In mammalian organisms, native tissue function depends on precise spatial organization down to the cellular level. Reconstituting tissue architectures in 2Din vitroplatforms can provide a means to study direct and indirect cell-cell interactions in a variety of tissue contexts while remaining compatible with high-throughput assays and high-resolution live imaging. We combine cost-effective stereolithography leveraging 3D printing with replica molding to stencil spatially defined, multicellular culture systems with sub-millimeter resolution onto planar substrates. The system is designed for ease of use, requires no complex fabrication setups and scales readily to 96-well plates. Sequential stencil application and removal under a biosafety cabinet enables controlled positioning of multiple cell types and supports the maturation of tissue assemblies. We demonstrate the utility of this stencil-based patterning strategy in three applications. First, we employ a combination of two circular stencils to recreate a structural feature characteristic of the tumor microenvironment of solid tumors: the encapsulation of colorectal cancer cells by cancer-associated fibroblast cells. Resulting cell patternings recapitulate key aspects of native tissue dynamics of the densely packed tumor tissues, in which cancer-associated fibroblast cells actively compress the cancer cells and confer targeted therapy resistance. Second, we probe an engineered synNotch-based signaling system in patterned cell patches that mimics morphogen gradient formation, where GFP-secreting sender cells generate a ligand-dependent gradient. Third, we recapitulate the characteristic crypt-villus architecture of the mammalian intestine by patterning intestinal organoids within a stencil-restricted crypt region and allowing differentiating cells to collectively migrate along a designed villus axis. The presented strategy allows for rebuilding multicellular tissue architecturesin vitrowith biologically relevant spatial precision for high-throughput drug screenings and dissection of tissue-specific cellular interactions.

Epidermal growth factor receptor (EGFR) is a validated therapeutic target in several human cancers harboring wild-type KRAS. However, intrinsic and acquired resistance to EGFR-targeted antibody therapies such as cetuximab remains a major limitation to achieving broad and durable treatment responses. Extensive clinical and translational studies have shown that resistance frequently arises from missense mutations within the extracellular domain (ECD) of EGFR. In this study, we developed novel antagonistic EGFR antibodies that bind epitopes overlapping but distinct from the cetuximab-binding site while retaining high affinity for all major EGFR ECD escape variants. Antibody binding effectively inhibited EGFR phosphorylation, downstream signaling, and tumor cell proliferation. The antibodies were further engineered into bispecific EGFR × CD3 T-cell engagers (TCEs) with either 1 + 1 or 2 + 1 stoichiometries. In contrast to cetuximab-based TCEs, the newly developed EGFR-directed TCEs efficiently induced T cell-mediated cytotoxicity against tumor cells expressing wild-type EGFR as well as the clinically relevant ECD escape variants S492R and G465R. Anti-tumor activity was additionally demonstrated in an EGFR-expressing CT-26 syngeneic tumor model in vivo. Collectively, these findings define a promising therapeutic strategy to overcome resistance to current EGFR-targeted therapies and provide a strong rationale for the development of next-generation T-cell - redirecting therapies in patients with EGFR-positive malignancies.

Abstract Cancer-associated fibroblasts (CAFs), the main stromal cell population within the tumor microenvironment (TME), play a pivotal role in cancer progression, metastasis, and immune evasion. To target this compartment, two antibody-drug conjugates (ADCs), OMTX705 and OMTX105, were developed against fibroblast activation protein (FAP) expressed on CAFs. Both share the same human IgG1 antibody backbone (OMTX005) but differ in linker-payload chemistry: OMTX705 carries the cytolysin A1B1, whereas OMTX105 includes monomethyl auristatin E (MMAE). Despite similar drug-antibody ratios (DAR ≈ 4), their distinct vc-PABA-derived linkers confer differential stability and intracellular processing characteristics.We performed a comprehensive characterization of their mechanisms of action, including internalization, intracellular trafficking, and bystander cytotoxicity, in vitro and in vivo, as well as plasma stability and maximum tolerated dose (MTD) analyses to assess their pharmacological and safety profiles. Both ADCs displayed potent antitumor effects but differed markedly in their intracellular fate and pharmacodynamic properties. OMTX105 demonstrated faster processing and stronger bystander killing, leading to higher antitumor activity in murine xenografts; however, its effective dose was close to the MTD in rat, limiting its therapeutic window. In contrast, OMTX705 exhibited slower intracellular processing, resulting in a sustained and controlled payload release. This translated into a longer-lasting effect in vivo, with CAFs acting as a drug reservoir prolonging cytotoxic activity in neighboring tumor cells, while maintaining a superior safety profile.Remarkably, both ADCs exhibited in humanized xenograft mice a shared immunomodulatory activity, promoting infiltration of CD4+ and CD8+ T lymphocytes within the TME—particularly CD8+ cells in treated tumors—suggesting that FAP-targeted ADCs facilitate immune cell recruitment compared to control. This was confirmed in clinical samples from patients treated with OMTX705, currently under clinical development. In these patients, CAF disruption, much lower FAP expression and higher CD8+ and CD4+ cell infiltration was detected in tumor areas co-localizing with the OMTX705 payload signal, providing evidence that OMTX705 enhances local immune activation and supports an antitumor immune response.Overall, these findings reveal both cytotoxic and immunomodulatory functions for FAP-targeted ADCs within the TME and provide molecular mechanistic insights into their bystander effect, emphasizing how differences in linker-payload chemistry critically determine efficacy, durability of response, immune engagement, and tolerability. This study highlights OMTX705 potential as a safer FAP-targeted ADC with long-lasting effect and underlines the importance of biology-driven linker-payload engineering for next-generation stroma-directed ADC anticancer therapies. Citation Format: Atanasio Pandiella, Monica Redondo-Puente, Maria del Carmen Gomez-Garcia, Patricia Gonzalez, Isabel Egaña, Roland Kontermann, Oliver Seifert, Jorge Galino, Sonia Montero-Molina, Jose Luis Hernandez, Juan Daniel Sanjuan, Guillermo Quintas, Ignacio Garcia, Manuel Hidalgo, Laureano Simon, Myriam Fabre. Mechanistic drivers of FAP-targeted ADC performance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5390.

Cost-effective, fluorescence-based biodiagnostic devices have the potential to expand point-of-care (POC) testing in resource-limited settings, thereby creating new opportunities for accessible and decentralised diagnostics. The objective of this study is to investigate the performance of a newly designed and simplified system that uses several cost-effective sensors for use in a fluorescence-based biodiagnostic setup. A collecting setup that includes an elliptical mirror to focus emitted fluorescence onto the semiconductor sensors was designed for an intensity-based readout. The readout was realised by various photodiodes, photoresistors, phototransistors and one multi-pixel photon counter (MPPC). Over a range of fluorophore concentrations using serial dilutions of Rhodamine B (RhB), the limit of detection (LOD) and limit of quantification (LOQ) at system level were evaluated. With the exception of the photodiodes, the system demonstrated promising performance with all sensors. The system using the photoresistors achieved the lowest LOD but showed limited repeatability, while the system using the MPPC and phototransistors exhibited high repeatability. A proof-of-concept demonstrated the feasibility of a photoresistor-based configuration by using a sandwich immunoassay for the detection of the antigen Human Epidermal growth factor Receptor 2 (HER2) (100 nM). While this study has not yet encompassed the full spectrum of clinically relevant concentrations, it has yielded valuable insights to enable further enhancements, without the necessity for highly specialised or costly equipment. The limitations and necessary improvements for further developments are discussed.

Abstract In mammalian organisms, native tissue function depends on precise spatial organization down to the cellular level. Reconstituting tissue architectures in 2D in vitro platforms can provide a means to study direct and indirect cell-cell interactions in a variety of tissue contexts while remaining compatible with high-throughput assays and high-resolution live imaging. We combine cost-effective stereolithography leveraging 3D printing with replica molding to stencil spatially defined, multicellular culture systems with sub-millimeter resolution onto planar substrates. The system is designed for ease of use, requires no complex fabrication setups and scales readily to 96-well plates. Sequential stencil application and removal under a biosafety cabinet enables controlled positioning of multiple cell types and supported the maturation of tissue assemblies. We demonstrate the utility of this stencil-based patterning strategy in three applications. First, we employ a combination of two circular stencils to recreate a structural feature characteristic for the tumor microenvironment of solid tumors: the encapsulation of colorectal cancer cells by cancer-associated fibroblasts. Resulting cell patternings reproduce native tissue dynamics of the densely packed tumor tissues, in which cancer-associated fibroblast cells actively compress the cancer cells and confer targeted therapy resistance. Second, we probe the synthetic, diffusible morphogen system synNotch in patterned cell patches, where GFP-releasing cells generate a ligand-dependent gradient. Third, we recapitulate the characteristic crypt-villus architecture of the mammalian intestine by patterning intestinal organoids within a stencil-restricted crypt region and allowing differentiating cells to collectively migrate along a designed villus axis. The presented strategy allows for rebuilding multicellular tissue architectures in vitro with biologically relevant spatial precision for high-throughput drug screenings and dissection of tissue-specific cellular interactions.

With this status report, we aim to provide a timely snapshot of the protein engineering field as a broad and rapidly advancing discipline that integrates computational, molecular biology, structure-guided, evolutionary, and synthetic approaches to create new and improved proteins with tailored structures and useful functions. The report is organized into eight thematic areas spanning core methodologies and major application domains, including enzymes, therapeutics, detection, synthetic biology, and materials. Contributions from experts across these areas highlight both the historical foundations and recent advances in their respective fields, with particular emphasis on the growing influence of machine learning and artificial intelligence-based methods. Emerging from this broad overview is a central message: protein engineering appears to be entering a golden age, defined by a rapidly accelerating pace of progress, even as significant challenges in design, screening, and real-world application remain. Looking ahead, the continued integration of computational and experimental strategies is poised to further accelerate the impact of protein engineering across an expanding range of economically and societally important sectors, from therapeutics and molecular imaging to diagnostics, plastic recycling, and industrial chemistry.

Trispecific antibodies have emerged as molecules for enhanced cancer immunotherapy by addressing the complexity of cancer cell biology and anti-cancer immune responses. Here, we present a novel approach to generate trispecific antibodies based on the previously developed eIg technology. These trispecific antibodies comprise one Fab and two eFab moieties, fused to obtain an asymmetric eFab-eIg molecule. The design principle employs two different eFab building blocks, characterized by divergent arrangements of heterodimerizing hetEHD2 domains. Specifically, the first (inner) eFab arm comprises the hetEHD2-1 domain in the heavy chain and the corresponding hetEHD2-2 domain in one of the light chains, while in the second eFab (outer) this arrangement is reversed. The feasibility of this approach was demonstrated for a trispecific eFab-eIg T-cell engager (TCE) targeting HER2, HER3, and CD3. Importantly, the trispecific TCE retained binding activity for all three antigens and was capable of recruiting T-cells to HER2 and/or HER3-expressing cancer cells and mediating effective cancer cell killing, as shown in 2D and 3D model systems. Due to the modular architecture, this approach should be suitable to generate trispecific antibodies of any specificity and for a multitude of applications.

ADCs targeting the tumor stroma offer a promising strategy to address tumor heterogeneity. Traditional tumor-targeting ADCs face limitations, as intratumor and interpatient variability in target protein expression can result in incomplete targeting. In contrast, the tumor stroma is more genetically stable and consistent across patients. CAFs, the predominant stromal cell type within the tumor microenvironment, play a critical role in cancer progression and immune evasion in multiple cancers. OMTX705, a clinical-stage ADC, targets FAP-positive CAFs in the tumor microenvironment. OMTX705 presents an optimized and highly stable linker and it is quickly internalized and transported to late endosomal compartments in FAP-expressing cells. Its novel cytolysin-based payload demonstrates strong cell killing activity upon specific ADC internalization in FAP positive cells. Indeed, enhanced cytotoxicity was observed when compared to MMAE, particularly in cells with high PgP expression, offering a potential solution for tumor drug resistance. OMTX705 has demonstrated preclinical robust efficacy, achieving 100% tumor growth inhibition and even regression, in PDX murine models for pancreatic, gastric, ovarian, triple negative breast and lung cancers, with or without humanized immune systems, and showing no significant toxicity. Higher anti-tumoral effect was evidenced over both classical chemotherapy and modern immunotherapy drugs, as a single agent or in combination therapies with Gemcitabine/Abraxane or immune checkpoint inhibitors. OMTX705 even displays superiority compared to other FAP-targeted ADCs bearing different payloads such as deruxtecan in pancreas cancer models. Moreover, it exhibits high plasma stability and excellent safety profile, with no adverse effects observed in non-human primates at doses up to 100 mg/kg. OMTX705 operates through a dual mechanism of action. First, upon ADC internalization, the bystander effect of the payload released from FAP-positive CAFs leads to efficient killing of neighboring tumor cells. This effect has been validated through different experimental techniques in vitro and in preclinical tumor samples. The second mechanism involves immune cell infiltration within the tumor microenvironment, further strengthening its therapeutic potential. This dual effect, combined with a favorable safety profile, positions OMTX705 as a promising candidate among cancer therapeutics, targeting the stroma in solid tumors and enhancing antitumor immune responses. Patricia González, Isabel Egaña, Cristina Ferrer, Roland Kontermann, Oliver Seifert, Klaus Pfizenmaier, Wolfgang Richter, Laureano Simon, Myriam Fabre. OMTX705: A dual-mechanism ADC targeting FAP-positive CAFs, to overcome chemo and immunotherapy resistance in solid tumors [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 6391.

Endoglin (CD105), a TGFb-RII co-factor, overexpressed in tumor microvasculature and endothelial-derived CAFs, has been extensively validated as a therapeutic target for cancer treatment. Endoglin-targeting ADCs have been developed in this work, using a human IgG1 antibody, OMTX003, conjugated to different microtubule inhibitors such as eribulin (OMTX603-3) or MMAE (OMTX103), with DAR values between 4 and 8. Target binding was maintained within subnanomolar values for each ADC, similar to the unconjugated antibody. Internalization was efficient, with 75% intracellular localization within 60 minutes, regardless of the conjugated payload. In vitro activity in Endoglin-expressing HT1080 cells was target- and concentration-dependent, with IC50 values of 6 and 95.5pM, respectively. ADCs were tested for their in vivo anti-tumor efficacy in a gastric cancer PDX murine model. Complete tumor regression was observed. No re-growth occurred up to 40 days after treatment end, showing the lack of resistance to treatment. ADCs were well tolerated with no weight loss. These encouraging results are postulating these anti-ENG ADCs for further development. Isabel Egaña, Patricia González-Callejo, Alvaro González, Cristina Ferrer, Roland Kontermann, Oliver Seifer, Klaus Pfizenmaier, Myriam Fabre. Comparative anti-tumoral efficacy of endoglin-targeting ADCs harbouring different payloads for the treatment of gastric cancer [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 2871.

Efficient delivery of therapeutics to the central nervous system (CNS) is one of the major challenges in treating neurological diseases due to brain barriers, which prevent entry of almost all potential therapeutic agents into the CNS. Targeting receptors that induce receptor-mediated transcytosis (RMT) across brain barriers has long been heralded as a potential solution to this problem, but this approach has yet to deliver clinical improvements for patients. Here, we set out to identify and characterize bivalent antibodies against the transferrin receptor 1 (TfR) as mediators of RMT. We identified the antibody YU904-F06 (hereafter referred to as F06) that showed efficient transcytosis as a bivalent IgG in two independent in vitro models of brain barriers. Despite its high affinity at extracellular pH levels, we determined that F06’s binding to TfR was greatly reduced at lower pH levels expected during endocytic acidification. We postulated, with the support of a validated predictive mathematical model of RMT, that the pH-sensitivity of F06 allowed it to overcome the lysosomal degradation that has been previously reported for high affinity bivalent binders of TfR. Finally, we demonstrated that F06 could mediate the transcytosis of scFvs that target TREM2 or EGFRvIII as potential therapeutic cargos. In conclusion, we present a proof-of-concept antibody and rationale for the design of high affinity bivalent anti-TfR antibodies that effectively induce RMT by exploiting pH-sensitivity in binding.

The development of therapeutics that enhances the regeneration of myelin sheaths following demyelination is predicted to prevent neurodegeneration. A promising target to enhance remyelination is the immunomodulatory cytokine tumor necrosis factor alpha (TNFα) and its receptors TNFR1 and TNFR2. TNFR2 on oligodendrocyte lineage cells and microglia coordinates different protective functions, such as proliferation of oligodendrocyte progenitor cells, survival of mature oligodendrocytes, and release of anti-inflammatory cytokines, in animal models of inflammation and demyelination. Here, we find in the cuprizone model that following demyelination, fewer axons are unmyelinated in the corpus callosum at an early stage of remyelination after single TNFR2 agonist delivery in the lateral ventricle, while astrocyte and microglia number and coverage are unchanged. Towards later stages of remyelination, TNFR2 agonist treatment maintains the number of oligodendrocyte lineage cells, and large caliber axons have thinner myelin. Hence, even short-term stimulation of TNFR2 has a positive impact on the remyelination processes. This study informs further on the beneficial implications of TNFR2 signaling on oligodendrocyte lineage cells and remyelination, emphasizing its potential therapeutic value for demyelinating diseases, including multiple sclerosis. KEY MESSAGES: Single TNFR2 agonist treatment in the lateral ventricle following cuprizone-induced demyelination impacts remyelination by: Leading to a lower percentage of unmyelinated axons at early stages. Preserving the number of oligodendrocyte lineage cells in the corpus callosum at later stages. Covering large calibre axons with thinner myelin sheaths at later stages.

Fusion proteins combining TNF-related apoptosis inducing ligand (TRAIL) and antibody building blocks have emerged as a strategy for the targeted treatment of cancer cells. Using a single-chain derivative of homotrimeric TRAIL (scTRAIL), several targeted and non-targeted scTRAIL fusion proteins of varying geometries and valencies for TRAIL receptors and target antigens, all comprising an Fc region, were generated. These fusion proteins comprised either 1 or 2 scTRAIL units, i.e. are tri- or hexavalent for TRAIL receptors and in the targeted versions, 1 or 2 binding sites for EGFR. These fusion proteins were analyzed for cell binding and cell death induction using the EGFR-expressing colorectal cancer cell lines Colo205 and HCT116. In line with previous findings, all fusion proteins that were hexavalent for TRAIL receptors exhibited a strongly increased cell killing activity compared to the trivalent ones. Interestingly, the fusion proteins comprising one scTRAIL unit, did not benefit from targeting to EGFR. In contrast, the hexavalent scTRAIL fusion proteins further benefited from EGFR targeting, resulting in an approximately 6- to 30-fold increase in cell killing. In summary, this study shed further light on the influence of geometry and valency of TRAIL fusion proteins and confirmed IgG-scTRAIL fusion proteins as highly potent cell death inducers.

Immunotherapies have emerged as a promising pillar for cancer therapy. However, due to their reliance on the individual, complex and dynamic tumor microenvironment, treatment success remains limited to a subset of patients. A deeper understanding of the response of individual tumors and their heterogeneous microenvironment is essential to improve patient stratification and develop novel treatment approaches. This requires suitable model systems which allow tracking of the tumor microenvironment‘s (TME) response to immunotherapy. In this study, we combined patient-derived precision-cut tumor slices (PCTS) with multiplex immunofluorescence staining and cytokine analyses to trace the treatment response of different cell types in their native TME while preserving their location. PCTS derived from ovarian cancer, lung cancer and colorectal cancer tissue (n=13) were treated with OMTX305, an anti-FAP T-cell engaging bispecific antibody, for up to three days. A co-culture system integrating PCTS with autologous PBMCs was established to mimic the in vivo environment, and enable monitoring of immune cell activities. While treatment responses were patient-specific, determination of overall ATP levels of PCTS showed a significant decrease in viability after three days of treatment. Multiplex immunofluorescence stainings were performed to spatially analyze therapeutic effects, including T cell infiltration and distribution, T cell activation (Granzyme B) and cell death induction (Cleaved Caspase-3) in different cell types. T cells were significantly activated after treatment, and tumor slices showed significant cell death induction on the second day of treatment. Some patients showed T cell infiltration into FAP+ regions in response to treatment. Analysis of over 10 cytokines in the culture medium provides deeper insights into how the treatment induced immune cell activation. By integrating spatial biology and cytokine analysis with our PCTS platform, we can map the relationship between the tumor, stromal, and immune cell components within the TME, visualize inter-patient differences, and predict individual therapy responses to immunotherapy. This makes our platform a valuable tool for personalized cancer medicine. Julia Thiel, Jan Schlegel, Adrian Kneer, Sascha Dreher, Annelie Schäfer, Oliver Seifert, Katrin S. Kurz, Marc-H Dahlke, Georg Sauer, Gerhard Preissler, Laureano Simon, Myriam Fabre, Isabel Egaña, Roland Kontermann, Monilola Olayioye, German Ott, Walter E. Aulitzky, Thomas E. Mürdter, Matthias Schwab, Meng Dong. Advancing personalized medicine using precision-cut tumor slices - assessing individual responses to immunotherapy in the tumor microenvironment [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 1602.

Multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system (CNS), predominately affects females compared to males. Tumor necrosis factor (TNF), a pro-inflammatory cytokine, signaling through TNF receptor 1 contributes to inflammatory disease pathogenesis. In contrast, TNF receptor 2 signaling is neuroprotective. Current anti-TNF MS therapies are shown to be detrimental to patients due to pleiotropic effects on both pro- and anti-inflammatory functions. Using a non-pertussis toxin (nPTX) experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice, we systemically administered a TNFR2 agonist (p53-sc-mTNFR2) to investigate behavioral and pathophysiological changes in both female and male mice. Our data shows that TNFR2 activation alleviates motor and sensory symptoms in females. However, in males, the agonist only alleviates sensory symptoms and not motor. nPTX EAE induction in TNFR2 global knockout mice caused exacerbated motor symptoms in females along with an earlier day of onset, but not in males. Our data demonstrates that TNFR2 agonist efficacy is sex-specific for alleviation of motor symptoms, however, it effectively reduces mechanical hypersensitivity in both females and males. Altogether, these data support the therapeutic promise TNFR2 agonism holds as an MS therapeutic and, more broadly, to treat central neuropathic pain.

Bispecific antibodies (bsAbs) enable novel mechanisms of action and/or therapeutic applications that cannot be achieved using conventional IgG-based antibodies. Consequently, development of these molecules has garnered substantial interest in the past decade and, as of the end of 2023, 14 bsAbs have been approved: 11 for the treatment of cancer and 3 for non-oncology indications. bsAbs are available in different formats, address different targets and mediate anticancer function via different molecular mechanisms. Here, we provide an overview of recent developments in the field of bsAbs for cancer therapy. We focus on bsAbs that are approved or in clinical development, including bsAb-mediated dual modulators of signalling pathways, tumour-targeted receptor agonists, bsAb–drug conjugates, bispecific T cell, natural killer cell and innate immune cell engagers, and bispecific checkpoint inhibitors and co-stimulators. Finally, we provide an outlook into next-generation bsAbs in earlier stages of development, including trispecifics, bsAb prodrugs, bsAbs that induce degradation of tumour targets and bsAbs acting as cytokine mimetics.

PDF - 61KB, Functional activity of RD_IL-15_scFv_m4-1BBL.

PDF - 49KB, Comparison of the cytokine activity of RD_IL-15_scFv and scFv_RD_IL-15.

PDF file - 132K, Supplementary Figure 1. Determination of melting points of the different fusion proteins by dynamic light scattering.

IL-15 activity of mono- and bivalent IL-15 fusion proteins