Advancing college food security: priority research gaps.

Despite over a decade of both quantitative and qualitative studies, food insecurity among US college/university students remains a pervasive problem within higher education. The purpose of this perspective piece was to highlight research gaps in the area of college food insecurity and provide rationale for the research community to focus on these gaps going forward. A group of food insecurity researchers from a variety of higher education institutions across the United States identified five thematic areas of research gaps: screening and estimates of food insecurity; longitudinal changes in food insecurity; impact of food insecurity on broader health and academic outcomes; evaluation of impact, sustainability and cost effectiveness of existing programmes and initiatives; and state and federal policies and programmes. Within these thematic areas, nineteen specific research gaps were identified that have limited or no peer-reviewed, published research. These research gaps result in a limited understanding of the magnitude, severity and persistence of college food insecurity, the negative short- and long-term impacts of food insecurity on health, academic performance and overall college experience, and effective solutions and policies to prevent or meaningfully address food insecurity among college students. Research in these identified priority areas may help accelerate action and interdisciplinary collaboration to alleviate food insecurity among college students and play a critical role in informing the development or refinement of programmes and services that better support college student food security needs.

Development of a Highly Sensitive Hybrid LC/MS Assay for the Quantitative Measurement of CTLA-4 in Human T Cells.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a check point protein expressed on the surface of T cells and plays a central role in regulating the immune response. In recent years, CTLA-4 has become a popular target for cancer immunotherapy in which blocking CTLA-4 can restore T-cell function and enhance the immune response against cancer. Currently, there are many CTLA-4 inhibitors in a variety of modalities, including cell therapies, which are being developed in both preclinical and clinical stages to further harness the potential of the target for the treatment of certain types of cancer. In drug discovery research, measuring the level of CTLA-4 in T cells is important for drug discovery and development because it provides key information for quantitative assessment of the pharmacodynamics, efficacy, and safety of the CTLA-4-based therapies. However, to our best knowledge, there is still no report of a sensitive, specific, accurate, and reliable assay for CTLA-4 measurement. In this work, an LC/MS-based method was developed to measure CTLA-4 in human T cells. The assay demonstrated high specificity with an LLOQ of 5 copies of CTLA-4 per cell when using 2.5 million T cells for analysis. As shown in the work, the assay was successfully used to measure CTLA-4 levels in subtype T-cell samples from individual healthy subjects. The assay could be applied in supporting the studies of CTLA-4-based cancer therapies.

Supplemental Nutrition Assistance Program (SNAP)-authorized retailers received a low score using the Business Impact Assessment for Obesity and population-level nutrition (BIA-Obesity) tool.

BACKGROUND: The Supplemental Nutrition Assistance Program (SNAP) supports Americans with lower income to purchase dietary products at authorized retailers. This research aimed to evaluate SNAP-authorized retailers' public commitments in support of nutrition security and to examine differences between traditional grocers and nontraditional (e.g., convenience, drug, dollar) SNAP-authorized retailers' public commitments. METHODS: Prominent United States (U.S.) SNAP-authorized retailers nationally and in two U.S. states (California and Virginia) were identified based on number of store locations (n = 61). Public information available in grey literature were reviewed and scored using the Business Impact Assessment for Obesity and population-level nutrition (BIA-Obesity) tool. SNAP-authorized retailers were classified as traditional (e.g., grocery) or nontraditional (e.g., non-grocery) retailers. Total BIA-Obesity from 0 to 615, representing low to optimal support) and category scores were calculated for corporate strategy, relationships with external organizations, product formulation, nutrition labeling, product and brand promotion, and product accessibility. Descriptive statistics were used to describe BIA-Obesity scores overall and by category. Mann-Whitney U was used to test for potential differences in median BIA-Obesity total scores between traditional and nontraditional SNAP-authorized retailers (a priori, p < 0.05). RESULTS: Average total BIA-Obesity scores for SNAP-authorized retailers ranged from 0 to 112 (16.5 ± 23.3). Total BIA-Obesity scores for traditional SNAP-authorized retailers (32.7 ± 33.6; median 25) were higher than nontraditional SNAP-authorized retailer scores (11.2 ± 16; median 5) (p = 0.008). For BIA-Obesity categories, average scores were highest for the category relationships with external organizations (8.3 ± 10.3) and lowest for promotion practices (0.6 ± 2.1). CONCLUSIONS: Results of this research underscore a dearth of available evidence and substantial opportunity for improvement regarding SNAP-authorized retailer strategies to support nutrition security among Americans with lower income.

Fc-independent functions of anti-CTLA-4 antibodies contribute to anti-tumor efficacy.

Ipilimumab, a monoclonal antibody that recognizes cytotoxic T-lymphocyte associated protein 4 (CTLA-4), was the first immune checkpoint inhibitor approved by the FDA to treat metastatic melanoma patients. Multiple preclinical studies have proposed that Fc effector functions of anti-CTLA-4 therapy are required for anti-tumor efficacy, in part, through the depletion of intratumoral regulatory T cells (Tregs). However, the contribution of the Fc-independent functions of anti-CTLA-4 antibodies to the observed efficacy is not fully understood. H11, a non-Fc-containing single-domain antibody (VHH) against CTLA-4, has previously been demonstrated to block CTLA-4-ligand interaction. However, in vivo studies demonstrated lack of anti-tumor efficacy with H11 treatment. Here, we show that a half-life extended H11 (H11-HLE), despite the lack of Fc effector functions, induced potent anti-tumor efficacy in mouse syngeneic tumor models. In addition, a non-Fc receptor binding version of ipilimumab (Ipi-LALAPG) also demonstrated anti-tumor activity in the absence of Treg depletion. Thus, we demonstrate that Fc-independent functions of anti-CTLA-4 antibodies contributed to anti-tumor efficacy, which may indicate that non-Treg depleting activity of anti-CTLA-4 therapy could benefit cancer patients in the clinic.

A mixed-methods study to test a tailored coaching program for health researchers to manage stress and achieve work-life balance.

Dissemination and implementation (D&I) researchers serve critical scientific, practical, and personal roles in translating science to public health benefit. However, they face multifaceted barriers that may erode their capacity to plan, lead, and evaluate implementation. Individualized coaching focused on human flourishing is an unexplored approach to fully actualize D&I researchers' capacity to bridge the research-practice gap. The purpose of this exploratory pilot study was to investigate a tailored coaching program to support human flourishing among D&I researchers. A pragmatic, mixed-methods approach guided by the RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) Framework was used to evaluate an individualized, nine session coaching program called FUEL (Focus, Unplug, Exercise, Love). Reach and Implementation were assessed through descriptive statistics and rapid qualitative analysis of surveys and coaching logs. Effectiveness and Maintenance were assessed through descriptive statistics and iterative content analysis of participant surveys, as well as iterative content analysis of proxy (e.g., colleague) semi-structured interviews. Reach results indicated that demand for coaching exceeded study enrollment capacity (n = 16 participants). Implementation results showed that the coach spent 12.96 ± 2.82 hr per participant over 3 months. Effectiveness and Maintenance results indicated that FUEL was well-received and provided participants with myriad psychological and professional benefits. Preliminary evidence suggests that the FUEL coaching program is a promising and feasible approach to enhance flourishing among D&I researchers. Future research is needed to evaluate Adoption and scalability. This pilot study may inform future D&I capacity-building initiatives that address researchers' holistic situatedness within the implementation process.

Direct and indirect freedom in addiction: Folk free will and blame judgments are sensitive to the choice history of drug users.

People view addiction as a source of diminished free will and moral responsibility. Yet, people are also sensitive to the personal histories of moral actors, including, perhaps, the way by which people became addicted. Across two studies (N = 806), we compare people's moral intuitions about cases in which the actor becomes addicted by force or by choice. We find that perceptions of reduced free will partially mediate an association between choice (vs. no choice) in addiction and moral blame for a bad act (Study 1). We replicate this pattern and show that blame judgments are stronger when the bad act is related (vs. unrelated) to obtaining the addictive substance (Study 2). Our work is novel in demonstrating that lay people evince relatively nuanced intuitions about the role of free will in addiction and morality-they track direct and indirect paths to choices when making free will and blame judgments.

Insights on Droplet Digital PCR-Based Cellular Kinetics and Biodistribution Assay Support for CAR-T Cell Therapy.

Characterizing in vivo cellular kinetics and biodistribution of chimeric antigen receptor T (CAR-T) cells is critical for toxicity assessment, nonclinical and clinical efficacy studies. To date, the standardized assay to characterize CAR-T cell distribution, expansion, contraction, and persistence profiles is not readily available. To overcome this limitation and increase comparability among studies, we have established a universal protocol for analysis. We established a duplexing ddPCR protocol for the CAR-T transgene and reference gene to normalize the genomic DNA input prepared from mouse blood and tissues. The high-throughput gDNA extraction method enabled highly reproducible gDNA extraction while eliminating labor-intensive steps. The investigational CAR-T cells were intravenously injected into immunodeficient mice bearing human colorectal cancer xenografts. The blood and tissue samples were collected to measure the cellular kinetics by ddPCR and flow cytometry. The standard curves were linear throughout the calibration range with acceptable intra- and inter-day precision and accuracy. The gDNA recovery study performed by spiking in the exo-gene plasmid DNA or CAR-T cells revealed that the recovery ranged from 60 to 100% in blood and tissue homogenates. The use of both units of copy/µg gDNA and copy/µL blood met the current regulatory requirement and allowed for a systematic understanding of CAR-T cell expansion and a direct comparison with the flow cytometry data. A standardized ddPCR assay, including automated gDNA extraction procedures, has been established for evaluating cellular kinetics and biodistribution in CAR-T cell therapies.

Supplemental Nutrition Assistance Program (SNAP)-Authorized Retailers' Perceived Costs to Use Behavioral Economic Strategies to Encourage Healthy Product Sales.

SNAP-authorized retailers could use marketing-mix and choice-architecture (MMCA) strategies to improve SNAP purchases, but associated costs are unknown. Perceived cost and inconvenience to implement eight MMCA strategies were assessed among 29 U.S. retailers. Differences in perspective were explored (owners vs. managers, corporate vs. independent retailers, and by format). Place changes (e.g., added refrigeration) were perceived more costly and prompting (e.g., shelf labeling) less costly. Managers rated the perceived inconvenience to make proximity changes higher than owners (3.78 ± 1.4 and 2.33 ± 1.2, respectively) (p < .05). Results can inform strategies to improve the adoption and implementation of healthy food retail programs.

Optogenetic Control Reveals Differential Promoter Interpretation of Transcription Factor Nuclear Translocation Dynamics.

Gene expression is thought to be affected not only by the concentration of transcription factors (TFs) but also the dynamics of their nuclear translocation. Testing this hypothesis requires direct control of TF dynamics. Here, we engineer CLASP, an optogenetic tool for rapid and tunable translocation of a TF of interest. Using CLASP fused to Crz1, we observe that, for the same integrated concentration of nuclear TF over time, changing input dynamics changes target gene expression: pulsatile inputs yield higher expression than continuous inputs, or vice versa, depending on the target gene. Computational modeling reveals that a dose-response saturating at low TF input can yield higher gene expression for pulsatile versus continuous input, and that multi-state promoter activation can yield the opposite behavior. Our integrated tool development and modeling approach characterize promoter responses to Crz1 nuclear translocation dynamics, extracting quantitative features that may help explain the differential expression of target genes.

Pharmacokinetic Characterization and Tissue Distribution of Fusion Protein Therapeutics by Orthogonal Bioanalytical Assays and Minimal PBPK Modeling.

Characterization of pharmacokinetic (PK) properties and target tissue distribution of therapeutic fusion proteins (TFPs) are critical in supporting in vivo efficacy. We evaluated the pharmacokinetic profile of an investigational TFP consisting of human immunoglobulin G4 fused to the modified interferon alpha by orthogonal bioanalytical assays and applied minimal physiologically based pharmacokinetic (PBPK) modeling to characterize the TFP pharmacokinetics in mouse. The conventional ligand binding assay (LBA), immunocapture-liquid chromatography/tandem mass spectrometry (IC-LC/MS) detecting the human IgG4 peptide or the interferon alpha peptide were developed to measure the TFP concentrations in mouse plasma and tumor. The minimal PBPK model incorporated a tumor compartment model was used for data fitting. The plasma clearance measured by LBA and IC-LC/MS was comparable in the range of 0.5-0.6 mL/h/kg. However, the tumor exposure measured by the generic human IgG4 IC-LC/MS was significantly underestimated compared with the interferon alpha specific IC-LC/MS and LBA. Furthermore, the minimal PBPK model simultaneously captured the relationship between plasma and tissue exposure. We proposed the streamlined practical strategy to characterize the plasma exposure and tumor distribution of a TFP by both LBA and IC-LC/MS. The minimal PBPK modeling was established for better understanding of pharmacokinetic profile of investigational TFPs in the biotherapeutic discovery.

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells.

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods for the assembly of mammalian DNA circuits are laborious, slow, and expensive. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible, and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be assembled into transcriptional units and further weaved into complex circuits. We showcase the capabilities of the MTK by using it to generate single-integration landing pads, create and deliver libraries of protein variants and sgRNAs, and iterate through dCas9-based prototype circuits. As a biological proof of concept, we demonstrate how the MTK can speed the generation of noninfectious viral circuits to enable rapid testing of pharmacological inhibitors of emerging viruses that pose a major threat to human health.

Rural independent and corporate Supplemental Nutrition Assistance Program (SNAP)-authorized store owners' and managers' perceived feasibility to implement marketing-mix and choice-architecture strategies to encourage healthy consumer purchases.

Supplemental Nutrition Assistance Program (SNAP) participants could benefit from exposure to marketing-mix and choice-architecture (MMCA) strategies that encourage healthy consumer purchases. However, the perceived feasibility of independent and corporate SNAP-authorized store owners and managers (e.g., retailers) to implement healthy MMCA strategies is understudied. The purpose of this study was to inform a healthy food retail program that meets both public health and business expectations by prioritizing retailer perspectives. A mixed methods approach was used. Retailers completed a card sorting exercise to determine perceived feasibility to implement MMCA strategies place, profile, portion, pricing, promotion, priming, prompting, and proximity. This process was audio-recorded. Chi-square was used to identify potential differences in perceived feasibility to implement healthy MMCA strategies between independent and corporate SNAP-authorized retailers. Qualitative data were coded among a panel to construct themes. Themes were organized by barriers and facilitators and coded for strategy acceptability, appropriateness, and feasibility. SNAP-authorized retailers' (n = 29) considered prompting (e.g., labeling; 83%) and proximity (e.g., location; 90%) strategies to encourage healthy consumer purchases highly feasible. Few differences were detected between independent and corporate retailers' perceived feasibility to implement healthy MMCA strategies. The largest barriers to implementing healthy MMCA strategies were related to strategy appropriateness. Priorities for healthy food retail initiatives included prompting and proximity changes that highlight products aligned with the DGA, without altering products available to consumers that are misaligned with the DGA. Future work is required to understand how other healthy MMCA strategies may be adapted to enhance their appropriateness for these settings.

Plate Waste in School Lunch: Barriers, Motivators, and Perspectives of SNAP-Eligible Early Adolescents in the US.

OBJECTIVE: To determine barriers, motivators, and perspectives regarding plate waste reduction of early adolescents. DESIGN: Trained interviewers conducted audio-recorded individual interviews with adolescents. SETTING: Elementary schools implementing the National School Lunch Program in Hawai'i, Montana, and Virginia. PARTICIPANTS: Early adolescents (n = 47, aged 9-13 years) from families receiving or eligible to receive Supplemental Nutrition Assistance Program benefits were recruited to participate. PHENOMENON OF INTEREST: Factors influencing plate waste among adolescents and potential plate waste reduction strategies. ANALYSIS: Coders analyzed content and thematic data to identify code categories and themes. RESULTS: Main barriers to reducing school lunch plate waste were unsupportive school policy, undesirable food quality, satiation, and social influences. Key motivators to reducing school lunch plate waste were supportive school policy, including allowing students to share food with peers and save food to eat later; and social influences. Participants found it acceptable to throw away disliked food and unacceptable to throw away wanted food; they perceived that their peers did not care whether food was thrown away; and they thought their parents disliked wasting food. CONCLUSION AND IMPLICATIONS: Results suggest that several factors might allow for minimization of school lunch plate waste in the National School Lunch Program, including improvements in food quality, food policy, and social influences. Under these important themes, strategies to employ may include improving food preparation and taste, allocating more time for students to finish lunch, allowing students to self-select food lunch items, and permitting them to share and save their leftover food.

Psychometric evaluation of the Informed Consent Process Scale in Chinese.

BACKGROUND: Informed consent is essential for the ethical conduct of clinical research and is a culturally sensitive issue. But, a measurable Chinese version of the scale to evaluate the informed consent process has not yet been explored in the existing literature. RESEARCH OBJECTIVES: This study aimed to develop and psychometrically test the Chinese version of the Informed Consent Process Scale. RESEARCH DESIGN: Back-translation was conducted to develop the Chinese version of the questionnaire. A cross-sectional survey was administered, after which an exploratory factor analysis was conducted. PARTICIPANTS: We recruited a total of 375 participants who had experience in signing an informed consent form within the previous 3 years in Taiwan. ETHICAL CONSIDERATIONS: This study was approved by two Institutional Review Boards and the autonomy of the participants was respected. FINDINGS: The Chinese version of the Informed Consent Process Scale is composed of three factors with 23 items showing evidence of acceptable reliability and validity. Three major factors were extracted and labeled: Factor 1 - 'Understanding of the research', Factor 2 - 'Trust and confidence' and Factor 3 - 'Doubt and uncertainty'. The three factors accounted for is 52.954 of the total variance with Cronbach's α of .917. DISCUSSION AND CONCLUSION: The finding corroborates previous studies showing that participants had too little understanding on the informed consent forms they signed and implied the need to clarify the critical points in clinical research. The psychometric results indicated good internal consistency and validity for this newly constructed instrument, and it was found worthy of conducting further testing and application.

Identification and Optimization of Novel Small c-Abl Kinase Activators Using Fragment and HTS Methodologies.

Abelson kinase (c-Abl) is a ubiquitously expressed, nonreceptor tyrosine kinase which plays a key role in cell differentiation and survival. It was hypothesized that transient activation of c-Abl kinase via displacement of the N-terminal autoinhibitory "myristoyl latch", may lead to an increased hematopoietic stem cell differentiation. This would increase the numbers of circulating neutrophils and so be an effective treatment for chemotherapy-induced neutropenia. This paper describes the discovery and optimization of a thiazole series of novel small molecule c-Abl activators, initially identified by a high throughput screening. Subsequently, a scaffold-hop, which exploited the improved physicochemical properties of a dihydropyrazole analogue, identified through fragment screening, delivered potent, soluble, cell-active c-Abl activators, which demonstrated the intracellular activation of c-Abl in vivo.

An LC/MS based method to quantify DNA adduct in tumor and organ tissues.

Highly potent DNA damaging agents have become a key class of toxins for antibody-drug conjugate (ADC) based targeted therapy. However, until recently, no quantitative bioanalytical method was available to measure the toxin in the form of DNA adducts. In this work, a novel microwave assisted organic solvent extraction and LC-MS/MS based bioanalytical method was developed to extract and quantify DNA-bound toxin IGN-P1 in tissue samples. Using ADC-1 as the model ADC, the method was orthogonally checked with a radioactive method for the recovery of free toxins from DNA adducts in biological matrices. It was found that the bioanalytical method can achieve a high recovery of the IGN-P1 toxin from DNA adducts. In further assessment, tumor and organ tissue samples collected at multiple time points from in vivo studies after dosing with two other ADCs, ADC-2 and ADC-3, were measured by the method. Given the generic nature of the established bioanalytical method without the need of radiolabels, the methodology could be broadly utilized to quantitatively assess the relationship between DNA adduct levels and pharmacological/toxicological effects.

Quantification of interobserver variability in image registration using cone beam CT for partial bladder radiotherapy-a comparison between lipiodol and bladder wall surface.

OBJECTIVE:: The use of lipiodol or bladder wall surface (BWS) for image guidance has improved the treatment quality for partial bladder irradiation. Currently, this procedure is manually performed by different users. This study assessed the interobserver variability of using image guidance for partial bladder irradiation. METHODS:: 7 observers were prospectively recruited to manually register 5 cone beam CTs (CBCT) from each of 20 bladder cancer patients with lipiodol injected for tumor demarcation. Lipiodol and BWS were used to register the CBCT to pre-treatment reference images, and displacement values in three directions were collected. Mean difference among observers and the 95% limit of agreement were calculated to measure interobserver variability. Margin required and the resultant treatment volume were compared between the surrogates. RESULTS:: A total of 4200 displacement values were collected for analysis. Lipiodol was superior to BWS, with a mean difference among observers of <2 mm and a 95% limit of agreement of <5 mm in all directions. Of the three directions, greatest variability was observed in the superior-inferior direction for both surrogates, hence requiring a larger margin than the other two directions. After applying the corresponding margin, the mean volume of BWS-planning target volume was calculated to be significantly larger than lipiodol-planning target volume (166 cm3vs 134 cm3, p < 0.05). CONCLUSIONS:: The use of lipiodol achieved a higher interobserver agreement than BWS. A larger margin in the superior-inferior direction is recommended due to greater interobserver variability observed in this direction for both surrogates. ADVANCES IN KNOWLEDGE:: The uncertainty associated with the image registration by multiple observers for bladder image-guided radiotherapy is quantified for two surrogates.

Efficient and Effective Personalization of PTV Margins During Radiation Therapy for Bladder Cancer.

INTRODUCTION: The magnitude of bladder filling variation during bladder cancer radiation therapy varies considerably between patients. Population-based approaches for planning target volume (PTV) margin calculation may be suboptimal for this disease site, and a strategy for personalizing PTV margins for each patient may be particularly beneficial. The purpose of this study was to identify the optimal number and sampling pattern of cone beam CT image data sets that are required when generating personalized PTV margins for whole bladder (WB) and partial bladder (PB) radiation therapy. METHODS AND MATERIALS: Personalized PTV margins were generated for 24 bladder cancer patients (15 WB and 9 PB) using nine experimental strategies that varied in the number and pattern of images incorporated into the margin generation process. These PTVs were compared to the standard-of-care (SoC) PTV at our institution (15 daily fractions included) using PTV volume (cohort-based and individual patient ranking), superior and posterior expansion, and clinical target volume (CTV) coverage. RESULTS: For WB CTV, strategies ES4 (first five fractions), ES7 (every other fraction), and ES8 (first 10 fractions), provided CTV coverage equivalent to, or better than the SoC (first 15 fractions). Of these three strategies, ES4 resulted in the smallest superior and posterior borders, the smallest volume and the lowest intrapatient volume ranking, all achieved with the smallest number of fractions. For the PB CTV, strategies ES4 (first five fractions), ES7 (every other fraction), ES8 (first 10 fractions), and ES9 (last 10 fractions), provided CTV coverage equivalent to, or better than SoC (first 15 fractions). There were no statistically significant differences in the superior and posterior borders between these strategies, but ES4 resulted in the smallest volume and the lowest intrapatient volume ranking, all achieved with the smallest number of fractions. CONCLUSIONS: This study suggests that using contours from images taken during the first five daily fractions generated a personalized "patient-specific" PTV that provided CTV coverage equivalent to the 15-fraction SoC but decreased the irradiated volume, reduced delineation workload, and reduced the superior and posterior borders for WB. It is now the SoC for whole and PB radiation therapy at our institution.

Immunocapture-LC/MS-Based Target Engagement Measurement in Tumor Plasma Membrane.

For targeted therapies, immunocapture-liquid chromatography mass spectrometry (IC-LC/MS) technology has become an important tool for determination of both drug exposures, target antigen densities, and engagement in the systemic circulation and/or in total target tissue homogenates. Although the information collected from the circulation and tissue homogenates is useful in establishing the correlations of the exposure and target engagement with the pharmacodynamic response and efficacy of a therapy, the measurement at the cell plasma membrane within the target tissue is preferred, since it is the primary action site for antigen and the target drug. However, to the best of our knowledge, IC-LC/MS-based methodologies to conduct the assays at the plasma membrane from tissue sample has been quite limited. In this paper, we reported an IC-LC/MS-based assay platform for assessing the target engagement in tumor plasma membrane prepared from the tumor tissue samples. In addition, tumor samples with guanylyl cyclase C (GCC) expression after fully human IgG1 monoclonal antibody-based antibody-drug conjugate (ADC) treatment were used as a case study. The methodology can differentiate between the total and target-drug bound fraction of GCC with minimal potential equilibrium shift between in-cell surface protein and organelle protein in tumor samples to calculate in vivo target engagement. This approach to determine in vivo target engagement in tumor plasma membrane will provide better understanding of pharmacokinetic/pharmacodynamic relationship to achieve the desired antitumor efficacy.