous tumor-specific T cells to have increased potency while mitigating challenges of manufacturing. Here, we present a strategy for pseudotyping retroviral vectors with peptide-major histocompatibility complexes (pMHC) for antigen-specific gene delivery to CD8 T cells and examine the efficacy of these transduced cells in immunocompetent mouse models. We demonstrate that pMHC-targeted viruses are able to specifically deliver function-enhancing cargoes while simultaneously activating and expanding anti-tumor T cells. The specificity of these viral vectors enables in vivo engineering of tumor-specific T cells, circumventing ex vivo manufacturing processes and improving overall survival in B16F10-bearing mice. Altogether, we have established that pMHC-targeted viruses are efficient vectors for reprogramming and expanding tumor-specific populations of T cells directly in vivo, with the potential to substantially streamline engineered cell therapy production for a variety of applications.

immunology↗

bioRxiv2024

De novo pyrimidine biosynthesis inhibition synergizes with BCL-XL targeting in pancreatic cancer

Oncogenic KRAS, the genetic driver of 90% of pancreatic adenocarcinoma (PDAC), induces a metabolic rewiring characterized, in part, by dependency on de novo pyrimidine biosynthesis. Pharmacologic inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, delays pancreatic tumor growth in vivo; however, limited monotherapy efficacy suggests compensatory pathways and that combinatorial strategies are required for enhanced efficacy. Here, we use an integrated metabolomic, quantitative temporal proteomic and in vitro and in vivo DHODH inhibitor anchored CRISPR/Cas9 genetic screening approach to identify compensatory pathways to DHODH inhibition (DHODHi) and targets for combination strategies. We demonstrate that DHODHi alters the apoptotic regulatory proteome thereby enhancing sensitivity to inhibitors of the anti-apoptotic BCL2L1 (BCL-XL) protein. Combinatorial regimens with DHODH and BCL-XL inhibition synergistically induce apoptosis in PDAC cell lines and patient-derived PDAC organoids. In vivo DHODH inhibition with Brequinar and BCL-XL degradation with DT2216, a proteolysis targeting chimera (PROTAC), significantly inhibits the growth of PDAC tumors. Our data defines mechanisms of adaptation to DHODH inhibition and identifies a combination therapy strategy in PDAC.

cancer biology↗

bioRxiv2021

Antigen identification and high-throughput interaction mapping by reprogramming viral entry

Deciphering immune recognition is critical for understanding a broad range of diseases and for the development of effective vaccines and immunotherapies. Efforts to do so are limited by a lack of technologies capable of simultaneously capturing the complexity of adaptive immune receptor repertoires and the landscape of potential antigens. To address this, we present RAPTR (Receptor-Antigen Pairing by Targeted Retroviruses). RAPTR combines viral pseudotyping and molecular engineering approaches to enable one-pot library on library interaction screens by displaying antigens on the surface of lentiviruses and encoding their identity in the viral genome. Antigen-specific viral infection of cells allows readout of both antigen and receptor identities via single-cell sequencing. The resulting system is modular, scalable, and compatible with any cell type, making it easily implemented. These techniques provide a suite of new tools for targeted viral entry, molecular engineering, and interaction screens with broad potential applications.

immunology↗

bioRxiv2019

Targeting EZH2 Increases Therapeutic Efficacy of Check-Point Blockade in Models of Prostate Cancer

Prostate cancers are considered immunologically cold tumors given the very few patients who respond to checkpoint inhibitor therapy (CPI). Recently, enrichment of interferon (IFN) response genes predicts a favorable response to CPI across various disease sites. The enhancer of zeste homolog-2 (EZH2) is over-expressed in prostate cancer and is known to negatively regulate IFN response genes. Here, we demonstrate that inhibition of EZH2 catalytic activity in prostate cancer models derepresses expression of double-strand RNA (dsRNA), associated with upregulation of genes involved in antigen presentation, Th-1 chemokine signaling, and interferon (IFN) response, including PD-L1. Similarly, application of a novel EZH2 derived gene signature to human prostate sample analysis indicated an inverse correlation between tumor EZH2 activity/expression with T-cell inflamed and IFN gene signatures and PD-L1 expression. EZH2 inhibition combined with PD-1 CPI significantly enhances antitumor response that is dependent on up-regulation of tumor PD-L1 expression. Further, combination therapy significantly increases intratumoral trafficking of activated CD8+ T-cells and M1 tumor associated macrophages (TAMs) with concurrent loss of M2 TAMs. Our study identifies EZH2 as a potent inhibitor of antitumor immunity and responsiveness to CPI. This data suggests EZH2 inhibition as a novel therapeutic direction to enhance prostate cancer response to PD-1 CPI.

cancer biology↗