Cell-based Functional Assays Research Articles

Comparison of Two Clinical Laboratory Assays for Measuring Serum Adalimumab and Antibodies to Adalimumab.

Adalimumab is a fully human monoclonal antibody developed against tumor necrosis factor (TNF), used for the treatment of autoimmune and chronic inflammatory diseases. Immunogenicity to this drug may lead to therapeutic failure. Various laboratory assays are used for measuring serum adalimumab and anti-drug antibodies (ADA) to adalimumab, for therapeutic monitoring and evaluation of clinical non-responsiveness. This study compared the performance of 2 clinical assays used by different reference laboratories. In total, 120 residual clinical samples were tested at both laboratories. A sandwich ELISA for adalimumab detecting free drug and a bridging ELISA capable of detecting both free and bound ADA were performed at the Mayo Clinic. A functional cell-based reporter gene assay (RGA) was used at ARUP Laboratories for measuring bioactive serum drug concentrations, and neutralizing ADA. Seventy-eight samples had measurable concentrations of adalimumab by both methods and yielded a correlation coefficient r = 0.93, slope = 0.886, and intercept = 0.950. Overall agreement of 92.5% was observed between the assays, with most discrepant drug results being attributed to a higher positivity rate with ELISA (8/9). One outlier positive with RGA and negative with ELISA was confirmed by LC-MS/MS to be attributed to infliximab. Overall agreement of 79.2% was observed between the ADA assays. Differences in ADA results may be due to the bridging ELISA detecting total ADA (free, drug-bound, neutralizing, and non-neutralizing), while RGA detects free, neutralizing ADA only. Although the assays are fundamentally different, the results show significant concordance between the clinically validated tests performed in different laboratories.

Mechanistic insights into the anti-tumor and anti-metastatic effects of Patrinia villosa aqueous extract in colon cancer via modulation of TGF-β R1-smad2/3-E-cadherin and FAK-RhoA-cofilin pathways

BackgroundPatrinia villosa, a traditional medicinal herb commonly used for treating intestinal-related diseases, has been commonly prescribed by Chinese medicine practitioners as a key component herb to treat colon cancer, although its anti-tumor effect and mechanisms of action have not been fully elucidated. Hypothesis/purposeThis study aimed to investigate the anti-tumor and anti-metastatic effects of Patrinia villosa aqueous extract (PVW), and its underlying mechanisms. MethodThe chemical profile of PVW was analysed by high-performance liquid chromatography with photodiode-array detection (HPLC-DAD) method. Cell-based functional assays MTT, BrdU, scratch, and transwell were conducted to evaluate the effects of PVW on human colon cancer HCT116 and murine colon26-luc cells, assessing cytotoxicity, cell proliferation, motility, and migration, respectively. Western blotting was performed to assess the effect of PVW on the expression of key intracellular signaling proteins. In vivo studies were conducted using zebrafish embryos and tumor-bearing mice to evaluate the anti-tumor, anti-angiogenesis, and anti-metastatic effects of PVW in colon cancer. ResultsFive chemical markers were identified and quantified in PVW. PVW exhibited significant cytotoxicity and anti-proliferative activity, as well as inhibitory effects on cell motility and migration in both HCT116 and colon 26-luc cancer cells via modulating protein expressions of TGF-β R1, smad2/3, snail, E-cadherin, FAK, RhoA, and cofilin. PVW (0.01–0.1 mg/ml) could significantly decrease the length of subintestinal vessels of zebrafish embryos through decreasing mRNA expressions of FLT1, FLT4, KDRL, VEGFaa, VEGFc, and Tie1. PVW (> 0.05 mg/ml) also significantly suppressed colon cancer cells migration in the zebrafish embryos. Furthermore, oral administration of PVW (1.6 g/kg) significantly inhibited tumor growth by decreasing the expressions of tumor activation marker Ki-67 and CD 31 in tumor tissues of HCT116 tumor-bearing mice. PVW could also significantly inhibit lung metastasis in colon 26-luc tumor-bearing mice by modulating their tumor microenvironment, including immune cells populations (T cells and MDSCs), levels of cytokines (IL-2, IL-12, and IFN-γ), as well as increasing the relative abundance of gut microbiota. ConclusionThis study revealed for the first time the anti-tumor and anti-metastatic effects of PVW through regulation of TGF-β-smad2/3-E-cadherin, and FAK-cofilin pathways in colon cancer. These findings provide scientific evidence to support the clinical use of P. villosa in patients with colon cancer.

Beyond Pattern Recognition: TLR2 Promotes Chemotaxis, Cell Adhesion, and Migration in THP-1 Cells.

The interaction between monocytes and endothelial cells in inflammation is central to chemoattraction, adhesion, and transendothelial migration. Key players, such as selectins and their ligands, integrins, and other adhesion molecules, and their functions in these processes are well studied. Toll-like receptor 2 (TLR2), expressed in monocytes, is critical for sensing invading pathogens and initiating a rapid and effective immune response. However, the extended role of TLR2 in monocyte adhesion and migration has only been partially elucidated. To address this question, we performed several functional cell-based assays using monocyte-like wild type (WT), TLR2 knock-out (KO), and TLR2 knock-in (KI) THP-1 cells. We found that TLR2 promotes the faster and stronger adhesion of monocytes to the endothelium and a more intense endothelial barrier disruption after endothelial activation. In addition, we performed quantitative mass spectrometry, STRING protein analysis, and RT-qPCR, which not only revealed the association of TLR2 with specific integrins but also uncovered novel proteins affected by TLR2. In conclusion, we show that unstimulated TLR2 influences cell adhesion, endothelial barrier disruption, migration, and actin polymerization.

Optimization and evaluation of pyridinyl vinyl sulfones as Nrf2 activator for the antioxidant and anti-inflammatory effects

Many studies have reported that chalcone-based compounds exhibit biological activities such as anticancer, antioxidant, anti-inflammatory and neuroprotective effects. Among the published chalcone derivatives, (E)-1-(3-methoxypyridin-2-yl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (VEDA-1209), which is currently undergoing preclinical study, was selected as a starting compound for the development of new nuclear factor erythroid 2-related factor 2 (Nrf2) activators. Based on our previous knowledge, we attempted to redesign and synthesize VEDA-1209 derivatives by introducing the pyridine ring and sulfone moiety to ameliorate its Nrf2 efficacy and drug-like properties. Among the synthesized compounds, (E)-3-chloro-2-(2-((3-methoxypyridin-2-yl)sulfonyl)vinyl) pyridine (10e) was found to have approximately 16-folds higher Nrf2 activating effects than VEDA-1209 (10e: EC50 = 37.9 nM vs VEDA-1209: EC50 = 625 nM) in functional cell-based assay. In addition, 10e effectively improved drug-like properties such as CYP inhibition probability and metabolic stability. Finally, 10e demonstrated excellent antioxidant and anti-inflammatory effects in BV-2 microglial cells and significantly restored spatial memory deficits in lipopolysaccharide (LPS)-induced neuroinflammatory mouse models.

Zafirlukast Is a Promising Scaffold for Selectively Inhibiting TNFR1 Signaling.

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory and autoimmune diseases such as rheumatoid arthritis and Crohn's disease. The biological effects of TNF are mediated by binding to TNF receptors, TNF receptor 1 (TNFR1), or TNF receptor 2 (TNFR2), and this coupling makes TNFR1-specific inhibition by small-molecule therapies essential to avoid deleterious side effects. Recently, we engineered a time-resolved fluorescence resonance energy transfer biosensor for high-throughput screening of small molecules that modulate TNFR1 conformational states and identified zafirlukast as a compound that inhibits receptor activation, albeit at low potency. Here, we synthesized 16 analogues of zafirlukast and tested their potency and specificity for TNFR1 signaling. Using cell-based functional assays, we identified three analogues with significantly improved efficacy and potency, each of which induces a conformational change in the receptor (as measured by fluorescence resonance energy transfer (FRET) in cells). The best analogue decreased NF-κB activation by 2.2-fold, IκBα efficiency by 3.3-fold, and relative potency by two orders of magnitude. Importantly, we showed that the analogues do not block TNF binding to TNFR1 and that binding to the receptor's extracellular domain is strongly cooperative. Despite these improvements, the best candidate's maximum inhibition of NF-κB is only 63%, leaving room for further improvements to the zafirlukast scaffold to achieve full inhibition and prove its potential as a therapeutic lead. Interestingly, while we find that the analogues also bind to TNFR2 in vitro, they do not inhibit TNFR2 function in cells or cause any conformational changes upon binding. Thus, these lead compounds should also be used as reagents to study conformational-dependent activation of TNF receptors.

Abstract 2356: Aberrant TIMP-1 secretion by tumor-associated fibroblasts is a major contributor to the selective positive response to nintedanib in lung adenocarcinoma

Abstract Adenocarcinoma (ADC) and Squamous Cell Carcinoma (SCC) are the most frequent histologic subtypes of lung cancer, and both are rich in activated/myofibroblast-like tumor-associated fibroblasts (TAFs). Nintedanib is a potent antifibrotic drug that targets the tumor stroma and is clinically approved to treat advanced lung ADC patients owing to the survival benefits observed in the LUME-1 clinical trial in ADC but not SCC patients. Although the mechanism underlying the ADC-selective therapeutic effects of nintedanib remained poorly understood, we have previously reported that nintedanib abrogates the pro-tumoral traits of the secretome of ADC-TAFs but not SCC-TAFs, suggesting that secreted factor(s) in ADC-TAFs may be implicated. In addition, we recently unveiled an ADC-specific tumor-promoting crosstalk between TAFs and cancer cells driven by TIMP-1 and CD63, whereas others have shown that TIMP-1 is a molecular target of nintedanib. However, it remains unknown if TIMP-1 is involved in the ADC-selective benefits of nintedanib. To address this question, we used patient-derived TAFs obtained with the patient’s informed consent and using protocols approved by the Ethics Committee of the Hospital Clinic. We treated TGF-β1-activated ADC-TAFs and SCC-TAFs with nintedanib and determined the content of TIMP-1 in their conditioned medium by ELISA. Moreover, we used cell-based functional assays and tumor xenografts after knocking-down TIMP-1 in TAFs by siRNA to examine how silencing TIMP-1 altered their response to nintedanib. By analyzing TCGA data and the human protein atlas, we found that TIMP-1 is consistently upregulated in ADC compared to SCC tumors both at the mRNA and protein levels. Moreover, TCGA data revealed that TIMP-1 is increased in tumors compared to paired control tissue in ADC but not SCC. In culture, we observed that TIMP-1 secretion was higher in ADC-TAFs than SCC-TAFs, and that this secretion was downregulated by nintedanib to a larger extent in ADC-TAFs compared to SCC-TAFs. In vivo analyses revealed that ADC cells co-injected with fibroblasts silenced for TIMP-1 as in SCC-TAFs into immunocompromised mice exhibited a less invasive growth pattern compared to tumors bearing control fibroblasts. In addition, we observed that the inhibition of the pro-tumoral secretome of ADC-TAFs elicited by nintedanib was abrogated upon knocking-down TIMP-1 in TAFs both in vitro and in vivo. Collectively, these results reveal that the high secretion of TIMP-1 in ADC-TAFs render them more responsive to nintedanib, whereas the low TIMP-1 secretion of SCC-TAFs may be a major contributor to the selective resistance of SCC patients to nintedanib. Moreover, our results identify TIMP-1 as a promising predictive biomarker of nintedanib responses. Citation Format: Paula Duch, Natalia Díaz-Valdivia, Marta Gabasa, Rafael Ikemori, Frank Hilberg, Noemí Reguart, Derek Radisky, Jordi Alcaraz. Aberrant TIMP-1 secretion by tumor-associated fibroblasts is a major contributor to the selective positive response to nintedanib in lung adenocarcinoma [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 2356.

Abstract 5526: State-of-the-art biobanking of gastroesophageal adenocarcinoma samples: Integrating non-viable biospecimens with 3D and 2D cell models and their characterization, validation, and utilization of cell models for precision oncology

Abstract Traditionally, biobanking platforms have collected and stored non-viable biological specimens such as serum, plasma, and fresh-frozen or formalin-fixed paraffin-embedded (FFPE) tissues. These constitute key resources for clinical and contemporary genomics, transcriptomics, and proteomics studies. However, such specimens cannot be used for studies involving drug testing, high throughput target validation, and implementation for personalized medicine. Next-generation biobanking strategies rectify this issue by combining the collection of non-viable samples from patients with the propagation of viable tissue fractions as in vitro 2D and 3D cell models or in vivo xenograft models. Such cutting-edge biobanking strategies enable downstream cell-based high-throughput functional assays promoting the discovery of therapeutic targets or assessing treatment responses and resistance to treatment. Here, we provide comprehensive details of our establishment of a state-of-the-art biobanking platform for gastroesophageal adenocarcinoma (GEA) samples (n=389). Our approach opens new directions for understanding disease biology and conducting translational cancer studies. Patient-derived 2D cells (n=376), organoids (PDOs) (n=185), and xenografts (PDXs) (n= 99) included in our pipeline retain crucial features of the original human tumors, serving as valuable tools for clinical and experimental analyses in the context of precision oncology. We also discuss the entire approach of next-generation biobanking and emphasize the importance of integrating the propagation of PDOs and PDXs simultaneously. In addition, we explain each protocol optimized to propagate patient tissue-derived cell models. We validate that these models recapitulate tissue heterogeneity and are relevant preclinical models. Taken together, we described each approach used to develop one of the largest next-level biobanks for GEA. Citation Format: Mingyang Iris Kong, Sanjima Pal, Julie Berube, France Bourdeau, Betty Giannias, Nicholas Bertos, Veena Sangwan, Lorenzo Ferri. State-of-the-art biobanking of gastroesophageal adenocarcinoma samples: Integrating non-viable biospecimens with 3D and 2D cell models and their characterization, validation, and utilization of cell models for precision oncology. [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 5526.

Abstract 1824: Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity

Abstract Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. Although recombinant mIL18 has demonstrated efficacy in mouse tumor models, recombinant human IL18 has not been efficacious in clinical studies, likely due to upregulation of IL18 binding protein (IL18BP), a negative regulator of the IL18 signaling axis. IL18BP is induced 10 - 100 fold after IL18 administration, binds to IL18 with high affinity, and prevents IL18 binding to the IL18 receptor. Analysis of expression data from the TCGA database as well as archived patient samples from select indications revealed that both IL18 and IL18BP are co-expressed in a number of tumor types, suggesting there may be a preexisting pool of IL18/IL18BP complex at the site of the tumor in some patients. Inhibition of IL18/IL18BP binding using a monoclonal antibody may therefore allow release of IL18 in situ, leading to IL18-mediated proinflammatory effects and enhanced anti-tumor immunity. Although the interaction of IL18 with IL18BP is a high affinity interaction, we have been able to identify very high affinity antibodies which can both inhibit complex formation and disrupt existing mouse IL18/IL18BP complexes. These antibodies are active in a number of cell-based functional assays, resulting in potent stimulation of interferon gamma production from cell lines or splenocytes. In vivo, we observed efficacy in a mouse syngeneic tumor model using an anti-mIL18BP antibody. RNA and protein analysis demonstrated expression of both IL18BP and IL18 within the tumors. Combined, these data suggest that inhibition of IL18/IL18BP binding may prove efficacious in the treatment of cancers where existing pools of IL18/IL18BP complexes prevent an IL18-mediated proinflammatory response. Experiments exploring anti-IL18BP therapy in combination with other checkpoint inhibitors such as anti-PD-1 are under current investigation. Citation Format: H. Toni Jun, Deborah A. Witherden, Eric K. Elliot, Christine M. Chidester, Maggie Willen, Bryan R. Peguero, Merrick Chai, Robert A. Horlick, David J. King. Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity [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 1824.

Abstract 3940: Validation of TFX1, a first-in-class target for cancer metastasis

Abstract Most of the drugs approved for metastatic epithelial carcinomas treat the proliferation of the tumor and not the biology of metastasis. To date, all clinical trials around metastasis that focused on proteases, kinases, or integrins have failed in clinics. As a result, metastasis biology-focused drug discovery has been deprioritized.We asked the question as to what the rate-limiting steps of metastasis were. To answer it, we created a cell-based functional assay platform, METAssay™, that dissected all metastasis biology steps into thirty cellular assays and characterization steps. The platform identified the functional differences between metastatic and proliferating cells. This platform was normalized with patient tumor cells, and a machine learning algorithm, METSCAN™, was subsequently applied to identify the weighted steps that contributed towards successful metastasis. This led to the identification of four first-in-class targets; two transcription factors, one cytokine receptor and one nuclear hormone receptor.We then validated the relevance of the first target, TFX1, by overexpressing it in both colorectal and triple-negative breast cancer cell lines. Interestingly, apart from the expected increase in migration and invasion, all cell lines showed an increase in ROS, Autophagy, PDL-1, CD-73, quiescence, exosome uptake and a decrease in apoptosis, mitosis, and exosome secretion. Conversely, the downregulation of TFX1 showed the opposite effect. TFX1 was purified, and a biochemical isothermal calorimetry (ITC) assay was standardized with a tool compound with a KD of 107 nM. The compound effectively reduced metastatic properties in the cell lines and in multiple patient samples, as analyzed by the METAssay™ platform. Colorectal cancer cell lines overexpressing TFX1 also showed increased platelet binding, which was reduced by the tool compound, but not triple-negative cancer cell lines. Also, the effect of overexpression in non-metastatic cells was more profound than in metastatic cells, suggesting a saturating effect. Further characterization of the tool compound is currently ongoing to help establish a baseline for a novel discovery programme.The ideal drug to delay metastasis should be given to primary tumor patients with no evidence of clinical metastasis, irrespective of their pathological grading. Therefore, it should have a high bar for safety. Our goal is to collaborate on a discovery journey to identify candidates and create a companion diagnostic that will identify primary tumor patients at high risk of metastasis, thereby selecting the right patient cohort for clinical trials. Citation Format: Arnab Roy Chowdhury, Manoj Pandre, Chinmaya Amarkanth, Debabani Roy Chowdhury, John Ellingboe. Validation of TFX1, a first-in-class target for cancer metastasis. [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 3940.

Abstract 6376: Discovery and preclinical characterization of dual antagonist antibodies targeting both LILRB1 and LILRB2 that enhance innate and adaptive anti-cancer immune responses

Abstract Background: One cause for the failure of checkpoint inhibitors is the immunosuppressive nature of the tumor microenvironment. LILRB1 (ILT2) and LILRB2 (ILT4) are ITIM-containing inhibitory receptors that recognize HLA Class 1 and nonclassical ligands (e.g., HLA-A, HLA-G, etc.). LILRB1 is expressed on myeloid cells and subsets of B, NK, and T cells, while LILRB2 expression is mostly restricted to myeloid cells. Interaction of LILRB1 and LILRB2 receptors with HLA ligands promotes an inhibitory milieu that prevents T cells from attacking cancer cells. The distinct pattern of expression and function of these lymphoid and myeloid checkpoints suggests complementary targeting approaches for cancer immunotherapy. Dual blockade of LILRB1 and LILRB2 receptors by a single antibody that restores both innate and adaptive immune responses is a promising strategy to enhance efficacy of checkpoint inhibitors. Methods: Dual LILRB1 and LILRB2 targeting antibodies were cloned from B cells derived from rabbits immunized with human LILRB2 recombinant protein, and subsequently humanized. Antibodies were evaluated for binding to human LILRB1 and LILRB2 proteins. Dual targeting antibodies were evaluated in a panel of functional and phenotypic assays. Selected antibodies were further tested for efficacy in a humanized NSG-SGM3 tumor model. Results: Dual antibodies were selected based on binding to recombinant human LILRB1 and LILRB2 protein, as well as blocking of HLA-G binding. These antibodies demonstrated binding to cells expressing LILRB1 and LILRB2, with no appreciable binding to other family members. Lead antibodies demonstrated activity in functional cell-based assays modeling LILRB1- or LILRB2-mediated immunosuppression. Dual antibodies also enhanced IFN-γ production by LPS-stimulated human PBMC. Selected clones restored T-cell function from M2c macrophage-mediated suppression in coculture with CD8+ T cell, and enhanced the tumoricidal activity of NK cells. Importantly, the lead antibody demonstrated in vivo efficacy with significant tumor growth inhibition and tumor regression in an SK-MEL-5 tumor model in humanized NSG-SGM3 mice. Conclusions: We have identified dual antagonist antibodies targeting both LILRB1 and LILRB2 antibodies that restore both innate and adaptive immune responses. Additionally, dual antibodies restored CD8+ T cell activation from macrophage-mediated suppression and enhanced NK cell cytotoxic activity. These data provide a strong rationale for further development of dual antibodies as an anti-cancer immunotherapy. Citation Format: Meghan Zuck, Myriam Bouchlaka, Huyen Dinh, Kevin Green, Francisco Zapata, Ramya Chandrasekaran, Tatyana Pisarenko, Lauren Loh, Gajendra S. Naika, Meilyn Sylvestre, Jacob Heit, Raymond Fox, Darbie Whitman, Tom Graddis, Kamal D. Puri, Peter Probst. Discovery and preclinical characterization of dual antagonist antibodies targeting both LILRB1 and LILRB2 that enhance innate and adaptive anti-cancer immune responses [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 6376.

The structural basis of the pH-homeostasis mediated by the Cl−/HCO3− exchanger, AE2

The cell maintains its intracellular pH in a narrow physiological range and disrupting the pH-homeostasis could cause dysfunctional metabolic states. Anion exchanger 2 (AE2) works at high cellular pH to catalyze the exchange between the intracellular HCO3− and extracellular Cl−, thereby maintaining the pH-homeostasis. Here, we determine the cryo-EM structures of human AE2 in five major operating states and one transitional hybrid state. Among those states, the AE2 shows the inward-facing, outward-facing, and intermediate conformations, as well as the substrate-binding pockets at two sides of the cell membrane. Furthermore, critical structural features were identified showing an interlock mechanism for interactions among the cytoplasmic N-terminal domain and the transmembrane domain and the self-inhibitory effect of the C-terminal loop. The structural and cell-based functional assay collectively demonstrate the dynamic process of the anion exchange across membranes and provide the structural basis for the pH-sensitive pH-rebalancing activity of AE2.

ATG16L1 adopts a dual-binding site mode to interact with WIPI2b in autophagy.

Macroautophagy plays crucial roles in the regulation of cellular physiology and requires de novo synthesis of double-membrane autophagosomes, which relies on a specific interaction between autophagy-related 16L1 (ATG16L1) and WD repeat domain phosphoinositide-interacting protein 2b (WIPI2b). However, the molecular mechanism governing the interaction of ATG16L1 with WIPI2b remains elusive. Here, we find that ATG16L1 has two distinct binding sites for interacting with WIPI2b, the previously reported WIPI2b-binding site (WBS1) and the previously unidentified site (WBS2). We determine the crystal structures of WIPI2b with ATG16L1 WBS1 and WBS2, respectively, and elucidate the molecular mechanism underpinning the recruitment of ATG16L1 by WIPI2b. Moreover, we uncover that ATG16L1 WBS2 and its binding mode with WIPI2b is well conserved from yeast to mammals, unlike ATG16L1 WBS1. Last, our cell-based functional assays demonstrate that both ATG16L1 WBS1 and WBS2 are required for the effective autophagic flux. In conclusion, our findings provide mechanistic insights into the key ATG16L1/WIPI2b interaction in autophagy.

Autoantibodies are highly prevalent in non-SARS-CoV-2 respiratory infections and critical illness.

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non-SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non-SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

A cell-based functional assay that accurately links genotype to phenotype in familial HLH

AbstractFamilial forms of the severe immunoregulatory disease hemophagocytic lymphohistiocytosis (HLH) arise from biallelic mutations in the PRF1, UNC13D, STXBP2, and STX11 genes. Early and accurate diagnosis of the disease is important to determine the most appropriate treatment option, including potentially curative stem cell transplantation. The diagnosis of familial HLH (FHL) is traditionally based on finding biallelic mutations in patients with HLH symptoms and reduced natural killer (NK)–cell cytotoxicity. However, patients often have a low NK-cell count or receive immunosuppressive therapies that may render the NK-cell cytotoxicity assay unreliable. Furthermore, to fully understand the nature of a disease it is critical to directly assess the effect of mutations on cellular function; this will help to avoid instances in which carriers of innocuous mutations may be recommended for invasive procedures including transplantation. To overcome this diagnostic problem, we have developed a rapid and robust method that takes advantage of the functional equivalence of the human and mouse orthologues of PRF1, UNC13D, STX11, and STXBP2 proteins. By knocking out endogenous mouse genes in CD8+ T cells and simultaneously replacing them with their mutated human orthologues, we can accurately assess the effect of mutations on cell function. The wide dynamic range of this novel system allowed us to understand the basis of, otherwise cryptic, cases of FHL or HLH and, in some instances, to demonstrate that previously reported mutations are unlikely to cause FHL. This novel approach provides valuable new information to enable more accurate diagnosis and treatment of patients with HLH or FHL who inherit mutations of undetermined pathogenicity.

Use of complementary biophysical methods to characterize ligand binding to IL-17A.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine produced by subset 17 of T helper cells and is emerging as one the key mediators for chronic inflammation and autoimmune disease. IL-17A signaling occurs through its membrane-bound receptors, IL-17RA and IL-17RC. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy but, no small molecule IL-17A antagonists have yet progressed. Small molecule inhibition of protein-protein interactions is difficult due to lack of well-defined pockets to target and inherent conformational flexibility of IL-17A makes designing inhibitors even more challenging. To characterize binding of small molecule ligands to IL-17A, we used a variety of biophysical methods including SPR, NMR and a relatively new method called Second Harmonic Generation (SHG). Conformational flexibility of IL-17A was a great hurdle in obtaining high quality X-ray structures. In absence of X-ray crystal structures, the team was dependent on biophysical data to make design decisions. Along with studying direct binding of compounds to IL-17A, we devised competition experiments to determine binding site specificity for ligands that bind to IL-17A. We eventually were successful in obtaining X-ray crystal structures to study the binding interactions in detail. Data obtained from these complementary methods matched very well boosting confidence in the chemical matter. The affinity of ligands to IL-17A correlated with inhibition of IL-17A:receptor interaction in the cell-based functional assays.

Abstract A016: Aging-dependent emergent mechanical properties of single epithelial cells exploited for detection of breast cancer susceptibility

Abstract Age is the major risk factor in most carcinomas, yet, little is known about the specific reasons aging increases cancer susceptibility. In the mammary gland, luminal epithelial cells rank high as the putative breast cancer cell of origin. Dysregulation of keratin intermediate filament proteins exemplifies a hallmark age-dependent change in luminal cells, which implicates mechanical states unique to cancer susceptible cells. We implemented mechano-node-pore sensing (mechano-NPS), a multi-parametric single-cell analysis that simultaneously measures cell diameter, resistance to compressive deformation, transverse deformation under constant strain, and recovery time after deformation. We demonstrated that the epithelial lineages, chronological ages, and stages of cancer progression of primary human mammary epithelial cells (HMEC) exhibited discrete mechanical phenotypes. We trained a machine learning model that accurately predicted the chronological age of average risk HMEC cells based exclusively on mechanical properties. Application of the model to cells from women who are germline carriers of high-risk cancer-causing mutations showed that they are mechanically old irrespective of their chronological age, suggesting that mechanical states could be a window into detection and prevention of cancer susceptible states. Indeed, this mechano-age model detected high-risk women with >90% accuracy. Mass spectrometry and cell-based functional assays in mammary epithelia revealed that cytoskeleton related proteins keratin 14 (KRT14) and pseudopodium enriched atypical kinase 1 (PEAK1) were key drivers of age-dependent mechanical signatures. Pharmacological and gene silencing approaches that targeted KRT14 and PEAK1 modulated the mechanical age of HMEC and, in the case of PEAK1 modulation, ablated luminal epithelial cells in an age- and lineage- dependent manner. We define an intersection between mechanical phenotypes and novel age-dependent changes in cytoskeleton-related proteins that we hypothesize can be exploited to assess an individual’s breast cancer susceptibility and provide new targets for cancer-prevention strategies. Citation Format: Stefan Hinz, Masaru Miyano, Antigoni Manousopoulou, Rosalyn W. Sayaman, Kristina Y. Aguilera, Michael E. Todhunter, Jennifer C. Lopez, Leo D. Wang, Lydia L. Sohn, Mark A. LaBarge. Aging-dependent emergent mechanical properties of single epithelial cells exploited for detection of breast cancer susceptibility [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A016.

CD3 Target Affinity Chromatography Mass Spectrometry as a New Tool for Function–Structure Characterization of T-Cell Engaging Bispecific Antibody Proteoforms and Product-Related Variants

T-cell engaging bispecific antibodies (TCBs) targeting CD3 and tumor-specific antigens are very promising therapeutic modalities. Since CD3 binding is crucial for the potency of TCBs, understanding the functional impact of CD3 antigen-binding fragment modifications is of utmost importance for defining critical quality attributes (CQA). The current CQA assessment strategy requires the integration of structure-based physicochemical separation and functional cell-based potency assays. However, this strategy is tedious, and coexisting proteoforms with potentially different functionalities may not be individually assessed. This increases the degree of ambiguities for defining meaningful CQAs, particularly for complex bispecific antibody formats such as TCBs. Here, we report for the first time a proof-of-concept study to separate and identify critically modified proteoforms of TCBs using functional CD3 target affinity chromatography (AC) coupled with online mass spectrometry (MS). Our method enabled functional distinction of relevant deamidated and glycosylated proteoforms and the simultaneous assessment of product-related variants such as TCB mispairings. For example, CD3 AC-MS allowed us to separate TCB mispairings with increased CD3 binding (i.e., knob-knob homodimers) within the bound fraction. The functional separation of proteoforms was validated using an established workflow for CQA identification based on thoroughly characterized ion-exchange fractions of a 2+1 TCB. In addition, the new method facilitated the criticality assessment of post-translational modifications in stress studies and structural variants in early stage clone selection. CD3 AC-MS has high impact for streamlining the integration of functional and structural characterizations of the large landscape of therapeutic CD3 targeting TCBs from early stage research to late stage characterization.

FAM111B dysregulation promotes malignancy in fibrosarcoma and POIKTMP and a low-cost method for its mutation screening

IntroductionMutations in the uncharacterised human FAM111B gene are associated with POIKTMP, a rare multi-organ fibrosing disease. Recent studies also reported the overexpression of FAM111B in specific cancers. Moreover, FAM111B mutation screening may prove expensive in under-resourced facilities. Therefore, this study investigated its cellular function and dysfunction and described an inexpensive mutation screening method. Materials and MethodsFAM111B expression was assessed in silico and validated in vitro in cell lines and primary skin fibroblasts from a South African POIKTMP-patient with the heterozygous FAM111B gene mutation: NM_198947.4: c.1861T>G (p. Tyr621Asp or Y621D) by qPCR and western blot. The cellular function of FAM111B was studied in HT1080 using various cell-based functional assays, and the Y621D mutation was genotyped by PCR-RFLP. ResultsExpression studies showed upregulated FAM111B mRNA and protein in the cancer cells. High FAM111B expression with robust nuclear localization occurred in HT1080. Additionally, expression data and cell-based assays indicated that FAM111B led to the upregulation of cell migration, decreased cell apoptosis, and modulatory effects on cell proliferation. Y621D mutation showed similar effects on cell migration but minimal impact on cell apoptosis. FAM111B mRNA and protein expression were markedly downregulated (p ≤ 0.05) in the POIKTMP-patient's fibroblasts. The PCR-RFLP method successfully genotyped Y621D gene mutation. DiscussionFAM111B is a cancer-associated nuclear protein: Its modulation by mutations or overexpression may contribute to the malignancy of cancers and POIKTMP/fibrosis and poor clinical outcomes and represents a viable prognostic marker or therapeutic target. Furthermore, the PCR-RFLP method could prove a valuable tool for FAM111B mutation validation or screening in resource-constrained laboratories.

Functional Assay for Measuring Bacterial Degradation of Gemcitabine Chemotherapy.

Drug biotransformation by the host microbiome can impact the therapeutic success of treatment. In the context of cancer, drug degradation can take place within the microenvironment of the targeted tumor by intratumor bacteria. In pancreatic cancer, increased chemo-resistance against the frontline chemotherapy gemcitabine is thought to arise from drug degradation by the tumor microbiome. This bacterial-drug interaction highlights the need for developing rapid assays for monitoring bacterial gemcitabine breakdown. While chemical approaches such as high-performance liquid chromatography are suitable for this task, they require specialized equipment and expertise and are limited in throughput. Functional cell-based assays represent an alternate approach for performing this task. We developed a functional assay to monitor the rate of bacterial gemcitabine breakdown using a highly sensitive bacterial reporter strain. Our method relies on standard laboratory equipment and can be implemented at high throughput to monitor drug breakdown by hundreds of strains simultaneously. This functional assay can be readily adapted to monitor degradation of other drugs. Key features Quantification of gemcitabine breakdown by incubating bacteria that degrades the drug and subsequently testing the growth of a reporter strain on filtered supernatant. Use of an optimized reporter strain that was genetically engineered to be a non-degrader strain and highly sensitive to gemcitabine. A high-throughput assay performed in microplates that can be adjusted for identifying bacteria with a fast or slow gemcitabine degradation rate. The assay results can be compared to results from a standard curve with known drug concentrations to quantify degradation rate.

Discovery of a small molecule ligand of FRS2 that inhibits invasion and tumor growth

PurposeAberrant activation of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases drives oncogenic signaling through its proximal adaptor protein FRS2. Precise disruption of this disease-causing signal transmission in metastatic cancers could stall tumor growth and progression. The purpose of this study was to identify a small molecule ligand of FRS2 to interrupt oncogenic signal transmission from activated FGFRs.MethodsWe used pharmacophore-based computational screening to identify potential small molecule ligands of the PTB domain of FRS2, which couples FRS2 to FGFRs. We confirmed PTB domain binding of molecules identified with biophysical binding assays and validated compound activity in cell-based functional assays in vitro and in an ovarian cancer model in vivo. We used thermal proteome profiling to identify potential off-targets of the lead compound.ResultsWe describe a small molecule ligand of the PTB domain of FRS2 that prevents FRS2 activation and interrupts FGFR signaling. This PTB-domain ligand displays on-target activity in cells and stalls FGFR-dependent matrix invasion in various cancer models. The small molecule ligand is detectable in the serum of mice at the effective concentration for prolonged time and reduces growth of the ovarian cancer model in vivo. Using thermal proteome profiling, we furthermore identified potential off-targets of the lead compound that will guide further compound refinement and drug development.ConclusionsOur results illustrate a phenotype-guided drug discovery strategy that identified a novel mechanism to repress FGFR-driven invasiveness and growth in human cancers. The here identified bioactive leads targeting FGF signaling and cell dissemination provide a novel structural basis for further development as a tumor agnostic strategy to repress FGFR- and FRS2-driven tumors.