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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

PGC1α regulates mitochondrial oxidative phosphorylation involved in cisplatin resistance in ovarian cancer cells via nucleo-mitochondrial transcriptional feedback

Mitochondria play an important role in effective cell energy production and cell survival under stress conditions, such as treatment with chemotherapeutic drugs. Mitochondrial biogenesis is increased in ovarian cancer tissues, which is accompanied by alteration of mitochondrial energy metabolism, structure, and dynamics. These factors are involved in tumorigenesis and apoptosis resistance, highlighting the role of mitochondria in resisting cisplatin toxicity. Cisplatin-resistant ovarian cancer cells are dependent on mitochondrial OXPHOS for energy supply, and intracellular PGC1α-mediated mitochondrial biogenesis levels are increased in this cell line, indicating the important role of mitochondrial oxidative phosphorylation in cisplatin resistance. As PGC1α is a key molecule for integrating and coordinating nuclear DNA and mitochondrial DNA transcriptional machinery, an investigation into the regulatory mechanism PGC1α in mitochondrial energy metabolism via transcription may provide new clues for solving chemotherapy resistance. In the present study, it was demonstrated that inhibiting the expression of PGC1α decreased nuclear and mitochondrial DNA transcription factor expression, leading to increased lactic acid production and decreased cellular oxygen consumption and mitochondrial oxidative phosphorylation. Furthermore, mitochondrial stress-induced ROS production, as a feedback signal from mitochondria to the cell nucleus, increased PGC1α expression in SKOV3/DDP cells, which was involved in mitochondrial oxidative phosphorylation regulation. Collectively, the present study provides evidence that PGC1α-mediated nuclear and mitochondrial transcription feedback regulates energy metabolism and is involved in ovarian cancer cells escaping apoptosis during cisplatin treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

The role of non-apoptotic cell death in the treatment and drug-resistance of digestive tumors

Tumor cell apoptosis evasion is one of the main reasons for easy metastasis occurrence, chemotherapy resistance, and the low five-year survival rate of digestive system tumors. Current research has shown that non-apoptotic cell death plays an important role in tumors of the digestive system. Therefore, increasing the proportion of non-apoptotic tumor cells is one of the effective methods of improving therapeutic efficacies for digestive system tumors. Non-apoptotic cell death modes mainly include autophagic cell death, pyroptosis, ferroptosis, in addition to other cell death modes. This review covers a systematic review relating to the research progress made into autophagic cell death, pyroptosis, ferroptosis, and other cell death modes in the treatment of digestive system tumors. It also highlights how treatment is a reasonable prospect based on clinical experience and provides reliable guidance for the further development of digestive system tumor treatments.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Enhancement of anticancer drug sensitivity in multidrug resistance cells overexpressing ATP-binding cassette (ABC) transporter ABCC10 by CP55, a synthetic derivative of 5-cyano-6-phenylpyrimidin

Highlights: • Multidrug resistance (MDR) in cancer is one of the major obstacles in chemotherapy. • ABCC10 mediates resistance to wide spectrum of chemotherapeutic agents, but only limited number of studies are available. • Overexpression of ABCC10 has been linked to various types of cancers including colorectal, gastric and lung cancer. • CP55 is a potent ABCC10 inhibitor which enhances the anticancer drug sensitivity in ABCC10-overexpression MDR cells. ATP-binding cassette (ABC) transporter C10 (ABCC10), also named multidrug resistance protein 7 (MRP7), is a member of ABC transporter superfamily and has been revealed to transport a wide range of chemotherapeutic agents including taxanes, epothilone B, Vinca alkaloids, and anthracyclines. In our previous study, a 5-cyano-6-phenylpyrimidin derivative CP55 was synthesized and found significantly reversal effect of multidrug resistance (MDR) mediated by ABCB1. In this study, we found CP55 also efficiently reversed MDR mediated by ABCC10. Our in vitro study showed that co-treatment with CP55 significantly increased the efficacy of ABCC10-substrate anticancer drugs in MDR cells overexpressing ABCC10. Furthermore, we showed that treatment with CP55 increased the intracellular accumulation of [{sup 3}H]-labeled anticancer drugs and in-turn decreasing drug efflux by inhibiting the transport activity, without altering ABCC10 protein ex-pression level or cellular localization. Potential CP55-ABCC10 interactions were predicted via docking analysis using human ABCC10 homology model and obtained high docking score. Therefore, CP55 represents a promising therapeutic agent in the combinational treatment of chemo-resistant cancer related to ABCC10.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Dichloroacetate enhances the anti-tumor effect of sorafenib via modulating the ROS-JNK-Mcl-1 pathway in liver cancer cells

Liver cancer is one of the most common and high recurrence malignancies. Besides radiotherapy and surgery, chemotherapy also plays an essential role in the treatment of liver cancer. Sorafenib and sorafenib-based combination therapies have been proven efficacy against tumors. However, previous clinical studies have indicated that some patients with liver cancer are resistant to sorafenib treatment and the existing strategies are not satisfactory in the clinic. Therefore, it is urgent to investigate strategies to improve the effectiveness of sorafenib for liver cancer and to explore effective drug combinations. In the present study, we found that dichloroacetate (DCA) could significantly enhance the anti-tumor effect of sorafenib on liver cancer cells, including reduced viability and dramatically promoted apoptosis in liver cancer cells. Moreover, compared to sorafenib alone, the combination of DCA and sorafenib markedly increased the degradation of anti-apoptotic protein Mcl-1 by enhancing its phosphorylation. Overexpression of Mcl-1 could significantly attenuate the synergetic effect of DCA and sorafenib on apoptosis induction in liver cancer cells. Furthermore, we found that the ROS-JNK pathway was obviously activated in the DCA combined sorafenib group. The levels of ROS and p-JNK were dramatically up-regulated in the two drug combination groups. Antioxidant NAC could alleviate the synergetic effects of DCA and sorafenib on ROS generation, JNK activation, Mcl-1 degradation, and cell apoptosis. Moreover, DCA and sorafenib's effects on Mcl-1 degradation and apoptosis could also be inhibited by JNK inhibitor ‘SP’600125. Finally, the synergetic effects of DCA and sorafenib on tumor growth suppression, Mcl-1 degradation and induction of apoptosis were also validated in liver cancer xenograft in vivo. These findings indicate that DCA enhances the anti-tumor effect of sorafenib via the ROS-JNK-Mcl-1 pathway in liver cancer cells. This study may provide new insights to improve the chemotherapeutic effect of sorafenib, which may be benecial for further clinical application of sorafenib in liver cancer treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Integrin α5 mediates intrinsic cisplatin resistance in three-dimensional nasopharyngeal carcinoma spheroids via the inhibition of phosphorylated ERK /caspase-3 induced apoptosis

Highlights: • Highly expressed integrin α5 was observed in in vitro three-dimensional NPC spheroids. • The overexpression of integrin α5 in NPC spheroids demonstrated a correlation with intrinsic cisplatin resistance. • The integrin α5-induced intrinsic cisplatin resistance was mediated by an inactivation of ERK/caspase-3-induced apoptosis. • ATN-161, an integrin α5β1 inhibitor, reduced integrin α5 expression and increased sensitization to cisplatin in NPC spheroids. • ATN-161 reversed the intrinsic cisplatin resistance in NPC spheroids by reactivating ERK/caspase-3-induced apoptosis. Nasopharyngeal carcinoma (NPC) originates in the nasopharynx epithelium. Although concurrent chemoradiation therapy followed by chemotherapy is considered as an effective treatment, there is substantial drug resistance in locally advanced NPC patients. One major contributor to the chemoresistance includes aberrant expression of cell adhesion molecules, such as integrin α and β subunits, giving rise to cell adhesion-mediated drug resistance. Thus, the aim of this study was to investigate the effect of integrin α5 on the development of intrinsic cisplatin resistance in NPC and the associated underlying mechanisms using in vitro three-dimensional (3D) spheroid models, as well as induced cisplatin-resistant NPC (NPCcisR). We demonstrated that established 3D highly- (5-8F) and lowly- (6-10B) metastatic NPC spheroids overexpressed integrin α5 and aggravated their resistance to cisplatin. Besides, enhanced integrin α5 resulted in substantially reduced growth, corresponding to G{sub 0}/G{sub 1} and G{sub 2}/M cell cycle arrest. In addition, 5-8FcisR and 6-10BcisR cells in 3D forms synergistically strengthened endurance of their spheroids to cisplatin treatment as observed by increased resistance index (RI) and decreased apoptosis. Mechanistically, the aberrantly expressed integrin α5 decreased drug susceptibility in NPC spheroids by inactivating ERK and inhibition of caspase-3 inducing apoptosis. Furthermore, the effect of integrin α5 inducing intrinsic resistance was verified via treatment with ATN-161, a peptide inhibitor for integrin α5β1. The results showed dramatic reduction in integrin α5 expression, reversal of ERK phosphorylation and caspase-3 cleavage, together with elevated cisplatin sensitivity, indicating regulation of innate drug resistance via integrin α5. Taken together, our findings suggest that integrin α5 could act as a promising target to enhance the chemotherapeutic sensitivity in NPC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Anticancer effect of AZD2461 PARP inhibitor against colon cancer cells carrying wt or dysfunctional p53

Colon cancer is one of the most common cancers, currently treated with traditional chemotherapies or alternative therapies. However, these treatments are still not enough effective and induce several side effects, so that the search of new therapeutic strategies is needed. The use of Poly-(ADP-ribose)-polymerase (PARP) inhibitors, although originally approved against BRCA-1 or BRCA-2 mutated cancers, has been extended, particularly in combination with other treatments, to cure cancers that do not display defects in DNA repair signaling pathways. The role of p53 oncosuppressor in the regulating the outcome of PARP inhibitor treatment remains an open issue. In this study, we addressed this topic by using a well-tolerated PARP 1/2/3 inhibitor, namely AZD2461, against colon cancer cell lines with different p53 status. We found that AZD2461 reduced cell proliferation in wtp53 and p53−/− cancer cells by increasing ROS and DNA damage, while R273H mutant (mut) p53 counteracted these effects. Moreover, AZD2461 improved the reduction of cell proliferation by low dose radiation (IR) in wtp53 cancer cells, in which a down-regulation of BRCA-1 occurred. AZD2461 did not affect cell proliferation of mutp53 colon cancer cells also in combination with low dose radiation, suggesting that only wt p53 or p53 null colon cancer cells could benefit AZD2461 treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Rutin is a potent senomorphic agent to target senescent cells and can improve chemotherapeutic efficacy

Aging is a major risk factor for most chronic disorders, for which cellular senescence is one of the central hallmarks. Senescent cells develop the pro-inflammatory senescence-associated secretory phenotype (SASP), which significantly contributes to organismal aging and age-related disorders. Development of senotherapeutics, an emerging class of therapeutic agents to target senescent cells, allows to effectively delay aging and alleviate chronic pathologies. Here we report preliminary outputs from screening of a natural medicinal agent (NMA) library for senotherapeutic candidates and validated several agents with prominent potential as senomorphics. Rutin, a phytochemical constituent found in a number of plants, showed remarkable capacity in targeting senescent cells by dampening expression of the full spectrum SASP. Further analysis indicated that rutin restrains the acute stress-associated phenotype (ASAP) by specifically interfering with the interactions of ATM with HIF1α, a master regulator of cellular and systemic homeostasis activated during senescence, and of ATM with TRAF6, part of a key signaling axis supporting the ASAP development toward the SASP. Conditioned media produced by senescent stromal cells enhanced the malignant phenotypes of prostate cancer cells, including in vitro proliferation, migration, invasion, and more importantly, chemoresistance, while rutin remarkably downregulated these gain-of-functions. Although classic chemotherapy reduced tumor progression, the treatment outcome was substantially improved upon combination of a chemotherapeutic agent with rutin. Our study provides a proof of concept for rutin as an emerging natural senomorphic agent, and presents an effective therapeutic avenue for alleviating age-related pathologies including cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Oxaliplatin–DNA Adducts as Predictive Biomarkers of FOLFOX Response in Colorectal Cancer: A Potential Treatment Optimization Strategy

FOLFOX is one of the most effective treatments for advanced colorectal cancer (CRC). However, cumulative oxaliplatin neurotoxicity often results in halting the therapy. Oxaliplatin functions predominantly via the formation of toxic covalent drug-DNA adducts. We hypothesize that oxaliplatin-DNA adduct levels formed in vivo in peripheral blood mononuclear cells (PBMC) are proportional to tumor shrinkage caused by FOLFOX therapy. We further hypothesize that adducts induced by sub-therapeutic "diagnostic microdoses" are proportional to those induced by therapeutic doses and are also predictive of response to FOLFOX therapy. These hypotheses were tested in CRC cell lines and a pilot clinical study. Four CRC cell lines were cultured with therapeutically relevant (100 µM) or diagnostic microdose (1 µM) concentrations of [ 14 C]oxaliplatin. The C-14 label enabled quantification of oxaliplatin-DNA adduct level with accelerator mass spectrometry (AMS). Oxaliplatin-DNA adduct formation was correlated with oxaliplatin cytotoxicity for each cell line as measured by the MTT viability assay. Six CRC patients received by IV a diagnostic microdose containing [ 14 C]oxaliplatin prior to treatment, as well as a second [ 14 C]oxaliplatin dose during FOLFOX chemotherapy, termed a "therapeutic dose." Oxaliplatin-DNA adduct levels from PBMC correlated significantly to mean tumor volume change of evaluable target lesions (5 of the 6 patients had measurable disease). Finally, oxaliplatin-DNA adduct levels were linearly proportional between microdose and therapeutically relevant concentrations in cell culture experiments and patient samples, as was plasma pharmacokinetics, indicating potential utility of diagnostic microdosing.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

The FBXW7-binding sites on FAM83D are potential targets for cancer therapy

Increasing evidence shows the oncogenic function of FAM83D in human cancer, but how FAM83D exerts its oncogenic function remains largely unclear. Here, we investigated the importance of FAM83D/FBXW7 interaction in breast cancer (BC). We systematically mapped the FBXW7-binding sites on FAM83D through a comprehensive mutational analysis together with co-immunoprecipitation assay. Mutations at the FBXW7-binding sites on FAM83D led to that FAM83D lost its capability to promote the ubiquitination and proteasomal degradation of FBXW7; cell proliferation, migration, and invasion in vitro; and tumor growth and metastasis in vivo, indicating that the FBXW7-binding sites on FAM83D are essential for its oncogenic functions. A meta-evaluation of FAM83D revealed that the prognostic impact of FAM83D was independent on molecular subtypes. The higher expression of FAM83D has poorer prognosis. Moreover, high expression of FAM83D confers resistance to chemotherapy in BCs, which is experimentally validated in vitro. We conclude that identification of FBXW7-binding sites on FAM83D not only reveals the importance for FAM83D oncogenic function, but also provides valuable insights for drug target.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

A Simple Route for Open Fluidic Devices with Particle Walls

Open fluidics, allowing liquid in a flow channel to interact with the external environment, is a revolutionary concept. However, fabricating a highly stable open fluidic device of arbitrary complexity, while maintaining reconfigurability, is still a challenge. This is achieved by the use of a patterned substrate and liquids that are covered with functional, readily available hydrophobic particles, providing great flexibility in the construction and use of open fluidic structures. Decorated with a coating of modified carbon nanotubes (CNTs) to encapsulate the fluids, the study capitalizes on the photothermal characteristics of CNTs to fabricate a device to probe the effects of temperature on tumor chemotherapy. The strategy substantially increases the availability and potential use of open fluidic devices.

Liu, Heng↗
DOE OSTI2022

Development of a prediction model for radiotherapy response among patients with head and neck squamous cell carcinoma based on the tumor immune microenvironment and hypoxia signature

Abstract Introduction The immune system and hypoxia are major factors influencing radiosensitivity in patients with different cancer types. This study aimed at developing a model to predict radiotherapy response in patients with head and neck squamous cell carcinoma (HNSCC) based on the tumor immune microenvironment and hypoxia signature. Materials and Methods We first evaluated the hypoxia status and tumor immune microenvironment in the Cancer Genome Atlas (TCGA) cohort by using transcriptomic data. Differentially expressed genes (DEGs) were identified between the “high immunity and low hypoxia” and “low immunity and high hypoxia” groups and those DEGs significantly associated with disease‐specific survival in the univariate Cox regression analysis were selected as the prognostic DEGs. We selected the immune hypoxia–related genes (IHRGs) by intersecting prognostic DEGs with immune and hypoxia gene sets. We used the IHRGs to train a multivariate Cox regression model in the TCGA cohort, based on which we calculated the IHRG prognostic index (IHRGPI) for each patient and validated its efficacy in predicting radiotherapy response in the Gene Expression Omnibus cohorts. Furthermore, we explored potential mechanisms and effective combinational treatment strategies for different IHRGPI groups. Results Five IHRGs were used to construct the IHRGPI, which was used to dichotomize the cohorts. The patients with lower IHRGPI showed a better radiotherapy response across different cohorts and endpoints, including overall survival, progression‐free survival, and recurrence‐free survival ( p < 0.05). Patients with higher IHRGPI showed greater hypoxia and lesser immune cell infiltration. A lower IHRGPI indicated a better immunotherapy response, while a higher IHRGPI indicated a better chemotherapy response. Conclusions IHRGPI is promising for predicting radiotherapy response and guiding combinational treatment strategies in patients with HNSCC.

Zhu, Guang‐Li↗
DOE OSTI2024

NCAPH drives breast cancer progression and identifies a gene signature that predicts luminal a tumour recurrence

Luminal A tumours generally have a favourable prognosis but possess the highest 10-year recurrence risk among breast cancers. Additionally, a quarter of the recurrence cases occur within 5 years post-diagnosis. Identifying such patients is crucial as long-term relapsers could benefit from extended hormone therapy, while early relapsers might require more aggressive treatment. We conducted a study to explore non-structural chromosome maintenance condensin I complex subunit H’s (NCAPH) role in luminal A breast cancer pathogenesis, both in vitro and in vivo, aiming to identify an intratumoural gene expression signature, with a focus on elevated NCAPH levels, as a potential marker for unfavourable progression. Our analysis included transgenic mouse models overexpressing NCAPH and a genetically diverse mouse cohort generated by backcrossing. A least absolute shrinkage and selection operator (LASSO) multivariate regression analysis was performed on transcripts associated with elevated intratumoural NCAPH levels. We found that NCAPH contributes to adverse luminal A breast cancer progression. The intratumoural gene expression signature associated with elevated NCAPH levels emerged as a potential risk identifier. Transgenic mice overexpressing NCAPH developed breast tumours with extended latency, and in Mouse Mammary Tumor Virus (MMTV)-NCAPH ErbB2 double-transgenic mice, luminal tumours showed increased aggressiveness. High intratumoural Ncaph levels correlated with worse breast cancer outcome and subpar chemotherapy response. A 10-gene risk score, termed Gene Signature for Luminal A 10 (GSLA10), was derived from the LASSO analysis, correlating with adverse luminal A breast cancer progression. The GSLA10 signature outperformed the Oncotype DX signature in discerning tumours with unfavourable outcomes, previously categorised as luminal A by Prediction Analysis of Microarray 50 (PAM50) across three independent human cohorts. This new signature holds promise for identifying luminal A tumour patients with adverse prognosis, aiding in the development of personalised treatment strategies to significantly improve patient outcomes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

2.5 Å-resolution structure of human CDK-activating kinase bound to the clinical inhibitor ICEC0942

The human CDK-activating kinase (CAK), composed of CDK7, cyclin H, and MAT1, is involved in the control of transcription initiation and the cell cycle. Because of these activities, it has been identified as a promising target for cancer chemotherapy. A number of CDK7 inhibitors have entered clinical trials, among them ICEC0942 (also known as CT7001). Structural information can aid in improving the affinity and specificity of such drugs or drug candidates, reducing side effects in patients. Here, we have determined the structure of the human CAK in complex with ICEC0942 at 2.5 Å-resolution using cryogenic electron microscopy. Our structure reveals conformational differences of ICEC0942 compared with previous X-ray crystal structures of the CDK2-bound complex, and highlights the critical ability of cryogenic electron microscopy to resolve structures of drug-bound protein complexes without the need to crystalize the protein target.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus

Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3'-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII 170-755 , as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Evaluating Exposures of Bay Mussels (Mytilus trossulus) to Contaminants of Emerging Concern through Environmental Sampling and Hydrodynamic Modeling.

Bay mussels (Mytilus trossulus) were transplanted to 18 locations representing a range of potential exposures throughout Puget Sound, WA. Tissues were analyzed for over 200 organic contaminants. Results indicated the widespread exposure of marine organisms to trace levels of organic contaminants including the synthetic opioid oxycodone, present at three urban sites, and the chemotherapy drug melphalan, present at nine locations, at levels that may be of biological concern. Land-use and wastewater outfalls were evaluated as potential sources of CECs to the nearshore. Exposure to alkylphenol ethoxylates was associated with increased impervious surfaces in upland watersheds. A hydrodynamic simulation was performed using the Salish Sea Model to integrate inputs from 99 wastewater sources to Puget Sound. Predictions were consistent with concentrations of several wastewater-associated contaminants and d15N enrichment. These results support the notion that Puget Sound nearshore biota suffer chronic exposures to a suite of contaminants from multiple sources and provide critical to focus future monitoring and management.

toxic contamination, Salish Sea, Sish Sea Model, m↗
DOE OSTI2020

Rapid and Simultaneous Characterization of Drug Conjugation in Heavy and Light Chains of a Monoclonal Antibody Revealed by High-Resolution Ion Mobility Separations in SLIM

Antibody-drug conjugates (ADCs) have recently gained traction in the biomedical community due to their promise for human therapeutics and an alternative to chemotherapy for cancer. Crucial metrics for ADC efficacy, safety, and selectivity are their drug-antibody ratios (DARs). However, DAR characterization (i.e., determining the average number of conjugated drugs on the antibody) through analytical methods remains challenging due to the heterogeneity of drug conjugation as well as the numerous post-translational modifications possible in the monoclonal antibody. In this work, we report on the use of high-resolution ion mobility spectrometry separations in our structures for lossless ion manipulations coupled to mass spectrometry (SLIM IMS-MS) platform for the rapid and simultaneous characterization of the drug load profile (i.e., stoichiometric distribution of the number of conjugated drugs present on the mAb), determination of the weighted average DAR in both the heavy and light chains of a model antibody-drug conjugate, and calculation of the overall DAR of the ADC. After chemical reduction of the ADC and a subsequent 31.5 meter SLIM IMS separation the various drug bound antibody species could be well resolved for both chains. Furthermore, we also show that higher resolution separations were possible with SLIM IMS as compared to ones performed on a commercially available (~1 meter) drift tube IMS-MS platform. Interestingly, additional gas-phase substructures become evident with longer path length SLIM IMS, potentially providing insight related to how drug conjugation may alter the structural properties of a monoclonal antibody and thus affect its overall potency and/or selectivity for a given antigen. We expect high-resolution SLIM IMS separations will augment the existing toolbox for ADC characterization, particularly for structural confirmation to enable the rapid optimization of DAR for a given ADC and thus better understand its potential toxicity and potency.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

The ELISA Detectability and Potency of Pegfilgrastim Decrease in Physiological Conditions: Key Roles for Aggregation and Individual Variability

PEGylated recombinant human granulocyte colony stimulating factor (pegflgrastim) is used clinically to accelerate immune reconstitution following chemotherapy and is being pursued for biosimilar development. One challenge to overcome in pegflgrastim biosimilar development is establishing pharmacokinetic (PK) similarity, which is partly due to the degree of PK variability. We herein report that commercially available G-CSF and PEG ELISA detection kits have different capacities to detect pegflgrastim aggregates that rapidly form in vitro in physiological conditions. These aggregates can be observed using SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and real-time NMR analysis and are associated with decreased bioactivity as reflected by reduced drug-induced cellular proliferation and STAT3 phosphorylation. Furthermore, individual variability in the stability and detectability of pegflgrastim in human sera is also observed. Pegflgrastim levels display marked subject variability in sera from healthy donors incubated at 37°C. The stability patterns of pegflgrastim closely match the stability patterns of flgrastim, consistent with a key role for pegflgrastim’s G-CSF moiety in driving formation of inactive aggregates. Taken together, our results indicate that individual variability and ELISA specificity for inactive aggregates are key factors to consider when designing and interpreting studies involving the measurement of serum pegflgrastim concentrations.

59 BASIC BIOLOGICAL SCIENCES↗