Immunotherapy has significantly improved treatment outcomes for cancer patients within the past decade, with breakthrough results using immune checkpoint inhibitors (ICIs), most notably those targeting the PD-1/PD-L1 inhibitory axis. Nevertheless, many patients and tumor types do not respond to current ICIs, and next-generation drugs are urgently needed. Dual Signaling Protein 216 (DSP216) is a new ligand-based immunotherapeutic-a dual HLA-G and CD47 ICI. DSP216 was designed to exclusively bind to cells co-expressing the immune checkpoints CD47 and HLA-G, thereby mitigating ICI activity towards normal cells expressing only CD47 or HLA-G and associated side effects. Computational chemistry was used to optimize DSP216 affinity to HLA-G with the aim to achieve the desired binding mode and DSP216 binding to CD47+/HLA-G+ and CD47+/HLA-G- cancer cells, PBMCs, and RBCs was tested. Functional blocking of the CD47 and HLA-G axis was investigated in macrophage polarization and phagocytosis assays and NK cytotoxicity assays. DSP216 with an 'active' (DSP216a), but not with an 'inactive' Fc (DSP216i), triggered CD16-signaling in a reporter cell line, and the combination of checkpoint blockade and ADCC by DSP216a potentiated NK-mediated cytotoxicity. These encouraging findings support the continued preclinical evaluation of DSP216.

Chimeric Antigen Receptor T (CAR-T) cell therapy is an established treatment for haematological malignancies, yet the environmental impact of its manufacturing and administration remains unexplored. As healthcare systems aim to reduce their carbon footprint, understanding the climate impact of CAR-T cell manufacturing is essential. The objective of this study was to quantify the carbon footprint, expressed in CO2-equivalents (CO2e), of CAR-T cell manufacturing and identify process steps that contribute most to CO2-emissions. A systematic life cycle assessment (LCA) comparing centralized and point-of-care (PoC) CAR-T cell manufacturing processes was conducted, according to ISO14044:2006 standards. All major processes were assessed, including leukapheresis, cryopreservation, transport, manufacturing, background facilities, quality controls, and infusion. Emissions were calculated using openLCA and open-access databases. Transport was the dominant contributor to total emissions, accounting for the higher footprint of centralized manufactured CAR-T cells (355.75 kg CO2e) compared to PoC CAR-T cells (72.85 kg CO2e). In addition, open system background facilities emitted substantially more CO2 compared to closed system background facilities (85.16 vs. 25.00 kg CO2e). Reducing travel distance and optimizing cleanroom use offer the greatest potential for emission reduction. This study provides the first assessment of differences in the carbon footprint of two established CAR-T cell manufacturing platforms and offers a framework that can be applied across a broad range of CAR-T products. It highlights transport and cleanroom energy use as key drivers of emissions, causing PoC manufacturing to substantially reduce the environmental impact by limiting long-distance transport.

Figure S4. Binding of DSP502 to A549 and SKOV-3 cells with and without IFN-γ pretreatment.

Potency is a critical specification for CAR-T cells and is typically determined by co-culture of CAR-T cells with target cell lines. However, cell line heterogeneity hampers standardization and can lead to analytical variation impacting potency test results. Further, both the assay and cell line maintenance are labor, time, and resource intensive. Here, we developed and validated a versatile, fully standardized, and GMP-compliant CAR-T cell potency assay. This assay utilizes antigen-loaded beads instead of target cell lines and was fully validated for CD19 and qualified for CD7 and HER2 CAR-T cells. Incubation of streptavidin beads with recombinant biotinylated antigen yielded a dose-dependent bead loading. Subsequent incubation of CAR-T cells with increasing amounts of antigen-loaded beads yielded a dose-dependent secretion of IFN-γ, whereas non-loaded or MOCK antigen-loaded beads did not significantly trigger IFN-γ secretion. Notably, as assessed for CD19 CAR-T cells, cryopreserved CAR-T cells yielded lower potency than fresh CAR-T cells and potency results correlated with the total amount of CAR-T cells in the test sample. Therefore, the assay was fully standardized using a fixed amount of 50 k CAR-T cells per test, a fixed amount of antigen loaded onto the beads (1 pg/bead), and a fixed amount of 500 k antigen-loaded beads. A quantitative and statistically substantiated potency threshold for batch release was established for fresh as well as cryopreserved CAR-T cell drug products This fully standardized assay protocol and validation strategy provides a facile potency assay for CAR-T cell drug products that can be implemented for essentially any antigen of interest.

Chimeric Antigen Receptor (CAR)-T cell therapy holds considerable promise for the treatment of CD7+ T cell malignancies. However, a major challenge limiting clinical development of CD7-targeted CARs has been fratricide, a process of self-cytotoxicity caused by the shared expression of CD7 on malignant and healthy cells. Current solutions, including CD7 gene editing, intracellular retention or cell-sorting strategies, add significant complexity and cost, thereby limiting the feasibility and cost-effectiveness of this therapy. In this study, we evaluated whether fratricide can instead be overcome by tailoring standard manufacturing protocols to generate effective CAR-T cell products. Specifically, we report the GMP-compliant development of UMCG-001, a non-proprietary, academically generated, third-generation ligand-based CD7-targeting autologous CAR-T product. Production of UMCG-001 was explored using standard manufacturing protocols with various cytokine mixtures and GMP-grade human plate lysate (HPL). A GMP-compliant process was optimized with both healthy donor and patient material and subsequently transferred to our GMP facility. The anti-leukemic activity of UMCG-001 was assessed in vitro and in vivo, followed by in-depth multi-omics characterization to evaluate the impact of the fratricide phase on product quality. Supplementation with human platelet lysate during the ex vivo expansion phase effectively restored expansion and improved viability of fratricide-driven CAR⁺CD7Low/Neg cells. These unedited cells exhibited robust CD7-specific antileukemic activity and achieved 90

Figure S1. Characterization of DSP502 by Size Exclusion Chromatography (SEC) and SEC-multi-angle light scattering (MALS).

Figure S5. Expression of DNAM-1 in T and NK cells and CD25 expressions in T and NK cells with or without DSP502 treatment co-cultured with ES-2.WT, ES-2.PD-L1KO, ES-2.PVRKO, or ES-2.PVR/PD-L1KO.

Adoptive T-cell therapies using tumour-specific T-cell receptors (TCRs) are limited by competition with endogenous receptors, which impairs efficacy and poses risks of off-target autoreactivity. Here we present a CRISPR-based platform that completely and selectively eliminates both endogenous TCR-α and -β chains without affecting introduced transgenic TCRs, irrespective of codon optimization. This approach achieves >90% deletion efficiency in Jurkat and primary human T cells, markedly enhancing the expression, pairing fidelity, and functional potency of transgenic receptors. Using a clinically relevant HLA-A*02:01-restricted DMF5 TCR, we show that dual TCR ablation boosts antigen-specific activation and cytotoxicity in vitro and significantly enhances tumor clearance in vivo in human immune system (HIS) mice, while preventing graft-versus-host disease (GVHD). Targeted locus amplification revealed that CRISPR-induced double-strand breaks did not alter lentiviral integration profiles, confirming genomic safety. Extending this approach to four insulin-reactive TCRs demonstrated that removal of endogenous receptors increased transduction efficiency and functional activity, with one (1E6) showing selective activation and infiltration of stem cell-derived islet grafts (SC-islets) in vivo. This study establishes a universal, safe, and scalable genome-editing platform for generating functionally precise human T cells. By integrating cancer immunotherapy and autoimmune disease modelling within a single framework, it provides a strong preclinical rationale for dual endogenous TCR removal as a route to improved specificity, safety, and therapeutic efficacy in TCR-based cell therapies.

PD-1 immune checkpoint inhibition (ICI) is ineffective in most patients with cancer. However, combination therapy can improve response rates, with the checkpoint TIGIT being a particularly interesting candidate as it is expressed on tumor-infiltrating exhausted T and NK cells. TIGIT's primary ligand, PVR, is overexpressed in many cancers, and both TIGIT and PVR correlate with poor prognosis. To therapeutically exploit this, we developed a novel therapeutic termed dual signaling protein 502 (DSP502). DSP502 is composed of the extracellular domains of TIGIT and PD-1, each fused to human IgG1 Fc containing knob-in-hole mutations. DSP502 was designed to simultaneously block PVR/TIGIT and PD-L1/PD-1 by binding to cancer cell-expressed PVR and PD-L1. Moreover, the human IgG1 domain can recruit FcR-positive effector cells to further reactivate anticancer immunity. Treatment with DSP502 potentiated NK cell activation and boosted the anticancer cytotoxicity of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes from patients with non-small cell lung cancer (NSCLC) and metastatic colorectal cancer toward cancer cells expressing both PD-L1 and PVR. Transcriptomic analysis confirmed NSCLC as a potential target, showing co-expression of TIGIT and PD-1 (PDCD1) on a high percentage of exhausted CD8+ T cells. Notably, treatment with DSP502 not only blocked checkpoint signaling but also preserved surface expression of the co-stimulatory PVR ligand, DNAM-1, on T and NK cells. Finally, DSP502 inhibited tumor growth by potentiating antitumor immunity in xenograft ovarian and lung cancer models. Collectively, these findings demonstrate that DSP502, by blocking PVR and PD-L1 pathways, has dual ICI activity and holds potential therapeutic benefits for cancers such as NSCLC.

Acute myeloid leukaemia is an aggressive hematologic malignancy that remains exceedingly difficult to treat despite recent advancements. High expression of CD47 on leukemic blasts interacts with the inhibitory receptor SIRP-alpha (SIRP-α) on innate immune cells, resulting in a strong "don't eat me" signal. Therefore, identifying AML patients who could benefit from immune-targeted therapies is crucial SIRP-β2 is predominantly expressed in myeloid cells and positively regulates innate anticancer immunity. Furthermore, endogenously expressed SIRP-β2 potentiates cancer cell trogocytosis by granulocytes. Here, we delineate the role of SIRP-β2 in AML. High expression of SIRP-β2 is independently associated with favorable overall survival (OS) and event free survival (EFS) independent of the ELN intermediate risk group. SIRP-β2 is more prevalent in the more committed FAB M4 and M5 subgroups. SIRP-β2 is also expressed on normal myeloid cells in patient samples, with higher expression on tumor-suppressive M1 macrophages than on adverse prognostic and tumor-supportive M2 macrophages. In line with this, co-culture of macrophages/neutrophils with ectopically expressed SIRP-β2 tumor cells results in an increased phagocytosis/trogocytosis treated with anti-CD47. These data indicate that AML patients with high SIRP-β2 AML expression could significantly benefit from innate immune-targeting therapies such as CD47 immune checkpoint inhibitor.

T-cell malignancies remain challenging to treat with standard therapies. Development of Chimeric Antigen Receptor (CAR) T cell therapies against CD7 holds promise to increase current therapeutic outcome. To address the shared expression of CD7 with normal T cells and resultant fratricide effect, several strategies including the natural selection towards CD7Low/Neg cells have been proposed. Here, three batches of a ligand-based 3rd generation CAR against CD7, termed K12CAR-T, were manufactured in a GMP bioreactor-setting (BR-batches) and compared to matched-donor batches generated in the lab with in-depth immunophenotyping using full spectrum flow cytometry. On day 6, the maturation state and expression of prominent markers of exhaustion/activation were assessed. On average, 80% of the cells retained a TCM phenotype (CD62L+/CD45RA-) in BR-batches, with an ∼48% increase in %TCM cells and ∼9-fold increase in CD62L MFI levels compared to Lab-batches. Contrary to Lab-batches, CD62L expression in BR-batches was not significantly different to CD19CAR-Ts. BR-batches cells also had a more advantageous phenotype, exemplified by 67% lower expression of PD-1 and 39% higher expression of CD28. BR-batches had prominent CD7-restricted cytotoxic activity towards T-ALL cell lines with slightly higher IFN-? secretion. Overall, GMP bioreactor manufacturing yielded improved K12CAR-Ts compared to lab-based production, with a good phenotype suitable for use in a subsequent phase I clinical trial. Just Transition Fund (JTF) by the European Union Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Introduction:The main cause of death for patients with non-Hodgkin lymphoma (NHL) remains therapy resistant relapses. Chemoresistance is commonly associated with apoptosis defects and upregulated autophagy. Therefore, novel therapeutic options that do not rely on apoptosis and target autophagy would be of interest to treat NHL. An agent that may fulfill these requirements is the glycan-binding protein Galectin-9 (Gal-9). Methods:A panel of B cell lymphoma NHL cell lines, including diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt's lymphoma (BL), and (chemoresistant) follicular lymphoma (FL), were treated with Gal-9 after which cell counts and cell viability were determined. Basal mRNA and protein expression levels were respectively determined by RTqPCR and western blot. The impact of Gal-9 treatment on the autophagy pathway was determined using lysotracker, Cyto-ID and western blot (targeting LAMP2, p62, LC3B-I/LC3B-II). Results:Treatment with Gal-9 reduced total cell counts and cell viability of various DLBCL, MCL, BL and FL cell lines. Gal-9-induced cell death was associated with the inhibition of autophagy, as demonstrated by the accumulation of LC3B-II and p62. In addition, Gal-9-sensitive cells expressed lower basal protein levels of LC3B-I as compared to cells that responded less to this lectin. Furthermore, Gal-9 was cytotoxic for chemoresistant Sc-1 cells (Sc-1-RES), which were even more sensitive toward Gal-9 treatment than the parental cells (Sc-1-PAR). Conclusion:Gal-9 is a potent inducer of B cell lymphoma cell dead by inhibiting the proper execution of autophagy.

Chimeric antigen receptor (CAR)-based therapy is of interest for relapsed or refractory (r/r) T cell acute lymphoblastic leukemia (T-ALL) and T cell lymphomas. A prominent target antigen for this is the receptor CD7, which is expressed in ∼95% of T-ALL, ∼50% of peripheral T cell lymphomas, as well as 10% of acute myeloid leukemias. Here, we preclinically evaluated and compared CD7-targeted ligand K12-based CAR-T to an scFvCD7-based CAR-T construct. K12 CAR-T cells produced significantly higher interferon gamma (IFN-γ) after CD7 activation compared to scFv-CD7 CAR-T cells. Similarly, in a JurkatNFAT-luc reporter cell line expressing the respective CAR, CD7-induced luminescence was significantly higher by the K12 CAR than the scFv-CD7 CAR. K12 CAR-T treatment selectively and specifically eliminated a panel of CD7-positive, but not CD7-negative, cell lines and eliminated acute-T-cell-leukemia-patient-derived and acute myeloid leukemia blasts in an effector-to-target ratio-dependent manner. Further, K12 CAR-T cells had prominent anti-leukemic activity in an intravenously (i.v.) injected Jurkat leukemia mouse model, with no detectable disease in three out of five mice treated with K12 CAR-T. Therefore, K12 CAR T cell therapy might be of use for the treatment of r/r patients with CD7-positive T cell leukemia/lymphoma and acute myeloid leukemia (AML).

Early insight into the strength of antigen-specific T cell immune responses is an important aspect for the screening and development of novel immunotherapies, including immune checkpoint inhibitors. Here, we describe a simple, rapid, and cost-effective luminescence-based protocol for the assessment of antigen-specific T cell responses in vitro. This method makes use of genetically engineered Jurkat reporter cells expressing a high-affinity T cell receptor (TCR) toward a commercially available human papillomavirus 16 E7 peptide (E7-TCR), in which luciferase activity is coupled to activation of the promoter of the Nuclear Factor of Activated T Cells (NFAT) transcription factor. With this method, luminescence is generated and can be detected within 6 h only upon antigen-specific activation of the E7-TCR transgenic Jurkat cells with cognate E7 antigenic peptide. This method can be easily modified to study the impact of potential co-stimulatory and immunosuppressive molecules that may be present within the tumor microenvironment (TME) and to monitor the impact of antagonistic antibody treatment on abrogating immunosuppression. We believe this method can be further exploited as valuable tool to study factors that may affect T cell immunity in the immunosuppressive TME and the impact of molecules, cytokines, or therapeutics in triggering effective antigen-specific T cell responses.

About 30% of the FDA approved drugs in 2021 were protein-based therapeutics. However, therapeutic proteins can be unstable and rapidly eliminated from the blood, compared to conventional drugs. Furthermore, on-target but off-tumor protein binding can lead to off-tumor toxicity, lowering the maximum tolerated dose. Thus, for effective treatment therapeutic proteins often require continuous or frequent administration. To improve protein stability, delivery and release, proteins can be encapsulated inside drug delivery systems. These drug delivery systems protect the protein from degradation during (targeted) transport, prevent premature release and allow for long-term, sustained release. However, thus far achieving high protein loading in drug delivery systems remains challenging. Here, the use of protein desolvation with acetonitrile as an intermediate step to concentrate monoclonal antibodies for use in drug delivery systems is reported. Specifically, trastuzumab, daratumumab and atezolizumab were desolvated with high yield (∼90%) into protein nanoparticles below 100 nm with a low polydispersity index (<0.2). Their size could be controlled by the addition of low concentrations of sodium chloride between 0.5 and 2 mM. Protein particles could be redissolved in aqueous solutions and redissolved antibodies retained their binding activity as evaluated in cell binding assays and exemplified for trastuzumab in an ELISA.

Viruses critically rely on various proteases to ensure host cell entry and replication. In response to viral infection, the host will induce acute tissue inflammation pulled by granulocytes. Upon hyperactivation, neutrophil granulocytes may cause undue tissue damage through proteolytic degradation of the extracellular matrix. Here, we assess the potential of protease inhibitors (PI) derived from potatoes in inhibiting viral infection and reducing tissue damage. The original full spectrum of potato PI was developed into five fractions by means of chromatography and hydrolysis. Individual fractions showed varying inhibitory efficacy towards a panel of proteases including trypsin, chymotrypsin, ACE2, elastase, and cathepsins B and L. The fractions did not interfere with SARS-CoV-2 infection of Vero E6 cells in vitro. Importantly, two of the fractions fully inhibited elastin-degrading activity of complete primary human neutrophil degranulate. These data warrant further development of potato PI fractions for biomedical purposes, including tissue damage crucial to SARS-CoV-2 pathogenesis. KEY MESSAGES: Protease inhibitor fractions from potato differentially inhibit a series of human proteases involved in viral replication and in tissue damage by overshoot inflammation. Protease inhibition of cell surface receptors such as ACE2 does not prevent virus infection of Vero cells in vitro. Protease inhibitors derived from potato can fully inhibit elastin-degrading primary human neutrophil proteases. Protease inhibitor fractions can be produced at high scale (hundreds of thousands of kilograms, i.e., tons) allowing economically feasible application in lower and higher income countries.

Aims Resistance to targeted therapy is one of the critical obstacles in cancer management. Resistance to trastuzumab frequently develops in the treatment for HER2+ cancers. The role of protein tyrosine phosphatases (PTPs) in trastuzumab resistance is not well understood. In this study, we aim to identify pivotal PTPs affecting trastuzumab resistance and devise a novel counteracting strategy. Methods Four public datasets were used to screen PTP candidates in relation to trastuzumab responsiveness in HER2+ breast cancer. Tyrosine kinase (TK) arrays were used to identify kinases that linked to protein tyrosine phosphate receptor type O (PTPRO)-enhanced trastuzumab sensitivity. The efficacy of small activating RNA (saRNA) in trastuzumab-conjugated silica nanoparticles was tested for PTPRO upregulation and resistance mitigation in cell models, a transgenic mouse model, and human cancer cell line-derived xenograft models. Results PTPRO was identified as the key PTP which influences trastuzumab responsiveness and patient survival. PTPRO de-phosphorated several TKs, including the previously overlooked substrate ERBB3, thereby inhibiting multiple oncogenic pathways associated with drug resistance. Notably, PTPRO, previously deemed “undruggable,” was effectively upregulated by saRNA-loaded nanoparticles. The upregulated PTPRO simultaneously inhibited ERBB3, ERBB2, and downstream SRC signaling pathways, thereby counteracting trastuzumab resistance. Conclusions Antibody-conjugated saRNA represents an innovative approach for targeting “undruggable” PTPs.

Despite clinical advances in immunotherapy, still many therapeutics cause dose-limiting (auto)immune-mediated toxicities. Nanoparticle-based drug delivery systems (DDS) can improve cancer immunotherapy through site-specific delivery and controlled release of immunotherapeutics in the tumor microenvironment (TME). However, DDS face several challenges, including unspecific release. To address this, vaterite nanoparticles (VNPs) that selectively release immunotherapeutic proteins at low pH conditions find in the TME, are established previously. In the current study, these VNPs are further modified for active targeting without affecting the loaded protein activity, exemplified with Tumor Necrosis Factor alpha (TNF). Specifically, VNPs are coated with gelatin, a matrix-metalloprotease sensitive polymer which provides functional groups for further conjugation. Subsequently, streptavidin is covalently linked to the gelatin shell by amine-epoxy chemistry, enabling coupling of any biotinylated ligand. Exemplified by biotinylated cetuximab and rituximab, targeted VNPs selectively bind to cells expressing epidermal growth factor receptor (EGFR) or CD20, respectively. Importantly, TNF remains functionally active after the modification steps, as VNP treatment increased ICAM-1 expression on FaDu cells and activated NF kappa B signaling in a Jurkat.NF kappa B-luciferase cell line model. In conclusion, a targetable vaterite-based DDS is produced that allows for easy surface modification with any biotinylated ligand that may find broad applications in tumor-selective immunotherapy. Protein-loaded, vaterite nanoparticles (VNPs) are modified for active targeting by any biotinylated ligand. Therefore, VNPs are coated with gelatin, which provides functional groups for further conjugation. Subsequently, streptavidin is covalently linked to the gelatin shell by amine-epoxy chemistry. Thus, allowing coupling of any biotinylated ligand. Importantly, loaded protein remains functionally active after the modification steps, exemplified by Tumor Necrosis Factor alpha. image