Site-Specific Conjugation of the Indolinobenzodiazepine DGN549 to Antibodies Affords Antibody-Drug Conjugates with an Improved Therapeutic Index as Compared with Lysine Conjugation.

Antibody-drug conjugates have elicited great interest recently as targeted chemotherapies for cancer. Recent preclinical and clinical data have continued to raise questions about optimizing the design of these complex therapeutics. Biochemical methods for site-specific antibody conjugation have been a design feature of recent clinical ADCs, and preclinical reports suggest that site-specifically conjugated ADCs generically offer improved therapeutic indices (i.e., the fold difference between efficacious and maximum tolerated doses). Here we present the results of a systematic preclinical comparison of ADCs embodying the DNA-alkylating linker-payload DGN549 generated with both heterogeneous lysine-directed and site-specific cysteine-directed conjugation chemistries. Importantly, the catabolites generated by each ADC are the same regardless of the conjugation format. In two different model systems evaluated, the site-specific ADC showed a therapeutic index benefit. However, the therapeutic index benefit is different in each case: both show evidence of improved tolerability, though with different magnitudes, and in one case significant efficacy improvement is also observed. These results support our contention that conjugation chemistry of ADCs is best evaluated in the context of a particular antibody, target, and linker-payload, and ideally across multiple disease models.

Synthesis and Evaluation of Camptothecin Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) that incorporate the exatecan derivative DXd in their payload are showing promising clinical results in solid tumor indications. The payload has an F-ring that also contains a second chiral center, both of which complicate its synthesis and derivatization. Here we report on new camptothecin-ADCs that do not have an F-ring in their payloads yet behave similarly to DXd-bearing conjugates in vitro and in vivo. This simplification allows easier derivatization of camptothecin A and B rings for structure-activity relationship studies and payload optimization. ADCs having different degrees of bystander killing and the ability to release hydroxyl or thiol-bearing metabolites following peptide linker cleavage were investigated.

Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers.

Although peptide linkers are used in multiple clinical-stage ADCs, there are only few reports on optimizing peptide linkers for efficient lysosomal proteolysis and for stability in circulation. We screened multiple dipeptide linkers for efficiency of proteolysis and compared them to the dipeptide linkers currently being evaluated in the clinic: Val-Cit, Val-Ala, and Ala-Ala. Lead dipeptide linkers selected from the initial screen were incorporated into ADCs with indolinobenzodiazepine dimer (IGN) payloads to evaluate cellular processing, in vitro cytotoxic activity, plasma stability, and in vivo efficacy. ADCs with several dipeptide linkers bearing l-amino acids showed faster lysosomal processing in target cancer cells compared to the l-Ala-l-Ala linked ADC. These variances in linker processing rates did not result in different in vitro and in vivo activities among peptide linker ADCs, presumably due to accumulation of threshold cytotoxic catabolite levels for ADCs of several peptide linkers in the cell lines and xenografts tested. ADCs with l-amino acid dipeptide linkers exhibited superior in vitro cytotoxic potencies in multiple cell lines compared to an ADC with a d-Ala-d-Ala dipeptide linker and an ADC with a noncleavable linker. This work adds to the toolbox of stable, lysosomally cleavable peptide linkers for ADCs.

Attitudinal Spillover from Misleading Natural Cigarette Marketing: An Experiment Examining Current and Former Smokers' Support for Tobacco Industry Regulation.

This research examined the influence of natural cigarette advertising on tobacco control policy support, and the potential for misbeliefs arising from exposure to cigarette marketing to affect such support. Ample research indicates that natural cigarettes such as Natural American Spirit (NAS) are widely and erroneously perceived as safer than their traditional counterparts because of their marketed "natural" composition. Yet regulatory action regarding natural cigarette marketing has been limited in scope, and little research has examined whether misleading product advertising affects support for related policy, an important component of the policy process. Here, we administered a large-scale randomized experiment (n = 1128), assigning current and former smokers in the United States to an NAS advertising condition or a control group and assessing their support for tobacco industry regulation. Results show that exposure to NAS advertising reduces support for policies to ban potentially misleading terminology from cigarette advertising, and these effects are stronger for daily smokers. Further, misinformed beliefs about the healthy composition of NAS partially mediate effects on policy support. Yet interestingly, exposure to NAS marketing does not reduce support for policies to establish standards for when certain terms are permissible in cigarette advertising. The results of this analysis indicate potential spillover effects from exposure to NAS advertising in the realm of support for regulatory action pertaining to tobacco industry marketing.

The Effects of Graphic Warning Labels' Vividness on Message Engagement and Intentions to Quit Smoking.

The current study examined the effects of manipulating the level of vividness through the presence of various textual and visual components in the context of tobacco warning labels. An online experiment was conducted (N=2165) to examine whether increasing the vividness of warning labels, using narrative and non-narrative components, increased engagement with the messages, and the subsequent effects of vividness and engagement on intentions to quit smoking. Results showed that more vivid warning labels led to increased engagement, which in turn was linked to increased intentions to quit smoking. Specifically, the indirect effect of vividness on intentions to quit smoking was largely driven by the emotional component of engagement. Indirect effects of cognitive engagement were only apparent at higher levels of vividness.

Effect of Linker Stereochemistry on the Activity of Indolinobenzodiazepine Containing Antibody-Drug Conjugates (ADCs).

Antibody-drug conjugates (ADCs) that incorporate potent indolinobenzodiazepine DNA alkylators as the payload component are currently undergoing clinical evaluation. In one ADC design, the payload molecules are linked to the antibody through a peptidase-labile l-Ala-l-Ala linker. In order to determine the role of amino acid stereochemistry on antitumor activity and tolerability, we incorporated l- and d-alanyl groups in the dipeptide, synthesized all four diastereomers, and prepared and tested the corresponding ADCs. Results of our preclinical evaluation showed that the l-Ala-l-Ala configuration provided the ADC with the highest therapeutic index (antitumor activity vs toxicity).

A Case Study Comparing Heterogeneous Lysine- and Site-Specific Cysteine-Conjugated Maytansinoid Antibody-Drug Conjugates (ADCs) Illustrates the Benefits of Lysine Conjugation.

Antibody-drug conjugates are an emerging class of cancer therapeutics constructed from monoclonal antibodies conjugated with small molecule effectors. First-generation molecules of this class often employed heterogeneous conjugation chemistry, but many site-specifically conjugated ADCs have been described recently. Here, we undertake a systematic comparison of ADCs made with the same antibody and the same macrocyclic maytansinoid effector but conjugated either heterogeneously at lysine residues or site-specifically at cysteine residues. Characterization of these ADCs in vitro reveals generally similar properties, including a similar catabolite profile, a key element in making a meaningful comparison of conjugation chemistries. In a mouse model of cervical cancer, the lysine-conjugated ADC affords greater efficacy on a molar payload basis. Rather than making general conclusions about ADCs conjugated by a particular chemistry, we interpret these results as highlighting the complexity of ADCs and the interplay between payload class, linker chemistry, target antigen, and other variables that determine efficacy in a given setting.

Design, synthesis and evaluation of novel, potent DNA alkylating agents and their antibody-drug conjugates (ADCs).

Antibody-drug conjugates (ADCs) incorporating potent indolinobenzodiazepine (IGN) DNA alkylators as the cytotoxic payload are currently undergoing clinical evaluation. The optimized design of these payloads consists of an unsymmetrical dimer possessing both an imine and an amine effectively eliminating DNA crosslinking and demonstrating improved tolerability in mice. Here we present an alternate approach to generating DNA alkylating ADCs by linking the IGN monomer with a biaryl system which has a high DNA binding affinity to potentially enhance tolerability. These BIA ADCs were found to be highly cytotoxic in vitro and demonstrated potent antitumor activity in vivo.

Regulating language, not inference: an examination of the potential effectiveness of Natural American Spirit advertising restrictions.

OBJECTIVE: In 2017, the United States Food and Drug Administration (FDA) reached an agreement with Santa Fe Natural Tobacco Company (SFNTC) stipulating that SFNTC will remove 'natural' and 'additive-free' from Natural American Spirit (NAS) marketing to combat misperceptions that NAS is a healthier cigarette. The purpose of this study was to assess experimentally the potential effectiveness of the agreement in addressing NAS misperceptions. METHODS: In an online experiment, 820 current and former smokers were assigned randomly to advertising conditions with existing claims from NAS advertisements, modified claims or a no-exposure control. Advertising conditions included (1) 'original' NAS advertising text before the agreement; (2) '2017 agreement' language permissible under the FDA-SFNTC agreement (removing 'natural', 'additive-free'); (3) more restrictive ('stricter') language representing additional regulation (removing 'natural' from the brand name and the phrases 'tobacco+water', 'no chemicals'). Participants completed outcome measures assessing misinformed beliefs and intentions towards NAS. RESULTS: One-way ANOVA showed that relative to the 'original' language, the '2017 agreement' language reduced misconceptions about NAS addictiveness, but not about health or constituent composition. Yet 'stricter' language significantly reduced all categories of misinformed beliefs, which in turn mediated effects on (lower) intentions to use NAS. CONCLUSION: The 2017 agreement helps dispel some misconceptions about NAS addictiveness, but does not sufficiently rectify misinformation about health or composition. Since 'stricter' language more effectively corrects misinformed beliefs, our results suggest the need for further regulations in addressing misinformation that drives intentions towards NAS.

Does Interest in Smoking Affect Youth Selection of Pro-smoking Videos? A Selective Exposure Experiment.

BACKGROUND: Many pro-smoking videos on YouTube reach view counts in the hundreds of thousands and more. Yet, there is limited information on who is viewing these potentially misleading videos. This study attempts to understand the viewership of online pro-smoking videos to examine if youth at high risk for smoking are more likely to watch these videos. METHODS: We conducted a selective exposure experiment with a national sample of youths (ages 15-21 years; n = 614) to identify characteristics that make individuals more likely to select pro-smoking videos. During a 10-min browsing session, participants were given a set of 16 videos (eight smoking and eight nonsmoking) and were asked to view video(s) of their choice. Exposure to videos was unobtrusively logged. View count was manipulated such that smoking videos had either high or low views. RESULTS: Behavioral data revealed that youth with higher interest in smoking were more likely to select and spend more time watching pro-smoking videos than youth with lower interest in smoking. The view count manipulation did not affect selection patterns. However, exposure to high view count smoking videos was associated with more positive attitudes toward smoking. CONCLUSIONS: The findings of this study call into question the existence and prominence of pro-smoking videos on YouTube and bring to attention the need for regulatory or monitoring efforts of such content. IMPLICATIONS: Given the presence and prevalence of misleading pro-smoking videos online, this is the first study to ask the practical and important question of who is viewing these videos. Using behavioral data, we are able to demonstrate that youth who are high at risk for smoking are more susceptible to select and spend more time viewing pro-smoking videos than youth who are low at risk for smoking. Findings also show that when pro-smoking videos appear to be "popular," they affect attitudes toward smoking. Our findings provide policy implications regarding regulation of smoking promotion videos online.

When Visual Cues Activate Moral Foundations: Unintended Effects of Visual Portrayals of Vaping within Electronic Cigarette Video Advertisements.

Within multimodal persuasive messages, the roles of visual cues in producing unintended effects have been understudied. In an experiment on a sample of former and current smokers (N = 991), we manipulated the presence of visual vaping cues within electronic cigarette video advertisements (N = 25) to evaluate opinions towards vape-free policies. Such cues diminished the effects of pro-vaping arguments to increase support for vape-free policies, inadvertently benefiting public health. Consistent with the moral foundations theory (MFT), endorsement of the care/harm moral foundation strengthened message effects. Furthermore, cognitions and emotions related to moral intuitions mediated the effects of visual vaping cues. These findings suggest that MFT can help explain unintended effects of visual cues when outcomes are related to morality.

A DNA-Interacting Payload Designed to Eliminate Cross-Linking Improves the Therapeutic Index of Antibody-Drug Conjugates (ADCs).

Tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADCs) is now a clinically validated approach for cancer treatment. In an attempt to improve the clinical success rate of ADCs, emphasis has been recently placed on the use of DNA-cross-linking pyrrolobenzodiazepine compounds as the payload. Despite promising early clinical results with this class of ADCs, doses achievable have been low due to systemic toxicity. Here, we describe the development of a new class of potent DNA-interacting agents wherein changing the mechanism of action from a cross-linker to a DNA alkylator improves the tolerability of the ADC. ADCs containing the DNA alkylator displayed similar in vitro potency, but improved bystander killing and in vivo efficacy, compared with those of the cross-linker. Thus, the improved in vivo tolerability and antitumor activity achieved in rodent models with ADCs of the novel DNA alkylator could provide an efficacious, yet safer option for cancer treatment. Mol Cancer Ther; 17(3); 650-60. ©2018 AACR.

Treatment decision making in early-stage papillary thyroid cancer.

OBJECTIVE: The purpose of this study was to develop an in-depth understanding of papillary microcarcinoma (PMC) patients' decision-making process when offered options of surgery and active surveillance. METHODS: Fifteen PMC patients and 6 caregivers participated in either a focus group or individual interview. Focus groups were segmented by patients' treatment choice. Primary themes were identified in transcripts using thematic text analysis. RESULTS: Four themes emerged from the surgery subsample: (1) Decision to undergo thyroidectomy quickly and with a sense of urgency; (2) Perception of PMC as a potentially life-threatening disease; (3) Fear of disease progression and unremitting uncertainty with active surveillance; and (4) Surgery as a means of control and potential cure. Three themes emerged from the active surveillance subsample: (1) View of PMC as a common, indolent, and low-risk disease; (2) Concerns about adjusting to life without a thyroid and becoming reliant on hormone replacement medication; and (3) Openness to reconsidering surgery over the long run. Two themes were identified that were shared by participants from both subsamples: (1) Deep level of trust and confidence in physician and cancer center; and (2) Use of physician and internet as primary sources of disease and treatment-related information. CONCLUSIONS: Several factors influenced participants' treatment choice, with similarities and differences noted between surgery and active surveillance subsamples. Many of the emergent themes are consistent with research on decision making among localized prostate cancer patients. Findings suggest that participants from both PMC treatment subsamples are motivated to reduce illness and treatment-related uncertainty.

Influence of Natural American Spirit advertising on current and former smokers' perceptions and intentions.

OBJECTIVE: This study sought to demonstrate causal effects of exposure to Natural American Spirit (NAS) advertising content on misinformed beliefs of current and former smokers, and to empirically establish these beliefs as a mechanism driving intentions to use NAS. METHODS: Our study employed a randomised experimental design with 1128 adult daily, intermittent and former smokers. We compared participants who were exposed to NAS advertisements or claims made in the advertisements with those in a no-message control group to test the effects of NAS advertising content on inaccurate beliefs about NAS and attitudes and intentions towards the product. RESULTS: One-way analysis of variance revealed that exposure to NAS advertisements produced inaccurate beliefs about the composition of NAS cigarettes among current and former smokers (p<0.05). Planned contrasts indicated a compilation of arguments taken directly from NAS advertisements resulted in significantly greater beliefs that NAS cigarettes are healthier/safer than other cigarettes (for former smokers, t(472)=3.63, p<0.001; for current smokers, t(644)=2.86, p=0.004), demonstrating that suggestive claims used in the brand's marketing have effects on beliefs not directly addressed in the advertisements. Regression and mediation analyses showed that health-related beliefs predict attitudes towards NAS for current and former smokers, and mediate intentions to use NAS. CONCLUSIONS: The findings of this study provide causal support for the need for further regulatory action to address the potentially harmful ramifications of claims used in NAS advertising.

A Randomized Trial of the Effect of Youth Appealing E-Cigarette Advertising on Susceptibility to Use E-Cigarettes Among Youth.

Introduction: Very little is known about how e-cigarette marketing is being perceived by youth, and the potential effect it will have on youth vaping and smoking behaviors. This limits the ability to identify youth-focused marketing efforts and to design effective policies for the regulation of e-cigarette marketing content and placement. Methods: A sample of 417 nonsmoking youth (mean age = 15, SD = 1.3) were randomly assigned to either view four e-cigarette ads with low youth appeal, four e-cigarette ads with high youth appeal or four control ads. After exposure, participants completed covert and overt measurements of e-cigarette and tobacco cigarette attitudes and susceptibility to use. Results: Youth in an e-cigarette ad condition were more likely to select an e-cigarette item in a product choice task compared to control, and had more positive e-cigarette beliefs. Contrary to hypotheses, youth in the low youth appeal condition reported greater susceptibility to trying e-cigarettes and tobacco cigarettes compared to control. Conclusions: Exposure to any e-cigarette advertising may play a role in teens' decision to initiate e-cigarette and tobacco cigarette use. As the Food and Drug Administration now has regulatory authority over the marketing of e-cigarettes, regulations on e-cigarette advertising are suggested. Implications: Teens are increasingly being exposed to e-cigarette advertising, and many places are considering e-cigarette regulations, yet we know very little about how e-cigarette advertisements might influence youth tobacco use. This study utilized a novel dataset of e-cigarette ads coded for youth appeal and presented them to a sample of 417 nonsmoking teens in a randomized controlled design to test the effect of features on youth susceptibility to initiating e-cigarette and tobacco cigarette use. The findings inform evidence-based recommendations for regulating the marketing of e-cigarettes.

Effects of Drug-Antibody Ratio on Pharmacokinetics, Biodistribution, Efficacy, and Tolerability of Antibody-Maytansinoid Conjugates.

Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an average drug-to-antibody ratio (DAR) of 3-4 (potentially ranging from 0 to 8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an average DAR of 3.5. To understand the effect of DAR on the preclinical properties of ADCs using maytansinoid cytotoxic agents, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor α (FRα)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (average of ∼2 and range of 0-4) to very high (average of 10 and range of 7-14) were prepared in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR at a constant antibody concentration. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic analysis showed that conjugates with an average DAR below ∼6 had comparable clearance rates, but for those with an average DAR of ∼9-10, rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with maximum localization for this organ at 24-28% percentage injected dose per gram (%ID/g) compared with 7-10% for lower-DAR conjugates (all at 2-6 h post-injection). Our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2 to 6 have a better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy, likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs but suggest that the exploration of lower or higher DAR may be warranted depending on the biology of the target antigen.

Youth-Targeted E-cigarette Marketing in the US.

OBJECTIVES: The purpose of this project was to explore the extent to which e-cigarette advertisements use youth-appealing content. METHODS: A sample of e-cigarette video ads (N = 154) were coded by 3 independent coders using the content appealing to youth (CAY) index, which measures over 40 youth-appealing features. To calculate a total CAY score for each ad, scores were normalized and summed such that a higher score represented the presence of more youth-appealing elements. RESULTS: All ads included some youth-appealing content, with frequent use of emotional appeals, including happiness (68%), friendship (41%), sex (24%), and success (24%). Over half featured animation. However, product appeals, known to be disliked by youth, were also prevalent. Eighty-seven percent used a direct appeal to e-cigarettes' value over tobacco cigarettes; 66% mentioned product composition; and 40% mentioned health. CONCLUSIONS: Given the unregulated context in the US, e-cigarette marketing currently contains many features that may be particularly appealing to youth.

Potential Effectiveness of Pictorial Warning Labels That Feature the Images and Personal Details of Real People.

INTRODUCTION: Pictorial warning labels (PWL) that use photographs and the personal details of real people whose health has been affected by smoking (testimonial PWL) provide factual information about the consequences of tobacco use. METHODS: Nine hundred and twenty-four adult current smokers participated in an online experiment that tested responses to four types of warning labels: (1) non-testimonial text warning labels (currently on packs in the United States); (2) non-testimonial PWL (previously proposed by the United States Food and Drug Administration); (3) image only testimonial PWL (created for study); (4) image + personal details testimonial PWL (created for study). Participants were randomly assigned to condition and then exposed to up to five warning labels addressing different health effects. Differences between conditions were assessed using emotional responses and a set of intention measures immediately following exposure, and self-reported behavior change at 5-week follow-up. RESULTS: Compared to the non-testimonial text warning labels, all PWL elicited stronger emotional responses and intentions to forgo cigarettes and avoid the warning labels. Non-testimonial PWL and image + personal details testimonial PWL elicited stronger intentions to quit, whereas image only testimonial PWL generated a greater amount of quitting activity in the weeks following exposure. There were no significant differences in responses when comparing the non-testimonial PWL with both types of testimonial PWL. CONCLUSIONS: PWL that use images of real people convey factual information about the health effects of tobacco use. These testimonial PWL may be a promising alternative to the images previously proposed for use on PWL in the United States. IMPLICATIONS: In the United States, the PWL developed by the Food and Drug Administration (FDA) in 2011 were found by the courts to be unconstitutional, in part because they were deemed to present an opinion rather than fact. Findings from this experimental study indicate that PWL that use the images and personal details of real people to convey factual information about the health effects of tobacco use may satisfy the FDA's requirement for a set of PWL that (1) have the potential to positively impact the determinants of smoking cessation behavior, (2) meet legislative requirements under the Family Smoking Prevention and Tobacco Control Act and (3) may be more acceptable to the courts than the previously proposed and now dismissed PWL that carried non-factual images.

A New Class of Antibody-Drug Conjugates with Potent DNA Alkylating Activity.

The promise of tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADC) has now been realized, evidenced by the approval of two ADCs, both of which incorporate highly cytotoxic tubulin-interacting agents, for cancer therapy. An ongoing challenge remains in identifying potent agents with alternative mechanisms of cell killing that can provide ADCs with high therapeutic indices and favorable tolerability. Here, we describe the development of a new class of potent DNA alkylating agents that meets these objectives. Through chemical design, we changed the mechanism of action of our novel DNA cross-linking agent to a monofunctional DNA alkylator. This modification, coupled with linker optimization, generated ADCs that were well tolerated in mice and demonstrated robust antitumor activity in multiple tumor models at doses 1.5% to 3.5% of maximally tolerated levels. These properties underscore the considerable potential of these purpose-created, unique DNA-interacting conjugates for broadening the clinical application of ADC technology. Mol Cancer Ther; 15(8); 1870-8. ©2016 AACR.

Does Vaping in E-Cigarette Advertisements Affect Tobacco Smoking Urge, Intentions, and Perceptions in Daily, Intermittent, and Former Smokers?

Visual depictions of vaping in electronic cigarette advertisements may serve as smoking cues to smokers and former smokers, increasing urge to smoke and smoking behavior, and decreasing self-efficacy, attitudes, and intentions to quit or abstain. After assessing baseline urge to smoke, 301 daily smokers, 272 intermittent smokers, and 311 former smokers were randomly assigned to view three e-cigarette commercials with vaping visuals (the cue condition) or without vaping visuals (the no-cue condition), or to answer unrelated media use questions (the no-ad condition). Participants then answered a posttest questionnaire assessing the outcome variables of interest. Relative to other conditions, in the cue condition, daily smokers reported greater urge to smoke a tobacco cigarette and a marginally significantly greater incidence of actually smoking a tobacco cigarette during the experiment. Former smokers in the cue condition reported lower intentions to abstain from smoking than former smokers in other conditions. No significant differences emerged among intermittent smokers across conditions. These data suggest that visual depictions of vaping in e-cigarette commercials increase daily smokers' urge to smoke cigarettes and may lead to more actual smoking behavior. For former smokers, these cues in advertising may undermine abstinence efforts. Intermittent smokers did not appear to be reactive to these cues. A lack of significant differences between participants in the no-cue and no-ad conditions compared to the cue condition suggests that visual depictions of e-cigarettes and vaping function as smoking cues, and cue reactivity is the mechanism through which these effects were obtained.