Antibody Therapeutics

Epitope mapping is central to rational antibody drug design, affinity optimization and the anticipation of therapeutic resistance mechanisms. Here, we demonstrate the use of Sensor Integrated Proteome on Chip (SPOC) technology for single amino acid resolution epitope mapping. By generating high throughput (HTP) binding kinetics data, we identify important residues within the target epitope whose mutations alter drug-target interactions. The SPOC platform integrates simultaneous HTP cell-free production of folded proteins in nanowells from immobilized plasmid DNAs or linear expression cassettes and capture onto biosensor chips for subsequent label-free binding kinetic analysis using surface plasmon resonance (SPR). The model system comprised the extracellular domain (ECD) of CD20, a membrane-spanning 4-domain family protein, screened against its FDA-approved therapeutic monoclonal antibodies (thAbs) - rituximab and ocrelizumab. Using our proprietary POC protein nanofactory system, a partial deep mutationally scanned (DMS) CD20 ECD mutant library of 79 variants was produced on SPOC biosensor chips via rational single amino acid substitutions of the epitope and surrounding residues with alanine, aspartic acid, lysine, and serine, collectively representing four broad classes of amino acid side chain chemistries: nonpolar, acidic, basic, and polar neutral. The SPOC protein biosensor chip was then screened with both thAbs using SPOC SPR to generate kinetic affinity data, evaluate mutations that led to affinity loss or gain, and ultimately identify critical epitope residues that interface with the antibodies. Most mutations within the rituximab and ocrelizumab epitopes - EPANPSEK and YNCEPANPSEKNSPST, respectively - resulted in complete loss of binding or >25% increase in apparent KD. Notably, N171, P172, and S173 mutations, irrespective of side chain substitution, resulted in complete loss of rituximab binding while at least three diverse side chain substitutions at E168, P169, N171, P172, S173, E174, K175, and T180, led to complete loss of binding for ocrelizumab. These outcomes identify the listed residues as the most critical contact points for their respective antibodies. Interestingly, we also found that functional side-chain substitutions at some residues flanking the epitope increased affinity. This indicates that these non-epitope residues contribute to antibody contact, and that polarity at these sites is a tractable lever for affinity modulation by targeting the corresponding contact residues on the antibody CDRs. The proposed SPOC approach of screening drug candidates against on-chip library of mutationally-scanned therapeutic targets is relevant in the early phase of drug development to resolve epitopes at the residue-level to support more informed down-selection of candidates. It facilitates cost-effective improvement of thAbs, enhancing therapeutic efficacy across a wide array of therapeutic targets, including rare variants that might otherwise lead to therapeutic resistance.

While Trastuzumab emtansine (T-DM1) and other HER2-targeting antibody-drug conjugates (ADCs) are used to treat cancer patients with HER2-amplified tumors, there is a need to improve the efficacy through the understanding of their mechanism of uptake into cells. Flotillin-2 (FLOT2) regulates the internalization of epidermal growth factor receptor (EGFR), leading us to investigate FLOT2 effects on HER2 internalization. Higher FLOT2 expression in nine HER2 amplified cell lines correlated with a higher T-DM1 IC50 in vitro, and breast cancer patients with high FLOT2 expression had worse survival when receiving either T-DXd (16.2 months (m) vs 18.3 m, p=0.04) or T-DM1 (38.0 m vs 41.3 m, p=0.1) in real-world Caris Life Sciences data. FLOT2 interacts with HER2 and positively regulates HER2 activation and downstream signaling, while FLOT2 knockdown reduces the viability of HER2 amplified cancer cells. FLOT2 knockdown results in increased HER2 internalization upon binding of T-DM1, mediated by ubiquitination by the Cbl ubiquitin ligases. We investigated the effects of various small molecules and discovered that zoledronic acid binds to FLOT2 and disrupts the HER2/FLOT2 interaction, which enhances T-DM1 internalization and cytotoxicity. In conclusion, FLOT2 regulates the internalization and cytotoxicity of T-DM1 mediated by Cbl-dependent ubiquitination of HER2. Zoledronic acid disrupts the HER2/FLOT2 interaction, therefore increasing the internalization and cytotoxicity of T-DM1, providing proof of principle that a small molecule inhibitor of the HER2/FLOT2 interaction can enhance the activity of the HER2-targeting ADC.

The complement of tumor cell surface proteins, or "surfaceome", is a rich source of potential immunotherapy targets. To move beyond expression-based target discovery, we previously described "structural surfaceomics," combining crosslinking mass spectrometry (XL-MS) with surface protein biotinylation to identify conformation-selective targets. In our prior work, we applied this method to a single model of acute myeloid leukemia (AML), identifying active integrin beta-2 as a promising target. Here, we expand structural surfaceomics to identify additional immunotherapy targets and surface protein biology across additional models of AML, multiple myeloma, and prostate cancer, as well as donor peripheral blood mononuclear cells. Utilizing these models and different chemical crosslinkers, we compile an extensive database of 5,209 crosslinks. We characterize both shared and unique crosslink-based features, identifying 1,612 disease model-specific crosslinks, including 212 potentially defining tumor-specific conformations based on distance constraint violations relative to AlphaFold predictions. We further implement a suite of emerging modeling tools to predict tumor-specific protein structures. We probe crosslinking patterns suggesting multiple myeloma-specific CD48 and AML-specific integrin 1/{beta}4 heterodimer conformations. This work establishes a resource for cancer structural biology by implementation of structural surfaceomics. Our findings also point toward more realistic protein design models, potentially enabling systematic detection of targetable cancer-specific epitopes for next-generation immunotherapies.

Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism of action for many FDA-approved therapeutic antibodies that is driven by interactions between the antibody Fc and Fc{gamma} receptors (Fc{gamma}Rs) on immune effector cells. Murine models used for preclinical antibody evaluation currently have limited predictive value for clinical ADCC performance due to interspecies differences in Fc-Fc{gamma}R interactions. The molecular determinants governing Fc-Fc{gamma}R engagement in mice remain poorly defined, complicating the interpretation of murine ADCC data and its clinical relevance. To address this, we present the high-resolution crystal structure of the receptor that regulates Fc-mediated cytotoxicity in mice, mouse Fc{gamma}RIV, alone and in complex with mouse IgG2a Fc. This complex preserves key features of the human IgG1 Fc-human Fc{gamma}RIIIa interface which mediates ADCC in humans including salt bridges, hydrogen bonds, and a proline sandwich. However, subtle variations in receptor orientation, Fc-Fc{gamma}R electrostatics, and glycan positions reduce human IgG1 Fc- mouse Fc{gamma}RIV binding affinity, resulting in species-restricted Fc-Fc{gamma}R mediated immune responses. Modeling of human IgG1 Fc interactions with mouse Fc{gamma}RIV predicted steric clashes, suggesting opportunities to modulate the interaction. One structure-guided substitution variant of human IgG1, Fchumo, maintains comparable human Fc{gamma}RIIIa engagement with enhanced binding to and activation of mouse Fc{gamma}RIV, relative to human IgG1 Fc. This study provides proof-of-concept for engineering human Fc domains for cross-species Fc{gamma}R recognition and provides a strategic framework to improve the predictive power of in vivo preclinical models.

BackgroundAntibody-mediated blockade of innate receptor-MHC-I interactions represents a promising strategy to enhance anti-tumor immunity, particularly against metastatic cancers resistant to conventional checkpoint inhibitors. In this study, we investigated the effects of the pan anti-MHC-I monoclonal antibody M1/42, which targets MHC-I interactions with Ly49, selectively expressed on murine NK cell subsets. MethodsWe administered M1/42 to mice and assayed the proliferation and activation immune cells. Anti-tumor activity of growth and metastasis of checkpoint inhibitor-resistant pancreatic ductal adenocarcoma (PDAC) and B16F10 melanoma were assessed, complemented by extensive cellular phenotypic and RNA expression analysis. Binding and cryo-electron microscopic (cryo-EM) and X-ray crystallographic structural studies of M1/42 complexed with the mouse MHC-I molecule, H2-Dd, examined the Ab interaction site in comparison with those of Ly49 inhibitory receptors. ResultsM1/42 administration in mice robustly unleashed the proliferation and activation of natural killer (NK) cells, memory CD4+ and CD8+ T cells, dendritic cells, and macrophages in both lymphoid and non-lymphoid tissues, independent of Fc{gamma} receptors. M1/42 significantly restricted the growth and metastasis of checkpoint inhibitor-resistant pancreatic ductal adenocarcinoma (PDAC) and B16F10 melanoma in the liver and lungs, accompanied by increased tumor infiltration of effector CD8+ T cells, reduction of T regulatory cells, and a pro-inflammatory cytokine milieu. The anti-tumor effects of M1/42 depend on NK cells and are associated with upregulation of genes involved in antigen processing, interferon gamma responsiveness, and Th1 cytokine production, while downregulating inhibitory PD1/11 signaling. Structural analysis indicated that the effect of M1/42 on Ly49/MHC-I interactions was not due to direct steric competition. ConclusionsCollectively, these findings demonstrate that M1/42 unleashes coordinated innate and adaptive immune responses, overcoming tumor-induced immunosuppression and resistance to checkpoint blockade. This approach represents a paradigm shift in cancer immunotherapy, offering potential for more effective treatment of metastatic cancers that evade immune surveillance through MHC-I modulation. KEY MESSAGESO_ST_ABSWhat is already known on this topicC_ST_ABSA pan anti-mouse MHC-I mAb (M1/42) blocks interaction with several NK inhibitory receptors (Ly49A or Ly49C) resulting in NK cell activation and anti-viral and anti-tumor responses in vitro and in vivo. Other pan anti-human MHC-I mAbs (DX17 and W6/32) function similarly, blocking LILRB inhibitory receptor interaction of myeloid cells and NK cells. These stimulate human immune cells in humanized mouse models. What this study addsThis study analyzes the effects of the pan anti-mouse MHC-I mAb on NK and myeloid cell activation in detail, in the absence of T or B cells, and independent of FcR interaction. Additionally we analyze several mouse models of metastatic tumor progression, indicative of the progressive activation not only of the innate immune response, but also adaptive responses. The molecular mechanism of the mAb blocking of inhibitory receptors is revealed by cryo-EM and X-ray structures of M1/42 Fab/MHC-I (H2-Dd) complexes. How this study might affect research, practice, or policyElucidation of the details of the inhibitory effects of the mouse pan anti-mouse MHC-I mAb provides not only a more advanced understanding of the murine model system, but suggests additional functional avenues to be explored using the parallel an anti-human MHC-I mAbs.

BackgroundCombinatorial fragment antigen-binding (Fab) libraries encode an immense heavy-light chain pairing space, often exceeding 10{superscript 1} possible combinations, which far surpasses the diversity that can be experimentally constructed and screened in display systems. As a result, direct Fab screening samples only a small fraction of the theoretical search space, creating a practical bottleneck for functional binder discovery. ResultsHere, we frame Fab discovery as a staged search problem by decoupling heavy-chain (HC) and light-chain (LC) exploration. We implemented a sequential HC preselection-remating workflow in yeast surface display, in which antigen-reactive HC variants are first enriched and subsequently recombined with a diverse LC repertoire to reconstruct a focused Fab library. In a SARS-CoV-2 spike-targeted campaign, HC and LC libraries of 2.05 x 10 and 2.33 x 10 members corresponded to a theoretical pairing space of approximately 4.8 x 10{superscript 1} combinations. Sequential HC enrichment followed by LC remating allowed recovery of multiple functional Fab clones from a tractable library scale of approximately 10, including clones that shared a common HC scaffold but carried distinct LC partners. A representative recombinant IgG output showed broad but heterogeneous spike/RBD binding, measurable pseudovirus neutralization activity (EC = 11.1 nM), and compatibility with standard early biophysical characterization after full-length IgG reformatting. ConclusionsThese results provide proof of principle that combinatorial Fab discovery can be approached as a staged exploration problem under realistic library-size constraints. By focusing downstream Fab reconstruction on an antigen-compatible HC subspace, sequential HC preselection followed by LC remating offers a practical strategy for exploring otherwise intractable antibody pairing landscapes in eukaryotic display systems.

{beta}4-galactosylation is a critical quality attribute of therapeutic monoclonal antibodies (mAbs), enhancing complement-dependent cytotoxicity, antibody-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Despite its therapeutic importance, galactosylation remains the most variable glycosylation motif due to its sensitivity to cell culture conditions. Here, we describe a dual genetic engineering strategy applied to two mAb-producing CHO cell lines, DP12 and VRC01, to simultaneously overcome the cellular machinery and metabolic bottlenecks that limit {beta}4-galactosylation. The first engineering event knocks out COSMC, the chaperone required for core 1 {beta}-1,3-galactosyltransferase 1 activity, to redirect UDP-Gal consumption from O-linked {beta}3-galactosylation towards mAb Fc N-linked {beta}4-galactosylation. The second event overexpresses {beta}-1,4-galactosyltransferase 1 ({beta}4GalT1) to augment cellular galactosylation machinery. Each modification was characterised individually (COSMC- and GalT+) and in combination (C-/GT+) across both cell lines in batch and fed batch cultures. The combined C-/GT+ strategy consistently achieved greater than 90% mAb Fc {beta}4-galactosylation, irrespective of host cell line or culture mode. Metabolic characterisation confirmed that both engineering events alleviate their respective bottlenecks: COSMC knockout redirects UDP-Gal flux and {beta}4GalT1 overexpression increases N-galactosylation capacity. The C-/GT+ strategy also reduced production of Man5 glycans, which accelerate serum clearance and pose immunogenicity risks. Metabolic profiling suggests that the COSMC knockout attenuates UTP consumption and contributes to reduced Man5 production. C-/GT+ glycoengineering had no negative impact on mAb titre. Our results establish the C-/GT+ dual glycoengineering strategy as a robust approach for consistently achieving high mAb galactosylation across diverse cell culture conditions, with the additional benefit of reduced Man5 glycans. HighlightsO_LIDual COSMC KO and {beta}4GalT1 overexpression achieves >90% mAb Fc galactosylation. C_LIO_LICOSMC KO redirects UDP-Gal from O-glycans to mAb Fc without impacting cell growth. C_LIO_LIDual glycoengineering reduces production of undesired Man5 glycans. C_LI

The development of clinically effective CAR-T cell therapies for solid tumors requires careful optimization of receptor design, functional fitness, and manufacturability. While advancing low-affinity HER2-targeting CAR-T cells toward clinical application, we found that the candidate with the strongest in vivo antitumor activity--comprising a CD8 hinge and transmembrane region and a 4-1BB co-stimulatory domain--exhibited measurable tonic signaling. This basal antigen-independent signaling, likely driven by high CAR surface expression, was associated with increased apoptosis and reduced ex vivo expansion under research-grade manufacturing conditions. Modification of the transmembrane domain reduced CAR surface expression but did not alleviate tonic signaling and instead impaired antitumor activity. By contrast, transient pharmacologic inhibition of CAR signaling with dasatinib rescued expansion and reduced apoptosis in small-scale research cultures. Notably, these tonic-signaling-associated defects were largely absent during large-scale, GMP-compliant manufacturing, which enabled robust CAR-T cell expansion without additional benefit from dasatinib supplementation. Together, these findings show that tonic signaling is not inherently detrimental to CAR-T cell performance and that its functional consequences are highly dependent on manufacturing context. Our study underscores the importance of evaluating CAR candidates within clinically relevant production platforms and supports the advancement of this 4-1BB-based HER2-specific CAR-T cell product toward clinical testing.

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated transformative efficacy in hematologic malignancies, but its broader use remains constrained by complex ex vivo manufacturing, prolonged production timelines, high cost, and dependence on lymphodepleting chemotherapy. Emerging in vivo CAR-T generation strategies aim to address these limitations, but they introduce additional safety concerns associated with systemic delivery of gene-modifying vectors, including off-target transduction and insertional mutagenesis. This paper describes a novel antigen-driven CAR T-cell expansion platform (adCAR-T) based on co-culture of CAR T cells with engineered target cells expressing defined antigen density and lacking the inhibitory checkpoint ligand PD-L1. This system induces immediate activation, rapid proliferation, and sustained cytotoxic differentiation of CAR T cells without reliance on artificial CD3/CD28 bead stimulation or exogenous cytokine-driven expansion. In contrast to conventional methods, the platform eliminates the lag phase of CAR T-cell expansion and enables rapid scaling to clinically relevant doses (108-109 cells) within several days, depending on the initial cell input. Mechanistically, antigen-driven CAR engagement and target-cell lysis trigger cytokine release and amplification of CAR T cells in a physiologically relevant manner. This process promotes coordinated expansion of both directly antigen-engaged and non-engaged CAR T cells. The platform preserves "functional fitness", minimizes exhaustion, and avoids systemic exposure to gene-delivery vectors. Taken together, this strategy defines a hybrid manufacturing paradigm that bridges the control of ex vivo production with the physiological logic of in vivo activation. Proposed method has a potential to reduce manufacturing complexity, improve safety, and possibly decrease or eliminate the need for lymphodepleting conditioning. This work presents a potential alternative to both standard ex vivo manufacturing and emerging in vivo CAR-T generation approaches, with important implications for improving the accessibility, safety, and cost-effectiveness of CAR T-cell therapies.

Immune checkpoint inhibitors (ICI) have revolutionized cancer therapy, yet response rates remain suboptimal across many solid tumors, and resistance mechanisms, particularly those involving glycans, are not fully understood. Recent studies have identified sialic acid-containing glycans and their interactions with Siglec receptors on tumor-associated macrophages as an important contributor to immune suppression within the tumor microenvironment (TME). Targeting this sialic acid-Siglec axis by glycan engineering with sialidases and other glycosidases has shown therapeutic potential in preclinical models. However, safe and effective delivery of sialidases to tumors remains a challenge. Here, we present a novel approach using adeno-associated virus (AAV)-mediated therapy to deliver sialidases (AAVSia) and other glycosidases, including fucosidase, directly to the TME. Intratumoral administration of AAVSia in mouse models resulted in significant tumor growth reduction, enhanced survival, and robust systemic antitumor immunity through improved cross-presentation and dendritic cell activation. Furthermore, combining local sialidase expression with fucosidase treatment and classical PD-1 blockade allowed a synergistic effect, amplifying antitumor response. Our findings highlight the therapeutic promise of glycoengineering the TME using local delivery systems and support the development of combination strategies to overcome glycan-mediated resistance in cancer immunotherapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/720097v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@dc9d72org.highwire.dtl.DTLVardef@1e4e455org.highwire.dtl.DTLVardef@4a8f93org.highwire.dtl.DTLVardef@11813a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

1.Prediction of conformational B-cell epitopes is critical for vaccine design, immunotherapy, and antibody engineering. To date, several host-independent computational methods have been developed for predicting antibody-interacting residues in antigen structures. However, it is well established that antigen-antibody (Ag-Ab) interactions vary depending on the host immune system indicating the importance of developing host-specific prediction models. In this study, we present, for the first time, a human host-specific method, HAIRpred2, that predicts antibody-interacting residues in an antigen from its tertiary structure. The dataset was derived from HAIRpred and comprises 277 human Ag-Ab complexes, with 221 structures used for training and 56 for independent testing. Preliminary analysis revealed that residues with a relative surface accessibility (RSA) below 0.05, corresponding to buried regions, are highly likely to be non-interacting, underscoring the importance of structural accessibility in antibody recognition. To identify the most informative features, we evaluated multiple feature representations, including RSA, large language model (LLM)-based embeddings, distance-based features, and physicochemical properties. A model trained on single-residue RSA features achieved an AUC of 0.72. Incorporating a sliding window of 15 residues to capture local structural context improved performance to an AUC of 0.75. The best performance (AUC = 0.78 on the independent test set) was achieved by integrating RSA with physicochemical descriptors. Benchmarking against existing antibody-interaction prediction methods on the same independent dataset demonstrated that HAIRpred2 outperforms current tools, further highlighting the advantage of host-specific modeling. HAIRpred2 is freely available as a web server at https://webs.iiitd.edu.in/raghava/hairpred2/. HighlightsO_LIDevelopment of HAIRpred2, the first human host-specific method for predicting antibody-interacting residues. C_LIO_LIAnalysis of 277 human antigen-antibody complexes to capture host-dependent interaction patterns. C_LIO_LIRelative surface accessibility (RSA) identified as a key determinant, with buried residues rarely participating in interactions. C_LIO_LIIntegration of RSA with physicochemical features achieved the best performance (AUC = 0.78) on an independent dataset. C_LIO_LIHAIRpred2 outperforms existing methods and is available as a web server for epitope prediction. C_LI

The precise de novo design of antibodies remains a therapeutic challenge. The AI platform, GaluxDesign, was evaluated in a high-efficiency Precision-Scale Workflow by synthesizing and testing only 50 full-length IgG candidates per epitope across eight distinct epitopes from six therapeutic targets. This campaign yielded a 10.5% binder rate (estimated EC50 < 100 nM), identifying target-specific binders for seven of eight epitopes, with multiple candidates exhibiting sub-nanomolar to single-digit nanomolar dissociation constants (Kd). We further assessed the same workflow on nine shared benchmark targets selected for external comparison, where GaluxDesign identified target-specific binders for eight of nine targets, demonstrating strong target-level performance relative to previously reported de novo antibody design approaches. Together, these results establish a high-efficiency, precision-scale workflow for generating novel, high-affinity therapeutic antibodies.

BackgroundCytokines are immunomodulatory proteins that play central roles in regulating immune responses and represent attractive targets for cancer therapy. However, as single agents, cytokines have shown limited clinical benefit due to systemic toxicities and a short in vivo half-life. Our group has focused on engineering fusion cytokines (fusokines) that couple two cytokines into a single biologic to reprogram immune cell responses by enforcing non-canonical receptor engagement and signaling. A chimeric IL-6/IL-1{beta} fusokine was engineered to test the hypothesis that enforced co-engagement of IL-6 and IL-1{beta} signaling pathways would confer a gain-of-function phenotype in T cells and promote robust anti-tumor immunity. Here, we describe the immunomodulatory properties of IL6/1 fusokine and a method to deliver this fusokine to produce inhibition of ovarian tumor growth in a pre-clinical mouse model. MethodsLentiviral vectors encoding murine or human IL6/1 were designed using Vector Builder and expressed in either HEK293, CHO or ID8-F3 (p53-/-) cells depending on the downstream experiment to be conducted. IL6/1 expression was validated by ELISA and flow cytometry. Effects of human IL6/1 (hIL6/1) on T cell function (proliferation, memory phenotype, activation induced apoptosis) were monitored by flow cytometry. For in vivo studies, ID8-F3 murine ovarian cancer cells expressing mouse IL6/1 (mIL6/1) were administered intraperitoneally (I.P.) as a cell-based therapy to C57BL/6 female mice bearing established ID8-F3 luciferase tumors. Tumor progression was monitored by bioluminescence (BLI) imaging, and overall survival was evaluated. ResultshIL6/1 significantly enhanced T cell survival and selectively promoted activation and expansion of CD45RO memory T cells. mIL6/1 expressing ID8-F3 cells (ID8IL6/1) demonstrated stable transduction and sustained cytokine secretion. In vivo, ID8IL6/1 cell therapy significantly reduced tumor growth and improved overall survival compared to control groups, with 2 of 8 mice achieving complete tumor clearance. ConclusionThese findings indicate that IL6/1 fusokine enhances T cell survival and proliferation while promoting memory responses. Engineered cancer cells (ID8-F3) expressing mIL6/1 fusokine induced a strong anti-tumor response when delivered as a therapeutic vaccine in ovarian cancer mouse model. What is already known on this topicO_LIFusokines are a class of bifunctional proteins designed to achieve synergistic immune modulation. Previous studies in our lab have shown fusokine exhibit gain-of-function immunomodulating activity. Individually, IL-6 and IL-1{beta} are recognized for their roles in promoting T-cell proliferation and effector function. However, the potential for a fused IL-6/1 fusokine to reprogram the immune system and elicit a superior anti-tumor response in vivo in ovarian cancer model is not yet studied. C_LI What this study addsO_LIThis study develops a novel fusion cytokine (fusokine), combining IL-6 and IL-1{beta}, and demonstrate robust activation of T cells. In a preclinical ovarian cancer model, engineered cancer cells expressing IL6/1 used as a therapeutic vaccine showed significant tumor reduction and improved overall survival. C_LI How this study might affect research, practice or policyO_LIThis study demonstrates that in comparison to individual cytokines, fusokines have greater potential to activate T cell function and when delivered as a cell therapy, achieve clear therapeutic efficacy in an ovarian cancer model. Further translational and clinical studies may enable the development of novel and more effective fusokine cell therapy approaches for patients with ovarian cancer. C_LI

Neoantigens are cancer-specific antigens arising from genomic alterations. Single Amino Acid Variants (SAAVs) represent a primary class of these neoantigens. To evaluate the therapeutic potential of Neurofibromin 1 (NF1)-derived SAAVs - given that NF1 is frequently mutated in malignant brain tumors - we prioritized the 40 NF1 SAAVs determined to be HLA-A*02:01 binders using computational prediction coupled with experimental validation. To validate these predicted neoepitopes, we employed a two-tiered experimental approach in HLA-A*02:01 homozygous U87-MG cells. We first synthesized minigene constructs encoding the predicted neoepitopes, introduced them via lentiviral transfection and confirmed their expression by mass spectrometry (MS). Subsequently, we performed endogenous validation using pan-HLA immunoprecipitation mass spectrometry (IP-MS), confirming 4 (10 neoepitopes) of the 40 candidate SAAVs. We observed a discrepancy between in silico predictions and the observed sequences. Our endogenous peptidomics further revealed conserved peptide motifs and demonstrated that peptide selection for HLA presentation is transient. While our study substantiates the therapeutic feasibility of T-cell immunotherapies targeting NF1 mutations, these results underscore a limitation in current computational prediction. Our study highlights the necessity of experimental validation to refine neoantigen prioritization strategies.

Multiplexed imaging approaches of various molecular modalities in tissues are becoming increasingly adopted in discovery and translational studies. For clinical implementation, novel instrumentation, complex analysis workflows and high costs per sample are bottlenecks that hinders broader introduction in the clinical setting. Here, we demonstrate a cost efficient integrated workflow that combines multiplexed immunofluorescence of a handful of protein markers, with in situ proximity ligation assay, to detect direct protein interactions between neighboring cells. As a proof of concept case of relevance for clinical adaptation, we target the major immunotherapy signalling axis of programmed death receptor 1 (PD-1) and its ligand PD-L1, to demonstrate the interaction between immune cells in germinal centers of tonsil tissue and in a tertiary lymphoid structure in bladder cancer tissue, respectively, from a patient treated with immunotherapy.

BackgroundPrimary and secondary resistance to immune checkpoint blockade (ICB) remains a critical challenge in advanced melanoma. Oncolytic Viruses (OV) selectively lyse tumor cells while generating systemic anti-tumor immune responses with minimal side effects. Yet their clinical use is limited to refractory melanoma patients and are only given in combination with second-line ICB regimens. ICB can both help and hinder OV efficacy depending on the source of checkpoint interactions across the tumor-immune microenvironment (TiME). However, functional checkpoint interactions are typically inferred from gene or protein expression and rarely contextualized within myeloid- and antigen presenting cell-associated immune niches during OV therapy, despite these populations dominating melanoma TiMEs and serving as key regulators of anti-viral immunity. MethodsAn integrated multi-omics framework combining Nanostring GeoMx digital spatial profiling (DSP), COMET sequential immunofluorescence (seqIF) and functional oncology mapping (FuncOmap) was applied to melanoma patient tissues collected pre- and post-neoadjuvant Talimogene Laherparepvec (T-VEC) to characterize immune remodeling and directly quantify checkpoint interaction dynamics associated with pathologic responses to OV therapy. ResultsT-VEC induced broad lymphocyte- and myeloid-associated immune transcriptional activation across melanoma TiMEs; however, pathologic responses could not be defined by bulk transcriptomics or cellular deconvolution alone. COMET seqIF analysis identified that HSV-associated M1/APC-like tumor-associated macrophages (TAMs) were enriched in complete pathologic response (CR) tissues and were a major source of PD-1/PD-L1 interaction niches. While partial (PR) and non-pathologic response (NR) tissues retained melanoma-centered PD-1/PD-L1 interaction niches and were enriched for B cell and M2-like TAM populations. FuncOmap analysis indicated that post-T-VEC PD-1/PD-L1 interaction states were consistently elevated in tumor bed, but not in lymph node tissues, across all pathologic response groups. Suggesting that immune checkpoint interactions may benefit T-VEC therapeutic responses depending on their spatial and immune context relative to OV infection. ConclusionsThese findings highlight the importance of integrated transcriptomic and functional proteomic analyses for resolving the spatial distribution and functional status of immune niches during OV therapy. Resolving PD-1/PD-L1 interaction states to specific M1/APC-like TAM and B cell niches may define mechanisms of responses and resistance to OV therapy.

Targeting membrane receptors underlies the success of antibody-drug conjugates (ADCs), yet single-receptor formats can be limited by heterogeneous expression, compensatory signaling, and variable internalization. Here we developed Multivalent Interchangeable Nanobody Degradation System (MINDS), a modular nanobody-Fc chassis that co-engages multiple membrane receptors, promotes their lysosomal co-depletion, and enables delivery of diverse intracellular payloads. As a proof of concept, we generated Tritazumab, a trispecific nanobody-Fc targeting three oncogenic receptors EGFR, cMET, and TfR1. Tritazumab incorporates a high-affinity, non-transferrin-competing anti-TfR1 nanobody that drives efficient uptake and lysosomal trafficking, enabling coordinated depletion of all three receptors. Across non-small cell lung cancer models, Tritazumab achieved rapid and sustained multi-receptor surface loss with picomolar degradation potency, reaching near-maximal depletion within approximately 1.5 hours. Conjugation of Tritazumab to MMAE preserved receptor binding and produced substantially greater antiproliferative activity and improved tumor selectivity relative to clinical ADCs in matched cell models, along with potent in vivo tumor growth inhibition and acceptable tolerability in a xenograft model. Extending the platform beyond cytotoxic payloads, a BRD4 molecular glue conjugate improved the selectivity window by > 100-fold and showed marked in vivo efficacy, while an EZH2-targeting PROTAC conjugate achieved an approximately 1,000-fold increase in intracellular degradation potency relative to the free PROTAC. These findings establish MINDS as a modular multispecific degrader-payload platform that integrates receptor co-depletion to enhance anticancer selectivity and efficacy.

Metastasis remains the major cause of cancer-related mortality, and circulating tumor cells (CTCs) are both candidate liquid-biopsy biomarkers and plausible intermediates of metastatic dissemination. Because CTCs are extremely rare in peripheral blood, platform comparisons have often focused solely on recovery. That focus is insufficient for applications that depend on the quality of the recovered material, including single-cell profiling, short-term culture, and functional testing. Here, we compared four CTC isolation approaches: TellDx CTC System, Genesis System, RosetteSep, and flow cytometry, using spike-in experiments in human blood. Capture efficiency was evaluated across all four platforms; purity was assessed for TellDx, Genesis, and RosetteSep; and post-isolation GFP signal persistence in culture was assessed for TellDx and Genesis as an exploratory proxy for short-term post-isolation preservation. Under the conditions tested, TellDx showed the highest recovery (88.1% {+/-} 3.7%), followed by Genesis (40.6% {+/-} 12.1%), RosetteSep (36.5% {+/-} 9.0%), and flow cytometry (7.6% {+/-} 4.5%). TellDx also showed the highest purity score (3.76), whereas Genesis (2.25) and RosetteSep (2.09) did not differ substantially. In the short-term culture assay, TellDx-derived samples retained a higher normalized GFP signal than Genesis-derived samples at 48 h and 72 h. To synthesize these readouts, we propose the Recovery Performance Index (RPI), a composite score integrating recovery, purity, and post-isolation signal persistence. Within this experimental framework, TellDx achieved the highest RPI. These data support two conclusions. First, platform benchmarking for CTC workflows benefits from multidimensional evaluation rather than recovery alone. Second, under this spike-in model and within the specific workflows used here, TellDx performed best among the platforms tested. The principal contribution of this study is therefore the establishment of a practical benchmarking framework that can be expanded in future work using clinical samples, multiple CTC phenotypes, and orthogonal viability assays.

Immune checkpoint inhibition (ICI) is clinically active against multiple cancers, including urothelial cancer at the non-muscle invasive, muscle-invasive, and metastatic stages. Despite this, large numbers of patients experience disease progression and relapse after treatment with ICI-containing regimens. Tumor antigen-specific T cells are critical to ICI response, however few studies have evaluated the breadth and magnitude of tumor antigen-specific T cell responses with ICI therapy. In this study, we mapped the tumor antigen immunodominance hierarchy in the BBN963 model of murine basal-like bladder cancer for endogenous tumor neoantigens expressed physiologically. We used a high-throughput matrixed ELISpot assay to detect CD8+ T cell responses to predicted BBN963 tumor antigens derived from multiple mutational genomic sources. We found CD8+ T cell responses were directed against a subset of tumor antigens forming a stable and reproducible immunodominance hierarchy across individual mice. Treatment with anti-PD-1 or anti-CTLA-4 did not substantially reshape this hierarchy or broadly shift dominant responses to previously defined subdominant epitopes. Predicted peptide MHC binding stability and affinity was associated with antigen immunogenicity. Cancer-testis antigens, endogenous retroviral antigens, and SNV-derived tumor antigens that were immunogenic were found across tumor subclones. By diversifying the immunogenic antigen repertoire beyond SNVs, we achieved nearly 100% tumor subclone coverage, suggesting that broader antigen selection could help immunotherapy target more tumor subclones. In conclusion, this study supports the stability of the immunodominance hierarchy under ICI therapy and a role for broadening antigen discovery to multiple expressional sources in immunotherapy design.

Bispecific fusion proteins represent a unique strategy for developing precision therapeutics. By linking functional domains from distinct proteins, these biomolecules can engage multiple targets, enhancing both therapeutic efficacy and safety. Unlike bispecific antibodies, low-molecular-weight fusion proteins offer distinct advantages, including reduced immunogenicity and superior tissue penetration due to their relatively compact size and structure. Such a profile is particularly valuable in managing complex inflammatory diseases, where modulating multiple pathways is required to impart an effective anti-inflammatory effect. Among the various regulators of immune signaling, the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and interleukin-10 (IL-10) play imperative roles in immune suppression through their interactions with CD80/86 and IL-10R, respectively. While Fc-fused CTLA-4 is a clinically approved drug (e.g., Abatacept), the clinical development of IL-10 has been hampered by unpredictable immunostimulatory side effects. Here, we engineered a bispecific fusion protein linking the extracellular domain of CTLA-4 to IL-10. We successfully expressed the protein in E. coli as an N-terminal GST-tagged variant and refolded it from the inclusion bodies. Additionally, we achieved soluble expression of an Fc-tagged variant in mammalian CHO cells. Both origins demonstrated binding to their cognate receptors, CD80 and IL-10R. Finally, the fusion protein demonstrated a T cell-inhibitory effect by reducing Interferon-{gamma} (IFN{gamma}) secretion levels in an in vitro human Virus-Specific T cells (VSTs) model. This innovative protein engineering offers a promising strategy for addressing unmet clinical needs in autoimmune and inflammatory diseases.