Potency Assay Research Articles

Abstract 3527: Application of a deep learning based drug sensitivity prediction model on a novel anticancer drug

Abstract Discovery of a candidate drug molecule through the preclinical stages is an iterative multiparametric process until a candidate is selected for investigational new drug submission enabling studies. This iterative approach takes up significant time and effort to design, synthesize, and test new compounds. A specific testing step is evaluation of half maximal inhibitory concentration (IC50) of compounds on cell line (CL) models. To augment CL testing, computational approaches predicting the IC50 of a drug can be used to guide choice of optimization as the molecule goes through hit to lead optimization onto candidate selection. DeepDSC [1] is a deep learning (DL) model developed to predict the IC50 in a specific CL of a given drug by integrating public data from large-scale drug screens and high-throughput RNAseq profiling. The Cancer Cell Line Encyclopedia (CCLE) [2] and Genomics of Drug Sensitivity (GDSC) [3] projects compiled pharmacological profiles of 23 and 139 drugs across 491 and 655 cancer CLs, respectively. A 10-fold cross validation (CV) approach was employed to train DeepDSC on the datasets independently and reported the average root mean squared error (RMSE) and the coefficient of determination (R2) on the held-out sets across the folds. However, the model’s generalizability to novel compounds not in the dataset, specifically compounds that are structurally dissimilar, is yet to be tested. In this regard, the DeepDSC model was retrained, with modifications, on a subset of the GDSC dataset - GDSC2 - to access the generalizability of DL models on BRG399, a proprietary microtubule targeting agent (MTA) developed by BPGbio Inc. GDSC2 was used since data generated for BRG399 and GDSC2 share the same potency assay (CellTiter-Glo [6]), The GDSC2 set was split into training and held-out sets using a 80-20 random split. Further, we chose to optimize the learning rate for both the autoencoder and feed forward networks (FFN), and the L2 regularization factor for the FFN only. Hyperparameters were optimized using 5-fold CV on the training set. Finally, we evaluated the predictive performance of the model on BRG399, which was not present in the training set. We encoded each compound in GDSC2 (n=234) using Circular Fingerprints [4] and observed an average Jaccard (or Tanimoto) distance (JD) [5] of 0.892 across 27261 drug pairs. BRG399 showed an average JD of 0.873 across 234 drug pairs, with the most similar compound JD of 0.785. Our retrained DeepDSC model had RMSE 1.15 and R2 0.82 on the training set and RMSE 1.19 and R2 0.86 on the held-out set. However, on BRG399 tested CLs (n=101) model had RMSE 1.84 and R2 < 0, which indicated that predictions were on average worse compared to a mean-predictor model for BRG399. These results suggest that DL models that can generalize predictions to a held-out set for compounds that were present in the training set may not accurately predict IC50 of novel compounds not in the training set. Citation Format: Nischal M. Chand, Vivek Vishnudas, Michael A. Kiebish, Anjan Thakurta, Niven R. Narain, Stephane Gesta, Gregory M. Miller. Application of a deep learning based drug sensitivity prediction model on a novel anticancer drug [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3527.

Abstract 4017: CAR T cell-mediated cytotoxicity and cytokine release in response to varying levels of antigen expression on target cells

Abstract The development of immunotherapies relies on the use of in vitro potency assays, which are crucial for understanding the complex interactions between immune cells (effectors) and cancer cells (targets). Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapeutic approach that exhibits promise; however, varying levels of antigen expression on the surface of tumor cells can influence the efficacy of CAR T cell-mediated killing. To fully understand the impact of target antigen density on CAR T cell activity, we utilized a matrix approach to assess both target cell death, using an impedance-based in vitro potency assay to quantify CAR T cell-mediated cytotoxicity, as well as cytokine release with homogeneous LumitTM immunoassays. SKOV3 (high HER2 expression), A549 (low HER2 expression), or MDA-MB-231 (no HER2 expression) target cells were seeded into a 96-well microplate with embedded electrodes in the substrate that detect the attachment and proliferation of target cells. HER2 CAR T cells were added after 24 hours at effector-to-target (E:T) ratios of 1:5 or 1:1 and target cell cytolysis was recorded continuously by the Maestro Z for 72 hours. Cytolysis of the target cells was calculated by comparing the treated wells to no treatment control wells and full lysis wells (1% TritonX). At 72 hours post CAR T cell addition, complete killing was observed in SKOV3 cells at the 1:1 ratio, while A549 cells exhibited only 80% cytolysis. MDA-MB-231 cells showed 20% cytolysis, likely due to nonspecific killing by non-engineered T cells, as approximately only 78% of the CAR T cell population was CAR positive. Because the cytolysis assay was label-free, cytokine analysis was able to be multiplexed with the same plates. To assess release of pro-inflammatory cytokines, TNF-α and IFN-γ, we collected supernatant post-antigen exposure at 24, 48, and 72 h. At the 1:1 E:T ratio, CAR T cells co-cultured with target cells expressing low (A549) or high (SKOV3) levels of HER2 had the highest TNF-α production at 24 hours, with CAR T cells co-cultured with SKOV3 cells releasing 30.6% more TNF-α when compared to A549 cells. The highest levels of IFN-γ were detected in both A549 and SKOV3 groups at the 1:1 E:T ratio, with the highest levels observed from CAR T cells co-cultured with SKOV3 cells at 72 hours, releasing approximately 1459.6 +/- 357.3 pg/mL of IFN-γ, compared to 1093.8 +/- 387.5 pg/mL IFN-γ released by CAR T cells co-cultured with A549 cells. As expected, CAR T cells co-cultured with MDA-MB-231 cells did not release any detectable TNF-α or IFN-γ at either E:T ratios during the duration of the experiment. Future work will continue to explore multiplexed potency assays for the evaluation of CAR T cell therapies. Citation Format: Sierra Barnes, Denise Sullivan, Dan Lazar, Kim Haupt, Danielle Califano, Stacie Chvatal, Daniel Millard. CAR T cell-mediated cytotoxicity and cytokine release in response to varying levels of antigen expression on target cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4017.

Abstract 88: Novel cell-based bioassays enable preclinical animal studies during immuno-oncology drug development

Abstract Translation of immuno-oncology research into novel therapies relies on animal models to advise target selection and portray mechanism of action (MoA) and pharmacokinetics of lead candidates. Mouse, monkey, and more recently, canine models of human cancers are being utilized to this end. However, one major challenge in developing and utilizing these models is the lack of technology to characterize non-human analogs of therapeutic antibodies. Herein, we report the development of novel cell-based bioassays for mouse, monkey, and canine targets to enable immuno-oncology preclinical studies. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are important MoAs for antibody-based cancer immunotherapies. We have developed a suite of cell-based bioassays, each expressing a corresponding animal Fcγ receptor (FcγR) and a luciferase reporter that responds to FcγR activation. These assays exhibit sensitive and quantitative luciferase readouts of FcγR activation. Animal models are further used to provide insight into the potential efficacy of immune checkpoint (IC) inhibitors. Programmed cell death protein 1 (PD-1) is an IC receptor that negatively regulates T cell function. Blockade of PD-1 has demonstrated clinical efficacy, but there remains a significant fraction of patients unresponsive to this therapy. Combination of PD-1 inhibitors with other interventions has been a significant focus for many programs. However, tools for functional validation of non-human PD-1 inhibitors are lacking. We report here the development of two new bioassays for functional characterization of PD-1/PD-L1 inhibitors for mouse and canine targets. Each of these assays consists of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), with modulation from PD-1, and an artificial antigen presenting cell line (aAPC). The bioassays are sensitive, quantitative, and have demonstrated characteristics required for potency assays for validation of antibodies in preclinical combination studies. Citation Format: Jun Wang, Denise Garvin, Aileen Paguio, Elizabeth Perrin, Jim Hartnett, Mei Cong, Jamison Grailer. Novel cell-based bioassays enable preclinical animal studies during immuno-oncology drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 88.

Abstract 92: Assessment of an in vitro potency assay for evaluation of immune cell-mediated cytotoxicity using the Omni Pro 12 automated imaging platform

Abstract The development of immunotherapies relies on the use of in vitro potency assays—which are key for understanding complex interactions between immune cells and cancer cells. Immune effector T-cells are a promising cancer therapy due to their innate cytotoxicity. In particular, CAR T-cell therapy uses genetically engineered T-cells that express a chimeric antigen receptor that binds to a specific antigen on tumor cells. Assessing the efficacy and potency of CAR T-cell therapies in vitro and at high throughputs is vital for the preclinical development of these promising therapies. Here, we describe an in vitro potency assay that uses an automated imaging platform to quantify immune cell-mediated cytotoxicity of cancer target cells by immune effector cells. A GFP fluorescent and HER-2 expressing lung cancer cell line, A549, was seeded into a 96-well cell culture plate. Then, HER-2 CAR T-cells were added at 24 hours post target cell seeding at various E:T ratios (1:10, 1:5, 1:2, 1:1, and 5:1). Fluorescent images of the target cells were captured every 6 hours by the Omni Pro 12, an automated, high-throughput, live-cell analysis platform designed for continuous multi-well imaging inside an incubator. Automated image analysis was performed using the Confluency Module in the Axion Portal to view attachment and proliferation and quantify cytolysis of fluorescent A549 target cells. Percent cytolysis of the target cells was calculated by comparing the green fluorescent confluency of treated wells to no treatment control wells. A549-GFP cells showed a dose-dependent decrease in confluency over time that correlated with increasing amounts of CAR T-cells. At 92 hours post HER-2 CAR T-cell addition, the 5:1 ET group demonstrated approximately 91.8% +/- 2.0% cytolysis of A549-GFP cells, while the 1:10 group demonstrated approximately 69.2% +/- 3.0% cytolysis. Future work will evaluate differences in CAR T-cell potency when co-cultured with target cells that express different levels of HER-2. Overall, the Omni Pro 12 platform enables continuous quantification of the potency and kinetics of immune cell-mediated cytolysis. Citation Format: Denise Sullivan, Lieke Stemkens, Inge Thijssen, Svenja Meiler, Danielle Califano, Stacie Chvatal, Daniel Millard. Assessment of an in vitro potency assay for evaluation of immune cell-mediated cytotoxicity using the Omni Pro 12 automated imaging platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 92.

Development of a robust cell-based potency assay for a coxsackievirus A21 oncolytic virotherapy

Oncolytic viruses (OV) are part of a burgeoning field of investigational oncolytic therapy (OT), in which lytic viruses dissolve advanced tumors productively and specifically. One such OT is a Coxsackievirus A21 (CVA21) based OV that is currently under clinical evaluation. A tissue culture infectious dose (TCID50) assay was used for CVA21 potency release and stability testing in early clinical development. The titer measured in this method was an extrapolated value from cytopathic effect (CPE) observed during the serial dilution but doesn't represent direct viral killing of cells. Moreover, the assay was not deemed to be optimal to carry into late phase clinical development due to limitations in assay precision, turn-around time, and sample throughput. To address these points, we developed a plaque assay to measure viral plaque forming units to measure the potency value for drug substance (DS), drug product (DP) and virus seed (master and working) stocks. In this manuscript, we describe the steps taken to develop this plaque assay for the late-stage clinical development, which include the assay qualification, validation, and robustness protocols, and describe statistical methods for data analysis. Moreover, the method was validated for linearity, accuracy, precision, and specificity. Furthermore, the plaque assay quantifies OV infectivity with better precision (32% vs 58%), with higher sample throughput (22 samples/week vs 3 samples/week) and shorter assay turnaround time (4 days vs 7 days) than the TCID50 method. This assay development strategy can provide guidance for the development of robust cell-based potency methods for OVs and other infectious viral products.

Development of a Potency Assay for Nous-209, a Multivalent Neoantigens-Based Genetic Cancer Vaccine.

Quality control testing of vaccines, including potency assessment, is critical to ensure equivalence of clinical lots. We developed a potency assay to support the clinical advancement of Nous-209, a cancer vaccine based on heterologous prime/boost administration of two multivalent viral vector products: GAd-209 and MVA-209. These consist of a mix of four Adeno (Great Ape Adenovirus; GAd) and four Modified Vaccinia Ankara (MVA) vectors respectively, each containing a different transgene encoding a synthetic polypeptide composed of antigenic peptide fragments joined one after the other. The potency assay employs quantitative Reverse Transcription PCR (RT-Q-PCR) to quantitatively measure the transcripts from the four transgenes encoded by each product in in vitro infected cells, enabling simultaneous detection. Results showcase the assay's robustness and biological relevance, as it effectively detects potency loss in one component of the mixture comparably to in vivo immunogenicity testing. This report details the assay's setup and validation, offering valuable insights for the clinical development of similar genetic vaccines, particularly those encoding synthetic polypeptides.

Analytical characterization of full, intermediate, and empty AAV capsids.

Manufacturing of recombinant adeno-associated virus (AAV) vectors produces three types of capsids: full, intermediate, and empty. While there are different opinions about the impact of intermediate and empty capsids on safety and efficacy of AAV products, they are generally considered impurities because they are not the intended fully intact vector product. The presence of these impurities could impact product efficacy due to potential competition with fully packaged AAVs for cellular transduction, as well as have potential implications to patient safety due to increased capsid load during dosing. To determine the impact of intermediate capsids on potency, an AAV preparation was separated into fractions enriched for full, intermediate, or empty capsids. Using a matrix of in vitro (infectivity, gene expression, biological activity) and in vivo potency assays to determine potency as a function of capsid content, our results indicate that while intermediate capsids contribute to the vector genome titer of the product and are equally as infectious as full capsids, they do not contribute to the potency of the AAV product. This study confirms the criticality of reducing and controlling the level of intermediate capsids to ensure a more efficacious AAV product.

Enhancing transduction efficiency of adeno-associated virus 9 by cell line engineering: implication for gene therapy potency assay

Enhancing transduction efficiency of adeno-associated virus 9 by cell line engineering: implication for gene therapy potency assay

Potency assay to predict the anti-inflammatory capacity of a cell therapy product for macrophage-driven diseases: overcoming the challenges of assay development and validation

BackgroundGiven the high level of product complexity and limited regulatory guidance, designing and implementing appropriate potency assays is often the most challenging part of establishing a quality control testing matrix for a cell-based medicinal product. Among the most elusive tasks are the selection of suitable read-out parameters, the development of assay designs that most closely model the pathophysiological conditions, and the validation of the methods. Here we describe these challenges and how they were addressed in developing an assay that measures the anti-inflammatory potency of mesenchymal stromal cells (MSCs) in an M1 macrophage-dominated inflammatory environment. MethodsAn in vitro inflammation model was established by coculturing skin-derived ABCB5+ MSCs with THP-1 monocyte-derived M1-polarized macrophages. Readout was the amount of interleukin 1 receptor antagonist (IL-1RA) secreted by the MSCs in the coculture, measured by an enzyme-linked immunosorbent assay. ResultsIL-1RA was quantified with guideline-concordant selectivity, accuracy and precision over a relevant concentration range. Consistent induction of the macrophage markers CD36 and CD80 indicated successful macrophage differentiation and M1 polarization of THP-1 cells, which was functionally confirmed by release of proinflammatory tumor necrosis factor α. Testing a wide range of MSC/macrophage ratios revealed the optimal ratio for near-maximal stimulation of MSCs to secrete IL-1RA, providing absolute maximum levels per individual MSC that can be used for future comparison with clinical efficacy. Batch release testing of 71 consecutively manufactured MSC batches showed a low overall failure rate and a high comparability between donors. ConclusionsWe describe the systematic development and validation of a therapeutically relevant, straightforward, robust and reproducible potency assay to measure the immunomodulatory capacity of MSCs in M1 macrophage-driven inflammation. The insights into the challenges and how they were addressed may also be helpful to developers of potency assays related to other cellular functions and clinical indications.

Fully automated high-throughput immuno-µPlaque assay for live-attenuated tetravalent dengue vaccine development.

Dengue fever has remained a continuing global medical threat that impacts half of the world's population. Developing a highly effective dengue vaccine, with live-attenuated tetravalent vaccines as leading candidates, remains essential in preventing this disease. For the development of live virus vaccines (LVVs), potency measurements play a vital role in quantifying the active components of vaccine drug substance as well as drug product during various stages of research, development, and post-licensure evaluations. Traditional plaque-based assays are one of the most common potency test methods, but they generally take up to weeks to complete. Less labor and time-intensive potency assays are thus called for to aid in the acceleration of vaccine development, especially for multivalent LVVs. Here, we introduce a fully automated, 96-well format µPlaque assay that has been optimized as a high-throughput tool to evaluate process and formulation development of a live-attenuated tetravalent dengue vaccine. To the best of our knowledge, this is the first report of a miniaturized viral plaque method for dengue with full automation via an integrated robotic system. Compared to the traditional manual plaque assay, this newly developed method substantially reduces testing time by approximately half and allows for the evaluation of over ten times more samples per run. The fully automated workflow, from cell culture to plaque counting, significantly minimizes analyst hands-on time and improves assay repeatability. The study presents a pioneering solution for the rapid measurement of LVV viral titers, offering promising prospects for advancing vaccine development through high-throughput analytics.

Advancing Extracellular Vesicle Therapeutics into Clinical Trials for Central Nervous System Disorders

In December 2023, the US Food and Drug Administration released a draft guidance document titled “Potency Assurance for Cellular and Gene Therapy Products Draft Guidance for Industry” for public comment [1]. Potency assays are arguably the most problematic analytic release assay to develop for cell and cell derivate products, including Extracellular Vesicles (EV). EVs are produced by many cell types and harbor parent cell-specific bioactive cargoes including proteins and nucleic acids. They are unique intercellular signaling particles that can act as both the messenger and/or effector in recipient cells. We and others have shown that Neural Stem Cell EVs (NSC EVs) derived in bioreactors have therapeutic potential for treating neurological disease and Acute Ischemic Stroke (AIS). New FDA Investigational New Drug (IND) applications are being filed and specifically, we have an IND application for AIS. As the field grows, new INDs will be filed for various other therapeutic indications. In order for EV therapeutics to move efficiently through the regulatory process to approval, there is a need for more emphasis on and development of analytical assays directly related to complex and likely multimodal, mechanisms of action. Research focused on this area will lead to new disease-specific potency assays and identification of critical quality attributes.

Nanobodies as potential tools for microbiological testing of live biotherapeutic products

Nanobodies are highly specific binding domains derived from naturally occurring single chain camelid antibodies. Live biotherapeutic products (LBPs) are biological products containing preparations of live organisms, such as Lactobacillus, that are intended for use as drugs, i.e. to address a specific disease or condition. Demonstrating potency of multi-strain LBPs can be challenging. The approach investigated here is to use strain-specific nanobody reagents in LBP potency assays. Llamas were immunized with radiation-killed Lactobacillus jensenii or L. crispatus whole cell preparations. A nanobody phage-display library was constructed and panned against bacterial preparations to identify nanobodies specific for each species. Nanobody-encoding DNA sequences were subcloned and the nanobodies were expressed, purified, and characterized. Colony immunoblots and flow cytometry showed that binding by Lj75 and Lj94 nanobodies were limited to a subset of L. jensenii strains while binding by Lc38 and Lc58 nanobodies were limited to L. crispatus strains. Mass spectrometry was used to demonstrate that Lj75 specifically bound a peptidase of L. jensenii, and that Lc58 bound an S-layer protein of L. crispatus. The utility of fluorescent nanobodies in evaluating multi-strain LBP potency assays was assessed by evaluating a L. crispatus and L. jensenii mixture by fluorescence microscopy, flow cytometry, and colony immunoblots. Our results showed that the fluorescent nanobody labelling enabled differentiation and quantitation of the strains in mixture by these methods. Development of these nanobody reagents represents a potential advance in LBP testing, informing the advancement of future LBP potency assays and, thereby, facilitation of clinical investigation of LBPs.

Microfluidics enabled multi-omics triple-shot mass spectrometry for cell-based therapies.

In recent years, cell-based therapies have transformed medical treatment. These therapies present a multitude of challenges associated with identifying the mechanism of action, developing accurate safety and potency assays, and achieving low-cost product manufacturing at scale. The complexity of the problem can be attributed to the intricate composition of the therapeutic products: living cells with complex biochemical compositions. Identifying and measuring critical quality attributes (CQAs) that impact therapy success is crucial for both the therapy development and its manufacturing. Unfortunately, current analytical methods and tools for identifying and measuring CQAs are limited in both scope and speed. This Perspective explores the potential for microfluidic-enabled mass spectrometry (MS) systems to comprehensively characterize CQAs for cell-based therapies, focusing on secretome, intracellular metabolome, and surfaceome biomarkers. Powerful microfluidic sampling and processing platforms have been recently presented for the secretome and intracellular metabolome, which could be implemented with MS for fast, locally sampled screening of the cell culture. However, surfaceome analysis remains limited by the lack of rapid isolation and enrichment methods. Developing innovative microfluidic approaches for surface marker analysis and integrating them with secretome and metabolome measurements using a common analytical platform hold the promise of enhancing our understanding of CQAs across all "omes," potentially revolutionizing cell-based therapy development and manufacturing for improved efficacy and patient accessibility.

A sensitive cell-based assay for testing potency of botulinum neurotoxin type A

Botulinum neurotoxin type A (BoNT/A) is a biopharmaceutic widely used for the treatment of neu­rological diseases and in aesthetic medicine to achieve months-long paralysis of target muscles and glands. Large numbers of mice are used in the mouse bioassay (MBA) for various botulinum-related applications including batch release potency testing, antitoxin testing, countermeasure development, and basic research. BoNT/A intoxication causes severe suffering to the mice used for testing, and application-specific, non-animal alternatives are urgently needed. Cell-based assays (CBA) need to replicate all the physiological steps of botulinum intoxication, comprising neuronal binding, internali­zation, endosomal escape, and cleavage of synaptosomal-associated protein of 25 kDa (SNAP25). However, the CBA currently in use have limitations. In this study we show that LAN5 cells, a human neuroblastoma-derived cell line, are sensitive to BoNT/A and can be engineered to express a recom­binant NanoLuciferase (NanoLuc)-tagged SNAP25 reporter molecule. On exposure, the reporter molecule is cleaved and releases a NanoLuc-SNAP25 fragment that can be captured on a 96-well plate for quantitative luminometry using a cleavage-specific SNAP25 antibody. We demonstrate, using purified BoNT/A and a commercial BoNT/A product, that the sensitivity of this new cell-based assay is in the fM range, comparable to that of the MBA. The new assay could replace the MBA in research and commercial testing of BoNT/A, benefiting both scientific progress and animal welfare.

A sensitive cell-based assay for testing potency of Botulinum neurotoxin type A_suppl

A sensitive cell-based assay for testing potency of Botulinum neurotoxin type A_suppl

TNF‐a/IL‐1B cytokine licensing enhances immunomodulatory potential of allogeneic mesenchymal stromal cells

Aims/Purpose: Mesenchymal stromal cells (MSCs) have shown great potential for therapeutic treatment of inflammatory diseases; however, their immunomodulatory potential can be further enhanced by cytokine licensing. The aim of this work was to assess changes in MSC surface characterization profile and immunomodulatory function following licensing with TNF‐α/IL‐1β pro‐inflammatory cytokines.Methods: MSCs were isolated from the bone marrow of FVB mice (H2kq) and licensed with a combination of 50 ng/mL TNF‐α and 50 ng/mL IL‐1β or cultured in growth medium alone. MSCs were harvested following 72‐h cytokine stimulation and added to immune cell potency assays. Characterization of MSC surface marker expression was assessed by flow cytometry. The immunology potential of FVB MSCs as an allogeneic cell therapy was assessed in vitro through co‐culture with Balb/c (H2kd) αCD3/CD28 activated T lymphocytes or Balb/c IFN‐γ/LPS‐stimulated macrophages.Results: TNF‐α/IL‐1β licensing was found to induce surface marker phenotype alterations compared with unlicenced MSCs, with increased expression of CD73, CD44, Sca‐1, CD29 and PD‐L1 observed. TNF‐α/IL‐1β licensed MSCs also secreted significantly higher quantities of nitric oxide compared with their untreated counterparts at two separate timepoints, 24‐h and 42‐h post‐stimulation. TNF‐α/IL‐1β MSCs significantly suppressed both CD4+ and CD8+ T cell proliferation at three different rations (1:5, 1:10, 1:50 MSCs: T cells) and significantly increased expansion of CD4+CD25++FoxP3+ regulatory T cells following co‐culture. TNF‐α/IL‐1β MSCs were found to significantly decrease levels of IFN‐γ from proliferating T cells. TNF‐α/IL‐1β MSCs were positive for CD80 expression had significantly reduced MHC I expression and could increase expansion of CD206 on M1‐like macrophages. Both T cell and macrophage co‐cultures with TNF‐α/IL‐1β MSCs showed a significant increase in IL‐6.Conclusions: Our results show the potential to enhance the therapeutic effects of MSC through cytokine licensing. Currently, TNF‐α/IL‐1β MSCs are being tested in ocular surface injury models for therapeutic efficacy.Keywords: Mesenchymal Stromal Cells, Cytokine Licensing, Immunomodulation, TNF‐α/IL‐1β.

The totality of evidence approach in the development of AVT02 (adalimumab), a biosimilar to Humira.

The development of a biosimilar is based on comparative structural, physicochemical, functional and clinical assessments. The sum of these analyses encompasses the 'totality of evidence', which demonstrates no clinically meaningful differences between the biosimilar and the reference product (RP). Once biosimilarity has been established, provided there is suitable scientific justification, clinical data may be extrapolated to other indications of the RP. AVT02 has been developed as a biosimilar to high-concentration, low-volume Humira (adalimumab), an anti-tumour necrosis factor-alpha monoclonal antibody approved for various chronic inflammatory indications. The totality of evidence for AVT02 is described, supporting its approval as an adalimumab biosimilar for all approved indications globally. Analytical similarity assessments using mass spectrometry methods demonstrated identical amino acid sequences for AVT02 and the RP, with high similarity in terms of primary structure, post-translational modifications and higher-order structural attributes. The mechanism of action was assessed by various cell-based potency assays and binding assays, and the results demonstrated that AVT02 is highly similar to the RP. No clinically meaningful differences in terms of purity, potency and safety were observed, and minor differences in a few physiochemical attributes did not impact the in vitro biologic activity and were not considered clinically relevant. Clinical similarity was demonstrated by comparing the pharmacokinetic, efficacy, safety and immunogenicity profiles of AVT02 with those of the RP. Clinical studies supported similar pharmacokinetic and comparable immunogenicity profiles between AVT02 and the RP in healthy participants and participants with moderate-to-severe chronic plaque psoriasis, with no new safety signals detected. The totality of evidence described demonstrates the biosimilarity of AVT02 to the RP, thereby fulfilling the scientific and regulatory requirements for AVT02 as a high-concentration biosimilar for the treatment of chronic plaque psoriasis and all approved indications of the RP.

Rational arguments for regulatory acceptance of consistency testing: benefits of non-animal testing over in vivo release testing of vaccines

ABSTRACTIntroductionThere are rational arguments to replace existing in vivo potency and safety assays for batch release testing of vaccines with more advanced non-animal techniques to measure critical quality attributes. However, the introduction of in vitro alternatives to replace in vivo release assays of authorized vaccines is challenging.Areas coveredThis report describes the hurdles encountered in substituting in vivo assays and ways to overcome these and provides arguments why more advanced in vitro alternatives are superior, not only as a tool to monitor the quality of vaccines but also from a practical, economical, and ethical point of view. The rational arguments provided for regulatory acceptance can support a strategy to replace/substitute any in vivo batch release test if an appropriate non-animal testing strategy is available.Expert opinionFor several vaccines, in vivo release assays have been replaced leading to an optimized control strategy. For other vaccines, new assays are being developed that can expect to be introduced within 5–10 years. From a scientific, logistical, and animal welfare perspective, it would be beneficial to substitute all existing in vivo batch release assays for vaccines. Given the challenges related to development, validation, and acceptance of new methods, and considering the relatively low prices of some legacy vaccines, this cannot be done without government incentives and supportive regulatory authorities from all regions.

Development of optimized cytotoxicity assays for assessing the antitumor potential of CAR-T cells

CAR-T cells are T cells expressing a chimeric antigen receptor (CAR) rendering them capable of killing tumor cells after recognition of a target antigen. CD19 CAR-T cells have revolutionized the treatment of hematological malignancies. Their function is typically assessed by cytotoxicity assays using human allogeneic cell lines expressing the target antigen CD19 such as Nalm-6. However, an alloreactive reaction is observed with these cells, leading to a CD19-independent killing. To address this issue, we developed a fluorescence microscopy-based potency assay using murine target cells to provide an optimized cytotoxicity assay with enhanced specificity towards CD19.Murine NIH/3T3 (3T3) fibroblast-derived cell line and EL4 T-cell lymphoma-derived cell line were used as targets (no xenoreactivity was observed after coculture with human T cells). 3T3 and EL4 cells were engineered to express eGFP (enhanced Green Fluorescent Protein) and CD19 or CD22 using retroviral vectors. CD19 CAR-T cells and non-transduced (NT) control T cells were produced from several donors. After 4 h or 24 h, alloreactive cytotoxicity against CD19+ Nalm-6-GFP cells and CD19− Jurkat-GFP cells was observed with NT or CAR-T cells. In the same conditions, CAR-T but not NT cells specifically killed CD19+ but not CD19− 3T3-GFP or EL4-GFP cells. Both microscope- and flow cytometry-based assays revealed as sensitive as impedance-based assay. Using flow cytometry, we could further determine that CAR-T cells had mostly a stem cell-like memory phenotype after contact with EL4 target cells.Therefore, CD19+ 3T3-GFP or EL4-GFP cells and fluorescence microscopy- or flow cytometry-based assays provide convenient, sensitive and specific tools to evaluate CAR-T cell function with no alloreactivity.

Mesenchymal stromal cells suppress microglial activation and tumor necrosis factor production

Background aimsWhite matter diseases are commonly associated with microglial activation and neuroinflammation. Mesenchymal stromal cells (MSCs) have immunomodulatory properties and thus have the potential to be developed as cell therapy for white matter disease. MSCs interact with resident macrophages to alter the trajectory of inflammation; however, the impact MSCs have on central nervous system macrophages and the effect this has on the progression of white matter disease are unclear. MethodsIn this study, we utilized numerous assays of varying complexity to model different aspects of white matter disease. These assays ranged from an in vivo spinal cord acute demyelination model to a simple microglial cell line activation assay. Our goal was to investigate the influence of human umbilical cord tissue MSCs on the activation of microglia. ResultsMSCs reduced the production of tumor necrosis factor (TNF) by microglia and decreased demyelinated lesions in the spinal cord after acute focal injury. To determine if MSCs could directly suppress the activation of microglia and to develop an efficient potency assay, we utilized isolated primary microglia from mouse brains and the Immortalized MicroGlial Cell Line (IMG). MSCs suppressed the activation of microglia and the release of TNF after stimulation with lipopolysaccharide, a toll-like receptor agonist. ConclusionsIn this study, we demonstrated that MSCs altered the immune response after acute injury in the spinal cord. In numerous assays, MSCs suppressed activation of microglia and release of the pro-inflammatory cytokine TNF. Of these assays, IMG could be standardized and used as an effective potency assay to determine the efficacy of MSCs for treating white matter disease or other neuroinflammatory conditions associated with microglial activation.