JoeyKelly

Nat Commun. 2025 Dec 17. doi: 10.1038/s41467-025-66132-w. Online ahead of print.

ABSTRACT

Covalent inhibitors of oncoprotein KRAS have initial efficacy, but responses lack durability. Covalently modified oncoproteins are presented as MHC-restricted hapten-peptides (p*MHC) on the cancer cell surface, enabling combination of targeted therapy with immunotherapy to overcome drug resistance. Building on indirect evidence of KRAS^G12C-derived p*MHCs, we use immunopeptidomics to identify and directly quantify these synthetic neoantigens. To address challenges by their low copy number, we develop AETX-R114, a T cell engaging bispecific antibody with picomolar affinity for MHC-restricted sotorasib-modified KRAS^G12C peptides presented by three HLA-A3 supertype alleles. AETX-R114 dramatically increases the half-life and thereby the number of presented p*MHCs, enabling selective and potent killing of resistant cancer cells both in vitro and in vivo. To broaden the therapeutic potential of creating and targeting synthetic neoantigens, we further develop AETX-R302, which recognizes divarasib-modified KRAS^G12C peptides presented on alleles from the HLA-A2 and A3 supertypes. Cryo-EM structure determination reveals the molecular basis for breaking HLA supertype restriction. Collectively, our study illustrates how engineered antibodies can transform synthetic neoantigens into actionable cancer immunotherapy targets.

PMID:41408054 | DOI:10.1038/s41467-025-66132-w

J Biol Chem. 2025 Dec 12:111049. doi: 10.1016/j.jbc.2025.111049. Online ahead of print.

ABSTRACT

A fraction of the major histocompatibility complex (MHC) class I proteins can bind N-myristoylated short lipopeptides rather than conventional long peptides. The molecular mechanisms underlying lipopeptide antigen presentation were recently delineated for N-myristoylated 4-mer lipopeptides (C14-Gly1-Gly2-Ala3-Ile4; C14nef4) derived from the retroviral Nef protein. The C14nef4 lipopeptides are captured by the rhesus MHC class I allomorph, Mamu-B*05104 and recognized by specific αβ T-cell receptors (TCRs). The crystal structure of the Mamu-B*05104:C14nef4:TCR complex indicates that both ends of C14nef4, namely, myristic acid and C-terminal Ile4, are anchored at the antigen-binding groove, leaving Gly1, Gly2 and Ala3 exposed. Among these residues, only the amide bond of Gly1 forms a hydrogen bond with TCRs and serves as a primary T-cell epitope. However, it remains unclear how antigenic and non-antigenic lipopeptides exist, both of which share the primary T-cell epitope. To gain insight into this enigma, we utilized C14nef4 and its analogs with an amino acid substitution for Ala3. Bio-layer interferometry experiments with immobilized TCRs and lipopeptide-bound Mamu-B*05104 indicated that the antigenic strength varied among these lipopeptides. The crystal structures of Mamu-B*05104 complexed with either C14nef4 or each of its five analogs showed a downward shift in the proximal part (C₁-C₄ carbons) of the hydrocarbon chain and the linked Gly1 residue for poorly antigenic analogs. Furthermore, molecular dynamics simulations indicated that lipopeptide ligands alter their conformation dynamically, with differential efficiency in exposing Gly1 externally. Thus, the antigenic strength of lipopeptides is affected by their intrinsic ability to sustain a T-cell epitope-exposed configuration. (248 words/250 words).

PMID:41391759 | DOI:10.1016/j.jbc.2025.111049

Proc Natl Acad Sci U S A. 2025 Dec 16;122(50):e2525911122. doi: 10.1073/pnas.2525911122. Epub 2025 Dec 5.

NO ABSTRACT

PMID:41348746 | PMC:PMC12718358 | DOI:10.1073/pnas.2525911122

bioRxiv [Preprint]. 2025 Nov 20:2025.11.19.689332. doi: 10.1101/2025.11.19.689332.

ABSTRACT

Mycobacterium tuberculosis (Mtb) remains one of the world's leading infectious killers. Although CD8 T cells contribute to immune control of tuberculosis, the pathways through which bacterial antigens access major histocompatibility complex class I (MHC-I) antigen presentation remain incompletely defined. Here, we show that the activity of an Mtb secretion system actively promotes antigen presentation on MHC-I. Using quantitative immunopeptidomics, host and bacterial genetic perturbations, and T cell activation assays, we demonstrate that presentation of Mtb-derived peptides on MHC-I requires the ESX-1 type VII secretion system. Presentation of these peptides proceeds in a manner dependent on the transporter associated with antigen processing (TAP) but independent of host cell mechanisms such as autophagy or MPEG1-mediated pore formation. Chemical induction of phagosomal membrane damage fails to restore antigen presentation in the absence of ESX-1 activity, suggesting that pathogen-encoded secretion, not nonspecific membrane rupture, governs access to MHC-I antigen processing pathways. These findings reveal a secretion system-driven mechanism of antigen presentation, redefining how mycobacteria interface with host MHC-I pathways, potentially informing tuberculosis vaccine design strategies, and highlighting a potential route for synthetic antigen delivery to the cytosol in therapeutics and vaccination.

PMID:41332634 | PMC:PMC12667757 | DOI:10.1101/2025.11.19.689332

J Clin Invest. 2025 Oct 2;135(23):e190851. doi: 10.1172/JCI190851. eCollection 2025 Dec 1.

ABSTRACT

Single-cell studies have revealed that intestinal macrophages maintain gut homeostasis through the balanced actions of reactive (inflammatory) and tolerant (noninflammatory) subpopulations. How such balance is impaired in inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC), remains unresolved. Here, we define colon-specific macrophage states and reveal the critical role of noninflammatory colon-associated macrophages (niColAMs) in IBD recovery. Through trans-scale analyses-integrating computational transcriptomics, proteomics, and in vivo interventional studies-we identified GIV (CCDC88A) as a key regulator of niColAMs. GIV emerged as the top-ranked gene in niColAMs that physically and functionally interacts with NOD2, an innate immune sensor implicated in CD and UC. Myeloid-specific GIV depletion exacerbates infectious colitis, prolongs disease, and abolishes the protective effects of the NOD2 ligand muramyl dipeptide in colitis and sepsis models. Mechanistically, GIV's C-terminus binds the terminal leucine-rich repeat 10 (LRR 10) of NOD2 and is required for NOD2 to dampen inflammation and clear microbes. The CD-associated 1007fs NOD2 variant, which lacks LRR 10, cannot bind GIV, which provides critical insights into how this clinically relevant variant impairs microbial sensing and clearance. These findings illuminate a critical GIV•NOD2 axis essential for gut homeostasis and highlight its disruption as a driver of dysbiosis and inflammation in IBD.

PMID:41321314 | PMC:PMC12646664 | DOI:10.1172/JCI190851

bioRxiv [Preprint]. 2025 Nov 25:2025.11.02.686125. doi: 10.1101/2025.11.02.686125.

ABSTRACT

Tuberculosis causes over one million deaths annually and remains the leading cause of death from a single infectious agent. The emergence of multidrug-resistant Mycobacterium tuberculosis strains highlights the urgent need for new antibiotics, a pursuit hindered by the bacterium's complex cell envelope. As most anti-tuberculosis agents act on intracellular targets, assessing cytosolic drug accumulation is critical. Conventional approaches generally quantify whole-cell association without resolving subcellular localization. Moreover, no current method permits real-time monitoring of drug accumulation in live mycobacterial cells. Here, we present a split-luciferin-based assay to quantify molecular accumulation in mycobacteria. Using this approach, we quantified the cytosolic accumulation of diverse small-molecule antibiotics and polyarginine peptides conjugated via a disulfide-linked D-cysteine tag. We also show the localization of a polyarginine peptide inside of mycobacteria in infected macrophage cells, demonstrating that these peptides can cross multiple accumulation barriers. Our findings establish the first assay for real-time quantification of cytosolic molecular accumulation in live mycobacteria, addressing a longstanding methodological gap and enabling mechanistic insights into intracellular drug uptake.

PMID:41279386 | PMC:PMC12636552 | DOI:10.1101/2025.11.02.686125

bioRxiv [Preprint]. 2025 Nov 7:2025.11.06.687013. doi: 10.1101/2025.11.06.687013.

ABSTRACT

For cytotoxic T-cells to identify and eliminate infected and transformed cells, the adaptive immune system requires the presentation of antigenic peptides by major histocompatibility complex class I (MHC-I) molecules. These peptides, typically derived from intracellular proteins, undergo a rigorous selection process to ensure self-tolerance. However, post-translational modifications (PTMs) and non-enzymatic chemical modifications can alter peptide structure and chemistry, potentially affecting immune recognition. In this study, we investigated the impact of non-enzymatic PTMs on antigen presentation and T cell recognition. Using the well-characterized model epitope SIINFEKL, we synthesized peptide variants incorporating common non-enzymatic PTMs and assessed their effects on MHC-I binding interactions and T cell recognition. We demonstrated that non-enzymatic PTMs markedly alter MHC-I affinity of peptides in a cancer-associated immunopeptidome. Additionally, we developed a novel enrichment strategy using an alkyne-modified probe to identify sites of non-enzymatic acylation prone to MHC-I display. These findings underscore the significance of non-enzymatic PTMs in altering the immunopeptidome and modulating immune responses.

PMID:41279913 | PMC:PMC12637712 | DOI:10.1101/2025.11.06.687013

J Immunol. 2026 Mar 17;215(3):vkaf312. doi: 10.1093/jimmun/vkaf312.

ABSTRACT

Peptide-based therapeutic vaccines exploit cross-presentation by dendritic cells for the induction of effective T cell responses. Their clinical success, however, has been limited due to incomplete understanding of antigen processing and presentation (APP). Bioorthogonal chemistry (BOC) uses chemical "click" reactions that can be performed selectively without interference of the cellular environment. A "click handle" can be incorporated into a biomolecule with only minor effects on its characteristics, allowing selective visualization and tracking of the biomolecule. We generated 10 analogues of the HLA-A2-restricted, immunogenic hepatitis B virus-derived epitope HBcAg18-27 by replacing each amino acid with the click handle homopropargylglycine. Each analogue was tested for (1) peptide binding affinity to HLA-A2, (2) preservation of immunogenicity, and (3) accessibility for on-cell click reactions. Lastly, we performed a proof-of-concept experiment in which we demonstrate the feasibility of BOC for APP studies. All amino acids could be modified with the click handle without loss of HLA binding. Furthermore, 7 out of 10 analogues retained cognate T cell recognition. Two of the most promising analogues were tested and demonstrated to be accessible for on-cell click as well as suitable for long peptide processing and presentation studies. To our knowledge, we are the first to show the feasibility of BOC to study APP in the human setting. With BOC techniques, we may now be able to sensitively follow antigen routing by visualizing the intracellular location of (long) peptides. Furthermore, this tool enables direct and quantitative epitope studies in a T cell-independent manner.

PMID:41267163 | DOI:10.1093/jimmun/vkaf312

Sci Transl Med. 2025 Nov 5;17(823):eadw9184. doi: 10.1126/scitranslmed.adw9184. Epub 2025 Nov 5.

ABSTRACT

No currently licensed vaccine reliably prevents pulmonary tuberculosis (TB), a leading cause of infectious disease mortality. Developing effective new vaccines requires identifying which Mycobacterium tuberculosis (Mtb) proteins are presented on major histocompatibility complex class II (MHC-II) by infected human phagocytes (target cells) and defining their capacity for recognition by CD4⁺ T cells. Vaccine designs must elicit T cell responses recognizing the same peptide-MHC complexes presented by infected cells. Although many human CD4⁺ T cell Mtb epitopes have been described, presentation on MHC-II by infected cells in most cases has not been directly evaluated. Using mass spectrometry (MS), we demonstrated that Mtb type VII secretion system (T7SS) substrates are enriched in the MHC-II repertoire of Mtb-infected human monocyte-derived phagocytes and that many of these antigens are immunogenic in people with prior evidence of Mtb infection. We next used MS to guide TB messenger RNA (mRNA) vaccine design, increasing the presentation of target MHC-II epitopes by orders of magnitude by incorporating design features that mirror aspects of antigen presentation dynamics in infected phagocytes. Our results provide a strategy for TB vaccine design that is guided by bottom-up unbiased discovery. Our approach combines targeted evaluation of antigen presentation in human cells paired with rapid iterative testing of mRNA vaccine designs to optimize antigen presentation before animal studies or human clinical trials.

PMID:41191777 | DOI:10.1126/scitranslmed.adw9184

Eur J Immunol. 2025 Oct;55(10):e70080. doi: 10.1002/eji.70080.

ABSTRACT

At birth, the mucosal immune system of neonates is not fully developed, making them more susceptible to respiratory and intestinal diseases. Previously described host-directed therapies using toll-like receptor (TLR) activation-based strategies have proven effective in controlling neonatal diseases, including cryptosporidiosis. In this study, we investigated the effect of nucleotide-binding oligomerization domain (NOD) receptors stimulation on the control of enteric infection by the protozoan Cryptosporidium parvum in neonatal mice. NOD2 stimulation by intraperitoneal injection of muramyl dipeptide (MDP) resulted in a rapid reduction in the parasite burden. The protective effect was associated with increased pro-inflammatory cytokine and antimicrobial peptide gene expression and a rapid influx of neutrophils to the site of infection, whereas NOD1 stimulation did not show a protective effect. The protective mechanism did not involve microbiota participation but involved IFN-γ and IL-22 cytokines and was associated with increased intestinal epithelium renewal in infected neonates. Our findings showed that stimulating neonatal mice with the bacterial ligand MDP, which targets the NOD2 receptor, actively contributes to the nonspecific clearance of C. parvum infection by eliminating or renewing infected epithelial cells.

PMID:41116307 | PMC:PMC12538031 | DOI:10.1002/eji.70080