Abstract Background CEACAM5 and CDH17 are highly co-expressed in certain solid tumors including colorectal cancer (CRC). Current single-target based immunotherapies, such as antibody-drug conjugates (ADC) and T cell engagers (TCE), have shown promising efficacy in clinical trials. In this study, we generated a novel tri-specific TCE (tsTCE) targeting CEACAM5, CDH17, and CD3, aiming to enhance tumor-targeting selectivity and therapeutic efficacy. Methods Various TCE molecule formats, including 1+1 monovalent (CEACAM5 or CDH17 x CD3), 2+1 bivalent or biparatopic (CEACAM5 x CEACAM5 x CD3, CDH17 x CDH17 x CD3) and 1+1+1 tsTCEs (CEACAM5 x CDH17 x CD3) were generated. A lead tsTCE molecule was selected and investigated by antigen binding affinity, cell binding, T-cell-dependent cellular cytotoxicity, and T-cell activation in vitro. In addition, its’ in vivo antitumor efficacy were evaluated in subcutaneous HT55, SW403, and LS174T tumor xenografts in NCG mice engrafted with human PBMCs. Results The CEACAM5 x CDH17 x CD3 tsTCE (tsTCE3) possesses a moderate binding affinity to human/cynomolgus CEACAM5 (Kd: 51.0 nM / 109 nM), CDH17 (Kd: 42.8 nM / 9.26 nM), and human CD3ε (Kd: 51.9 nM). The tsTCE3 effectively kills tumor cells with variable expression levels of CEACAM5 and/or CDH17 and induces significant tumor growth inhibition in multiple CDX models. In the CEACAM5-high / CDH17-high HT55 tumor model, tsTCE3 was more efficacious than cibisatamab analogue and a 2+1 CEACAM5 x CEACAM5 x CD3 bivalent bispecific TCE, whereas in the LS174T tumor model (CEACAM5-high / CDH17-low) tsTCE3 was more potent than a 2+1 CDH17 x CDH17 x CD3 biparatopic TCE molecule. Notably tsTCE3 elicited minimal cytokine release in HT55 xenograft model. Conclusions Our tsTCE3 exhibits potent in vitro tumor cell killing and superior in vivo efficacy with a favorable safety profile, supporting its potential as a promising therapeutic agent for the treatment of CEACAM5 / CDH17 positive solid tumors.
Background:Epidermal growth factor receptor (EGFR)-targeted near-infrared photoimmunotherapy (NIR-PIT) is an emerging drug-device modality that enables spatially selective tumor cell killing through antibody-photoabsorber conjugation and localized light activation. Although clinical translation has advanced in selected settings, particularly head and neck squamous cell carcinoma (HNSCC), barriers and enablers influencing broader implementation across solid tumors have not been systematically mapped. This review focuses specifically on the IR700 (IRDye700DX)-based conjugate cetuximab sarotalocan (RM-1929/ASP-1929), which is the only NIR-PIT construct to have advanced into clinical use, while acknowledging the broader and rapidly expanding photoimmunotherapy landscape. Objective:To synthesize and characterize the biological, technical, clinical, and system-level factors that facilitate or hinder the clinical translation of EGFR-targeted NIR-PIT across EGFR-expressing advanced solid tumors. Methods:A scoping review was conducted in accordance with PRISMA-ScR guidelines. Comprehensive searches of PubMed, Embase, Scopus, ClinicalTrials.gov, and the Cochrane Library identified 28 eligible studies, including preclinical investigations, early-phase clinical trials, observational studies, and case reports or series. Data were charted and synthesized thematically, with study quality appraised to inform interpretation. Results:Preclinical studies consistently demonstrated light-dependent, EGFR-selective cytotoxicity and identified non-linear dose-response relationships that support standardized light dosing strategies. Clinical translation has been most successful in recurrent or unresectable HNSCC, facilitated by tumor accessibility, multidisciplinary workflows, and regulatory approval in Japan. Technical enablers included endoscopic and navigation-assisted light delivery, real-time fluorescence imaging, and the feasibility of repeat treatment without cumulative toxicity. Key barriers included limited light penetration, anatomical constraints, reliance on specialized equipment, and restricted applicability to deep-seated or diffuse disease. Although most adverse events were localized and manageable, rare but serious complications reported across multiple case studies represent an important translational challenge. Evidence outside HNSCC remains sparse and is largely confined to preclinical models. Conclusions:EGFR-targeted NIR-PIT has achieved meaningful clinical translation within a narrow but well-defined therapeutic niche, supported by strong mechanistic rationale and growing real-world experience. Broader adoption is constrained by biological, technical, regulatory and system-level barriers, as well as gaps in comparative effectiveness, long-term outcomes, and health-economic evidence. Addressing these challenges through targeted clinical trials, improved patient selection, and implementation-focused research will be essential to advance NIR-PIT toward wider clinical integration.
Abstract Background Tumor heterogeneity has been identified as a major roadblock for cancer immunotherapy. To overcome this, universal effector cell engagers with interchangeable tumor-targeting adaptors have been developed. Current concepts in this field consist of antibody-based adaptors. Small-molecule (SM) ligands, on the other hand, infiltrate tissues rapidly, have short half-lives, and are potentially orally available. Therefore, we hypothesized that utilizing SM adaptors, combined with effector antibodies, could represent an attractive off-the-shelf therapy. Methods Here, we introduce the development of target-agnostic, small-molecule-guided hapten- and T cell-bispecific (TCB) antibodies with high affinity between the adaptor-effector pair. Specifically, we designed SM adaptors based on known tumor-targeting ligands with specificity to the antigens folate receptor 1 (FOLR1), prostate-specific membrane antigen (PSMA) and carbonic anhydrase IX (CAIX), and conjugated them to Ca2+-loaded 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM) as a hapten. As an effector antibody, we designed DOTAM-specific T cell bispecific antibodies (DOTAM-TCB) with femtomolar (fM) affinity for the hapten. Results In vitro experiments showed in-solution assembly of stable adaptor-effector complexes. Additionally, T cell-mediated tumor killing was initiated by the universal DOTAM-TCB when combined with adaptors across various tumor cells. Ex vivo study of CAIX-directed Ca-DOTAM-acetazolamide (Ca-DOTAM-AAZ) with DOTAM-TCB showed potent activation of T cells against murine-engrafted human HT-29 tumor. Conclusions The studies described here demonstrate proof-of-concept for using hapten-containing small molecules as adaptors for effective universal T cell engager-based cancer immunotherapy.
Background:Gastric cancer (GC) remains a major cause of cancer mortality worldwide, particularly in metastatic stages. N-formyl peptide receptor 2 (FPR2), a class A G protein-coupled receptor activated by the Helicobacter pylori-derived peptide Hp(2-20), promotes GC progression by stimulating oncogenic signaling pathways. However, no clinically applicable agents selectively targeting FPR2 have been developed. Methods:We generated fully human antagonistic antibodies targeting FPR2 using a two-step discovery strategy. A CDR-H3-focused synthetic single-chain variable fragment library was screened against an extracellular loop 3 (ECL3) peptide, followed by light-chain engineering to confer dual engagement of extracellular loop 2 (ECL2). Results:The engineered antibody exhibited dose-dependent binding to both ECL2 and ECL3 peptides, selectively recognized FPR2 over other FPR family members, and showed nanomolar binding to FPR2-expressing cancer cells. The antibody inhibited Hp(2-20)-induced calcium mobilization and suppressed migration and invasion of Adenocarcinoma Gastric (AGS) gastric cancer cells. Structural modeling indicated a lid-like binding mode occluding the orthosteric pocket. Conclusions:We developed a fully human antagonistic antibody that selectively binds and inhibits FPR2 through dual extracellular-loop engagement. The antibody potentially suppresses FPR2-driven signaling and invasive behaviors in gastric cancer cells, supporting its development for therapeutic application. Further assessment of its efficacy in vivo is warranted.
Background:Cysteine-engineered monoclonal antibodies (mAbs), or ThiomAb, enable site-specific conjugation and form the basis of several next-generation antibody-drug conjugates (ADCs). However, the presence of intentionally unpaired cysteine residues introduces reactive free thiols that pose challenges for manufacturing consistency, including variable capping by cysteine/glutathione or uncapped free thiol during cell culture. This increases the charge heterogeneity of protein and may compromise stability due to increased risk of oxidation, disulfide scrambling, or aggregation. Methods:To address these risks, we developed an on-column capping strategy, in which the antibody is immobilized on chromatography resin during the capture step and subjected to selective thiol masking under optimized redox conditions. This in-process approach allows for early and efficient stabilization of engineered thiol sites prior to polishing and formulation. High-throughput condition screening and analytical techniques-including mass spectrometry and charge variant profiling-were employed to fine-tune buffer conditions and understand on-column redox chemistry. Results:The optimized thiol protection conditions substantially reduce the population of free thiols, resulting in more consistent charge profiles and improved stability during downstream processing and storage. Conclusion:This strategy offers a scalable and platform-adaptable solution for enhancing the manufacturability and alternative control strategy of ThiomAb intermediates used in site-specific ADC processes.
Background:Mesothelin (MSLN) is a therapeutic target for antibody-drug conjugates (ADCs) due to its tumor-selective overexpression. However, soluble MSLN (sMSLN) in circulation compromises efficacy by acting as a decoy. RC88 is a clinical-stage ADC composed of a humanized anti-MSLN antibody conjugated to a monomethyl auristatin E (MMAE) payload. This study elucidates the unique binding mechanism that allows RC88 to maintain superior efficacy despite sMSLN interference. Methods:We characterized the RC88-MSLN interaction via structural analysis and mutagenesis. The impact of sMSLN on binding was assessed using competitive cellular assays. In vitro internalization kinetics and cytotoxicity were evaluated to determine the therapeutic potential. Results:RC88 targets a high-affinity N-terminal epitope overlapping the MUC16/CA125-binding site, competitively inhibiting this interaction. Notably, due to the structural flexibility of MSLN, RC88 also engages a secondary, low affinity juxtamembrane epitope at the C-terminus. This conformational binding confers resistance to sMSLN interference and enhanced tumor cell retention. In vitro, RC88-ADC maintained near-complete cytotoxic potency even in the presence of physiologically relevant sMSLN concentrations. In vivo, RC88 suppressed tumor growth in ovarian cancer xenografts. Conclusion:Our findings demonstrate that RC88 utilizes a novel conformational binding strategy to overcome the limitations of soluble antigen interference. These findings provide a mechanistic rationale for the encouraging clinical activity of RC88 and suggest that conformational epitope targeting represents a promising strategy for overcoming sMSLN interference in MSLN-positive cancers.
Abstract Monoclonal antibodies (mAbs), engineered to recognize disease-associated antigens, are central to cancer immunotherapy due to their ability to block or eliminate malignant cells by engaging immune effector mechanisms. Yet many tumors evade or suppress these responses, and many patients fail to achieve durable clinical benefit, highlighting the need to optimize antibody-based strategies. A critical determinant of mAb activity is antibody isotype, which governs interactions with Fc receptors, complement pathways, and immune effector cells. While most approved therapeutics employ human IgG1 for its robust immune-stimulating capacity, growing evidence suggests that deliberate, context-dependent isotype selection could improve therapeutic efficacy. Here, we review the structural and functional properties of human IgG and IgA subclasses, with a focus on anti-CD20 rituximab in two- and three-dimensional models of human B-cell lymphoma. We further discuss how antigen density, tumor architecture, and host variables influence antibody-mediated effector functions. Together, these considerations highlight opportunities to refine antibody isotype selection for more individualized cancer immunotherapy.
Background:CD93 is an emerging immune checkpoint. Blocking its interaction with insulin-like growth factor-binding protein-7 (IGFBP7) restores antitumor immunity, yet discovering high-affinity antibodies against this specific functional epitope remains a formidable challenge for traditional methods. Methods:We employed a proprietary generative artificial intelligence (AI) model, GeoFlow, to de novo design antibodies targeting the predicted binding epitope of a reference antibody 7F3. A library constructed from 105 sequences was screened via phage display, and the lead candidate, 7-4_com10, was validated through biolayer interferometry, flow cytometry, and in vivo efficacy studies. Results:To dissect the contribution of endothelial CD93 to immunotherapy resistance, we utilized an endothelial-specific conditional knockout model in MB49 bladder cancer, where the loss of endothelial CD93 significantly sensitized tumors to PD-1 inhibition. The genetic findings identified endothelial CD93 as a fundamental driver of immunotherapy resistance and provided the rationale for our GeoFlow generative AI platform, which was employed to de novo design ~105 candidates targeting the IGFBP7-binding interface on CD93. Our lead candidate, 7-4_com10, demonstrated superior affinity and more potent functional blockade of the CD93-IGFBP7 axis compared to the reference 7F3. Crucially, 7-4_com10 sensitized H22 solid tumors to anti-PD-1 therapy in vivo, which confirmed that 7-4_com10 successfully targeted the identified immunosuppressive axis and pharmacologically phenocopied the benefits of genetic endothelial ablation. Conclusions:This study validated generative AI for designing epitope-specific antibodies with superior potency, offering a robust candidate for next-generation immunotherapy.
Immune cell engagers have emerged as a powerful class of multi-specific therapeutics that redirect immune effector cells toward tumor cells to induce targeted cytotoxicity. Among these, T cell engagers (TCEs) represent the most clinically advanced platform, with multiple approved agents demonstrating substantial efficacy in hematologic malignancies. However, their broader application remains limited by systemic toxicities, antigen heterogeneity, and reduced efficacy in solid tumors. To address these challenges, next-generation TCEs are being engineered with improved selectivity, and optimized signaling properties. In parallel, increasing attention has shifted toward engaging alternative immune effectors, including γδ T cells, natural killer cells, and myeloid populations, which provide complementary mechanisms of tumor recognition and immune modulation. In this Review, we summarize the biological principles underlying T or other immune cell engagers, highlight emerging alternative platforms, and discuss evolving engineering strategies that are shaping the future of programmable cancer immunotherapy.
Antibody-drug conjugates (ADCs) are a rapidly growing class of targeted therapeutics designed to deliver potent payloads while minimizing systemic toxicity. Despite significant progress, all approved ADCs are administered intravenously (IV). In contrast, subcutaneous (SC) delivery is well established for antibodies, offering shorter administration time, improved convenience, and the potential for outpatient or home-based treatment. Extending SC delivery to ADCs is therefore attractive but introduces unique challenges related to structural complexity, linker-payload instability, and cytotoxic payloads. This review evaluates the feasibility of subcutaneously administered ADCs by integrating insights from pharmacokinetic and pharmacodynamic studies, emerging clinical data, and advances in molecular design, formulation, and delivery technologies. Key challenges include limited clinical comparisons between IV and SC routes, uncertain bioavailability and tumor exposure, linker-payload instability, injection-site reactions, and high-concentration formulation constraints. Enabling strategies, such as rational molecular design, hyaluronidase-enabled delivery, polymer-based systems, and conversion of IV ADCs to SC dosing, are discussed. Recent advances, exemplified by JSKN033, demonstrate the feasibility of high-concentration liquid co-formulation approaches for SC administration and may represent a future direction. Evidence across oncology and non-oncology programs suggests that while SC delivery is achievable, success is highly molecule dependent. Overall, transitioning ADCs from IV to SC requires case-by-case evaluation and coordinated innovation across rational molecular design, formulation, and nonclinical and clinical development to realize the full patient-centric benefits of this approach.
The rapid evolution of antibody-based biologics is transforming the management of chronic conditions and driving innovation in drug-delivery devices and container closure systems (CCS). Combination therapy enabled through coformulation or coadministration offers the potential for synergistic outcomes that are not achievable with single-drug products. This review examines development pathways and emerging strategies for delivery devices and CCS that support antibody-biologic coformulations and coadministration, from clinical trials through commercialization. We evaluate established technologies with an emphasis on design and delivery mechanisms, and on key technical considerations, including compatibility with dual-molecule formulations, protein-protein interactions, sterilization approaches, container closure integrity, and human factors in CCS and device design. We also highlight emerging technologies and future opportunities for advancement. Overall, this review provides insights into the challenges and innovations shaping CCS and delivery systems for co-formulated or co-administered antibody biologics.
Abstract Background As a clinical vaccine platform, lipid nanoparticle (LNP)-formulated mRNA has demonstrated potent and broad antibody responses, prompting speculation about its potential for antibody discovery. Membrane proteins remain among the most challenging targets for antibody development, highlighting the urgent need for technologies that preserve their native conformation during immunization and screening. Nanobodies (VHHs), the single-domain fragments of heavy-chain-only antibodies naturally found in camelids, offer unique advantages over conventional antibodies, including high solubility, stability, and the ability to access cryptic or membrane-proximal epitopes. Methods Here, we report the first demonstration of target specific VHH discovery in a llama using an mRNA–LNP immunization platform. A llama was immunized with LNPs encapsulating mRNA encoding the severe acute respiratory syndrome coronavirus 2 spike protein, a well-characterized model antigen. The resulting immune VHH library was screened using a cell-based panning strategy that maintains antigen conformation and enables recovery of binders recognizing native epitopes. Results This approach yielded multiple high-affinity spike-specific VHHs, including two clones that effectively blocked the spike–angiotensin-converting enzyme 2 interaction in a surrogate cell-based assay. Conclusions These findings demonstrate the feasibility of mRNA–LNP immunization as a rapid and efficient method for isolating target specific, functionally promising VHHs from camelids. When combined with conformationally relevant screening, this platform may provide a versatile and accelerated route for generating high-quality VHHs against complex or recalcitrant targets, particularly membrane proteins, thereby expanding the potential of VHH-based therapeutics and diagnostics.
Abstract Background Helicobacter pylori infection is a global health concern associated with gastritis, peptic ulcer disease, and gastric cancer. With increasing antibiotic resistance, egg yolk immunoglobulin (IgY) has emerged as a promising candidate owing to its stability and pathogen-neutralizing effects. This clinical trial evaluated the efficacy of IgY-enriched egg yolk powder (anti-H. pylori IgY) in eliminating the bacterium and improving clinical symptoms as an adjunctive. Methods In this randomized, double-blind trial, 60 patients with confirmed H. pylori infection received a 14-day standard therapy regimen (omeprazole, amoxicillin, clarithromycin) supplemented with IgY-enriched egg yolk powder (intervention) or plain egg yolk powder (control). The primary outcome was bacterial eradication, assessed by the 14C-urea breath test (14C-UBT) performed 4 weeks after completion of therapy (i.e. 6 weeks from baseline). Secondary outcomes included symptom improvement (abdominal pain, nausea, bloating, and anorexia) measured using the Gastrointestinal Symptom Rating Scale (GSRS), via telephone interviews conducted three times weekly for 3 weeks. Data were analyzed using analysis of variance (ANOVA) and t-tests (SPSS v24). Results Baseline demographic characteristics were homogeneous. The median UBT value in the intervention group decreased from 77 to 11, whereas that of the control group remained unchanged (median = 55). Bacterial eradication was achieved in 33.3% of IgY recipients versus 13.3% of controls (difference, 20%; p = 0.241). IgY was well tolerated, with no treatment-related adverse events observed in the intervention group. Conclusion Clinical symptoms showed trends toward faster improvement, but these did not reach statistical significance. IgY may be a promising safe adjunct for the treatment of H. pylori infection. Clinical Trial Registration NCT06973889.
Therapeutic peptides have become an important class of pharmaceuticals, driven in part by the clinical success of long-acting incretin therapies. Despite their potency and specificity, peptides are intrinsically limited by rapid proteolytic degradation and renal clearance, resulting in short plasma half-lives and frequent dosing. Antibody-peptide conjugates represent an emerging class of biotherapeutics that combine the long half-life and target specificity of monoclonal antibodies (mAbs) with the chemical tunability and pharmacological activity of peptides. Unlike fusion proteins, antibody-peptide conjugates decouple peptide synthesis from antibody expression, enabling incorporation of non-proteinogenic amino acids, cyclic structures, and other chemical features that enhance peptide stability, potency, and half-life. This review examines the biological rationale, molecular design principles, and enabling technologies underlying antibody-peptide conjugates development, using the GIPR antagonist/GLP-1R agonist conjugate maridebart cafraglutide (AMG133) as a central case study. We illustrate how antibody-peptide conjugates can integrate complementary mechanisms of action into a single molecular entity and achieve sustained exposure profiles that are difficult to realize with fusion-based approaches. The discussion is extended to antibody-peptide conjugates programs across metabolic disease, oncology, neurology, pain, ocular, and infectious disease, highlighting recurring design themes such as half-life extension, targeted delivery, receptor-mediated internalization, and synergistic activity. Collectively, antibody-peptide conjugates represent a biology-driven and strategically important therapeutic modality that bridges antibody engineering and peptide medicinal chemistry. When guided by strong mechanistic rationale, antibody-peptide conjugates offer a powerful solution for therapeutic challenges that cannot be adequately addressed by peptides or antibodies alone.
Backgroud:Nephrotic syndrome (NS) is a major cause of end-stage renal disease. Treating NS relies on immunosuppressants, which have numerous side effects. Therefore, there is an urgent need to identify effective and safe alternative treatments for NS. Angiopoietin-like protein 3 (ANGPTL3) exacerbates proteinuria, whereas interleukin (IL)-22 has a reparative effect on renal cells. Methods:In the present study, we developed a bifunctional anti-ANGPTL3/IL-22 fusion protein and validated its efficacy in an adriamycin-induced nephropathy in mice. Results:The fusion protein significantly decreased the urinary albumin-to-creatinine ratio, serum creatinine, blood urea nitrogen, and total cholesterol levels while increasing serum albumin levels. Pathological renal damage was also alleviated. These therapeutic effects were accompanied by the preservation of mitochondrial integrity, reduced apoptosis, and inhibited autophagy. Finally, we humanized the fusion protein to facilitate its potential clinical translation. Conclusions:In conclusion, our results revealed that the anti-ANGPTL3/IL-22 bifunctional fusion protein ameliorates NS by protecting mitochondria, inhibiting apoptosis, and suppressing autophagy, highlighting a novel therapeutic approach for NS.
Abstract In 2025, the 50th anniversary of the invention of hybridoma technology, the United States Food and Drug Administration (US FDA) approved a total of 44 new molecular entities (NMEs), of which 10 were therapeutic antibody-based molecules. Additionally, the US FDA approved 31 small molecules (of which one was a peptide), and 3 new nonantibody therapeutic proteins, but no new chimeric antigen receptor (CAR)-T-cell therapeutics. Of the new antibody-based biologics, two were antibody–drug conjugates, one was a T-cell engager–bispecific antibody, two were Fc-modified, half-life extended antibodies, and two targeted the complex coagulation pathway. The eighth novel anti-PD-1 antibody was also approved, along with the newest entry of biologics targeting the neonatal Fc receptor (FcRn). Additionally, two new entries in which hyaluronidase was coformulated with an antibody to provide rapid subcutaneous dosing were FDA-approved. Finally, the first antibody-like scaffold-fusion protein was approved that will compete in an antibody-rich environment.
Background:In vitro display technologies, especially phage and yeast display, are revolutionary tools in antibody discovery, enabling high-throughput screening and engineering. Previous efforts to integrate phage and yeast display for synergistic antibody discovery have predominantly focused on scFvs and sdAbs. Methods:Here, we developed an integrated phage-yeast display platform for efficient Fab antibody discovery. We transferred paired Fab libraries from phage output into yeast display vectors while preserving VH-VL pairings with the engineered compatible Fab display vectors that we developed for both phage and yeast. Specifically, we identified restriction enzyme sites that are rare in antibody variable regions and designed novel signal peptides compatible with these sites for Fab display. These signal peptides performed at least as well as commonly used benchmarks (such as PelB or StII for phage display, and AGA2 or MFAL1 for yeast display) in bio-panning experiments. Our modular vector architecture supports straightforward library shuttling across mouse-derived, fully human, and naïve Fab libraries. Results:Using a mouse immune Fab library, we applied our integrated platform to isolate antibodies against a soluble protein. Following two rounds of liquid-phase phage display and four rounds of fluorescence-activated cell sorting combined with a yeast-based receptor-blocking screening method developed herein, we successfully isolated antigen-specific Fabs with sub-nanomolar affinities and potent receptor-blocking activity. Conclusions:Our integrated approach leverages the capacity of phage display to screen large Fab libraries while harnessing the strength of yeast display for functional screening, exemplified here by the successful identification of receptor-blocking antibodies, thereby streamlining the discovery of high-quality Fab candidates.
Background:Trophoblast cell surface antigen 2 (TROP2) is frequently overexpressed in various cancers and is associated with poor prognosis. While TROP2-targeted antibody-drug conjugates (ADCs), such as sacituzumab govitecan, sacituzumab tirumotecan, and datopotamab deruxtecan (DXD), achieved clinical success, the mechanisms underlying resistance to these therapies remain incompletely understood. Consequently, the full therapeutic potential of TROP2 ADCs has yet to be comprehended. Methods:We developed R7059-DXD, a novel TROP2-targeted ADC featuring an antibody that binds an epitope that is distinct from those recognized by sacituzumab and datopotamab. The structural basis of epitope recognition was elucidated using cryo-electron microscopy (cryo-EM), complemented by binding affinity analyses across multiple surface-expressed TROP2 variants. Functional activity was systematically evaluated through in vitro antibody-dependent cellular cytotoxicity (ADCC) assays, in vitro ADC-mediated killing assays, and in vivo efficacy studies in tumor models, including resistant lines. Results:The R7059 antibody exhibited high binding affinity across multiple TROP2 variants, addressing a key resistance mechanism. The cryo-EM structure analysis revealed that the R7059 epitope lies in the N-terminal domain of TROP2 and is distinct from the sacituzumab and datopotamab epitopes. Functionally, R7059 demonstrated potent antitumor activity through both ADCC-dependent and independent mechanisms, suggesting additional signaling effects. The R7059 ADC demonstrated robust efficacy in multiple preclinical tumor models, including those resistant to approved TROP2 ADCs. Conclusions:R7059-DXD is a differentiated, epitope-distinct TROP2-targeted ADC with the potential to overcome resistance to existing therapies. These findings support its further clinical development as a novel treatment for TROP2-expressing malignancies, including in patients previously treated with sacituzumab- or datopotamab-based regimens.
Precise control over how and where small-molecule drugs are covalently attached to monoclonal antibodies is increasingly vital for creating consistently efficacious and safe antibody-drug conjugates (ADCs) as powerful targeted therapies. Enzymatic conjugation methods have gained considerable attention for their abilities to efficiently install payloads at defined locations under mild and predictable conditions. This work provides a comprehensive review of current enzymatic technologies for site-specific ADC construction, organized by the biological origin of enzymes. Among various enzyme-based conjugating approaches, a special focus is given to the emerging ADP-ribosyl cyclase-enabled ADC (ARC-ADC) platform. By utilizing genetically fused CD38, a member of the ARC family, together with its dinucleotide-derived inhibitor, site-specific ADCs with defined drug-to-antibody ratios in varied formats could be facilely produced with demonstrated efficacy and specificity in preclinical models of different types of cancer. Unlike most enzymatic methods requiring recognition tags or external catalytic steps, ARC-ADC provides a fully integrated, modular strategy for streamlined ADC discovery and development.
Background:Cadherin-17 (CDH17) is a cell-adhesion molecule physiologically expressed along the intestinal epithelial tight junctions. Aberrant overexpression of CDH17 in gastrointestinal (GI) cancers promotes tumor growth and metastasis and is associated with poor patient prognosis. Due to its restricted expression in normal tissues and strong association with malignancy, CDH17 represents an emerging therapeutic target for GI tract cancers. Methods:A high-affinity anti-CDH17 monoclonal antibody (TAVO307) was generated and conjugated with auristatin-derived cytotoxic payloads to obtain CDH17-directed antibody-drug conjugates (ADCs). In parallel, a VHH antibody capable of activating γδ T cell receptors from both Vδ1 and Vδ2 subsets was identified and combined with the anti-CDH17 antibody to generate CDH17-targeted T cell engager (TCE) to recruit γδ T cells, which are abundant in the intestinal mucosa and play a critical role in tumor immunosurveillance. An attenuated interleukin-15 (IL-15) fused with the IL-15 receptor α sushi domain was further incorporated on TCE to enhance γδ T cell expansion and activation. Results:CDH17-based ADCs exhibited potent and selective cytotoxicity in multiple GI cancer cell lines and significant tumor regression in xenograft models. The CDH17 γδ TCE induced tumor antigen-dependent γδ T cell degranulation and redirected both Vδ1 and Vδ2 T cells to effectively kill CDH17-expressing cancer cells. IL-15 fusion further augmented γδ T cell expansion and activation. Conclusions:Both CDH17-targeted ADCs and γδ TCEs demonstrated promising potency and efficacy to control GI cancers. They could offer complementary therapeutic options that could be used in combination therapy.
