BackgroundEngineered T cell therapies, such as Chimeric Antigen Receptor (CAR) T cell therapies offer great promise in becoming a new wave of highly specific therapies against solid tumors. Solid ...tumors generally lack the expression of tumor specific target antigens. The alternatively selected target antigens are often expressed at low levels in healthy tissues throughout the human body as well, which poses the risk of activation of engineered T cell therapies against these tissues resulting in severe side effects. Therefore, assessing the safety of engineered T cell therapies is a critical step during early development and before filing for Investigational New Drug (IND) status.MethodsUsing a set of assays, we have generated an in vitro safety profile for CAR-T cells targeting the ‘Human Epidermal growth factor Receptor 2’ (HER2) which is amplified and/or overexpressed in 20–30% of invasive breast carcinomas and ovarian cancers. To determine which tissues are most at risk for unwanted CAR-T cell reactivity, in silico analysis for expression of the HER2 gene was performed. Subsequently, HER2 protein expression in various tissues was validated by flow cytometry using a HER2 targeting antibody. Primary tissues and hiPSC-derived cells with high and low HER2 protein expression were selected, characterized, and utilized for in vitro co-culture assays to evaluate on-target off-tumor and/or off-target cytotoxicity of CAR-T cells. As readout we measured target cell viability by flow cytometry and/or high content analysis and T cell activation by cytokine release.ResultsOur study generated high quality data that provided insight into the safety of the HER2 targeting CAR-T cells. Cytotoxicity of HER2 CAR-T cells against low HER2 expressing human healthy cardiomyocytes and renal cells was observed with a clear E:T ratio dependent effect which was confirmed by IFNγ secretion. HER2 negative neurons showed a clear absence of CAR-T response. This data suggests a safety risk of our HER2 targeting CAR-T cells against heart and renal tissue.ConclusionsEngineered T cell therapies have the capacity to fill tremendous unmet medical needs and are moving into the clinic at a high pace. Shortening the timeframe towards clinical application, requires assay panels which can be conducted quickly and in are in line with the rigorous safety tests required before FDA approval. Our strategy of using primary tissues and hiPSC derived cells to generate a safety profile for these therapies in vitro is robust and can be applied during both early-stage development and late-stage testing of the therapeutic product.
BackgroundCell therapies such as Chimeric Antigen Receptor T cells (CAR-T) and T Cell Receptor (TCR) T cells are immune-therapeutic approaches showing great momentum in research and the clinic. To ...date, four anti-CD19 CAR-T products and one anti-BCMA CAR-T products have been approved by the FDA for the treatment of lymphoid malignancies. Many more CAR-T cell products are currently being explored, targeting a wide variety of tumor antigens directed towards both liquid and solid tumors as well other clinical indications. In early-stage pre-clinical development, the use of in vivo animal models has presented significant hurdles in translatability of cell therapies. As a result, the establishment of high-quality in vitro efficacy and safety studies to foster the development of such therapies has become critical. The purpose of this study was to develop several in vitro efficacy experiments aimed at determining cell therapy activity, specificity and potency.MethodsWe have generated CAR-T cells targeting the Human Epidermal growth factor Receptor 2 (HER2) as a model system. In vitro cytotoxicity co-culture assays were developed using flow cytometry-, high content analysis- or impedance-based read-outs.ResultsHER2 CAR-T cells efficiently reduced the viability of the HER2-positive cell line ZR-75-30 in an effector:target cell ratio-dependent manner but had a limited effect on the viability of the HER2-negative cell line MDA-MB-468, confirming the activity and selectivity of the T cell therapy. A more complex three-way co-culture system (HER2 CAR-T cells co-cultured with both HER2-positive and -negative target cells) confirmed HER2 CAR-T specificity under activating conditions. Finally, following several rounds of antigen stimulation, the HER2 CAR-T cells persistently killed HER2-positive tumor cells, indicative of ‘cellular fitness’.ConclusionsTo conclude, we developed several in vitro proof of concept assays for the assessment of cell therapy activity, specificity, and potency during early-stage development. (Three-way) co-culture or repeated antigen stimulation assays can be used to aid cell therapy discovery research and lead optimization. These in vitro assays will provide the possibility to select the best therapies to further progress to clinic.
Abstract
Neutrophils represent the first line of defense against pathogens by using multiple mechanisms, including phagocytosis, degranulation, and the release of neutrophil extracellular traps ...(NETs). NET formation can lead to a unique form of programmed cell death in neutrophils, called NETosis. In addition to its microbicidal function, compelling evidence has linked NETosis to the pathogenesis of diseases including autoimmune diseases, atherosclerosis and thrombosis.
To facilitate drug discovery programs for NETosis modulators, a high content biology-based method was developed and applied to blood derived primary human neutrophils. In summary, NETosis was triggered by PMA or BSA-Immune Complexes and the loss of membrane integrity together with spreading of NET formation beyond cell membranes as well as nuclear decondensation were captured in a live-cell setting on a CellVoyager (CV8000) platform. Furthermore, after addition of NETosis modulators (DPI and R406) to the triggered-neutrophils, a dose-dependent NETosis reduction was observed with optimal assay windows between 2.5 and 4 hours after trigger addition. Based on their distinct changes in nuclear morphology and loss of membrane integrity, this method was also able to distinguish various mechanisms of neutrophil cell death including NETosis and apoptosis. This procedure was successfully applied in medium-throughput screenings (96-well plate format), assessing dose-dependent inhibitory effects of selected large molecules, small molecules, and lipids on NETosis processes.
In conclusion:
We developed a high content biology-based approach that can be employed to advance drug discovery for therapeutic areas associated with NETosis.
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Abstract
Engineered T cell therapies, such as Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR) T cells, but also bi-specific antibody therapies, play a major role in the field of ...immuno-oncology and offer great promise in becoming a new wave of highly specific therapies against solid tumors. Solid tumors generally lack the expression of tumor specific target antigens, posing a significant safety challenge. Selected solid tumor target antigens are often expressed at low levels in healthy tissues throughout the human body, risking the activation of the engineered T cells against these tissues. This can potentially result in life-threatening side effects, as such, assessing the safety of engineered T cell therapies is a critical step during early development and before filing for Investigational New Drug (IND) status. We have generated an in vitro safety profile for CAR-T cells targeting the ‘Human Epidermal growth factor Receptor 2' (HER2). The gene encoding this receptor is found to be amplified and/or overexpressed in 20-30% of invasive breast carcinomas and ovarian cancers. To determine which tissues are most at risk for unwanted reactivity by the CAR-T cells, in silico analysis for expression of the HER2 gene was performed. Subsequently, HER2 protein expression in various tissues was validated by staining cells with a HER2 antibody and determining the percentage of positive cells by flow cytometry. Primary tissues and iPSC-derived cell with high and low HER2 protein expression were selected, characterized and utilized for in vitro co-culture assays to evaluate on-target off-tumor and/or off-target cytotoxicity of the HER2 targeting CAR-T cells. Readouts for the in vitro cytotoxicity assays included measuring target cell viability by flow cytometry and/or HCA and T cell activation by cytokine release. Our study generated high quality data that provided insight into the safety of the HER2 targeting CAR-T cells. Moreover, we were able to demonstrate the value of using iPSC-derived cells in de-risking selected tissues against unwanted reactivity of engineered T cell therapies. Our strategy to generate a safety profile for T cell therapies and bi-specific antibodies is robust can be applied during both early stage development and late stage testing of the therapeutic product. Engineered T cell therapies have the capacity to fill tremendous unmet medical needs and are moving into the clinic at a high rate. This expedited timetable, although good news for patients with late stage disease, results in less time to conduct the rigorous safety tests required before FDA approval. The use of primary tissues and iPSC derived cells to test the safety of these therapies in vitro has been and will be essential in getting these novel drugs on the market.
Citation Format: Sanne L. Holt, Sophie Vermond, Monique Hazenoot, Rene McLaughlin, Marco Guadagnoli, Marijn Vlaming. Primary or iPSC-derived cell-based cytotoxicity assays to assess potential safety risks of engineered T cell therapies in vitro 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 1373.
Abstract
Cell therapies, such as Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR) T cells, are some of the latest immune-therapeutic approaches, showing great momentum in research and now ...the clinic. Only recently, two anti-CD19-CAR-T products have been approved by the FDA and EMA for treatment of CD19+ lymphoid malignancies, including Acute Lymphoblastic Leukemia (B-ALL) and Diffuse Large B cell Lymphoma (DLBCL). Many more CAR-T cell products are being explored, targeting a wide variety of tumor antigens, directed towards both liquid and solid tumors as well other clinical indications such as autoimmune diseases or organ transplantations. In early stage development (pre-clinical research), the use of in vivo animal models has presented significant hurdles in translatability of cell therapies. As a result, the establishment of high-quality in vitro efficacy and safety studies to foster the development of such therapies has become critical. The purpose of this study is to show a number of in vitro efficacy experiments aimed at determining cell therapy activity, specificity and potency using an anti-HER2 CAR-T as a model system. To asses therapeutic activity, a proof of concept (PoC) study must be in place in which the therapy is tested for its ability to kill a reactive control cell line. Activity tests can be used to aid disease indications (including a panel of cell lines or primary material) and importantly, to aid lead optimization. Especially for cell therapies, activity experiments should be combined with “cellular fitness” tests, which are able to determine a therapy's ability to expand and persist following serial rounds of tumor challenges. Here we have developed an in vitro CAR-T activity and Repeated Antigen Stimulation (RAS) assay. Furthermore, specificity studies are aimed at quantifying the absence of killing of control cells that are negative for the target antigen. Here we used a three-way co-culture system in which effector cells are incubated with positive target cells and negative control cells in order to determine specificity under activating cellular conditions. Finally, to determine cell therapy potency we have used cytotoxicity co-culture experiments in which different effector-target ratios are tested at different time points to allow quantification of lower and upper limits of the cellular therapy to be tested. Our studies can generate a robust and essential efficacy data package that defines and describes a cell therapy's activity, specificity and potency, and is applicable to CAR-T cells and other engineered cell therapy approaches. One lesson learnt the hard way is that the most active and potent drug is not always the safest; for all cell therapies efficacy and safety will need to be balance and taken into considering during early development and lead optimization. Having developed a robust in vitro efficacy and safety platform, we keep strengthening our abilities to select the best therapies to further progress to clinic.
Citation Format: Sanne L. Holt, Sophie Vermond, Monique Hazenoot, Rene McLaughlin, Marco Guadagnoli, Marijn Vlaming. In vitro efficacy studies to support engineered T cell therapies 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 294.
Background
Microglia are resident immune effector cells in the CNS and play an essential role in neuroinflammation, ischemic and neurodegenerative disease. Therefore, microglia cells are considered a ...potential therapeutic target for neurodegenerative diseases. To fully understand the role of microglia, the preferred strategy would be to study primary human microglia isolated from post‐mortem human brain tissue. Microglia can be isolated from both control and diseased human brain tissue with confirmed neuropathology. However, the obvious limitation on brain collection and yield of isolated cells restricts the ability to perform screening studies. Induced pluripotent stem cells (iPSCs)‐derived microglia, may provide a suitable alternative for screening studies and large‐scale compound validation. Yet, to effectively use iPSC‐derived microglia, one must characterize the extent to which these cells faithfully represent biological processes in primary brain tissue.
Method
Here, we compared the gene expression and cytokine release from primary human microglia cells obtained from tissue provided by the Netherlands Brain Bank and iPSC‐derived microglia.
Result
Exposure of primary and iPSC‐derived microglia to LPS resulted in increased TNF‐α secretion in a concentration and time dependent manner. LPS‐mediated TNF‐α secretion was strongly inhibited by dexamethasone. Priming of primary and iPSC‐derived microglia with LPS and treatment with nigericin, a potent inflammasome activator, resulted in robust secretion of IL‐1β and IL‐18. Furthermore, nigericin induced IL‐1β and IL‐18 release was blocked by the inflammasome inhibitor MCC950 in both cell types. In addition, similarly to in‐house differentiated microglia, commercially available iPSC‐derived microglia (Bit.bio) showed a strong expression of specific markers as well as cytokine response upon LPS treatment.
Conclusion
Taken together, we successfully demonstrated that primary and iPSC‐derived microglia respond similarly to LPS and nigericin treatment. For these reasons, these cell types could serve as a reliable tool for evaluating the potency and efficacy of prospective drugs for multiple neurological diseases associated with microglia activation, such as Alzheimer’s and Parkinson’s Disease.
Background
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons. Many ALS cases are associated with nuclear‐to‐cytosolic mislocalization and phosphorylation ...of the transactivation response DNA binding protein (TDP)‐431. This is caused by several mutations such as M337V in the TAR DNA binding protein (TARDBP) gene1. In addition, proteasomal degradation is often affected, resulting in aggregation of TDP‐432. TDP‐43 is involved in many mRNA processes, and its depletion results in mis‐splicing of the neuronal growth associated factor, stathmin 2 (STMN‐2)3. This mis‐splicing contributes to axonal degeneration. To model the ALS phenotypes in vitro, proteasomal inhibition is often used to trigger TDP‐43‐associated phenotypes2.
Methods
In this report we assessed if homozygous or heterozygous CRISPR‐edited TPD‐43 M337V mutations in induced pluripotent stem cell (iPSC)‐derived glutamatergic neurons, generated by bit.bio using the opti‐ox technology4‐5, alone, or in combination with an MG‐132 proteasomal inhibition trigger, would result in TDP‐43 mislocalization and/or STMN‐2 splicing, and could therefore be used as an ALS‐relevant disease model. This was assessed in highly mature (DIV21), and less mature (DIV7) neurons.
Results
Immunocytochemistry, high‐content imaging, and automated image analysis were used for visualization, localization, and quantification of proteins of interest in individual cells. Here, increase in TDP‐43 mislocalization, phosphorylation (and aggregation) was observed upon acute proteasomal inhibition in all cell lines compared to their respective untreated sample, with more extreme phenotypes on DIV7. More interestingly, the homozygous M337V cell line D5 showed showed TDP‐43 associated phenotypes in untreated conditions at DIV7. Digital droplet PCR was used to evaluate mRNA expression, using absolute quantification of nucleic acid molecules. This showed decrease of full length STMN‐2 and the generation of truncated STMN‐2 upon MG‐132 treatment in most cell lines. Additionally, preliminary functional data generated using Multi Electrode Arrays (MEA) showed that all neuronal lines demonstrated formation of synchronous activity at late stage of maturation, suggesting these neurons are electrophysiologically active and amenable to functional studies.
Conclusion
These results suggest that wildtype and mutant clones show TDP‐43 dysregulation and mis‐splicing of STMN‐2 upon acute proteasomal inhibition, thus establishing an ALS relevant phenotype that can be used for in vitro drug discovery.
Background
Tauopathies, such as frontotemporal dementia (FTD) and Alzheimer’s dimentia (AD), are neurodegenerative diseases characterized by the pathological aggregation of paired helical filaments ...(PHFs) or neurofibrillary tangles (NFTs) within neurons and glia, leading to cell death1. PHFs and NFTs are formed by aggregation of hyperphosphorylated tau1. Mutations in the microtubule‐associated protein tau (MAPT) gene result in tauopathies. Moreover, FTD is a common clinical syndrome of 4 repeat (4R) tauopathies, as defined by aggregation of tau protein isoforms with four microtubule binding domains2. Here, we aimed to develop and characterize a physiologically relevant and robust in vitro FTD model, to aid the future development of FTD disease therapeutics.
Method
Using CRISPR‐Cas9 gene editing technology, familial mutations MAPT P301S and N279K underlying AD/FTD3 were engineered into an iPSC line that carries the opti‐oxTM technology and can rapidly be reprogrammed into glutamatergic neurons4. By means of immunocytochemistry we characterized b‐amyloid oligomer treated neurons, and neurons with the MAPT mutations to assess tau hyperphosphorylation.
Result
The CRISPR‐edited MAPT neurons can mature and show a healthy morphology. b‐amyloid oligomers (dose dependently) induce tau hyperphosphorylation in wild type glutamatergic neurons. Total tau expression is reduced in MAPT P301S homozygous and N279K heterozygous cells when compared to wild type cells. The MAPT P301S cells also show hyperphosphorylation for p‐tau 217, p‐tau202/205, and p‐tau404, and the N279K cells show hyperphosphorylation for p‐tau202/205, and p‐tau404.
Conclusion
The elevated p‐tau to total tau ratio measured by the immunocytochemistry assay indicates the potential of at least two cell lines as possible disease models to aid future research into developing AD/FTD disease therapeutics.
References:
1. Silva MC, eLife, 2019
2. Seward ME, et al, 2013. Journal Cell Sci 126(5):1278‐1286
3. Hutton M, et al, 1998, Nature 393:702‐705
4. PawlowskiM, et al, 2017. Stem cell reports 8(4), 803‐812
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