US Biologic (United States)

Abstract Background: Despite their therapeutic potential, T-cell engagers (TCEs) encounter significant hurdles in solid tumor therapy, primarily manifesting as on-target off-tumor toxicities, cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS). To address these limitations, we have developed a novel TCE prodrug platform that utilizes a proprietary cleavable substrate linker and a VHH masking domain (VHHm) specific for SP34-derived anti-CD3 antibodies. Dual masking is achieved through the binding of VHHm to anti-CD3 scFv, which simultaneously induces conformational 'locking' of tumor-associated antigen (TAA)-targeting Fab domains, ensuring minimal off-tumor activity. An albumin-binding domain (ABD) is fused to the N-terminus of VHHm to prolong the half-life of prodrug, with a single-site cleavage releasing ABD-VHHm in circulation. This platform eliminates the need to screen specific masking peptides for different TAA-targeting antibodies during dual masking, distinguishing it from previously reported TCE prodrugs. Leveraging these advantages, three prodrugs (DR510 targeting hEGFR, DR507 targeting hTROP-2, and DR508 targeting hEpCAM) have been characterized. Methods: Binding interactions of DR510, DR507 and DR508 to hCD3 and to hEGFR/hTROP-2/hEpCAM were evaluated via flow cytometry. Enzymatic cleavage-induced T-cell activation was evaluated via T-cell-dependent cytotoxicity (TDCC) reporter bioassays using different tumor cell lines. Then, the in vivo efficacy of DR510 was evaluated in human PBMC-engrafted mice harboring HT29, PC9, and HNSCC PDX tumors. ELISA and Western blotting (WB) were conducted to identify the protease cleavage efficiency of DR510 in serum and tumors. A single-dose pharmacokinetic (PK) study and a pilot toxicity study (once-weekly dosing with two doses of 0.65 mg/kg followed by two doses of 1.3 mg/kg) were performed in cynomolgus monkeys, respectively. Results: Flow cytometry analysis confirmed negligible binding of the prodrugs to hCD3 and target antigens (hEGFR, hTrop-2, and hEpCAM). Consistent with this binding profiles, TDCC reporter assays demonstrated minimal basal bioactivity but revealed a significant functional restoration following enzymatic cleavage. In all mouse models, DR510 exhibited dose-dependent tumor growth inhibition that outperformed a clinical-stage comparator across the 0.066 to 0.2 mg/kg dose range without any impact on body weight. In cynomolgus monkey studies, DR510 exhibited favorable PK profiles and good tolerability at two doses of 0.65 mg/kg and 1.3 mg/kg, respectively, accompanied by minimal IL-6 release and undetectable levels of other cytokines including IL-2, IL-4, IL-10, TNF-α, and IFN-γ, suggesting a reduced risk of CRS. Conclusions: These preclinical data highlight our platform's ability to balance the therapeutic efficacy of TCEs with safety. Supported by these data, DR510 is now advancing toward clinical development for the treatment of EGFR-positive solid tumors. Citation Format: Xiaofang Wen, Gaofeng Yao, Wenwen Duan, Zhenxing Zhou, Yonglu Chen, Xin Zhang, Jingjin Fang, Changkui Li, Mingjie Shao, Chengcheng Yang, Dan Cheng, Yanshan Huang. A novel T-cell engager prodrug platform achieves dual masking by a single VHH domain for balancing efficacy and safety in solid tumor therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C010.

Equines are parasitized by complex communities of Strongylidae (Nematoda) comprising multi-species infections. Currently, Cyathostominae are most prevalent, while Strongylus species are only rarely detected. Since eggs and, in most cases, infective larvae cannot be differentiated to species level, except for Strongylus spp., species-specific knowledge of the pathology, epidemiology and ecology of these parasitic nematodes is limited. Reference sequence data for several cyathostomin species are limited or missing. Deep amplicon sequencing of internal transcribed spacer 2 (ITS-2) regions of nematodes has been used in equines previously, although barcoding studies demonstrated a better species resolution for the cytochrome c oxidase subunit I (COI) region. The present study introduces a nemabiome method based on sequencing of COI fragments. This method was applied to compare third stage larvae, representing strongyle communities, derived from regularly treated (RT) and never treated (NT) equine populations from Brazil, France (only RT), Germany, Ukraine, the UK, and the USA. Samples were predominantly from horses, but some were obtained from Przewalski's horses (Ukraine), donkeys (Germany, Ukraine) and kulans (Ukraine). Most sequence reads (87.7%) were identified to species level, but unclassified reads occurred more frequently in donkeys and kulans than horses. No obvious difference in species diversity and richness was observed between RT and NT equines. However, there were significant differences in species composition between the RT and NT groups. Strongylus spp. were more common in NT groups but also showed unexpectedly high abundances in RT horses. Cylicocyclus nassatus, Cylicostephanus longibursatus, and Cyathostomum catinatum were more abundant in RT groups, suggesting that strongyle communities in domestic equines may have been shaped by anthelmintic treatments during last decades. The decreased classification success for reads from non-caballine equines suggests that there are more strongyle species specific for this rarely-investigated group which requires additional efforts to improve the sequence database, particularly for these hosts.

Pseudomonas aeruginosa bloodstream infections carry mortality rates exceeding 60%, with escalating antibiotic resistance dramatically limiting therapeutic options. The type III secretion system (T3SS), a virulence apparatus delivering cytotoxic effectors via PcrV-dependent translocation pores, represents a therapeutic target. Here, we developed a monoclonal antibody (5C8) targeting the central domain (H106-D173) of PcrV, which regulates translocation pore size. 5C8 demonstrated sub-nanomolar affinity (KD = 0.32 nM) via biolayer interferometry and broad neutralization efficacy against clinical isolates (IC50: 0.32-1.47 μg/mL). In murine bloodstream infection models, 5C8 conferred improved survival against cytotoxic (exoU+) and invasive (exoS+) strains (P < 0.01 vs controls), reducing bacterial loads in lungs/kidneys by 1.5-log10 colony-forming unit (P < 0.01) and suppressing interleukin-6 levels by 60-82% (P < 0.01). Mechanistic studies revealed 5C8's dual action: blocking effector release (ExoU/ExoT reduced by 41-88% via liquid chromatography-mass spectrometry) and constricting T3SS pores below 1.2 nm (carbohydrate exclusion assay). Molecular docking identified D125/K129/Y145 as critical binding residues, validated by alanine scanning and mutant construction. Humanized Hu5C8 retained potency (KD = 0.55 nM) with extended half-life (t1/2 = 91.26 h) through Fc receptor engineering. As an inhibitor targeting the pore size-determining domain of PcrV, 5C8 disrupts virulence through a novel dual mechanism, providing a paradigm-shifting strategy against multidrug-resistant P. aeruginosa, bridging a critical gap in sepsis management.

High-resolution protein structures are essential for understanding biological mechanisms and drug discovery. While cryoEM has revolutionized structure determination of large protein complexes, most disease-related proteins are small (<50 kDa) and challenging to resolve due to low signal-to-noise ratios and alignment difficulties. Current scaffold protein strategies increase target size but suffer from inherent flexibility, resulting in poorly resolved targets compared to scaffolds. We present an iteratively engineered molecular design transforming antibody fragments (Fabs) into conformationally Rigid Fabs that enable high-resolution structure determination of small proteins (~20 kDa). This design introduces strategic disulfide bonds, creating well-folded, rigidly constrained Fabs applicable across various species, frameworks, and chimeric constructs. Rigid Fabs enabled high-resolution cryoEM structures (2.3-2.5 Å) of two small proteins: Ang2 (26 kDa) and KRAS (21 kDa). Our disulfide-constrained Rigid Fab strategy provides a general approach for overcoming target size limitation of single-particle cryoEM.

Extracellular matrix (ECM) bioscaffolds have demonstrated therapeutic potential across a variety of clinical and preclinical applications for tissue repair and regeneration. In parallel, these scaffolds and their components have shown the capacity to modulate the immune response. Unlike synthetic implants, which are often associated with chronic inflammation or fibrotic encapsulation, ECM bioscaffolds interact dynamically with host cells, promoting constructive tissue remodeling. This effect is largely attributed to the preservation of structural and biochemical cues—such as degradation products and matrix-bound nanovesicles (MBV). These cues influence immune cell behavior and support the transition from inflammation to resolution and functional tissue regeneration. However, the immunomodulatory properties of ECM bioscaffolds are dependent on the source tissue and, critically, on the methods used for decellularization. Inadequate removal of cellular components or the presence of residual chemicals can shift the host response towards a pro-inflammatory, non-constructive phenotype, ultimately compromising therapeutic outcomes. This review synthesizes current basic concepts on the innate immune response to ECM bioscaffolds, with particular attention to the inflammatory, proliferative, and remodeling phases following implantation. We explore how specific ECM features shape these responses and distinguish between pro-remodeling and pro-inflammatory outcomes. Additionally, we examine the impact of manufacturing practices and quality control on the preservation of ECM bioactivity. These insights challenge the conventional classification of ECM bioscaffolds as medical devices and support their recognition as biologically active materials with distinct immunoregulatory potential. A deeper understanding of these properties is critical for optimizing clinical applications and guiding the development of updated regulatory frameworks in regenerative medicine.

Abstract Bacterial vesicles have emerged as therapeutic drug candidates to treat a wide range of diseases, including cancer. However, critical concerns remain regarding their safety, in view of inducing systemic inflammation. To address this, semi‐synthetic bacterial vesicles (SyBV) have recently been developed, directly derived from bacterial cell membranes. These vesicles have reduced toxicity but retained immunomodulatory ability, which is important for immuno‐oncology purposes. In this study, the manufacturing process of SyBV has been further refined to meet Good Manufacturing Practice (GMP) standards. Multiple steps are reconsidered in the optimized method, including adaptation of centrifugation steps, benzonase treatment, and elimination of sonication steps, thereby producing a new set of optimized SyBV, designated as SyBVOpti. Similarly to SyBV isolated using the previous protocol, SyBVOpti do not activate macrophages but stimulate dendritic cells to produce IL‐12 in a dose‐dependent manner—moreover, these vesicles attenuate tumor growth in vivo in a model of malignant melanoma. Further, the optimized process applies to a GMP‐compatible Escherichia coli source for producing SyBVOpti under GMP conditions. Collectively, these findings describe a reliable technique for large‐scale production of SyBV with preserved therapeutic potential, allowing GMP manufacturing and translation of these bioactive vesicles into clinical practice.

Abstract Cyclic peptides are promising therapeutics, but their flexible docking remains challenging. We present a protocol based on the well-established CABS-dock method, enhanced with cyclic restraints and Rosetta refinement. The approach was evaluated on 38 benchmark complexes previously used in other docking method studies. While selecting the truly best model remains difficult, near-native solutions are frequently sampled. CABS-dock offers global, unbiased docking without prior binding site knowledge, making it valuable for pose generation, structural ensemble modeling, and integration into AI-driven peptide–protein docking workflows.

Abstract With several well-known large-volume subcutaneous (SC) oncology drugs, such as daratumumab, trastuzumab, rituximab, nivolumab, and pembrolizumab, either approved or in trials, understanding how administration volume impacts drug development is critical. Patients generally prefer SC over intravenous administration. However, SC adoption is often limited by the perception that delivery volumes must stay below 3 milliliters without a permeation enhancer. This misconception hampers SC oncology drug prioritization and stifles innovation. To better understand these challenges, a survey was conducted to investigate decision-making processes surrounding large-volume SC oncology drug candidates. The online survey, conducted November 20-26, 2024, targeted 80 drug development professionals with SC drug development experience equally representing new product planning, drug formulation, combination products, and clinical development. Of the participants, 43 primarily worked in oncology. The 23-item survey explored perceptions of SC volume thresholds, factors influencing the deprioritization of oncology drug candidates, the potential of large-volume delivery solutions in oncology, and how organizations track and revisit deprioritized candidates. The oncology respondents stated that 32% of drugs are abandoned due to challenges with drug volume. The most common reason for abandonment of the asset was lack of experience with large-volume formulations or devices. Similarly, the respondents stated that 29.5% of drugs are deprioritized due to difficulties in reducing subcutaneous drug volumes. When it came to deprioritizing assets, the primary reason was that large-volume drugs are only pursued if they address a unique unmet need regardless of the competitive landscape. Participants rated the commercial potential of these abandoned or deprioritized drugs at a 3 on a scale of 1-4 (1 = low potential, 4 = high potential) indicating that these drugs had worthwhile commercial potential. Fortunately, participants ranked the organizations willingness to revisit these drug candidates at a 3 on a scale of 1-4 (1 = not open at all, 4 = extremely open). The results suggest that oncology molecules with worthwhile commercial potential remain unexplored within companies due to misconceptions around large-volume SC delivery, with a high willingness to revisit these candidates. Citation Format: Mehul Desai,1 Omar Rahman,1 Beate Bittner,2 Wei Chen,3 Maxwell Koobatian,4 Deepa Rajagopalan5. Insights from drug development experts: Surveying challenges and outcomes for large-volume subcutaneous oncology drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1855.

Abstract Comparative studies of salamander limbs and fish fins may reveal shared components of an ancestral vertebrate appendage regeneration toolkit. However, traditional fish models, such as zebrafish, regenerate only dermal fin rays, which are neither homologous to limbs nor as cellularly complex. To address this, we employed a multi-omics approach leveraging the regenerative capacity ofPolypterus senegalus, a fish species capable of full fin regeneration. Single-nucleus RNA-sequencing revealed extensive erythrocyte and myeloid cell infiltration duringPolypterusfin regeneration. Spatial transcriptomics identified key cellular contributors to the blastema, while cross-species comparisons with axolotl limbs uncovered both common and divergent expression patterns of wound epidermis and blastema markers. Fin amputation triggered a conserved injury response, including sequential activation of pro- and anti-inflammatory programs and DNA damage repair in cycling cells. Hypoxia responses involvedhif1amodulation, consistent with other regenerative models, but uniquely included the expression ofhif4ain erythrocytes and the upregulation of a myoglobin gene in the wound epidermis. Furthermore, genome-wide chromatin profiling identified candidate regeneration-responsive elements. Together, these findings establishPolypterusas a powerful comparative model for identifying shared and derived mechanisms of limb and fin regeneration.