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The CD40 receptor is an attractive target for cancer immunotherapy. Although a modest pharmacodynamic effect is seen in patients following administration of CD40-targeting monoclonal antibodies (mAb), the doses that could be safely administered do not result in a meaningful clinical response, most likely due to the limited therapeutic window associated with systemic CD40 activation. To overcome this issue, we developed a multispecific DARPin construct, α-FAPxCD40, which has conditional activity at the site of disease. α-FAPxCD40 activation of CD40 depends on binding to fibroblast activation protein (FAP), a cell-surface protease overexpressed in the stroma of solid tumors. In vitro studies demonstrated that α-FAPxCD40 potently activates human antigen-presenting cells in the presence, but not in the absence, of FAP-positive cells. After intravenous injection, a murine surrogate construct (α-mFAPxCD40) accumulated in FAP-positive tumors, elicited rejection of 88% of these tumors, and induced memory antitumor immunity. Importantly, in contrast to the mouse anti-CD40 tested in parallel, the in vivo antitumor activity of α-mFAPxCD40 was associated neither with elevated blood cytokines nor with hepatotoxicity, both of which contribute to the clinical dose-limiting toxicities of several CD40 mAb. This study demonstrates that α-(m)FAPxCD40 engages CD40 in an FAP-restricted manner, leading to tumor eradication without signs of peripheral toxicity. This distinct preclinical profile suggests that a favorable therapeutic index may be achieved in humans. It further supports the development of α-FAPxCD40, currently tested in a first-in-human clinical study in patients with solid tumors (NCT05098405).

Supplementary Figure from A Multispecific Anti-CD40 DARPin Construct Induces Tumor-Selective CD40 Activation and Tumor Regression

The CD40 receptor is an attractive target for cancer immunotherapy. Although a modest pharmacodynamic effect is seen in patients following administration of CD40-targeting monoclonal antibodies (mAb), the doses that could be safely administered do not result in a meaningful clinical response, most likely due to the limited therapeutic window associated with systemic CD40 activation. To overcome this issue, we developed a multispecific DARPin construct, α-FAPxCD40, which has conditional activity at the site of disease. α-FAPxCD40 activation of CD40 depends on binding to fibroblast activation protein (FAP), a cell-surface protease overexpressed in the stroma of solid tumors. In vitro studies demonstrated that α-FAPxCD40 potently activates human antigen-presenting cells in the presence, but not in the absence, of FAP-positive cells. After intravenous injection, a murine surrogate construct (α-mFAPxCD40) accumulated in FAP-positive tumors, elicited rejection of 88% of these tumors, and induced memory antitumor immunity. Importantly, in contrast to the mouse anti-CD40 tested in parallel, the in vivo antitumor activity of α-mFAPxCD40 was associated neither with elevated blood cytokines nor with hepatotoxicity, both of which contribute to the clinical dose-limiting toxicities of several CD40 mAb. This study demonstrates that α-(m)FAPxCD40 engages CD40 in an FAP-restricted manner, leading to tumor eradication without signs of peripheral toxicity. This distinct preclinical profile suggests that a favorable therapeutic index may be achieved in humans. It further supports the development of α-FAPxCD40, currently tested in a first-in-human clinical study in patients with solid tumors (NCT05098405).

Background Over 2,000 people a year in the United Kingdom need a bone marrow or blood stem cell transplant. It is important to accurately quantify the hematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification. The current gold standard method is single-platform flow cytometry using internal reference counting beads to determine the concentration of CD34 cells. However, volumetric flow cytometers have the ability to measure the acquisition volume, which removes the need for reference beads for calculation of cell concentrations. Method In this study, we compared both methods for calculating CD34 cell concentrations in volumetric cytometers, using either the volume reading or the number of reference beads for calculation. In addition, the uncertainty of measurement for each method was estimated. Results The results show that both methods have similar uncertainties of measurement. Regression analysis showed low to no statistical difference in CD34 cell concentrations obtained with each method. Conclusions Overall, this study suggests that the volumetric method is a valid approach but that the adoption of this technology may be hindered without some form of external calibration of volume readings to increase confidence in the measurement. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Flow cytometry immunophenotyping is a sensitive technique allowing rapid characterization of single cells within heterogeneous populations, but it includes several subjective steps during sample analysis that impact the development of standardized methodology. Proposed strategies to overcome these limitations include fluorescent cell barcoding (FCB), which facilitates multiplexed sample evaluation with increased data reproducibility whilst reducing labeling variation, materials, and time. To date, the FCB assay has found utility for analyzing the phosphorylation status of intracellular targets but has not been intensively employed for cellular immunophenotypic analyses using cell surface markers. In this study we developed a modified FCB assay for multiplexed analysis of human mesenchymal stromal cells (hMSCs) to evaluate the quality of these cells during bioprocessing. A panel of fluorochrome-conjugated antibodies was used to target 15 ubiquitously expressed or stage-specific markers together with a fixable viability dye eFluor 506 acting as the cell barcoding agent. Critical technical considerations and validation steps were presented in the context of monitoring hMSC status, defined by generic, and specific surface markers for cell identity and quality. It was found that at discrete passages, inter-analyst expression patterns between hMSCs cultures were similar, but in contrast, diverse marker expression was evident between passages. A side-by-side analysis of barcoded and non-barcoded cells demonstrated the potential of this technique for the rapid phenotypic characterization of cells exposed to different bioprocessing conditions. Additionally, the method incorporates fewer subjective factors; including sample preparation and instrument day-to-day variations and is customizable across a diversity of cell types. © 2017 International Society for Advancement of Cytometry

Therapeutic agonists targeting CD40 have shown encouraging signs of anti-tumor efficacy in patients. Despite their initial promise however, optimal dosing of systemically active agents has been limited by toxicity and their full therapeutic potential not achieved. We have previously demonstrated in vitro the ability of MP0317, a novel multi-specific DARPin® therapeutic, to selectively activate CD40 bearing B-cells, dendritic cells and macrophages in the presence of fibroblast activation protein (FAP). FAP is highly expressed in the stroma of many solid tumors and is found only at lower levels in other tissues. MP0317 utilizes FAP and CD40 binding domains to selectively cross-link and activate CD40 in the presence of FAP and thus avoid systemic CD40 activation classically associated with toxicity. Here we present new data which confirm the unique therapeutic potential of MP0317 in ex vivo model systems, demonstrate modulation of macrophage phenotype and reveal a release of T-cells from macrophage-mediated suppression. Ex vivo functional assays performed with MP0317 on dissociated human tumors demonstrated FAP-dependent activation of CD40-expressing B-cell and myeloid cell populations. Immunohistochemical analysis of tissue micro-arrays demonstrated FAP levels consistent with those required for immune cell activation in a broad range of solid tumor indications. Furthermore, by mining publicly available data, we classified tumor indications based on their immune cell content, pathway activation and MP0317 ex vivo mode of action (T cell/macrophage ratio, FAP and CD40 expression) and proposed patient populations and indications for the upcoming clinical trials. To further investigate the functional impact of MP0317, macrophages were differentiated in vitro using canonical polarising cytokines. Suppressive tumor-associated macrophage (TAM)-like cells, were repolarized by MP0317 to an anti-tumor-like phenotype. Furthermore, we explored whether modulation of macrophage phenotype by MP0317 could relieve their suppressive effect on T-cells. TAM-like macrophages potently supressed anti-CD3/28 driven T-cell activation and indeed the addition of MP0317 was found to restore T-cell response. Together these data further support MP0317's FAP targeted multimodal mechanism of action in human tumors, provide evidence for an improved therapeutic window over existing CD40 agonists and support progression to the clinic with an informed target patient profile. Citation Format: Kyriaki Ioannou, Simone Ragusa, Joanna Roquette, Ana Florescu, Mariam Gachechiladze, Eliane Müller, Julia M. Martinez-Gomez, Sophie Barsin, Sarah Jetzer, Nicolo Rigamonti, Clara Domke, Tamara Lekishvili, Karolin Rommel, Ivana Tosevski, Anne Goubier, Philippe Legenne, Vladimir Kirkin, Mitch Levesque, Hong Ji, Rupert Kenefeck. MP0317, a CD40xFAP targeting multi-specific DARPin® therapeutic, drives immune activation and reverts myeloid-mediated T-cell suppression in vitro and ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1733.

Platinum–DNA adducts, and in particular Pt–GG, have been identified as the major cytotoxic species during chemotherapy treatment with platinum containing drugs. This paper reports for the first time a strategy based on the use of double species-specific isotope dilution analysis (IDA) for the quantification of carboplatin–GG adducts formed by exposing human lung carcinoma cells to carboplatin. The main challenge posed by the use of carboplatin in this pre-clinical application, in comparison with most previously reported studies using cisplatin, includes the relatively low reactivity of this drug, thus demanding for improved limits of detection to be achieved in order to perform accurate quantification of the adducts at low ng Pt per mg DNA levels with relatively small uncertainty in micro-volumes of the biological sample. This was alleviated by developing micro-flow HPLC reversed phased methodology coupled to sector field ICP-MS (R = 300), showing a limit of detection of 0.2 ng Pt per mg DNA. To perform IDA, carboplatin–GG calibrants and spikes (194Pt-enriched GG adducts) were synthesized in house and characterised for Pt mass fraction, Pt distribution and structural composition. In order to assess the accuracy of the developed procedure, in the absence of certified reference materials, a reference sample prepared by incubation of calf thymus DNA with carboplatin and characterised in house (e.g. for its total P and Pt contents and Pt–GG concentration) was analysed in parallel. The use of this sample as a quality control of the cleavage of DNA and recovery of Pt adducts from real samples makes the described strategy particularly novel. Moreover, spike recovery experiments on the cell samples with the reference carboplatin–DNA sample were undertaken. The validated methodology was applied to cultured human lung carcinoma cells exposed to carboplatin; Pt–GG adducts were found to be at a level of 5.54 ng Pt per mg DNA with a relatively expanded combined uncertainty (k = 2) of approximately 20%. The major contributing factors to the overall measurement uncertainty were the mass fraction of Pt in the natural carboplatin–GG standard, the measured isotope ratio precision of sample and calibration blends and the blend to blend variation. The SI traceable methodology presented here will be invaluable for the provision of reference values to clinical measurements and cancer clinical trials.

This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units. © 2015 International Society for Advancement of Cytometry

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) μL−1 CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment. © 2015 The Authors. Published by Wiley Periodicals, Inc.

Flow cytometry immunophenotyping is a sensitive technique allowing rapid characterization of single cells within heterogeneous populations, but it includes several subjective steps during sample analysis that impact the development of standardized methodology. Proposed strategies to overcome these limitations include fluorescent cell barcoding (FCB), which facilitates multiplexed sample evaluation with increased data reproducibility whilst reducing labeling variation, materials, and time. To date, the FCB assay has found utility for analyzing the phosphorylation status of intracellular targets but has not been intensively employed for cellular immunophenotypic analyses using cell surface markers. In this study we developed a modified FCB assay for multiplexed analysis of human mesenchymal stromal cells (hMSCs) to evaluate the quality of these cells during bioprocessing. A panel of fluorochrome-conjugated antibodies was used to target 15 ubiquitously expressed or stage-specific markers together with a fixable viability dye eFluor 506 acting as the cell barcoding agent. Critical technical considerations and validation steps were presented in the context of monitoring hMSC status, defined by generic, and specific surface markers for cell identity and quality. It was found that at discrete passages, inter-analyst expression patterns between hMSCs cultures were similar, but in contrast, diverse marker expression was evident between passages. A side-by-side analysis of barcoded and non-barcoded cells demonstrated the potential of this technique for the rapid phenotypic characterization of cells exposed to different bioprocessing conditions. Additionally, the method incorporates fewer subjective factors; including sample preparation and instrument day-to-day variations and is customizable across a diversity of cell types. © 2017 International Society for Advancement of Cytometry.

The increased use of nanoparticles in industrial and medical products is driving the need for accurate, high throughput in vitro testing procedures to screen new particles for potential toxicity. While approaches using standard viability assays have been widely used, there have been increased reports of the interactions of nanoparticles with their soluble labels or optical readouts which raise concerns over the potential generation of false positive results. Here, we describe the use of an impedance spectroscopy approach to provide real-time reagent free detection of toxicity for a panel of metal oxide nanoparticles (ZnO, CuO, and TiO(2)). Using this approach, we show how impedance measurements can be used to track nanoparticle toxicity over time with comparable IC(50) values to those of standard assays (ZnO-55 μg/mL, CuO-28 μg/mL) as well as being used to identify a critical 6 h period following exposure during which the nanoparticles trigger rapid cellular responses. Through targeted analysis during this response period and the use of a novel image analysis approach, we show how the ZnO and CuO nanoparticles trigger the active export of intracellular glutathione via an increase in the activity of the ATP dependent MRP/1 efflux pumps. The loss of glutathione leads to increased production of reactive oxygen species which after 2.5 h triggers the cells to enter apoptosis resulting in a dose dependent cytotoxic response. This targeted testing strategy provides comprehensive information beyond that achieved with standard toxicity assays and indicates the potential for cell-nanoparticle interactions that could occur following in vivo exposure.

Flow cytometry is one of the most versatile and powerful approach for the quantitative analysis of cell suspensions. With widespread applications in basic and clinical research, its commonest use is in the detection of cell populations labelled against markers specific for a particular phenotype. In this study, we aimed to expand the potential of flow cytometry by describing a method based on robust automated quantification of ubiquitous cell surface markers. We demonstrate that automatic fluorescence standardization combined with whole cell population analysis yields highly reproducible results and can alleviate many of the difficulties associated with conventional analyses. This new generic approach is very flexible for quantifying the uniqueness of entire cell populations regardless of their composition. It provides quantitative phenotypic fingerprints rapidly, does not incorporate subjective factors, is more amenable to standardization, and is easily transferable across a wide diversity of applications, such as quality control for cell manufacture and authentication of cell products.

Tumour-associated antigen L6 (L6-Ag, also known as TM4SF1) regulates tumour cell motility and invasiveness. We found that L6-Ag is abundant on the plasma membrane and on intracellular vesicles, on which it is co-localised with the markers for late endosomal/lysosomal compartments, including Lamp1/Lamp2 proteins and LBPA. Antibody internalisation and live-imaging experiments suggested that L6-Ag is targeted to late endocytic organelles (LEO) predominantly via a biosynthetic pathway. Mapping experiments showed that the presence of transmembrane regions is sufficient for directing L6-Ag to LEO. On the plasma membrane, L6-Ag is associated with tetraspanin-enriched microdomains (TERM). All three predicted cytoplasmic regions of L6-Ag are crucial for the effective recruitment of the protein to TERM. Recruitment to TERM correlated with the pro-migratory activity of L6-Ag. Depletion of L6-Ag with siRNA has a selective effect on the surface expression of tetraspanins CD63 and CD82. By contrast, the expression levels of other tetraspanins and β1 integrins was not affected. We found that L6-Ag is ubiquitylated and that ubiquitylation is essential for its function in cell migration. These data suggest that L6-Ag influences cell motility via TERM by regulating the surface presentation and endocytosis of some of their components.