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Purpose: This phase Ib trial evaluated fibroblast activation protein-α-targeted IL2 variant (FAP-IL2v), a novel immunocytokine engineered to minimize CD25-mediated toxicities, in combination with cetuximab, in patients with recurrent, unresectable, or metastatic head and neck squamous cell carcinoma (HNSCC)., Patients and Methods: Patients received FAP-IL2v either on a continuous weekly (QW) schedule or QW for 4 weeks and then every 2 weeks (Q2W). Cetuximab was dosed at QW or Q2W schedules. The primary objectives were to evaluate the safety and tolerability, maximum tolerated dose, pharmacokinetics, and clinical activity for the combination of FAP-IL2v with cetuximab. Exploratory objectives included pharmacodynamic analyses., Results: A total of 58 patients were enrolled, 19 patients into the dose-escalation part, and 39 patients into the expansion part. The maximum tolerated dose of FAP-IL2v was defined as 10 mg (QW/Q2W) in combination with cetuximab (500 mg/m2, Q2W), which was further tested in the expansion part. The most common FAP-IL2v-related adverse events with a grade 3 or 4 severity were hypophosphatemia (19%), lymphopenia (16%), and infusion-related reaction (14%). The pharmacokinetics of FAP-IL2v in combination with cetuximab was similar to that after administration as monotherapy. Consistent with the proposed mode of action, FAP-IL2v preferentially expanded intratumoral NK and CD8 T cells. Four patients achieved a partial response, and the objective response rate was 7% (95% confidence interval, 3.2-14.7)., Conclusions: The safety profile of FAP-IL2v in combination with cetuximab was acceptable, and pharmacodynamic markers support the proposed mode of action of this combination, but the overall low antitumor activity does not warrant further clinical exploration in HNSCC. [Part C of Study BP29842 (NCT02627274).]., (©2024 The Authors; Published by the American Association for Cancer Research.)

Despite the clinical validation and unequivocal benefit to patients, the development of cancer immunotherapies is facing some key challenges and the attrition rate in early phases of development remains high. Identifying the appropriate patient population that would benefit most from the drug is on the critical path for successful clinical development. We believe that a systematic implementation of patient enrichment strategies early in the drug development process and trial design, is the basis for an innovative, more efficient, and leaner clinical development to achieve earlier a clear proof of concept or proof of failure. In this position article, we will describe and propose key considerations for the implementation of patient enrichment strategies as an opportunity to provide decision-enabling data earlier in the drug development process. We introduce an innovative multidimensional tool for immuno-oncology drug development that focuses on facilitating the identification and prioritization of enrichment-relevant biomarkers, based on the drug mechanism of action. To illustrate its utility, we discuss patient enrichment examples and use a case in the field of cancer immunotherapy, together with technical and regulatory considerations. Overall, we propose to implement fit for purpose enrichment strategies for all investigational drugs as early as possible in the development process. We believe that this will increase the success rate of immuno-oncology clinical trials, and eventually bring new and better medicines to patients faster., (©2023 American Association for Cancer Research.)

To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution., Competing Interests: Declaration of interests E.M.U., M.G. and R.J. have filed a patent on the method published in this paper. F.O. is a shareholder of NanoTag Biotechnologies GmbH. The other authors declare no competing financial interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice. In addition, the Fc-engineered variant improves on-target complement-mediated killing of cancer cells as well as both gram-positive and gram-negative bacteria. Hence, this versatile Fc technology should be broadly applicable in antibody design aiming for long-acting prophylactic or therapeutic interventions., (© 2024. The Author(s).)

Purpose: To examine whether CD8+ T-cell numbers in paired tumor biopsies in early-stage clinical trials can be used as an early indicator of clinical benefit for cancer immunotherapies., Experimental Design: Paraffin sections of tumor biopsies were stained immunohistochemically for CD8+ T cells, which were digitally enumerated. The tumor biopsies were from cancer patients in early-phase trials testing novel immunotherapeutic agents. Paired biopsies taken before the start of treatment and on-treatment were compared. A total of 155 patients were used as the training set and an additional 221 patients were used as the validation set., Results: Using the Cox proportional hazard model, a ≥0.9- increase in fold change (FC) on a ln scale in CD8+ T cells (corresponding to a 2.5-fold increase on the linear scale), from baseline, demonstrated a greater association with prolonged progression-free survival and allowed improved differentiation between groups above and below the threshold. Similarly, a ≥6.2 threshold in geometric mean of the on-treatment density (OTD) of T cells, which approximately corresponds to 500 cells/mm2, correlated with longer PFS. The combination of both criteria (FC and OTD) provided the best discrimination between clinically nonactive and active compounds., Conclusions: We propose that a composite score of CD8+ T-cell density in paired biopsies taken before and on-treatment may be a new biomarker to inform on clinical outcomes in early immunotherapy clinical trials., (©2023 American Association for Cancer Research.)

Therapeutic antibodies are widely used to treat severe diseases. Most of them alter immune cells and act within the immunological synapse; an essential cell-to-cell interaction to direct the humoral immune response. Although many antibody designs are generated and evaluated, a high-throughput tool for systematic antibody characterization and prediction of function is lacking. Here, we introduce the first comprehensive open-source framework, scifAI (single-cell imaging flow cytometry AI), for preprocessing, feature engineering, and explainable, predictive machine learning on imaging flow cytometry (IFC) data. Additionally, we generate the largest publicly available IFC dataset of the human immunological synapse containing over 2.8 million images. Using scifAI, we analyze class frequency and morphological changes under different immune stimulation. T cell cytokine production across multiple donors and therapeutic antibodies is quantitatively predicted in vitro, linking morphological features with function and demonstrating the potential to significantly impact antibody design. scifAI is universally applicable to IFC data. Given its modular architecture, it is straightforward to incorporate into existing workflows and analysis pipelines, e.g., for rapid antibody screening and functional characterization., (© 2023. The Author(s).)

Drug-target-drug complexes (DTDCs) are phenomena newly observed in patients who switch from the complement component 5 (C5) inhibitor eculizumab to crovalimab, a novel, anti-C5 antibody in development for paroxysmal nocturnal hemoglobinuria (PNH), because these agents bind to different C5 epitopes. In Part 3 of the four-part, phase I/II COMPOSER study, 19 patients with PNH switching from eculizumab received 1,000-mg crovalimab intravenously, then subcutaneous maintenance doses from Day 8 (680 mg every 4 weeks (q4w), 340 mg every 2 weeks, or 170 mg every week). Crovalimab exposure was transiently reduced, and size-exclusion chromatography and crovalimab-specific enzyme-linked immunosorbent assays revealed DTDCs in all 19 patients' sera. Additionally, self-limiting mild to moderate symptoms suggestive of type III hypersensitivity reactions occurred in two patients. Mathematical modeling simulations of DTDC kinetics and effects of dosing on DTDC size distribution using Part 3 data predicted that increased crovalimab concentrations could reduce the proportion of large, slow-clearing DTDCs in the blood. A simulation-guided, optimized crovalimab regimen (1,000 mg intravenously; four weekly, subcutaneous 340-mg doses; then 680 mg q4w from Day 29) was evaluated in Part 4. Confirming the model's predictions, mean proportions of large DTDCs in patients who switched from eculizumab to this optimized regimen decreased by > 50% by Day 22, and target crovalimab concentrations were maintained. No type III hypersensitivity reactions occurred in Part 4. Optimizing crovalimab dosing thus reduced the proportion of large DTDCs, ensured adequate complement inhibition, and may improve safety. Model-based dosing optimization to mitigate DTDC formation offers a useful strategy for patients switching to novel antibody treatments targeting soluble epitopes., (© 2023 F. Hoffmann-La Roche Ltd and The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

There are few treatments that slow neurodegeneration in Alzheimer's disease (AD), and while therapeutic antibodies are being investigated in clinical trials for AD treatment, their access to the central nervous system is restricted by the blood-brain barrier. This study investigates a bispecific modular fusion protein composed of gantenerumab, a fully human monoclonal anti- amyloid-beta (Aβ) antibody under investigation for AD treatment, with a human transferrin receptor 1-directed Brainshuttle™ module (trontinemab; RG6102, INN trontinemab). In vitro , trontinemab showed a similar binding affinity to fibrillar Aβ 40 and Aβ plaques in human AD brain sections to gantenerumab. A single intravenous administration of trontinemab (10 mg/kg) or gantenerumab (20 mg/kg) to non-human primates (NHPs, Macaca fascicularis ), was well tolerated in both groups. Immunohistochemistry indicated increased trontinemab uptake into the brain endothelial cell layer and parenchyma, and more homogeneous distribution, compared with gantenerumab. Brain and plasma pharmacokinetic (PK) parameters for trontinemab were estimated by nonlinear mixed-effects modeling with correction for tissue residual blood, indicating a 4-18-fold increase in brain exposure. A previously developed clinical PK/pharmacodynamic model of gantenerumab was adapted to include a brain compartment as a driver of plaque removal and linked to the allometrically scaled above model from NHP. The new brain exposure-based model was used to predict trontinemab dosing regimens for effective amyloid reduction. Simulations from these models were used to inform dosing of trontinemab in the first-in-human clinical trial.

The impact of antibody glycoforms on FcγRIIa activation and immune responses is poorly understood. Yet, glycoform binding assessment remains one of the major analytical challenges requiring long enrichment or glycoengineering steps. Here, we developed and applied an affinity capillary electrophoresis-mass spectrometry approach to selectively assess the binding of different antibody glycoforms to the FcγIIa receptor without the need of glycoengineering. The approach required only low microgram amounts of antibody and receptor and enables assessing the binding of high and low-abundance glycoforms. The approach indicated clear differences in binging between doubly-, hemi-glycosylated and non-glycosylated antibodies as well as for mutated (Leu234Ala, Leu235Ala - Pro329-Gly (LALA-PG)) IgG1 antibodies silenced for Fcγ binding. The LALA-PG mutated antibody showed no binding to the FcγIIa receptor (excluding potential non-specific binding effects) while the non-glycosylated IgG1 showed a strongly reduced, but still minor binding. The highest binding affinity was for the antibody carrying two complex-type glycans. Man5 glycans resulted in decreased binding compared to complex-type glycans, with the lowest binding for the IgG containing two Man5. For complex-type glycans, galactosylation showed a subtle increase in binding to the FcγIIa receptor, and sialylation showed an increase in binding for lower sialylated species. Fucosylation did not influence binding to the FcγIIa receptor. Finally, the assay was evaluated for the two variants of the FcγRIIa receptor (allotypes H131 and R131) showing highly comparable glycoform selectivity. Overall, the proposed approach allows the direct comparison of binding affinities of different antibody species in mixtures promising a fast establishment of their structure-function relationships., Competing Interests: Authors DR, TS, AK are employed by the company Roche Diagnostics GmbH, Penzberg, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gstöttner, Knaupp, Vidarsson, Reusch, Schlothauer, Wuhrer and Domínguez-Vega.)

Advances in imaging techniques enable high-resolution three-dimensional (3D) visualization of vascular networks over time and reveal abnormal structural features such as twists and loops, and their quantification is an active area of research. Here, we showcase how topological data analysis, the mathematical field that studies the "shape" of data, can characterize the geometric, spatial, and temporal organization of vascular networks. We propose two topological lenses to study vasculature, which capture inherent multiscale features and vessel connectivity, and surpass the single-scale analysis of existing methods. We analyze images collected using intravital and ultramicroscopy modalities and quantify spatiotemporal variation of twists, loops, and avascular regions (voids) in 3D vascular networks. This topological approach validates and quantifies known qualitative trends such as dynamic changes in tortuosity and loops in response to antibodies that modulate vessel sprouting; furthermore, it quantifies the effect of radiotherapy on vessel architecture.

Background: The COVID-19 pandemic represents a major public health threat. Risk of death from the infection is associated with age and pre-existing comorbidities such as diabetes, dementia, cancer, and impairment of immunological, hepatic or renal function. It remains incompletely understood why some patients survive the disease, while others do not. As such, we sought to identify novel prognostic factors for COVID-19 mortality., Methods: We performed an unbiased, observational retrospective analysis of real world data. Our multivariable and univariable analyses make use of U.S. electronic health records from 122,250 COVID-19 patients in the early stages of the pandemic., Results: Here we show that a priori diagnoses of fluid, pH and electrolyte imbalance during the year preceding the infection are associated with an increased risk of death independently of age and prior renal comorbidities., Conclusions: We propose that future interventional studies should investigate whether the risk of death can be alleviated by diligent and personalized management of the fluid and electrolyte balance of at-risk individuals during and before COVID-19., Competing Interests: Competing interestsS.N. and A.B.-M. are Roche employees. A.B.-M. holds Roche stock options. T.B. and V.S. are contractors paid by Roche. S.M. is not paid by Roche. The authors declare no other competing interests., (© The Author(s) 2021.)

Background: The pathways that control protein transport across the blood-brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development., Methods: We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis., Results: BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis., Conclusions: Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies., (© 2021. The Author(s).)

Today, there is a huge effort to develop cancer immunotherapeutics capable of combating cancer cells as well as the biological environment in which they can grow, adapt, and survive. For such treatments to benefit more patients, there is a great need to dissect the complex interplays between tumor cells and the host's immune system. Monitoring mechanisms of resistance to immunotherapeutics can delineate the evolution of key players capable of driving an efficacious antitumor immune response. In doing so, simultaneous and systematic interrogation of multiple biomarkers beyond single biomarker approaches needs to be undertaken. Zooming into cell-to-cell interactions using technological advancements with unprecedented cellular resolution such as single-cell spatial transcriptomics, advanced tissue histology approaches, and new molecular immune profiling tools promises to provide a unique level of molecular granularity of the tumor environment and may support better decision-making during drug development. This review will focus on how such technological tools are applied in clinical settings, to inform the underlying tumor-immune biology of patients and offer a deeper understanding of cancer immune responsiveness to immuno-oncology treatments., (©2021 American Association for Cancer Research.)

Biotherapeutics have revolutionized our ability to treat life-threatening diseases. Despite clinical success, the use of biotherapeutics has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs). The multifactorial nature of immunogenicity has prevented a standardized approach for assessing this and each of the assessment methods developed so far does not exhibit high enough reliability to be used alone, due to limited predictiveness. This prompted the Roche Pharma Research and Early Development (pRED) Immunogenicity Working Group to establish an internal preclinical immunogenicity toolbox of in vitro/in vivo approaches and accompanying guidelines for a harmonized assessment and management of immunogenicity in early development. In this article, the complex factors influencing immunogenicity and their associated clinical ramifications are discussed to highlight the importance of an end-to-end approach conducted from lead optimization to clinical candidate selection. We then examine the impact of the resulting lead candidate categorization on the design and implementation of a multi-tiered ADA/immunogenicity assay strategy prior to phase I (entry into human) through early clinical development. Ultimately, the Immunogenicity Toolbox ensures that Roche pRED teams are equipped to address immunogenicity in a standardized manner, paving the way for lifesaving products with improved safety and efficacy., Competing Interests: Declarations of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: the following authors (KBF, KH, GS, CMD, JB, MFF, KGS, CS, TS, AD) declare to be paid employees of Roche Pharmaceutical Research and Early Development. NC declares to be a paid employee of F. Hoffmann-La Roche AG., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The purpose of this work is to assess the heterogeneity across organs of response to treatment in metastatic colorectal patient based on longitudinal individual target lesion diameters (ILD) in comparison to sum of tumor lesion diameters (SLD). Data were from the McCAVE trial, in which 189 previously untreated patients with metastatic colorectal carcinoma (mCRC) received either bevacizumab (control, C) or vanucizumab (experimental, E), on top of standard chemotherapy. Bayesian hierarchical longitudinal non-linear mixed effect models were fitted to the data using Hamilton Monte Carlo algorithm to characterize the time dynamics of the tumor burden, and to obtain estimates of the tumor shrinkage and regrowth rates. The ILD model brought more nuanced results than to the SLD model. Besides substantial differences in tumor size at baseline (with lesions located in liver more than twice as large as the ones in lungs), it revealed a more durable response in lesions located in lymph nodes and 'other organs' compared to liver and lungs. Specifically, in lymph nodes and 'other organs', the projected time to nadir was doubled in group E (2.12 and 2.44 years respectively) compared to group C (1.07 and 1.20 years respectively). This long period of tumor shrinkage associated with a slightly larger change from baseline at nadir (- 51.4% in lymph nodes and - 62.6% in 'other organs' in the group E, compared to - 46.2% and - 46.9% in group C) resulted in a clinically meaningful difference in the tumor dynamics of patients in group E compared to the group C. The proportion of variance explained by the inter-lesion variability for each model parameter was large (ranging between 10 and 56%), reflecting the heterogeneity in tumor dynamics across organs. These findings suggest that there is value in understanding both within- and between-patient variability in tumor size's time dynamics using an appropriate modeling framework, as this information may help in pairing the right treatment with individual patient profile.

Background: By understanding prognostic biomarkers, we gain insights into disease biology and may improve design, conduct, and data analysis of clinical trials and real-world data. In this context, we used the Flatiron Health Electronic Health Record-derived deidentified database that provides treatment outcome and biomarker data from >280 oncology centers in the USA, organized into 17 cohorts defined by cancer type., Patients and Methods: In 122 694 patients, we analyzed demographic, clinical, routine hematology, and blood chemistry parameters within a Cox proportional hazard framework to derive a multivariable prognostic risk model for overall survival (OS), the 'Real wOrld PROgnostic score (ROPRO)'. We validated ROPRO in two independent phase I and III clinical studies., Results: A total of 27 variables contributed independently and homogeneously across cancer indications to OS. In the largest cohort (advanced non-small-cell lung cancer), for example, patients with elevated ROPRO scores (upper 10%) had a 7.91-fold (95% confidence interval 7.45-8.39) increased death hazard compared with patients with low scores (lower 10%). Median survival was 23.9 months (23.3-24.5) in the lowest ROPRO quartile Q1, 14.8 months (14.4-15.2) in Q2, 9.4 months (9.1-9.7) in Q3, and 4.7 months (4.6-4.8) in Q4. The ROPRO model performance indicators [C-index = 0.747 (standard error 0.001), 3-month area under the curve (AUC) = 0.822 (0.819-0.825)] strongly outperformed those of the Royal Marsden Hospital Score [C-index = 0.54 (standard error 0.0005), 3-month AUC = 0.579 (0.577-0.581)]. We confirmed the high prognostic relevance of ROPRO in clinical Phase 1 and III trials., Conclusions: The ROPRO provides improved prognostic power for OS. In oncology clinical development, it has great potential for applications in patient stratification, patient enrichment strategies, data interpretation, and early decision-making in clinical studies., Competing Interests: Disclosure JW, WVS, AF, MW, FS, DR, and AB-M are employed by Roche. All remaining authors have declared no conflicts of interest., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Digital pathology enables computational analysis algorithms to be applied at scale to histological images. An example is the identification of immune cells within solid tumours. Image analysis algorithms can extract precise cell locations from immunohistochemistry slides, but the resulting spatial coordinates, or point patterns, can be difficult to interpret. Since localisation of immune cells within tumours may reflect their functional status and correlates with patient prognosis, novel descriptors of their spatial distributions are of biological and clinical interest. A range of spatial statistics have been used to analyse such point patterns but, individually, these approaches only partially describe complex immune cell distributions. In this study, we apply three spatial statistics to locations of CD68+ macrophages within human head and neck tumours, and show that images grouped semi-quantitatively by a pathologist share similar statistics. We generate a synthetic dataset which emulates human samples and use it to demonstrate that combining multiple spatial statistics with a maximum likelihood approach better predicts human classifications than any single statistic. We can also estimate the error associated with our classifications. Importantly, this methodology is adaptable and can be extended to other histological investigations or applied to point patterns outside of histology.

Reducing Amyloid β (Aβ) in the brain is of fundamental importance for advancing the therapeutics for Alzheimer`s disease. The endogenous metallopeptidase neprilysin (NEP) has been identified as one of the key Aβ-degrading enzymes. Delivery of NEP to the brain by utilizing the Brain Shuttle (BS) transport system offers a promising approach for clearing central Aβ. We fused the extracellular catalytic domain of NEP to an active or inactive BS module. The two BS-NEP constructs were used to investigate the pharmacokinetic/pharmacodynamics relationships in the blood and the cerebrospinal fluid (CSF) in dose-response and multiple dosing. As previously shown, NEP was highly effective at degrading Aβ in blood but not in the CSF compartment after systemic administration. In contrast, the NEP with an active BS module led to a significant CSF exposure of BS-NEP, followed by substantial Aβ reduction in CSF and brain parenchyma. Our data show that a BS module against the transferrin receptor facilitates the transport of an Aβ degrading enzyme across the blood-brain barriers to efficiently reduce Aβ levels in both CSF and brain., Competing Interests: All authors, Christoph Campos, Alicia M Kemble, Jens Niewoehner, Per-Ola Freskgard and Eduard Urich, are paid employment by the company F. Hoffmann-La Roche This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Purpose: The Göttingen minipig is a relevant non-rodent species for regulatory toxicological studies. Yet, its use with therapeutic antibodies has been limited by the unknown binding properties of human immunoglobulins (huIgG) to porcine Fc gamma receptors (poFcγR) influencing safety and efficacy readouts. Therefore, knowing IgG-FcγR interactions in the animal model is a prerequisite for the use of minipigs in preclinical safety and efficacy studies with therapeutic antibodies., Methods: Here, we describe the cloning and expression of poFcγRs and their interactions with free and complexed human therapeutic IgG1 by surface plasmon resonance and flow cytometry., Results: We show here that poFcγRIa, poFcγRIIa, and poFcγRIIb bind huIgG1 antibodies with comparable affinities as corresponding huFcγRs. Importantly, poFcγRs bind huIgG immune complexes with high avidity, thus probably allowing human-like effector functions. However, poFcγRIIIa binds poIgG1a but not to huIgG1., Conclusions: The lack of binding of poFcγRIIIa to huIgG1 might cause underestimation of FcγRIIIa-mediated efficacy or toxicity as mediated by porcine natural killer cells. Therefore, the suitability of minipigs in preclinical studies with human therapeutic antibodies has to be assessed case by case. Our results facilitate the use of Göttingen minipigs for assessment of human therapeutic antibodies in preclinical studies.

Purpose: Administration of therapeutic monoclonal antibodies (mAbs) is frequently accompanied by severe first infusion reactions (FIR). The mechanism driving FIR is still unclear. This study aimed to investigate the cellular and molecular mechanisms causing FIR in humanized mouse models and their potential for evaluating FIR risk in patients., Methods: Mice humanized for Fc gamma receptors (FcγRs) were generated by recombination-mediated genomic replacement. Body temperature, cytokine release and reactive oxygen species (ROS) were measured to assess FIR to mAbs., Results: Infusion of human mAb specific for mouse transferrin receptor (HamTfR) into FcγR-humanized mice, produced marked transient hypothermia accompanied by an increase in inflammatory cytokines KC and MIP-2, and ROS. FIR were dependent on administration route and Fc-triggered effector functions mediated by neutrophils. Human neutrophils also induced FIR in wild type mice infused with HamTfR. Specific knock-in mice demonstrated that human FcγRIIIb on neutrophils was both necessary and sufficient to cause FIR. FcγRIIIb-mediated FIR was abolished by depleting neutrophils or blocking FcγRIIIb with CD11b antibodies., Conclusions: Human FcγRIIIb and neutrophils are primarily responsible for triggering FIR. Clinical strategies to prevent FIR in patients should focus on this pathway and may include transient depletion of neutrophils or blocking FcγRIIIb with specific mAbs.

Background: Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a gene silencing approaches, have shown this receptor being a major driver target of fibrosis and glomerulosclerosis., Methods: The present study investigated the role and relevance of DDR1 in human crescentic glomerulonephritis (GN). Detailed DDR1 expression was first characterized in detail in human GN biopsies using a novel selective anti-DDR1 antibody using immunohistochemistry. Subsequently the protective role of DDR1 was investigated using a highly selective, novel, small molecule inhibitor in a nephrotoxic serum (NTS) GN model in a prophylactic regime and in the NEP25 GN mouse model using a therapeutic intervention regime., Results: DDR1 expression was shown to be mainly limited to renal epithelium. In humans, DDR1 is highly induced in injured podocytes, in bridging cells expressing both parietal epithelial cell (PEC) and podocyte markers and in a subset of PECs forming the cellular crescents in human GN. Pharmacological inhibition of DDR1 in NTS improved both renal function and histological parameters. These results, obtained using a prophylactic regime, were confirmed in the NEP25 GN mouse model using a therapeutic intervention regime. Gene expression analysis of NTS showed that pharmacological blockade of DDR1 specifically reverted fibrotic and inflammatory gene networks and modulated expression of the glomerular cell gene signature, further validating DDR1 as a major mediator of cell fate in podocytes and PECs., Conclusions: Together, these results suggest that DDR1 inhibition might be an attractive and promising pharmacological intervention for the treatment of GN, predominantly by targeting the renal epithelium.

Purpose: Dogs have been proposed as spontaneous animal models of human breast cancer, based on clinicopathologic similarities between canine and human mammary carcinomas. We hypothesized that a better knowledge of the natural history and prognostic factors of canine invasive mammary carcinomas would favor the design of preclinical trials using dogs as models of breast cancer., Methods: The 2-year outcome of 350 female dogs with spontaneous invasive mammary carcinoma was studied. The investigated prognostic factors included age at diagnosis, pathologic tumor size, pathologic nodal stage, lymphovascular invasion, histological grade, and expression of Estrogen Receptor alpha (ERα), Progesterone Receptor, Ki-67, Human Epidermal Growth Factor Receptor 2, basal cytokeratins 5/6, and Epidermal Growth Factor Receptor. Multivariate survival analyses were performed using the Cox proportional hazards model., Results: The overall survival after mastectomy was 11 months. Within 1 year post mastectomy, 41.5% of dogs (145/350) died from their mammary carcinoma. By multivariate analysis, the significant prognostic factors for overall survival included a pathologic tumor size larger than 20 mm [HR 1.47 (95% confidence interval 1.15-1.89)], a positive nodal stage [pN+, HR 1.89 (1.43-2.48)], a histological grade III [HR 1.32 (1.02-1.69)], ERα negativity [HR 1.39 (1.01-1.89)], a high Ki-67 proliferation index [HR 1.32 (1.04-1.67)], and EGFR absence [HR 1.33 (1.04-1.69)]., Conclusion: The short natural history of spontaneous canine invasive mammary carcinomas and high rate of cancer-related death allow for rapid termination of preclinical investigations. The prognostic factors of invasive mammary carcinomas are remarkably similar in dogs and humans, highlighting the similarities in cancer biology between both species.

Purpose: Relevant animal models of human breast cancer are currently needed, especially for the aggressive triple-negative breast cancer subtype. Recent studies and our results (Part 1) indicate that spontaneous canine invasive mammary carcinomas (CMCs) resemble human breast cancer by clinics and pathology as well as behavior and prognostic indicators. We hypothesized that the current molecular classifications of human breast cancer, used for therapeutic decision, could be relevant to dogs., Methods: Three hundred and fifty female dogs with spontaneous CMC and a 2-year follow-up were retrospectively included. By immunohistochemistry, CMCs were classified according to Nielsen (Clin Cancer Res 10:5367-5374, 2004) and Blows (PlosOne doi: 10.1371/journal.pmed.1000279, 2010) into the subtypes of human breast cancer., Results: Four immunophenotypes were defined either according to Nielsen classification (luminal A 14.3%, luminal B 9.4%, triple-negative basal-like 58.6%, and triple-negative nonbasal-like 17.7% CMCs); or to Blows classification (luminal 1-: 11.4%, luminal 1+: 12.3%, Core basal phenotype: 58.6%, and five-negative phenotype: 17.7%). No HER2-overexpressing CMC as defined by a 3 + immunohistochemical score was observed in our cohort. By univariate and multivariate analyses, both immunophenotypical classifications applied to CMCs showed strong prognostic significance: luminal A or luminal 1+ CMCs showed a significantly longer disease-free interval (HR = 0.46), Overall (HR = 0.47), and Specific Survival (HR = 0.56) compared to triple-negative carcinomas, after adjustment for stage., Conclusions: In our cohort, triple-negative CMCs largely predominated (76%), were much more prevalent than in human beings, and showed an aggressive natural behavior after mastectomy. Dogs are thus potent valuable spontaneous models to test new therapeutic strategies for this particular subtype of breast cancer.

Objectives: To investigate whether the glycoengineered type II anti-CD20 monoclonal antibody obinutuzumab (GA101) combined with the selective MDM2 antagonist idasanutlin (RG7388) offers superior efficacy to monotherapy in treating B-lymphoid malignancies in preclinical models., Methods: The combined effect of obinutuzumab or rituximab plus idasanutlin on direct cell death/apoptosis induction and antibody-dependent cellular cytotoxicity (ADCC) was evaluated using p53 wild-type Z-138 and DoHH-2 lymphoma cells. Furthermore, whole blood B-cell depletion was analysed, and tumour growth inhibition was evaluated in subcutaneous xenograft models., Results: Idasanutlin induced concentration-dependent death of Z-138 and DoHH-2 cells. At concentrations >10-100 nm, idasanutlin enhanced obinutuzumab-induced death of DoHH-2 and Z-138 cells without negatively impacting obinutuzumab-mediated ADCC, natural killer cell activation or whole blood B-cell depletion. In the Z-138 xenograft model, a suboptimal dose of obinutuzumab with idasanutlin yielded substantial tumour growth inhibition and prolonged survival in a time-to-event analysis. In the DoHH-2 model, idasanutlin plus obinutuzumab showed superior tumour growth inhibition to idasanutlin plus rituximab., Conclusions: Obinutuzumab plus idasanutlin enhanced cell death of p53 wild-type tumour cells vs. rituximab plus idasanutlin without affecting obinutuzumab-mediated ADCC or B-cell depletion and showed robust antitumour efficacy in xenograft models, strongly supporting the investigation of this combination in clinical trials., (© 2016 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd.)

Bifunctional antibody fusion proteins engaging effector T cells for targeted elimination of tumor cells via CD3 binding have shown efficacy in both preclinical and clinical studies. Different from such a polyclonal T-cell recruitment, an alternative concept is to engage only antigen-specific T-cell subsets. Recruitment of specific subsets of T cells may be as potent but potentially lead to fewer side effects. Tumor-targeted peptide-MHC class I complexes (pMHCI-IgGs) bearing known antigenic peptides complexed with MHC class I molecules mark tumor cells as antigenic and utilize the physiologic way to interact with and activate T-cell receptors. If, for example, virus-specific CD8(+) T cells are addressed, the associated strong antigenicity and tight immune surveillance of the effector cells could lead to efficacious antitumor treatment in various tissues. However, peptide-MHC class I fusions are difficult to express recombinantly, especially when fused to entire antibody molecules. Consequently, current formats are largely limited to small antibody fragment fusions expressed in bacteria followed by refolding or chemical conjugation. Here, we describe a new molecular format bearing a single pMHCI complex per IgG fusion molecule characterized by enhanced stability and expression yields. This molecular format can be expressed in a full immunoglobulin format and can be designed as mono- or bivalent antibody binders. Mol Cancer Ther; 15(9); 2130-42. ©2016 AACR., (©2016 American Association for Cancer Research.)