People experiencing homelessness are at risk for gaps in care after an emergency department (ED) or hospital visit, which leads to increased use, poor health outcomes, and high health care costs. Most people experiencing homelessness have a mobile phone of some type, which makes mobile health  interventions a feasible way to connect a person experiencing homelessness with providers.This study aims to investigate the accuracy, acceptability, and preliminary outcomes of a GPS-enabled mHealth  intervention designed to alert community health paramedics when people experiencing homelessness are in the ED or hospital.t tests compared scores on the Patient Health Questionnaire-9, Medical Outcomes Study Social Support Survey, and Adherence Starts with Knowledge-12 adherence survey at baseline and exit. Semistructured exit interviews examined the perceptions and benefits of the intervention.This study was a pre-post design with baseline and 4-month postenrollment assessments. People experiencing homelessness, taking at least 2 medications for chronic conditions, scoring at least 10 on the Patient Health Questionnaire-9, and having at least 2 ED or hospital visits in the previous 6 months were eligible. Participants were issued a study smartphone with a GPS app programmed to alert a community health paramedic when a participant entered an ED or hospital. For each alert, community health paramedics followed up via telephone to assess care coordination needs. Participants also received a daily email to assess medication adherence. GPS alerts were compared with ED and hospital data from the local health information exchange (HIE) to assess accuracy. Paired t19=2.9; P=.009) and a significant difference in adherence barriers between baseline  and exit . Participants agreed that the app was easy to use , and the email helped them remember to take their medications . Qualitative data indicated that unlimited smartphone access allowed participants to meet social needs and maintain contact with case managers, health care providers, family, and friends.In total, 30 participants were enrolled; the mean age was 44.1 (SD 9.7) years. Most participants were male , White , and not working . Only 19% (3/16) of the ED or hospital visit alerts aligned with HIE data, mainly because of patients not having the smartphone with them during the visit, the smartphone being off, and gaps in GPS technology. There was a significant difference in depressive symptoms between baseline  and exit (mean 12.7, SD 8.2; mHealth interventions are acceptable to people experiencing homelessness. HIE data provided more accurate ED and hospital visit information; however, unlimited access to reliable communication provided benefits to participants beyond the study purpose of improving care coordination. Mobile phone ownership is nearly ubiquitous among American adults, with 96% owning a mobile phone of some type, and most 81%) mobile phones are smartphones % mobile , diseaseRecent estimates of mobile phone use among the homeless population indicate that 89% of the people report having and using a mobile phone , and resEvidence suggests that the health service experiences of people experiencing homelessness are often interrupted and involve extensive barriers, including unmet physical needs, lack of affordable and available services, and lack of compassion that prevents people experiencing homelessness from accessing appropriate community-based services -17. ThesThis study focuses on the significant gap along the continuum of care that begins at the point of an emergency department (ED) visit or hospitalization for people experiencing homelessness. The study intervention was created on the basis of feedback from health care providers and case managers who deliver care to homeless individuals, and the fact that fragmented communication among various health care organizations limits the ability to provide real-time information about ED or hospital visits. When a person experiencing homelessness enters the ED or hospital, they are at high risk of losing contact with community-based health care providers and case managers . This isWhat is the accuracy of GPS technology in terms of tracking participant visits to the ED or hospital?How do depression symptoms, medication adherence, social support, and experience with and perceptions of GPS and mobile phone technology compare at baseline and exit?What is the number and type of community health paramedic encounters?What concerns do participants express regarding technology or privacy?This study used a pre-post design with assessments at baseline, 1 month, 2 months, 3 months, and 4 months after enrollment to evaluate the acceptability and preliminary outcomes of a GPS-enabled mHealth intervention. Participants were recruited from 2 churches that provided services to people experiencing homelessness. The first serves breakfast at 5:45 AM two mornings each week and is open to anyone in the community. The research staff attended this breakfast once per week for the study duration. Potential participants were referred to study staff for eligibility screening by either the meal program coordinator or the police officer assigned to the downtown Homeless Outreach Service team, whose job function includes attending these twice weekly breakfasts. The second church site doubles as a navigation center for people experiencing homelessness during weekdays. Services at the navigation center include coordinated assessments for housing, assistance with obtaining IDs, and case management. The research staff were on site at the navigation center 2 to 3 days per week for the study duration. Similar to the first site, potential participants were referred by the director of the navigation center or by navigation center volunteers to study staff for study eligibility screening.Recruitment occurred between October 2018 and April 2019. Community partners assisted with recruitment by distributing flyers to clients and by referring potential participants to research staff. Participants also referred peers who were potentially eligible to the study staff. Potential participants were screened for study eligibility on site at the churches by a member of the research team. The eligibility criteria included (1) being at least 18 years old, (2) currently experiencing homelessness defined as where the person had slept most nights in the past 30 days, (3) score of at least 10 on the Patient Health Questionnaire-9 (PHQ-9), (4) currently prescribed at least 2 medications for chronic medical conditions, (5) diagnosed with at least 1 chronic medical condition, and (6) experienced at least 2 hospitalizations or ED visits in the past 6 months. Exclusion criteria included (1) onset in the past 3 months of depressive symptoms and (2) suicide attempts or suicidal ideation in the past 6 months.This study was approved by the institutional review board of the university. Individuals interested in participating were screened by research staff, and, if eligible to continue, study details, including the purpose, procedures, risks, and benefits of study participation, were explained. If participants remained interested, informed consent was obtained. None of the participants who were eligible for the study declined to participate after being informed of the study details. After obtaining informed consent, a researcher administered a series of baseline assessments to collect information about demographics, health history, medication adherence, social support, and recent ED visit and hospitalizations. After completion of the surveys, participants were provided with a smartphone activated with a plan for unlimited texting, calling, and data; a hard-plastic smartphone case; and an armband to use for securing the smartphone. Participants were also given US $25 cash for the time spent enrolling in the study and a 31-day unlimited use bus pass to ensure their ability to attend the monthly follow-up assessment. They then received training on the intervention. The training described the expectations of participants, including keeping track of the smartphone, keeping it turned on and charged, attending monthly check-in visits, answering the daily email regarding medication adherence, and responding to community health paramedics or research staff as applicable. The training also included how to use the smartphone, set up voicemail, access email and SMS text messages, and access and use the bus pass. Participants were also informed that one replacement smartphone would be issued if their study smartphone was lost, stolen, or broken during the 4-month study.For this study, a mobile app was used to establish and monitor geofences around the 10 EDs located within the city limits where this study took place. The geofences were established using the mobile app so that when a participant entered a local ED or hospital, the research staff and the commander of the community paramedic team would receive an email notification. The email notification sent a secure link to view the participant’s name, geofence location, date, and time of entry and exit. On receipt, the commander tasked a community health paramedic member of his team to contact the participant via their smartphone within 2 business days of the geofence entry to follow-up on the visit and any identified health or social needs. The community health paramedic completed an event form documenting the participant-reported reason for the hospital or ED visit, admission, and discharge dates; if the ED or hospital visit was potentially preventable; what intervention may have prevented the ED and hospital visit; and the duration of the community health paramedic visit with the participant.In addition to the geofencing and care provided by community health paramedics as needed, the intervention had two additional components: (1) monthly in-person meetings and (2) daily adherence reminder emails. In-person meetings occurred between each participant and research staff at enrollment; 1-, 2-, and 3-month follow-up appointments; and at the exit. Monthly follow-up visits  were scheduled to maintain contact with participants and to identify any issues with the technology. Participants were also asked at these monthly meetings if they had visited the ED or been hospitalized in the past 30 days. At months 1, 2, and 3, participants received US $10 cash and an additional 31-day bus pass. Next, participants received an email every evening at 8 PM asking if they had taken their medications that day. Response options were “yes” or “no,” with a follow-up question requesting a short reason why they had not taken their medication if applicable. During the exit interviews, participants responded to a series of questionnaires before engaging in a semistructured interview to assess the overall acceptance of the intervention. Questions about the studyPlease describe your experience with this research study (probe 1: Did you experience benefits from participating in this study? probe 2: Was participating in this study helpful to you? probe 3: Were there any difficulties that you experienced with this study?)Can you share any barriers to study participation that you experienced? (Examples may include keeping the smartphone secure or charged)What strategies did you use to successfully complete the study requirements? What concerns did you have about your visits to the emergency room and hospital being monitored with GPS technology?Can you describe any experiences or interactions you had with community health paramedics?What suggestions do you have for us to improve this intervention for people in the future?At baseline, sociodemographic characteristics, including sex, race, highest education obtained, veteran status, and income, were collected. Participants were also asked a series of six questions from the American Community Survey designed to identify individuals who may experience functional limitations . ResponsHealth literacy was measured using the Brief Health Literacy Screening Tool , which cThe accuracy of the GPS technology was measured in 2 ways. First, when community health paramedics received an alert indicating that a participant had entered a geofence at an area hospital, a community health paramedic attempted to make contact with the participant within 2 business days. If contact was established, the community health paramedic confirmed the visit to the ED and hospital, as indicated by the geofence alert. Second, at the end of the study, the research staff obtained use records from the HIE. These records provided the dates of participants’ ED and hospital visits during the study period. Use records for the 25 participants for whom HIE data were collected were triangulated with geofence entry notifications to measure the accuracy of the GPS technology.The 9-item PHQ-9 was used to establish participant eligibility and as a baseline measure for depression symptoms. The PHQ-9 is a reliable and valid tool for diagnosing and grading depressive symptom severity . Each itMedication adherence was measured using a modified version of the Adherence Starts with Knowledge-12 (ASK-12). The ASK-12 is a brief, 12-item scale with 3 subscales that measure medication behavior, health beliefs, and inconvenience/ forgetfulness . For thiSocial support was measured using the Medical Outcomes Study Social Support Survey, a valid and reliable tool that has been used in multiple groups across various conditions . It inclAt baseline, experience with mobile phone technology was measured using a series of questions asking about current mobile phone ownership, mobile phone service, length of time owning a mobile phone, ability to charge the mobile phone, and if the participant had had a mobile phone stolen before. Acceptance of technology was measured at baseline and exit. At baseline, acceptance of technology was measured using a modification of the Technology Acceptance Questionnaire . At baseSelf-reported ED and hospital use were assessed at baseline, monthly visits, and exit. If participants indicated that they had visited the ED or hospital within the past month, their experience and perception of patient-centeredness of their care were assessed using the care transitions measure (CTM), a 15-item measure reflecting 4 content domains . The domt tests were used to compare scores at baseline and exit on the PHQ-9, ASK-12, Medical Outcomes Study, and technology acceptance scales.All statistical analyses were performed using IBM SPSS Statistics for Windows . Descriptive statistics were used to describe the sociodemographic and health characteristics of the sample and all study measures. Accuracy of the geofence entry notifications was determined by calculating the percentage of notifications that aligned with HIE use data. Paired sample Qualitative content analysis was used to identify participants’ acceptance of the intervention. All interviews were audio recorded and transcribed verbatim to facilitate coding and analysis. After a thorough reading and deductive coding of 5 representative transcripts by 2 members of the study team (LRM and WT), a consensus meeting was held to discuss and agree upon the codes. Discrepancies were resolved by discussing the context for each phrase being analyzed. After the meeting, a codebook was developed. The remaining interviews were divided between the 2 authors and coded separately. After coding was complete, the study team organized the codes into categories.Between October 2018 and April 2019, research staff screened 39 individuals for participation; of the 39 individuals, 32 (82%) met the eligibility criteria, and 30 (77%) were enrolled in the study. The 2 individuals who were eligible to participate but did not enroll did not return for the subsequent enrollment visit in the study after screening. The reasons for ineligibility for the study were not scoring at least 10 on the PHQ-9 , not having been to the ED or hospital at least twice in the past 2 months , not being prescribed a medication , and endorsing suicidal ideation . The participant who endorsed suicidal ideation was referred to the public safety officer on site at the community entity for appropriate follow-up and mental health services. Of the 30 participants, 10 (33%) were screened and enrolled at the first church with 2 weekly breakfasts, and the remaining 20 (67%) were screened and enrolled at the navigation center housed in a church.Of the 30 participants enrolled, 19 (63%) completed the 4-month intervention, with a completion rate of 63%. Of the 11 participants who did not complete the intervention, 6 (55%) were withdrawn from the study after they reported their second smartphone lost or stolen, 2 (18%) notified the research staff that they were moving to a different town, 2 (18%) were lost to follow-up, and 1 (9%) voluntarily withdrew from the study after losing his first smartphone. Of these 11 participants, 4 (36%) completed all but the exit data collection.Participants comprised 30 people experiencing homelessness. On average, participants were male 20/30, 67%), aged 44.1 years (SD 9.7 years), White , never married , and not working because of disability or other medical reasons . At baseline, participants reported a mean of 2.8 (SD 1.4) chronic conditions, and most  experienced multiple chronic conditions. All participants were prescribed at least 2 medications at baseline; 53% (16/30) were prescribed 4 or more medications. 0/30, 67%Accuracy of the GPS technology was calculated for the 25 participants who completed a release of information form, giving permission for the research team to access data in the HIE. During the 4-month study period, HIE use data indicated that these participants made 16 hospital or ED visits. Community health paramedics received 14 total geofence entry notifications during the study period; of these 14 notifications, 2 (14%) were from participants without a release of information for the HIE. Thus, community health paramedics received 12 geofence entry notifications for the 25 participants from whom HIE data were available. However, only 3 of the geofence entry notifications were consistent with the HIE use data for an overall accuracy rate of 19%.Of the 16 ED and hospital visits reported by the HIE data for which community health paramedics did not receive geofence entry notifications, 4 (25%) occurred during the first month of the intervention, a time during which the research staff identified a technical issue with the mobile app and geofence entries were not being received. Of these 16 visits, 3 (19%) occurred in the window of time during which the participant was without the study-assigned smartphone as the smartphone had been stolen or misplaced but not yet replaced. It is unclear why the remaining 43% (6/14) ED and hospital visits reported by the HIE data did not result in a geofence notification entry.Community health paramedics successfully reached participants to conduct follow-up and provide care coordination assistance after 79% (11/14) of geofence notifications. Of these 11 contacts, 10 (91%) lasted ≤10 minutes, and 1 (9%) contact lasted between 11 and 20 minutes. Of these 11 contacts, 3 (27%) participants reported having accompanied a friend or family member to the ED and were not seen themselves, and 1 (9%) participant reported having visited the hospital campus for a scheduled medical visit. Thus, 36% (4/11) of these geofence notifications were classified as false positives. Of the remaining 7 contacts, 3 (43%) aligned with the HIE notification data. Of the remaining 4 contacts, 3 (75%) did not align with the HIE data, as the participants did not have a release of information form on the file. It is unclear why the remaining contact did not register with the HIE.Community health paramedics determined that 43% (3/7) of the ED visits were emergent and likely unavoidable. Reasons for the emergent ED visits included chronic pulmonary obstructive disease exacerbation, a physical altercation at a local shelter, and uncontrolled epigastric pain. Reasons for the remaining ED visits were skin irritation because of scabies infection, shoulder pain, and 2 visits for gastrointestinal illness. Community health paramedics judged each of these 4 visits to be due to ambulatory care–sensitive conditions that could have been appropriately managed in the outpatient setting.t19=2.892; P=.009), indicating fewer depressive symptoms at the 4-month exit.There was a significant difference in depressive symptoms between baseline  and exit , indicating that at the 4-month exit visit, there were fewer barriers to taking medications.At baseline, scores on the ASK-12 ranged from 14-30 . Among those who completed the 4-month intervention, there was a significant difference in medication behavior between baseline  and exit .There was no significant difference in social support between baseline  and exit  of participants reported having a mobile phone. Of these 15 patients, 12 (80%) had current wireless service . Of the 15 participants with mobile phones, 13 (87%) reported that their mobile phones could support both SMS text messaging and mobile apps. At the exit interview, participants agreed that the smartphone app was easy to use , that they had the knowledge to use the smartphone app , and that they planned to continue using both a smartphone  and GPS technology . The acceptance of technology questionnaire indicated that participants had a high level of agreement at baseline and exit with items such as having the resources and knowledge to use smartphone technology and being comfortable with the health care team being alerted about ED or hospital use. There was a significant increase in agreement level from baseline  to exit  for the item, “My friends would encourage me to use this Smartphone app.” Participants’ agreement level increased for several other items, such as having the knowledge and resources to use GPS technology from baseline to exit, but not significantly. At baseline, 57% (17/30) of participants self-reported at least one ED or hospital visit in the previous 30 days and completed the CTM-15. At months 1, 2, 3, and exit, 33% (10/30), 13% (4/30), 13% (4/30), and 17% (5/30) of participants, respectively, self-reported at least 1 ED or hospital visit in the previous 30 days and completed the CTM-15 for their most recent visit. The mean score for the critical understanding and management preparation domains was 4.1, indicating that participants generally agreed that they left the hospital or ED understanding how to manage medications and their health. The mean score for the preferences important domain was 4.0 (SD 0.1), which means that participants agreed that hospital staff took their preferences for health care needs into account when planning for discharge. The lowest level of agreement was with the care plan domain . Of the 30 participants, 17 (57%) completed an exit interview. During data analysis, the first 2 authors of this study organized the codes into the following categories: (1) benefits of study participation, (2) challenges to study participation, (3) perceptions of GPS technology, and (4) suggestions for improvement.Overall, participants reported positive experiences with study participation. They also identified several benefits, defined as any real or perceived aid or assistance from participating in the research study or having access to the unlimited use of a smartphone. Benefits included self-management support, improved social connections, and improved well-being. An example of how study participation provided self-management support is demonstrated by this quote:[...] there was a time when I [...] would be confused as to whether or not I took my medicine. Sometimes I would go days without even thinking about it, you know? But now, I am confident knowing that every morning you know “Bam!”, you know it’s [daily email] right there and I had my medication and had taken it. There was never any more confusion.Social connections were facilitated by the ability to call friends and family, to stay up to date on current events by reading the news on the internet, and to use social media sites. Several participants described using the smartphone to reconnect with the family from out of state. One participant put it succinctly as follows:[...] being homeless, you can be very bored sometimes with nothing to do. And, [with the phone] I had something to do. You can read the news and find out what’s going on in the world. Or, you know, keep in touch with my friends with email.Participants also described improved well-being as they did not have to worry about paying for their smartphone, were able to travel to appointments because of bus pass on the smartphone, and felt more secure in their environments with the ability to contact the police or emergency medical services in the case of an emergency. An example of how study participation improved well-being is demonstrated by the following quote:It was a godsend. It really was, I mean because I didn’t have to worry about a lot of things. I could make phone calls when I needed to. It just took a lot of burden off me, knowing that I had a bus pass. I had a phone I could use you know if I got in trouble or something or was in a bad situation.Challenges to study participation were defined as circumstances in which participants had to navigate to access, use, and benefit from services and resources, including the research study itself. Challenges included differential treatment because of homelessness, difficulty with technology, and keeping the smartphone secure. For example, differential treatment resulted in participants having difficulty keeping their smartphones charged as business owners do not allow people experiencing homelessness to spend time charging smartphones in their establishments. Some participants also described trouble with technology, such as short battery life and slow internet service. Finally, keeping the smartphone secure required constant vigilance on the part of participants, and even with creative solutions for safekeeping, many experienced theft or damage to their smartphones. One participant expressed his desire for smartphones to be replaced up to 4 times, saying as follows:[...] the fact is, anything can happen out here. Like you know, I was charging my phone at Starbucks. I fell asleep, and when I woke up, my phone was stolen. Got my second phone…but I forgot to put the case back on and water hits it and its out.Perceptions of the GPS technology were uniformly positive, as each participant who completed the exit interview denied having concerns about the community health paramedics or research staff knowing when they visited the ED or hospital. One participant clearly articulated this by saying the following:...you know, that kind of thing right now is the least of my concerns. If you’re sleeping in an alley or somewhere else, you’re not really worried about somebody knowing that you’ve been to the hospital, or at least I’m not.Suggestions for improvement included two main subcategories: helping to complete the study requirements and tailoring the intervention. Participants suggested more teaching about using the smartphone and its functions and providing portable battery chargers to help overcome some of the technical challenges to study completion that participants faced. Participants also suggested sending daily messages via text instead of email and indicated that personalized and tailored medication reminders for their individual medication regimens would be helpful.The results of this study contribute to a small but growing body of literature documenting the utility of mHealth interventions among people experiencing homelessness. First, our findings suggest that GPS technology is not a reliable method for tracking visits to the ED or hospital among people experiencing homelessness. The geofence notifications aligned with objective HIE use data only 18.8% of the time, indicating that the community health paramedics were unable to connect with participants to provide follow-up assistance with care coordination after most participant ED and hospital visits. This finding was surprising given recent evidence that a smartphone app used by 12 patients with low income had 75% accuracy in detecting real-time ED or hospital use over a 3-month period . It is lDespite findings that GPS technology is not reliable for real-time ED or hospital use data, overall, participants expressed positive views of GPS technology. Participants embraced the idea of GPS being used by health care and other service providers to locate them if needed and described feeling more secure with the knowledge they could be found. This is similar to findings by Liss et al , who fouThere was a significant decrease in depression symptoms from baseline to exit, which aligns with the qualitative findings where participants reported improved well-being and an overall positive experience with the intervention at the study exit. In contrast, a 1-month, pre-post study of homeless young adults (aged 18-24 years) who participated in a remote mental health intervention, which included SMS text messaging, did not find a difference in depression symptoms . This maAlthough there was no significant difference in social support between baseline and exit, the qualitative data suggest that the smartphone had an impact on participants’ social connections. Prior evidence clearly indicates that social support can have a protective influence on multiple health outcomes among people experiencing homelessness  and thatOverall, the participants rated care transitions from the ED or hospital to the community fairly high. However, the results suggest that specific aspects of transitions could be improved. For example, in the critical understanding domain, two items related to understanding what and how to manage health on discharge and one item related to medication side effects scored lower than the remaining domain items. Future studies could investigate adapting the mHealth intervention to provide targeted follow-up post-ED or hospital discharge as well as specific guidance related to medication side effects to maximize adherence and optimize outcomes. Furthermore, the care plan domain scored the lowest among the four domains. This further supports the need to adapt the intervention to provide two-way communication between people experiencing homelessness and service providers to ensure that needed follow-up care is received in a timely and accessible manner.t21=–2.17; P=.02) with the group that did not complete the study having a higher mean score at baseline than the group that did finish. The small sample size, although sufficient for answering this study’s research questions, may further limit the generalizability of the findings.The findings of this study should be interpreted with caution because of the study’s limitations. Participants were recruited from one city in a large, southern state using convenience sampling; therefore, the generalizability of the findings is unknown. Furthermore, participants were recruited from community sites, which may have biased the results. The pre-post design is subject to bias, and as study participants were selected on the basis of their PHQ-9 score, it is possible that regression to the mean occurred for the depression symptom outcome. There were also baseline differences in PHQ-9 scores between the groups that did and did not complete the study (The findings from this study point to several directions for future research. First, based on participants’ responses to the daily email medication adherence message and their stated preferences for SMS text messages, a subsequent study tested an expanded SMS text messaging intervention. That study also included testing the use of remote location services preinstalled on the smartphone to locate participants during business hours. The findings also suggest that in addition to unlimited access to a smartphone, access to unlimited transportation can facilitate the ability of people experiencing homelessness to self-manage chronic illness. Thus, future research could investigate the impact of providing accessible transportation on health outcomes and use. Finally, because of the shortcomings of GPS technology in communicating real-time health care use information for people experiencing homelessness and as there is an operational HIE in the local area, future research investigating care coordination should incorporate the HIE to ensure transmission of objective use data. Qualitative findings also suggest that mHealth interventions, particularly unlimited access to a smartphone and bus pass transportation, have numerous benefits for well-being and the ability of people experiencing homelessness to meet social needs. These concepts need to be explored quantitatively in future studies. Furthermore, coupling access to a smartphone and transportation with health care programs should be pursued at a policy level for local programs ,43.mHealth interventions are acceptable to people experiencing homelessness and positively affected depression symptoms and medication adherence. Objective data from the HIE provided more accurate ED and hospital use information compared with alerts relying on predefined geofences. Despite this, participants favorably viewed GPS technology, warranting further exploration of GPS technology as a tool for facilitating care coordination among people experiencing homelessness.


Biologically-based therapies increasingly rely on the endocytic cycle of internalization and exocytosis of target receptors for cancer therapies. However, receptor trafficking pathways  and lateral membrane movement) are often dysfunctional in cancer. Antibody-drug conjugates (ADCs) have revitalized the concept of targeted chemotherapy by coupling inhibitory antibodies to cytotoxic payloads. Significant advances in ADC technology and format, and target biology have hastened the FDA approval of nine ADCs (four since 2019). Although the links between aberrant endocytic machinery and cancer are emerging, the impact of dysregulated internalization processes of ADC targets and response rates or resistance have not been well studied. This is despite the reliance on ADC uptake and trafficking to lysosomes for linker cleavage and payload release. In this review, we describe what is known about all the target antigens for the currently approved ADCs. Specifically, internalization efficiency and relevant intracellular sorting activities are described for each receptor under normal processes, and when complexed to an ADC. In addition, we discuss aberrant endocytic processes that have been directly linked to preclinical ADC resistance mechanisms. The implications of endocytosis in regard to therapeutic effectiveness in the clinic are also described. Unexpectedly, information on endocytosis is scarce (absent for two receptors). Moreover, much of what is known about endocytosis is not in the context of receptor-ADC/antibody complexes. This review provides a deeper understanding of the pertinent principles of receptor endocytosis for the currently approved ADCs. Cancer is the second leading cause of mortality worldwide, resulting in >1 million deaths annually. The unmet medical need for more effective anticancer therapeutics, especially for strategies that can localize cytotoxicity to tumor cells and reduce toxicity to normal tissues, has led to many years of development and ultimately successful realization of antibody-drug conjugates (ADCs), designed to provide the selective elimination of antigen-positive cells. At the time of this review, nine ADCs have been approved by the U.S. Food and Drug Administration for the treatment of six types of blood cancers and two types of solid tumors . HoweverThis recent success is perhaps a turning point in the field and a strong indication that the time for ADCs may have finally arrived. From 2000 to 2018, only five ADCs showed sufficient efficacy to gain approval. Just one, T-DM1, approved in 2013 for human epidermal growth factor receptor 2 (HER2)-positive breast cancer was approved for the treatment of solid tumors. ADCs are engineered to target overexpressed receptors on the surface of tumor cells and then release cytotoxins that deliver up to 10,000 times the potency of traditional chemotherapy within tumors, while minimizing toxicity to healthy tissues. The approach has been challenged for decades by the difficulty in the selection of a cytotoxic payload, the method of conjugation, and pairing with a monoclonal antibody (mAb) specific for the tumor-associated antigen. Several ADCs were abandoned, as their potency was too weak to effectively kill tumors or too harmful to healthy tissues. However, the ADC field is now in exciting times, as four new ADCs have been approved since 2019 ,5. In adApart from the preferential tumor overexpression of the eight cell surface antigens, which serve as binding platforms for the nine currently approved ADCs, another important element of these antigens is that they are intended to be entry portals into the intracellular space, which is required for drug release and activity. In fact, ADC efficacy is dependent on the efficiency of target-mediated internalization to deliver the payload inside tumor cells ,7. MoreoThis review is motivated by the surprisingly limited information concerning the mechanistic endocytic processes of target receptors, including when bound to antibodies or ADCs. Numerous reviews exist that provide excellent general overviews of ADCs for cancer therapy ,14,15,16It is essential to understand endocytosis, as it is the first stage of the ADC intracellular delivery process. This review does not attempt to provide a comprehensive description of normal endocytosis pathways, which can be found in ,27. In tTherefore, this review seeks to consolidate information and provide insight into important aspects of endocytosis, providing (1) a description of the endocytic pathways (or lack thereof) utilized by the receptors in normal and/or tumor cells targeted by the nine currently approved ADCs; (2) a description of unique endocytosis features when receptors are bound by ADCs ; (3) an overview of the fragmentary information available concerning these endocytosis pathways as ADC-specific resistance mechanisms; and (4) our perspective on endocytosis and its clinical implications. Target-independent uptake is a significant occurrence due to the Fc portion of mAbs and further information on its implications for ADC efficacy and toxicity is reviewed in . The preIn general, normal endocytosis can be categorized into three stages, (1) bud formation, (2) membrane curvature and vesicle maturation, and (3) scission and release of the membrane into the cytoplasm. There are multiple endocytosis pathways that have overlapping aspects, and hence the general process of endocytosis is highly flexible and complex. As a result, current ADC research is only scratching the surface of the relationship between endocytosis and ADC efficacy. Here, we briefly describe the implicated endocytosis pathways and their components that are pertinent to our findings regarding endocytosis in relation to the nine approved ADCs.Clathrin-mediated endocytosis (CME) is conceptually a straightforward process that includes a few sequential and partially overlapping steps. CME can occur constitutively for certain receptors at the plasma membrane, whereas other receptors require ligand and/or antibody binding to initiate CME or enhance its activity. CME commences when endocytic coat proteins from the cytoplasm begin to cluster on the inner leaflet of the plasma membrane. The protein coat continues to assemble and grow by recruiting and interacting with additional protein adaptors from the cytoplasm. Key adaptor proteins enable membrane bending and hence concentrate the internalizing receptors/ligands into a ‘clathrin-coated pit’ (CCP). Separation from the plasma membrane occurs through a scission process at the point where the CCP neck becomes constricted due to the CCP invagination growing larger. Actin polymerization contributes to pulling the CCP ‘inwards’ into the cytoplasm until scission is complete and the CCP is released and becomes a clathrin-coated vesicle (CCV). Finally, the CCV coat disassembles and the CCV fuses with endosomes for sorting to defined subcellular locations, or can be recycled back to the cell surface.Clathrin is the key component of CME and is composed of a heavy and light chain. Three clathrin heavy and light chains form a trimeric clathrin ‘triskelion’ that interacts with other triskelions and forms a polygonal lattice around the emerging CCP. More information on clathrin’s structure and function is reviewed in . There aClathrin-independent endocytosis mediates the cellular uptake of several extracellular ligands, receptors, pathogens, and as we will describe, potentially ADCs. The understanding of clathrin-independent endocytic systems is not nearly as comprehensive as our understanding of CME . FurtherCaveolae are small flask-shaped invaginations of the plasma membrane, characterized by high levels of cholesterol and glycosphingolipids that mediate endocytosis via clathrin-independent pathways and are found in most cell types . The priAlthough caveolae share an invagination-like morphology that is similar to that of CCPs, they are distinct and are reviewed in . BrieflyCLIC/GEEC is an endocytic compartment that occurs mainly in ligand-activated cells, which can be caused by growth factors, receptor crosslinking by antibodies, or by bacterial toxins and viruses . In addiMacropinocytosis describes a form of larger-scale endocytosis that frequently involves highly ruffled regions/protrusions of the plasma membrane that subsequently fuse with one another or back with the plasma membrane . MembranIn the following sections we describe the current understanding of the endocytosis activities of the targets for the currently approved ADCs. In addition, we summarize what has been demonstrated in relation to mAb/ADC binding and endocytosis and intracellular trafficking.N-terminal V-set immunoglobulin domain  family and is also known as Siglec-3. The characteristic structural feature of Siglec cells is their extracellular n domain , which rHowever, the ITIMs also regulate CD33 endocytosis. ITIM binding by SHP activates endocytosis via CME but in the absence of AP-2  59,61,6,661,62. For internalization efficiency, there is no correlation between the expression level of CD33 and its internalization rate in AML cells . CD33 isWalter et al. surmised that a potential cause of the slowness of CD33 endocytosis was its low abundance of tyrosine-based motifs . For exaImpaired CD33 internalization is strongly suggested as a significant reason underlying the ineffectiveness of GO. AML cells taken from patients administered with GO showed only 25–40% internalization of CD33 3–6 h post-injection . This inInterestingly, Paubelle et al. demonstrated that patients with mutations H63D or C282Y in a gene called HFE (most common cause of hereditary hemochromatosis) had poor overall survival rates compared to patients with wild-type HFE . Their sCD33 clearly undergoes endocytosis and, hence, GO is also internalized and routed to lysosomes, where it can successfully release its payload and kill AML cells. Nonetheless, overall it appears that CD33 undergoes slow internalization or degradation, which is further exacerbated by the presence of CD33-associated proteins that harbor mutations relevant to poor internalization and most likely contributes to the controversial effects of GO .CD30 is a 120-kDa transmembrane glycoprotein and belongs to the tumor necrosis factor receptor (TNFR) superfamily. Its extracellular portion is comprised of six cysteine-rich domains (CRD) in an extended conformation  69]. Th. Th69]. CD30 is expressed on activated T and B cells and on various lymphoid neoplasms, including Hodgkin lymphoma and ALCL . The CD3CD30 is not known for endocytosis and, in contrast, is well known for shedding due to proteolytic cleavage, and its release is enhanced upon interaction with CD30L-positive cells. CD30 shedding is mediated by matrix metalloproteinases (MMPs), including TNF-α converting enzyme  and ADAM10 in lymphoma cells ,77. LympInhibiting CD30 shedding has been investigated to improve the efficacy of CD30-targeted therapeutics. The hydroxamic acid-based inhibitor BB-3644 reduced soluble CD30 levels in the blood in preclinical studies . HoweverShedding is such a feature of CD30 biology that elevated concentrations of circulating soluble CD30 are sufficiently abundant that they can be used as a serum marker for monitoring tumor progression . CD30 maThere is a disconnect between CD30 endocytosis/Ab internalization and therapeutic efficacy. Previous reviews focusing on CD30 as a therapeutic target have claimed that CD30 undergoes rapid endocytosis ,84. HoweAn additional complication is that there are several anti-CD30 mAbs reported in the literature that bind different domains. The mAbs Ki-4, Ber-H2, and 5F11 were first shown to be able to bind to CD30 CRD 2 and 5 and reduce CD30 shedding ,79. In cAs previously mentioned, Sutherland et al. did show that humanized AC10 conjugated to monomethyl auristatin A and F  during the early preclinical development of what became BV, and did show internalization, colocalization with lysosomes, and highly cytotoxic effects . In addiStructural data on mAb binding to distinct CRDs would be valuable in order to determine the unique interactions that could explain why certain mAbs–CRD interactions promote CD30 shedding and to clarify which interactions could promote endocytosis, supposedly via receptor clustering.CD22 is a 140-kDa transmembrane glycoprotein, and like CD33 it is also a member of the Siglec family and shares multiple structural features with this family, specifically extracellular Ig domains capped by a V-set Ig ligand binding domain, and an intracellular region containing ITIM motifs . Key difCD22 undergoes endocytosis via CME  89,90,9,990,91. The Paulson group published a collection of papers that elegantly evaluated CD22 endocytosis in a more natural setting ,90,92. TTaken together, natural-like ligands accumulate within cells via the constitutive and rapid internalization of CD22. These ligands are sorted for degradation in lysosomes, while CD22 recycles back to the cell surface. In addition, CD22 ligand-induced endocytosis activates intracellular pools, which replenish or increase the level of CD22 expression on the cell surface. Hence, CD22 has excellent endocytosis properties for ADCs.The rapid internalization of anti-CD22 mAbs on a variety of B cell lines has been known for the past two decades ,94, yet The mAbs RFB4 and epratuzumab have been previously studied in the context of endocytosis and intracellular routing. O’Reilly et al. showed that, unlike the ligand NeuAcα2-6Galβ1-4GlcNAc, RFB4:CD22 did not separate after endocytosis and recycled back to the cell surface . Erena-OPseudomonas exotoxin. It was shown that when 100 nmol/L of the RFB4 immunotoxin was incubated with CD22-positive cells, there was an increase of up to 240% in the level of immunotoxin that accumulated inside the cells. This indicated that the amount of CD22 internalized was greater than the original cell surface levels by 2–3-fold. Quantification of the intracellular CD22 revealed levels exceeding cell surface levels by up to 140%. Shan et al. showed that the mAb HD39 did colocalize with early endosomes at early time points . P. P208]. Based on these indirect studies, nectin-4 exhibits the robust endocytosis activities that a viral receptor would need. In addition, at least partially, it appears to traffic to lysosomes. These are the essential qualities of an attractive target for an ADC. Further studies will be required in order to understand the endocytosis activity of nectin-4 as it relates to ADCs.A functional endocytic vesicle requires cargo recruitment adaptors, membrane curvature effectors and a membrane scission component. Endophilins have all these characteristics in one protein. Endophilins are well-characterized BAR-containing proteins that also contain an SH3 domain that is critical for their function. The endophilin BAR domains induce and stabilize membrane curvature and are able to ‘sense’ and bind to already curved membranes . In addiNoting that endophilins are important in the endocytosis process, there are numerous findings associating these proteins with cancer. Endophilin A1 (Endo A1) has shown decreased expression in several tumor types . Endo A1Endophilin 2 (Endo A2) is vital as it acts as a scaffold that tethers the membrane as actin pulls the budding endocytic vesicle downward to facilitate scission. When Endo A2 is overexpressed, ligand-induced endocytosis is increased . Endo A2MLL) gene encodes a methyl transferase involved in normal gene regulation. However, the translocation of MLL frequently occurs and leads to genetic fusions and chimeric proteins associated with leukemias prevalent in infants of poor prognosis [Endo A2 dysregulation and its effects have a more pronounced role in endocytosis and ADC resistance. The myeloid-lymphoid leukemia (rognosis . One sucrognosis . The redSh3gl1) was associated with reduced relapse-free and overall survival. Lamellipodin is an adaptor protein that interacts with various scaffold proteins and actin and is crucial for various processes related to plasma membrane protrusions [RAPH1) transcription was found to be increased and associated with significantly worse relapse-free survival in node-positive HER2-positive patients [Endo A2 was also observed to be overexpressed in tumors and metastatic lymph nodes, with the highest expression in HER2-positive cases . Increastrusions . Lamellipatients . NotablyBaldassare et al. reported that Endo A2 is a critical factor that promotes sensitivity to T-DM1 and that, when silenced, cells have reduced sensitivity . The HERIn summary, variable expression levels of endophilins are associated with tumor aggressiveness and poor patient outcomes. Low levels of Endo A2 may lead to T-DM1 resistance. Further efforts to stratify HER2-positive patients with endocytic protein markers such as endophilin may be required in order to avoid resistance to T-DM1.Sung et al. reported a novel mechanism for trastuzumab ADCs based on caveolae-mediated endocytosis . HER2-poSmith et al. reported on an investigational ADC specific for p97, a glycosylphosphatidyl inositol-linked receptor internalized via the caveolae pathway . In melaBiopharmaceuticals targeting cell surface receptors are frequently subject to target-mediated clearance, resulting in nonlinear pharmacokinetic (PK) profiles. Tumor uptake efficiency is highly dependent on PK. Endocytosis of a biopharmaceutical upon binding to a cell surface receptor is the underlying cause of target-mediated drug clearance. ADC internalization kinetics most likely have a significant impact on efficacy and off-tumor side effects. Moreover, the internalization of receptors with expression as low as 5000 molecules per cell can affect PK and increased internalization has been associated with more pronounced receptor-mediated clearance . The cliInformation on the clinical effects with respect to internalization is scarce and, consequently, at this time it is difficult to determine relationships between all or most vital variables . In terms of endocytosis, target-mediated clearance can be tumor-specific or non-specific. Non-specific endocytosis and degradation involve target receptors expressed on healthy tissues. The level of expression and the rate of internalization relative to the tumor further complicate matters.2 doses with 14 days between administrations. GO was withdrawn from the market in 2010 when its efficacy was found to be poor and its toxicity appeared excessive. The new dosing regimen of 3 mg/m2 on days 1, 4, and 7 allows for the safe administration of higher cumulative doses and significantly improved outcomes in patients with AML [Deslandes analyzed 21 ADCs with reported information on PK parameters at multiple doses or information on dose ranges and dosing regimens to elucidate dose–clearance relationships . GO dispwith AML . Importa50 values) and is coupled to a pH-labile linker (CL2A) with moderate stability. The linker is essential as it enables SG to readily release its payload in a rapid fashion due to the high internalization rate of Trop2 and thereby accelerates the drug delivery to tumor cells. Interestingly, given the sensitivity of the CL2A linker to acidic pH, SG releases SN-38 over a period of about 3 days, and the authors surmise that some portion of the therapeutic activity of SG could result from bystander effects, due to the local release of the payload in the tumor microenvironment. This ultimately results in effective cytotoxicity of tumor cells. This demonstrates that endocytosis is vital but also that the interplay between the linker type and internalization is also important.Recently, Goldenberg and Sharkey rationalized a link between the excellent internalization of Trop2 and/or SG  is just as important as the preferential overexpression status of target receptors for ADCs and additional pertinent parameters such as PK. Although there are now nine approved ADCs and the field is experiencing a resurgence due to technical advancements in ADCs, there have been >100 ADCs in clinical trials at one time or another over the last 25 years, and most of those agents have failed . MoreoveInterest in ADC endocytosis has increased with the discovery of additional overexpressed receptors on tumor cells and high-affinity-binding mAbs. Most investigational ADCs will demonstrate a minimum capability to exploit target receptor internalization processes as a means to justify the ADC concept for delivery payloads inside tumor cells. Despite this, there is still little understanding of the endocytosis of target receptors. Given the large number of differences between targets, ADC constructs, dosing regimens, and patient populations, dedicated streams of internalization and endosomal sorting processes—in parallel with traditional ADC development areas —will increase the understanding of ADCs and inform researchers about how best to develop their therapeutic potential. In addition, due to the vital importance of many core endocytic components and key adaptors, these proteins are likely to be found to be commonly mutated in cancer and this too will affect ADC endocytosis and efficacy. Lastly, as the field has evolved, and after decades of experience, each individual aspect of an ADC is likely to be vital to ADC efficacy, and measuring all of these components is important. In this review, endocytosis has been described as an important additional aspect.