Amlenetug (Lu AF82422) is a human monoclonal antibody targeting α-synuclein in clinical development for multiple system atrophy. We describe a series of studies that characterize its functional properties and supported its selection as a viable clinical candidate. Amlenetug inhibits seeding induced in mouse primary neurons by various α-synuclein fibrillar assemblies and by aggregates isolated from MSA brain homogenate. In vivo, both co-injection of amlenetug with α-synuclein assemblies in mouse brain and peripheral administration inhibit α-synuclein seeding. Amlenetug inhibits uptake of α-synuclein seeds as well as accumulation of C-terminal truncated α-synuclein seeds and demonstrates binding to monomeric, aggregated, and truncated forms of human α-synuclein. The epitope of amlenetug was mapped to amino acids 112-117 and further characterized by crystallographic structure analysis. Based on our data, we hypothesize that targeting α-synuclein will potentially slow further disease progression by inhibiting further pathology development but be without impact on established pathology and symptoms.

Pharmacokinetics and pharmacodynamics of GEN1046 in patients with advanced solid tumors. A, Mean plasma concentration of GEN1046 during the first two dosing cycles with administration Q3W. B and C, Maximal fold change from baseline in pharmacodynamic markers measured in peripheral blood during cycle 1 in patients receiving low (≤200 mg) and high (≥400 mg) doses of GEN1046. P values from the Wilcoxon–Mann–Whitney test. LLOQ, lower limit of quantification.

GEN1046 promotes TIL expansion from patient-derived tumor tissue. Tumor tissues resected from patients with NSCLC were cut into pieces of 1 to 2 mm3 and cultured in the presence of IL2 (10–50 U/mL) and GEN1046 (or a GEN1046 surrogate comprising the PD-L1–specific Fab arm of GEN1046 and a nonhumanized variant of the 4-1BB–specific Fab arm or atezolizumab (0.2 µg/mL), or with IL2 only for 14–17 days. A, Cell numbers after expansion were determined by flow cytometry, and total TILs, CD8+ T cells, CD4+ T cells, and NK cells are shown for three patients. Tumor PD-L1 expression and 4-1BB expression by CD8+ T cells in the specimen at baseline are indicated. B, TCR repertoire analysis was performed by TRB RNA sequencing of the expanded TILs and the tumor fragments. Cumulative frequency of shared clonotypes, the 20 most abundant clonotypes in the GEN1046 surrogate–treated cultures, is shown. C, TILs expanded as in A were restimulated with enzymatically digested autologous tumor (TC) in the presence or absence of an MHC I–blocking antibody. Expression of 4-1BB and intracellular expression of IFNγ and CD107a in CD8+ T cells were analyzed by flow cytometry.

Apoptosis can be induced through activation of death receptors (DRs), which, upon binding to their natural ligands, hypercluster to initiate caspase-dependent cell death. This process can be mimicked by agonistic antibodies, making it a promising approach for anticancer therapy. However, DR-targeting cancer therapies evaluated in the clinic have shown limited success, due to insufficient clinical activity and/or safety concerns primarily related to hepatotoxicity. We hypothesize that conditional activation of DR4 in the tumor microenvironment (TME) can improve the therapeutic window of DR agonism. Fibroblast activation protein (FAP)α-expressing cancer-associated fibroblasts (CAFs) are abundant in the stroma of many solid cancers, making FAPα an attractive target to direct DR4 agonism to the TME. Here we present the novel Fc-silenced bispecific antibody DuoBody-FAPαxDR4 (GEN1057) designed to simultaneously bind to DR4 expressed on tumor cells and FAPα expressed on CAFs, to conditionally activate DR4 on the tumor cells. Functional activity of DuoBody-FAPαxDR4 was assessed in tumor cell lines and colorectal carcinoma (CRC) patient-derived organoids (PDOs) using viability and bioluminescent reporter assays and potential hepatotoxicity was evaluated using human primary liver spheroids in vitro. In vivo, antitumor activity of DuoBody-FAPαxDR4 was evaluated in patient-derived xenograft (PDX) models from different tumor types and in a model for spontaneous multiorgan metastasis of CRC. DuoBody-FAPαxDR4 induced dose-dependent caspase-8 activation and cytotoxicity in DR4-expressing tumor cell lines and CRC PDOs, when cocultured with FAPα-expressing cells in vitro. Cytotoxicity was strictly dependent on the presence of FAPα-expressing cells, with no direct effect on their viability. Furthermore, no cytotoxicity was observed in cocultures treated with monospecific control antibodies containing either the FAPα- or DR4-specific Fab arm of DuoBody-FAPαxDR4, confirming that binding to both DR4 and FAPα is required for DR4 transactivation. DuoBody-FAPαxDR4 induced caspase-8 activation and cytotoxicity in the cocultures to levels in the same range as an antibody that induced unconditional DR4 hyperclustering. Importantly, DuoBody-FAPαxDR4 did not induce cytotoxicity in human primary liver spheroids. In vivo, DuoBody-FAPαxDR4 showed potent antitumor activity in PDX models and against multiorgan metastases in a CRC PDO cecum transplantation mouse model. In conclusion, DuoBody-FAPαxDR4 is a novel bispecific antibody that activates DR4 on cancer cells exclusively in the presence of FAPα-expressing cells, leading to targeted caspase activation and tumor cell death. DuoBody-FAPαxDR4 represents a promising therapeutic agent for the treatment of cancer and is currently under evaluation in a first-in-human trial in patients with solid tumors (NCT06573294). Citation Format: Ilse Jongerius, Jamila Laoukili, Bart de Goeij, Edouard Souteyrand, Grietje Andringa, Madelon Paauwe, Monique Luijten, David Satijn, Esther Breij, Onno Kranenburg, Marije Overdijk. DuoBody®-FAPαxDR4 induces tumor cell death through FAPα-dependent, DR4 transactivation-mediated apoptosis [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 3424.

Binding of bsHER2xCD63his to SK-OV-3 cells

PDF file - 27K, Antibody cross-competition studies; Alphascreen Assay to detect ERK phosphorylation.

Supplementary Figure from Preclinical Characterization and Phase I Trial Results of a Bispecific Antibody Targeting PD-L1 and 4-1BB (GEN1046) in Patients with Advanced Refractory Solid Tumors

Immuno-oncology (IO) has substantially improved the survival of cancer patients over the past several years encouraging the discovery of novel IO targets which are typically proteins expressed on the surface of immune cells. Sensitive quantification of proteins in complex biological samples is routinely achieved by immunoassays that use antibodies specific to target proteins. Such approaches can be a limitation in IO drug discovery and development as de novo development of antibodies is associated with long lead times, high costs, and high failure rates. Protein quantification using mass spectrometry (MS) is agnostic to species and matrices and removes the barriers of availability or specificity of antibody-based assay. Further, MS proteomics workflows can support both large scale discovery studies but also represent an attractive alternative to targeted quantitative studies. The main purpose of this work is to assess the performance of the TrueDiscovery™ and TrueSignature™ MS-proteomics platforms for the deep proteome and surfaceome profiling of human primary and immortalized immune cells compared to flow cytometry solutions. We assess the number of quantified proteins and specifically the coverage of immune cell marker proteins in primary human immune cells across cell count groups from 2 million down to 2500 immune cells. In addition, we compared the quantification of a multiplexed surface antigens panel using TrueSignature™ and QIFI® flow cytometry platforms. We found that the applied MS-based proteomics workflows achieve high sensitivity and robustness in detection and quantification of immune cell markers down to 2500 primary immune cells. Additionally, we observed a strong correlation of the quantitative data derived from our MS-based proteomics workflows with flow cytometry supporting the substitution of immunoassays by MS-based proteomics workflows in target discovery and validation. Citation Format: Yu-Wah Au, David Satijn, Martin Mehnert, Amaury Lachaud, Yuehan Feng, Jakob Vowinckel, Hélène Bon. Discovery and validation of therapeutic targets in immune cells by mass spectrometry-based proteomics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3974.

Supplementary Figure legends for figures S1, S2 and S3

Immuno-oncology (IO) has substantially improved the survival of cancer patients over the past several years encouraging the discovery of novel IO targets which are typically proteins expressed on the surface of immune cells. Sensitive quantification of proteins in complex biological samples is routinely achieved by immunoassays that use antibodies specific to target proteins. Such approaches can be a limitation in IO drug discovery since the development of de novo antibodies is associated with long lead times, high costs, and high failure rates, often resulting in the substitution of the protein readout by a surrogate transcript readout using RNA sequencing methods. Protein quantification using mass spectrometry (MS) is agnostic to any capture agent and removes the barriers of availability or specificity of antibody-based assays. Furthermore, MS proteomics workflows can support large scale discovery studies with wide proteome coverage, and also represent an attractive antibody-free alternative in targeted quantitative studies for protein measurement. The main purpose of this work was primarily to assess the sensitivity of DIA-based MS-proteomics platform for the deep proteome profiling of human primary immune cells by measuring the maximum protein coverage obtained across cell inputs ranging from 2 million down to single cells ( performance assessment). The secondary purpose of this work was to assess the accuracy of the quantification of targeted MS proteomics workflow in comparison with the gold standard QIFI® flow cytometry approach, and other methods using mRNA readouts in immortalized human hematological cell lines ( accuracy assessment). We found that our MS-based proteomics workflows achieve high sensitivity in detection of immune cell markers down to few immune cells as input. Additionally, we report a strong correlation of the quantitative data derived from our MS-based proteomics workflows compared to the flow cytometry gold standard (r 2= 0.7354). Notably, we observed that this correlation was significantly higher than using transcriptomics approaches, emphasizing the benefit of substituting immunoassays for MS-based proteomics platforms in IO target discovery and validation.

Supplementary Method S1: Quantitative determination of cell surface antigens. Supplementary Method S2: Synthesis of Duostatin-3. Supplementary Table S1: Apparent antibody affinities. Supplementary Figure S1: Distribution of HER2, EGFR and TF in unstimulated HCC1954 cells. Supplementary Figure S2: Endosomal and lysosomal colocalisation of TF, EGFR and HER2 in tumor cells after treatment with target-specific antibodies. Supplementary Figure S3: Flow cytometry analysis of SK-OV-3 cells after ADC-treatment.

Background Immuno-oncology (IO) has substantially improved the survival of cancer patients over the past several years encouraging the discovery of novel IO targets which are typically proteins expressed on the surface of immune cells. Sensitive quantification of proteins in complex biological samples is routinely achieved by immunoassays that use antibodies specific to target proteins. Such approaches can be a limitation in IO drug discovery since the development of de novo antibodies is associated with long lead times, high costs, and high failure rates. The main purpose of this work is to test whether it is possible to substitute antibody based validation for mass spectrometry (MS) based since protein quantification using MS is agnostic to species and matrices and removes the barriers of availability or specificity of antibody-based assays. Methods For parallel reaction monitoring (PRM)-MS, proteins were extracted from cells, denatured, digested with trypsin prior to LC-MS analysis. Results We first tested the accuracy of the PRM targeted MS True Signature™ (PRM-MS) advanced proprietary proteomics workflow developed by Biognosys by comparing the quantification of a multiplexed panel of known B cells surface antigens in hematopoietic cell lines using TrueSignature™ with the gold-standard QIFI® measurement by flow cytometry. We observed a strong correlation (r2=0.7534) between the quantitative data derived from our MS based proteomics workflow and the gold-standard QIFI® measurement, demonstrating the accuracy of the MS based method for protein quantification. We then designed two multiplexed panels of known and novel targets in activated T cells or in Macrophages and quantified their expression using targeted MS. We could use the relationship established between the MS and QIFI® measurement to interpolate the values obtained by MS to values in molecules per cell, a typical unit for the absolute quantification of therapeutic targets. Conclusions In conclusion, we observed a strong correlation between the quantitative data derived from our TrueSignature™ MS-based proteomics workflow with the gold-standard QIFI® measurement by flow cytometry, supporting the possibility to substitute immunoassays by mass spectrometry for the targeted multiplexed quantification of proteins. This opens new avenues in the way we can apply mass spectrometry to our research since it becomes an attractive alternative to antibody based assays. It will enable to reduce workload, increase speed, address problem of antibody availability or specificity, and provide an absolute quantification of novel IO targets.

PDF file - 27K, Antibody cross-competition studies; Alphascreen Assay to detect ERK phosphorylation.

Lysosomal transport of a bispecific antibody targeting integrin-β1 and CD63

Background HexaBody & REG;-CD38 (GEN3014) is a hexamerization-enhanced human IgG1 that binds CD38 with high affinity. The E430G mutation in its Fc domain facilitates the natural process of antibody hexamer formation upon binding to the cell surface, resulting in increased binding of C1q and potentiated complement-dependent cytotoxicity (CDC).Methods Co-crystallization studies were performed to identify the binding interface of HexaBody-CD38 and CD38. HexaBody-CD38-induced CDC, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), trogocytosis, and apoptosis were assessed using flow cytometry assays using tumour cell lines, and MM patient samples (CDC). CD38 enzymatic activity was measured using fluorescence spectroscopy. Anti-tumour activity of HexaBody-CD38 was assessed in patient-derived xenograft mouse models in vivo.Findings HexaBody-CD38 binds a unique epitope on CD38 and induced potent CDC in multiple myeloma (MM), acute myeloid leukaemia (AML), and B-cell non-Hodgkin lymphoma (B-NHL) cells. Anti-tumour activity was confirmed in patient-derived xenograft models in vivo. Sensitivity to HexaBody-CD38 correlated with CD38 expression level and was inversely correlated with expression of complement regulatory proteins. Compared to daratumumab, HexaBody-CD38 showed enhanced CDC in cell lines with lower levels of CD38 expression, without increasing lysis of healthy leukocytes. More effective CDC was also confirmed in primary MM cells. Furthermore, HexaBody-CD38 efficiently induced ADCC, ADCP, trogocytosis, and apoptosis after Fc-crosslinking. Moreover, HexaBody-CD38 strongly inhibited CD38 cyclase activity, which is hypothesized to relieve immune suppression in the tumour microenvironment.Interpretation Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of HexaBody-CD38 in patients with MM.Funding Genmab.Copyright & COPY; 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2023;93: Published 2023 https://doi.org/10. 1016/j.ebiom.2023. 104663

PDF file - 166K, Supplementary Table S1: TF HuMab: target binding characteristics and inhibition of FXa generation. Supplementary Table S2: Cross-competition between TF HuMab. Supplementary Table S3: Thromboelastography: coagulation of LPS-stimulated whole blood in the presence of 20 microg/mL TF-011, TF-013 or TF-098. Supplementary Figure S1: TF HuMab inhibit TF:FVIIa-induced ERK1/2 phosphorylation. Supplementary Figure S2: TF:FVIIa-induced IL-8 production in MDA-MB-231 cells is reduced in the presence of TF HuMab. Supplementary Figure S3: Binding of TF HuMab is unaltered after conjugation with vcMMAE or mcMMAF. Supplementary Figure S4: Anti-tumor activity of TF-ADCs in the A431 and HCT-116 xenograft models. Supplementary Figure S5: Anti-tumor activity of unconjugated TF HuMab in vivo. Supplementary Figure S6: Post-paclitaxel treatment with TF-011-MMAE in PDX models. Supplementary Figure S7: Full-length blots for Figure 1C.

Schematic overview of the mechanism of action of bsHER2xCD63his-ADC

Background Clustering of CD27 on the plasma membrane of T cells induces T-cell activation, proliferation, and differentiation. Therefore, this costimulatory receptor represents a target for cancer immunotherapy. Multiple monoclonal antibodies (mAbs) targeting CD27 are being explored in the clinic, which require Fc gamma receptor (FcγR)-mediated crosslinking to induce CD27 agonism. HexaBody-CD27 (GEN1053/BNT313) is a novel anti-CD27 mAb with an IgG Fc domain engineered to induce CD27 agonist activity independently of FcγR-bearing cells, which may be scarce in tumors. The Fc domain was further modified to silence Fc-mediated antibody effector functions, with the aim to prevent T-cell depletion. Here we present preclinical characterization of the mechanism of action of HexaBody-CD27. Methods Target binding characteristics and functional activity of HexaBody-CD27 were analyzed in vitro using flow cytometry, cell-based reporter assays and primary human lymphocyte assays. The capacity of HexaBody-CD27 to induce tumor-infiltrating lymphocyte (TIL) proliferation was assessed ex vivo using non-small cell lung cancer (NSCLC) tissue resected from patients. HexaBody-CD27 activity in vivo was investigated in human CD27 knock-in mice that were immunized with ovalbumin and treated with HexaBody-CD27 by characterizing peripheral blood and splenic T cells using flow cytometry. Results HexaBody-CD27 exhibited dose-dependent CD27 agonist activity in reporter assays, independent of crosslinking via FcγR-expressing cells. In contrast, agonist activity of benchmark anti-CD27 antibody analogs was dependent on FcγR-mediated crosslinking. HexaBody-CD27 did not functionally engage with FcγRs, and membrane-bound HexaBody-CD27 was unable to bind C1q, confirming functional silencing of the IgG Fc domain. In vitro, HexaBody-CD27 enhanced activation, proliferation, and proinflammatory cytokine secretion of TCR-stimulated human CD4+ and CD8+ T cells as well as CD8+ T-cell mediated cytotoxic activity towards cognate antigen-expressing tumor cells. In TIL assays with human NSCLC tumor tissue, HexaBody-CD27 promoted expansion of CD8+ T cells. In human CD27 knock-in mice, HexaBody-CD27 enhanced expansion and IFN-γ secretion of antigen-specific CD8+ T cells. No decrease in percentages of circulating or splenic T cells was detected in vivo after treatment with HexaBody-CD27, whereas treatment with a benchmark anti-CD27 mAb analog resulted in a marked reduction of T cells. Conclusions HexaBody-CD27 has a functionally inert Fc domain and exhibits FcγR-crosslinking-independent CD27 agonist activity, a unique mechanism of action that distinguishes HexaBody-CD27 from benchmark mAbs targeting CD27. In preclinical studies in vitro and in vivo, HexaBody-CD27 increased T-cell activation, proliferation, cytokine secretion, and cytotoxic activity. A first-in-human clinical trial has been initiated to evaluate HexaBody-CD27 in patients with advanced solid tumors (NCT05435339). Ethics Approval The use of resected tumor tissue was approved by BioNTech SE's Ethics Board, approval number 837.309.12 (8410-F). All mouse studies were performed at Crown Bioscience Inc. in China. Animals were housed and handled in accordance with good animal practice as defined by the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

CD3 bispecific antibodies (bsAbs) show great promise as anticancer therapeutics. Here, we show in-depth mechanistic studies of a CD3 bsAb in solid cancer, using DuoBody-CD3x5T4. Cross-linking T cells with tumor cells expressing the oncofetal antigen 5T4 was required to induce cytotoxicity. Naive and memory CD4+ and CD8+ T cells were equally effective at mediating cytotoxicity, and DuoBody-CD3x5T4 induced partial differentiation of naive T-cell subsets into memory-like cells. Tumor cell kill was associated with T-cell activation, proliferation, and production of cytokines, granzyme B, and perforin. Genetic knockout of FAS or IFNGR1 in 5T4+ tumor cells abrogated tumor cell kill. In the presence of 5T4+ tumor cells, bystander kill of 5T4- but not of 5T4-IFNGR1- tumor cells was observed. In humanized xenograft models, DuoBody-CD3x5T4 antitumor activity was associated with intratumoral and peripheral blood T-cell activation. Lastly, in dissociated patient-derived tumor samples, DuoBody-CD3x5T4 activated tumor-infiltrating lymphocytes and induced tumor-cell cytotoxicity, even when most tumor-infiltrating lymphocytes expressed PD-1. These data provide an in-depth view on the mechanism of action of a CD3 bsAb in preclinical models of solid cancer.