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.

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.

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 improved outcomes for patients with multiple myeloma (MM), but its broader use is restricted by manufacturing complexities and treatment-related toxicities. AZD0120 is a dual-targeting B-cell maturation antigen (BCMA)/CD19 CAR-T cell therapy manufactured via the rapid FasTCAR process. We developed a dual-targeting "loop" CAR that incorporates a novel humanized anti-BCMA single-chain variable fragment (scFv), clone SG, and an FMC63-derived anti-CD19 scFv. This AZD0120 CAR preserved functional binding to both antigens and conferred robust in vitro and in vivo cytotoxicity while maintaining single-antigen reactivity. Conventional manufacture of CAR-T cells with the AZD0120 CAR (AZD0120C) yielded cells with minimal tonic signaling, limited responsiveness to soluble BCMA, and preservation of naive/stem cell memory-enriched phenotypes, yet robust cytokine production upon BCMA+ target engagement. AZD0120C demonstrated cytotoxicity comparable to benchmark BCMA CAR-Ts across MM lines in vitro and showed strong in vivo expansion and tumor control in xenograft models. FasTCAR manufacturing - designed to shorten vein-to-vein timelines and enrich less-differentiated phenotypes - further enhanced in vivo performance: AZD0120 consistently achieved superior tumor control and greater CAR-T expansion vs AZD0120C across disseminated MM.1S, NALM-6, and JeKo-1 models, with superior efficacy observed at lower cell doses. Collectively, these data support clinical evaluation of AZD0120 as a differentiated BCMA/CD19 CAR-T cell therapy with the potential to improve disease control and patient access in MM. Key PointsO_LIAZD0120 is a dual-targeting CAR-T that displays a favorable anti-myeloma functional profile and co-targets a source of potential relapse C_LIO_LIThe FasTCAR process yields TN/SCM-rich CAR-T populations, promotes in vivo expansion and achieves potent tumor control in xenograft models C_LI

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.

Human epidermal growth factor receptor 2 (HER2) is an oncogenic receptor tyrosine kinase in breast cancer and other malignancies. A subset of HER2-positive tumours expresses 611-CTF-p95HER2, a tumour-specific, hyperactive truncated isoform associated with metastasis and treatment resistance that lacks most of the extracellular domain targeted by conventional HER2-directed antibodies. We previously developed NAZ-mAb (formerly known as Oslo-2), a monoclonal antibody against 611-CTF-p95HER2. Here, we describe a computational antibody-engineering workflow for designing variants of NAZ-mAb. Starting from the sequence alone, we modeled the NAZ-mAb-611-CTF-p95HER2 complex, generated a combinatorial mutational landscape using FoldX 5.0, and prioritized candidate variants using predicted interaction energy and developability criteria. Two variants representing distinct design strategies were selected for validation: an aromatic double mutant, NAZ-mAb v1 (L:S31W/L:H107W), and a conservative single mutant, NAZ-mAb v2 (L:S31M). Both variants were successfully expressed as recombinant IgGs; NAZ-mAb v2 achieved a five-fold higher recombinant expression yield than parental NAZ-mAb, while both variants retained antigen binding with a higher apparent signal than the parental antibody in indirect ELISA. However, Biacore two-state kinetic analysis revealed weaker affinities than the parental antibody (KD NAZ-mAb v1: 32.6 nM, NAZ-mAb v2: 9.45 nM vs. parental NAZ-mAb: 5.33 nM). These findings show that the computational workflow can generate experimentally tractable, antigen-engaging NAZ-mAb variants, while also highlighting the limitations of fixed-backbone interaction-energy ranking as a predictor of binding affinity and yield. This study provides a practical framework for computationally driven, developability-aware antibody optimization in the absence of experimental structural data.

Pancreatic cancer remains a highly lethal malignancy with limited therapeutic options. Chimeric antigen receptor (CAR) therapy has revolutionized the treatment of hematological cancers but still faces major limitations in solid tumors, particularly due to the scarcity of tumor-specific targets. Cutaneous lymphocyte antigen (CLA) recently emerged as a promising PDAC target due to its high tumor expression and limited presence in healthy tissues. However, previously reported CLA-directed CAR constructs lacked antitumor functionality. Here, we investigated multiple strategies to generate functional CLA-targeting CAR approaches. We first hypothesized that impaired activity resulted from fratricide caused by CLA expression on activated T cells. CLA knockout was successfully achieved through deletion of fucosyltransferase-7, but not by knockout of the major CLA carrier backbones CD162, CD44 or CD43, suggesting additional CLA carriers or compensatory regulation. As CLA knockout alone did not restore CAR-mediated killing, we explored whether insufficient binding affinity limited CAR activity. Affinity maturation was performed in silico and in vitro using yeast surface display, identifying 39 candidate mutations, although none restored cytotoxicity. We finally switched to an AdCAR strategy using anti-biotin CAR T cells combined with biotinylated anti-CLA scFv-Fc adapters. This approach enabled efficient, concentration-dependent cytotoxicity with both CLA-targeting binders. Additionally, we identified a dynamic, cell density-dependent regulation of CLA expression. Finally, glycan profiling of CLA binders further revealed broader-than-expected glycan interactions, suggesting a potentially wider definition of the CLA family. Overall, our findings establish CLA as a functional PDAC immunotherapy target while revealing unexpected complexity in its regulation and molecular presentation.

Antibody Fc glycosylation is modulated in a variety of disease and immune response contexts, altering downstream functional responses including antibody-dependent cellular cytotoxicity through modified immune cell Fc receptor binding. Accessible, high-throughput glycosylation assays such as enzyme-linked lectin assays (ELLAs) are essential to advance understanding of glycosylation regulation and function. However, current ELLA protocols lack standardization and optimization, and results are reported out in arbitrary absorbance units, limiting reproducibility and cross-study comparability. We developed an optimized multi-lectin parallel ELLA with three specific improvements: systematic optimization of incubation times and reagent concentrations; incorporation of Protein A for IgG specificity; and use of commercially available bovine fetuin B as a quantitative surrogate standard for cross-study reproducibility. Our panel of 8 lectins, SNA, RCA, LCA, PHA-E, PHA-L, MAL-I, WGA, and DSL, cover the major IgG glycoforms. We demonstrate that our ELLA panel can reveal biologically relevant cytokine-induced plasticity of IgG glycosylation profiles in immortalized B cells.

Fc receptor-like protein 5 (FCRL5) is a low-affinity IgG receptor expressed on B cells, with emerging therapeutic relevance due to its expression on multiple myeloma cells, and a potential role in regulating B cell responses. Previous reports on the FCRL5-IgG interaction vary widely in reported affinities, binding differences across IgG subclasses, and molecular requirements for maximal binding. Furthermore, the impact of Fc-engineering strategies, as used in (therapeutic) monoclonal antibodies, remains poorly understood. Here, we provide a comprehensive biochemical analysis of the FCRL5-IgG interaction. We demonstrate that FCRL5 is a true IgG Fc-receptor, binding with very low affinity (60-80 M). FCRL5 binds IgG in a manner involving primarily the two N-terminal domains of FCRL5, and the third domain for maximal binding, but with distinct essential residues in the IgG Fc-tail. Surface plasmon resonance analysis of the binding of FCRL5 to the various IgG subclasses revealed a preference for IgG1 and IgG4. Interestingly, various Fc-engineered IgG variants commonly used for silencing or enhancing of Fc receptor binding do not impact FCRL5 binding. Screening the binding of a set of IgG antibodies carrying defined sets of Fc-mutations to FCRL5 revealed E293 as a key binding determinant and led to the discovery of E293R as a mutation that selectively abrogates FCRL5 binding while preserving binding to other classical Fc{gamma}Rs. Lastly, we show that FCRL5 has considerable preference for binding afucosylated IgG. Together, our results define the essential characteristics of the IgG-FCRL5 interaction and demonstrate the potential of both naturally occurring IgG variants as well as therapeutically explored bioengineered IgG formats to differentially engage FCRL5.

Antibody- and cell-mediated immune responses against biologics, should they occur, can impact treatment efficacy and potentially pose severe risks to patient safety. Therefore, developers have focused on advancing strategies to mitigate such unwanted immunogenicity. Opportunities to address immunogenicity early in the development process, particularly during the drug design phase, have been identified. In vitro and in silico tools that facilitate the identification and removal of sequence liabilities have been established. For example, human cell-based in vitro T cell assays can be used to identify and remove CD4+ T cell epitopes, which are known to play a critical role in the development of anti-drug antibodies against recombinant proteins products as well as the transgenes of gene and therapy. Despite their widespread use in the industry, most of these assays lack thorough characterization, which undermines confidence in the results and comparability across laboratories. In this study, concepts of immunogenicity bioanalytical assay validation for study design and analysis were applied to characterize an internal CD4+ T cell proliferation assay as fit-for-purpose. A statistical path was applied to establish data acceptance criteria for handling of replicates, positivity and negativity of a signal, and donor cohort size. A Bayesian analysis was also performed and is proposed as an approach for sequence de-risking decision making. The in-depth characterization of the CD4+ T cell proliferation assay described here allows for accurate interpretation of the assay outcomes, thereby enhancing confidence in using this approach for mitigating the immunogenicity of biologics by design.

BackgroundLate-stage attrition in therapeutic antibody discovery is dominated by developability liabilities: aggregation, polyspecificity, charge-driven non-specific binding, and chain-mispairing artefacts. Bispecific antibodies amplify these risks because each additional binding arm adds a new biophysical envelope that must be jointly satisfied. The existing in-silico ecosystem addresses individual axes of this problem (humanization, structure prediction, single-metric developability scoring) but few platforms integrate them end-to-end. PTIm-mAb (SANSHI Bio Solutions Pvt Ltd) is a multi-objective, AI/ML-driven antibody design platform that jointly optimizes sequence liabilities, surface aggregation, charge balance, humanness, and predicted binding affinity, and recommends a bispecific architecture in a single workflow. MethodsWe applied PTIm-mAb to the published sequences of eleven FDA-approved bispecific antibodies using the platforms default-parameter Pareto-acceptance optimization loop, run to convergence or to the internal iteration ceiling, with no human curation between the platform run and the external profiler. Both wild-type and platform-optimized sequences were profiled independently with three publicly available developability tools: Aggrescan, CamSol, and the Therapeutic Antibody Profiler (TAP). Paired-sample tests (Wilcoxon signed-rank, exact binomial sign test, McNemar exact test) evaluated the direction and significance of changes. ResultsAcross the 17 evaluable paired arms profiled by TAP, PTIm-mAb cleared four wild-type CDR-vicinity Positive Charge Patch (PPC) flags Blinatumomab-Arm1 (1.9952 [-&gt;] 0.6885), Mosunetuzumab-Arm1 (1.3391 [-&gt;] 0.0568), Linvoseltamab-Arm2 (0.8060 [-&gt;] 0.0), and the headline Elranatamab-Arm1 case (1.7981 [-&gt;] 0.5799) achieved without trading off any other in-range metric and corroborated by Aggrescan and CamSol on the same arm. Total CDR length was significantly shortened across the cohort (Wilcoxon two-sided p = 0.0075, one-sided p = 0.0037, effect size r = 0.65): significant improvement on the metric most directly under the optimizers control. The directional shift on Aggrescan integrated aggregation propensity was also significant by sign test (24 of 36 chains improved, 2 unchanged, 10 worsened; p = 0.021). On the already-clean Zenocutuzumab profile the optimizer identified residual headroom (PPC 0.1191 [-&gt;] 0.0; SFvCSP 12.5 [-&gt;] 6.0), demonstrating that the platforms value extends to candidates that pass all flags. Three results: Teclistamab Arm-1, Emicizumab, and Talquetamab Arm-2 did not clear all flags and are presented as candidates for iterative re-invocation of the platform pipeline on the optimized output (planned follow-up; Section 5). The remaining TAP metrics (PSH, PPC magnitude, PNC, |SFvCSP|) trended in the improvement direction without reaching significance in this cohort, a pattern consistent with the expected statistical signature of a multi-objective optimizer applied to molecules already within the clinical-stage envelope. The platform reported a mean of 12.8 months and USD 723,889 of computational front-loading per project across the nine-project cohort (range 9.0-16.0 months; USD 510,000-960,000); the underlying cost assumptions are tabulated in Supplementary Table S3. ConclusionPTIm-mAb produces externally verifiable, literature-aligned improvements on the metrics most directly under its control, clears CDR-vicinity charge-patch flags on a meaningful fraction of flagged candidates, and front-loads substantial design-iteration work. The cohort-level pattern is consistent with a calibrated multi-objective optimizer operating at the edge of detectable headroom on a deliberately hard benchmark. We position the platform as an early-stage triage and lead-optimization layer in bispecific antibody discovery. For molecules whose first-pass result does not clear all flags, iterative re-invocation of the pipeline on the optimized output is a natural follow-up direction.

Integrin v{beta}8 is a major activator of latent transforming growth factor-{beta} (TGF-{beta}) and an emerging therapeutic target in cancer and immune regulation. However, functional assays that directly measure v{beta}8-mediated activation of latent TGF-{beta} in a physiologically relevant context remain limited. Here, we report a co-culture cell-based reporter assay for quantitative measurement of v{beta}8-mediated activation of latent TGF-{beta}1. NIH/3T3 reporter cells were engineered to express a SMAD-responsive NanoLuc reporter, constitutive firefly luciferase for internal normalization, and cell-surface GARP-latent TGF-{beta}1. When co-cultured with v{beta}8-expressing LN-229 cells, reporter cells produced a robust signal that directly reflected localized latent TGF-{beta}1 activation. The assay demonstrated stable expression of the required biological components, reproducible signal-to-background performance, and sensitivity to benchmark v{beta}8-blocking antibodies. Inhibition studies showed potent dose-dependent blockade by an anti-v{beta}8 antibody. In contrast, pan-TGF-{beta} neutralizing antibody displayed markedly weaker apparent potency, suggesting that targeting localized v{beta}8-mediated activation is more effective than neutralizing released TGF-{beta} in this assay context. The assay also enabled screening and ranking of anti-v{beta}8 antibodies, identifying several high-potency clones, and detected v{beta}8-mediated activation of a non-cleavable latent TGF-{beta}1 mutant. This platform provides a sensitive, internally normalized, and scalable approach for mechanistic studies and therapeutic discovery targeting the v{beta}8-TGF-{beta} axis.

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.

Colorectal cancer (CRC) remains a significant contributor to cancer-associated deaths worldwide, indicating the need for new therapeutic targets and modalities. Antibody-drug conjugates (ADCs) have demonstrated remarkable potential for the treatment of various cancer types, although their efficacy as monotherapies is often limited by insufficient targeting of tumor heterogeneity, dose-limiting toxicities, and drug resistance. Accordingly, multi-targeting therapeutic strategies, such as bispecific ADCs (bsADCs), which simultaneously target two cancer-associated antigens or non-overlapping epitopes on the same antigen, may prove more effective at overcoming resistance and eliminating tumors compared to monospecific ADCs. In this work, we describe the development of EGFR:LGR5 bispecific antibodies (bsAbs) and bsADCs. EGFR:LGR5 bsAbs were shown to internalize to the lysosome to a greater extent than EGFR- and LGR5-targeting monoclonal antibodies (mAbs) and drive EGFR lysosomal degradation in an LGR5-mediated fashion. However, EGFR:LGR5 bsAbs exerted suboptimal cytotoxicity in CRC cell lines. We therefore engineered an EGFR:LGR5 bsADC that demonstrated 100- to 1000-fold enhanced efficacy over a previously developed LGR5-targeting monospecific ADC (8E11-CPT2) with an identical linker-payload in CRC cell lines of various genetic backgrounds and EGFR and LGR5 expression levels. EGFR:LGR5 bsADC potency was strongly correlated with cell line sensitivity to the CPT2 payload. EGFR:LGR5 bsADC induced tumor regression in select RASMUT CRC xenograft models and demonstrated superior antitumor activity and prolonged survival benefit in all evaluated models versus EGFR mAb cetuximab (CTX), bsAb, and 8E11-CPT2. These findings strongly support the further development of EGFR and LGR5 dual-targeting approaches for CRC and other EGFR- and LGR5-expressing malignancies. One Sentence SummaryEGFR:LGR5 bsADCs exert robust antitumor activity and outperform EGFR:LGR5 bsAb and LGR5 monospecific ADC in RASWT and RASMUT colorectal cancer 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.

{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 role of antigen-specific T cells responding to antigen is a topic of intense studies, and critical for mechanistic insight of diseases and development of therapeutic strategies. Methods for broad-scale detection of antigen-specific CD4 T cells are lacking, while such methods have demonstrated great value in exploring CD8 T cell response in health and disease. Furthermore, major histocompatibility complex II (MHCII) assays are technically challenging due to high HLA diversity, lower binding affinities, low frequencies of ex vivo antigen-specific CD4 T cells and several bottlenecks in production and peptide exchange of MHCII monomers. Here we use peptide-loaded MHCII (pMHCII) proteins multimerized on a barcode- and fluorophore-labelled dextran backbone to provide a method for the detection of peptide-specific CD4 T cells by using a large display of MHCII-associated peptides. We have established a protocol for MHCII production and peptide-exchange suitable for the generation of large libraries of peptide-MHCII complexes. We validate the use of such pMHCII complexes in the form of barcode-labelled MHCII multimers to detect antigen-specific CD4 T cells. We demonstrate that we can identify antigen specific CD4 T cells, using these DNA barcoded peptide-MHCII multimer. The multimer bound CD4 T cells were selected based on the fluorochrome signal, and the co-attached DNA barcodes were hereafter amplified and used to identify the peptide-MHCII response/binding. In cases where the peptide-specific CD4 T cells frequencies are very low, we expanded the cell population with peptide-pools and in the presence of IL2. The given CD4 T cell populations hereby reach a cell number allowing for the DNA-barcoded pMHCII multimers to detect responses otherwise missed out. Applying this technology, we utilized a panel of 150 peptides derived from human cytomegalo virus (CMV), Epstein barr virus (EBV), Influenza (Flu), SARS CoV 2 and SARS CoV1, Hepatitis B virus (HBV), and Hepatitis C virus (HCV) loaded onto HLA-DRB1*01:01 and DRB1*04:01 to screen peripheral blood mononuclear cells (PBMC). We assessed ex vivo responses in 16 participants with HCV infection, and successfully detected naturally occurring viral-specific CD4 T cells at frequencies as low as 0.004% of total CD4 T cells. The low-frequency responses, identified via the barcode screen, were rigorously validated using individual fluorophore-labelled tetramer staining after a peptide-driven expansion in 15 participants. Furthermore, we assessed the recognition of novel HCV epitopes in 11 additional participants. Through this, we identified a total of 12 distinct HCV epitopes, including 9 that have not been previously utilized in assays to detect CD4 T cells. Overall, this barcoded-multimer platform provides a powerful tool for the large-scale discovery of class II epitopes and the broad profiling of CD4 T cell specificities. This method will allow for in-depth analyses of immune interactions, provide a better understanding of the antigen-driven associations between CD4 and CD8 T cell responses, and help dissect the complexities of CD4 T cell protection in HCV infection.

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.

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 rapid emergence of SARS-CoV-2 variants that evade neutralizing antibodies underscores the need for next-generation antiviral biologics that combine molecular precision with scalable, cost-effective manufacturing. Computationally designed miniproteins targeting the receptor-binding domain (RBD) of the spike protein offer a compelling alternative to monoclonal antibodies due to their small size, high thermal stability, and compatibility with microbial expression systems. Here we report the end-to-end development and cGMP production of IPD-52520, a de novo antiviral miniprotein, using an optimized E. coli platform. Two miniprotein candidates, a homotrimeric construct (Trimer is referred to as IPD-52520, 17 kDa) and a tandem fusion (Daisy is referred to as IPD-52521, 25 kDa), were evaluated in parallel through systematic optimization of strain selection, media composition, fed-batch fermentation, inclusion-body solubilization, refolding, and chromatographic purification. The Trimer was downselected as the lead molecule based on superior preclinical efficacy, favorable pharmacokinetic properties, and higher volumetric manufacturing yields. The optimized process delivers approximately 2 g/L of purified protein at greater than 90% purity. Scale-up from 5 L to 50 L under cGMP conditions demonstrated excellent batch-to-batch reproducibility across six independent batches, supporting nonclinical and Phase 1 clinical supply. Comprehensive biophysical characterization confirmed a well-folded, predominantly alpha-helical trimer (Tm = 73.4 {degrees}C; polydispersity = 1.005) with an intact primary structure and strong target-binding affinity (KD < 1 pM). Real-time stability studies indicate that the drug substance is stable at 2-8 {degrees}C for at least 12 months, with ongoing stability studies. These results demonstrate the feasibility of translating computationally designed antiviral miniproteins into manufacturable biologics and provide a platform applicable to rapid-response therapeutics against current and future pandemic threats.