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Supplementary methods

Supplementary figure 1. Sym015 inhibits viability of cell lines in a synergic manner; Supplementary figure 2. The Sym015 antibodies Hu9338 and Hu9006 bind to 2nd or 3rd blades of MET and block HGF binding; Supplementary figure 3. Sym015 induces MET internalization and degradation in MKN-45 cells; Supplementary figure 4. Sym015 induces MET internalization and degradation in EBC-1 cells; Supplementary figure 5. Sym015 induces MET degradation in MKN-45 and EBC-1 cells; Supplementary figure 6. Sym015 inhibits signaling by MET in EBC-1 cells; Supplementary figure 7. Sym015 inhibits motility of EBC-1

Increased MET activity is linked with poor prognosis and outcome in several human cancers currently lacking targeted therapies. Here, we report on the characterization of Sym015, an antibody mixture composed of two humanized IgG1 antibodies against nonoverlapping epitopes of MET. Sym015 was selected by high-throughput screening searching for antibody mixtures with superior growth-inhibitory activity against MET-dependent cell lines. Synergistic inhibitory activity of the antibodies comprising Sym015 was observed in several cancer cell lines harboring amplified MET locus and was confirmed in vivo. Sym015 was found to exert its activity via multiple mechanisms. It disrupted interaction of MET with the HGF ligand and prompted activity-independent internalization and degradation of the receptor. In addition, Sym015 induced high levels of CDC and ADCC in vitro. The importance of these effector functions was confirmed in vivo using an Fc-effector function–attenuated version of Sym015. The enhanced effect of the two antibodies in Sym015 on both MET degradation and CDC and ADCC is predicted to render Sym015 superior to single antibodies targeting MET. Our results demonstrate strong potential for use of Sym015 as a therapeutic antibody mixture for treatment of MET-driven tumors. Sym015 is currently being tested in a phase I dose escalation clinical trial (NCT02648724). Mol Cancer Ther; 16(12); 2780–91. ©2017 AACR.

Short description of methods and materials used for supplemmentary figures. BioLayer Interferometry, ADCC and CDC assays.

4–1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4–1BB on tumor-specific cytotoxic T cells makes 4–1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4–1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4–1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4–1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4–1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4–1BB–mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.

Supplementary Tables and Figures. Table S1 contains affinities for antibodies binding to HER2. Table S2 contains results from epitope bin analysis of the antibodies. Figure S1 shows a comparison of Perjeta and the pertuzumab analogue used in this paper. Figure S2 shows sensograms for the anti-HER2 antibodies. Figure S3 contains dose-response curves for anti-HER2 lead mixtures in the cell lines BT474, SK-BR3, HCC202 and NCI-N87. Figure S4 shows immunoblot data on the levels of HER2, pHER2, EGFR, pEGFR, HER3 and pHER3 in the eight cell lines OE19, N87, MCF7, MDA-MB-175, BT474, HCC202 SK-BR3 and ZE-75-30. Figure S5 shows activity of anti-HER2 mAbs and mixtures in ADCC and CDC assays. Figure S6 demonstrates synergy of the tripartite mixture in OE19 cell line. Figure S7 shows quantification of HER2 and pHER2 levels in OE19 and HCC202 cell lines upon treatment with anti-HER2 mAbs and mixtures. Figure S8 shows activity of tripartite mixtures of trastuzumab, pertuzumab and lead anti-HER2 antibodies in a cell viability assay

Supplementary figures and methods

Supplementary Figures S1-8. Figure S1 - Analysis of nonlinear blending synergy for the pairs of antibodies that constitute Pan-HER. Figure S2 - In vitro comparison of Pan-HER and a combination of cetuximab, trastuzumab and MM-121. Figure S3 - In vivo assessment of nonlinear blending synergy for the target specificities in the Pan-HER mixture. Figure S4 - Dose titration of Pan-HER in the BxPC3 xenograft model. Figure S5 - IHC analysis of Calu-3 tumors. Figure S6 - Assessment of cell death and cell cycle arrest. Figure S7 - Assessment of ADCC in a panel of cell lines. Figure S8 - Analysis of the effect of receptor internalization on effector functions.

Supplementary Methods. Description of methods used for assessment of synergy, immunohistochemistry, cell death, cell cycle arrest, ADCC and CDC.

Purpose: Activation of the receptor tyrosine kinase MET is associated with poor clinical outcome in certain cancers. To target MET more effectively, we developed an antagonistic antibody mixture, Sym015, consisting of two humanized mAbs directed against nonoverlapping epitopes of MET.Experimental Design/Results: We screened a large panel of well-annotated human cancer cell lines and identified a subset with highly elevated MET expression. In particular, cell lines of lung cancer and gastric cancer origin demonstrated high MET expression and activation, and Sym015 triggered degradation of MET and significantly inhibited growth of these cell lines. Next, we tested Sym015 in patient- and cell line–derived xenograft models with high MET expression and/or MET exon 14 skipping alterations, and in models harboring MET amplification as a mechanism of resistance to EGFR-targeting agents. Sym015 effectively inhibited tumor growth in all these models and was superior to an analogue of emibetuzumab, a monoclonal IgG4 antibody against MET currently in clinical development. Sym015 also induced antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggesting that secondary effector functions contribute to the efficacy of Sym015.Retrospectively, all responsive, high MET-expressing models were scored as highly MET-amplified by in situ hybridization, pointing to MET amplification as a predictive biomarker for efficacy. Preclinical toxicology studies in monkeys showed that Sym015 was well tolerated, with a pharmacokinetic profile supporting administration of Sym015 every second or third week in humans.Conclusions: The preclinical efficacy and safety data provide a clear rationale for the ongoing clinical studies of Sym015 in patients with MET-amplified tumors. Clin Cancer Res; 23(19); 5923–35. ©2017 AACR.

Supplementary Materials and Methods and Figure Legends

Suppl. Figure 1 In vivo efficacy of Sym015 treatment at various dose levels in the EBC-1 xenograft model. Suppl. Figure 2 In vivo efficacy of Sym015 and unbalanced mixtures of the two constituent antibodies Hu9006 and Hu9338, applied at 10 mg/kg or 50 mg/kg doses in the EBC-1 xenograft model. Suppl. Figure 3 In vivo efficacy of Sym015 treatment in the H596 CDX model, which harbors a MET exon 14 deletion without MET gene amplification. Suppl. Figure 4 2 Map of clinically relevant mutations, obtained from the Cancer Cell Line Encyclopedia(31), in 64 cell lines. Suppl. Table S1 List of the 64 cell lines used in the study with information on tissue of origin, supplier, and growth medium used for propagation. Suppl. Table S2 Yellow columns: FISH scores for CDX and PDX models, denoting the average number of MET and CEP7 signals per cell, the MET/CEP7 ratio, and the percentage of tumor cells amplified. Suppl. Table S3 Prediction of human pharmacokinetics based on one-species allometric scaling(33).

Supplementary Tables S1-2. Table S1 - Source, origin, subtype and growth medium for each cell line. Table S2 - Characteristics of tested patient-derived xenograft models.

Purpose: Accumulating evidence indicates a high degree of plasticity and compensatory signaling within the human epidermal growth factor receptor (HER) family, leading to resistance upon therapeutic intervention with HER family members.Experimental Design/Results: We have generated Pan-HER, a mixture of six antibodies targeting each of the HER family members EGFR, HER2, and HER3 with synergistic pairs of antibodies, which simultaneously remove all three targets, thereby preventing compensatory tumor promoting mechanisms within the HER family. Pan-HER induces potent growth inhibition in a range of cancer cell lines and xenograft models, including cell lines with acquired resistance to therapeutic antibodies. Pan-HER is also highly efficacious in the presence of HER family ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. All three target specificities contribute to the enhanced efficacy, demonstrating a distinct benefit of combined HER family targeting when compared with single-receptor targeting.Conclusions: Our data show that simultaneous targeting of three receptors provides broader efficacy than targeting a single receptor or any combination of two receptors in the HER family, especially in the presence of HER family ligands. Pan-HER represents a novel strategy to deal with primary and acquired resistance due to tumor heterogeneity and plasticity in terms of HER family dependency and as such may be a viable alternative in the clinic. Clin Cancer Res; 21(18); 4110–22. ©2015 AACR.See related commentary by Yarden and Sela, p. 4030

Background Even when palliative care is an integrated part of the healthcare system, the quality is still substandard for many patients and often initiated too late. There is a lack of structured guidelines for identifying and caring for patients; in particular for those with early palliative care needs. A care guide can act as a compass for best practice and support the care of patients throughout their palliative trajectory. Such a guide should both meet the needs of health care professionals and patients and families, facilitating discussion around end-of-life decision-making and enabling them to plan for the remaining time in life. The aim of this article is to describe the development and pilot testing of a novel Swedish palliative care guide. Methods The Swedish Palliative Care Guide (S-PCG) was developed according to the Medical Research Council framework and based on national and international guidelines for good palliative care. An interdisciplinary national advisory committee of over 90 health care professionals together with patient, family and public representatives were engaged in the process. The feasibility was tested in three pilot studies in different care settings. Results After extensive multi-unit and interprofessional testing and evaluation, the S-PCG contains three parts that can be used independently to identify, assess, address, follow up, and document the individual symptoms and care-needs throughout the whole palliative care trajectory. The S-PCG can provide a comprehensive overview and shared understanding of the patients’ needs and possibilities for ensuring optimal quality of life, the family included. Conclusions Based on broad professional cooperation, patients and family participation and clinical testing, the S-PCG provides unique interprofessional guidance for assessment and holistic care of patients with palliative care needs, promotes support to the family, and when properly used supports high-quality personalised palliative care throughout the palliative trajectory. Future steps for the S-PCG, entails scientific evaluation of the clinical impact and effect of S-PCG in different care settings – including implementation, patient and family outcomes, and experiences of patient, family and personnel.

4–1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4–1BB on tumor-specific cytotoxic T cells makes 4–1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4–1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4–1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4–1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4–1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4–1BB–mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.

Background Airway obstruction is a key feature of asthma and chronic obstructive pulmonary diseases (COPD). Smoking habits and workplace exposures to vapors, gas, dusts, and fumes (VGDF) could cause or exacerbate airway obstruction. The aim of this study is to evaluate the risk of airway obstruction due to smoking and workplace exposure, and their interaction, in a large population-based study. Methods In this cross-sectional study, a sample (n = 6153) of the Swedish population aged between 24 and 76 years underwent a questionnaire, clinical examination, blood test, and spirometry to gather information on airway obstruction classified by Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria or American Thoracic Society (ATS)/European Respiratory Society (ERS) criteria, risk factors, and confounders. Occupational exposures to VGDF were rated according to a specific job-exposure matrix. Adjusted logistic regression models were used to evaluate risk factors for airway obstruction in smokers and nonsmokers. Results In total, 9.8% had airway obstruction by GOLD criteria and 10.3% by ATS/ERS. Smokers with a high likelihood of exposure to VGDF had a higher risk of airway obstruction than those not exposed (odds ratio [OR]: 1.74, 95% confidence interval [CI]: 1.15-2.65 by GOLD; OR: 1.58, 95% CI: 1.06-2.37 by ATS/ERS) especially those >50 years of age. In smokers highly exposed to VGDF, risk estimates were higher than in the whole population, and the interaction between high exposure to VGDF and smoking further increased the risk of airway obstruction. Conclusions This study suggests a possible role for interaction between cigarette smoking and VGDF exposure on the risk of airway obstruction.

Background Water based paint has improved the work environment during occupational as well as domestic painting. Development of water-based paint formulations demanded an addition of anti-fungal/anti-microbial preservatives, and isothiazolinones (ITZ) have been frequently utilized over the last two decades. Isothiazolinones are known to cause skin sensitization and is furthermore suspected to cause problems from the respiratory tract. To assess professional painters’ exposure to air-borne ITZ, there is a need to develop an easy-to-use light-weight portable sampling technique of the three most abundant ITZ. Method Three different designs of SPE-samplers and three different chemistries were evaluated regarding resistance to flow, handling during sampling, analyte break-through and sample elution. The quantification was performed using RP-HPLC-UV. Results SPE-samplers are easy-to-use in a field setting. However, only the novel disc-type designs of SPE worked with the portable pumps because of the relatively low pressure drop over the disc. SPE disks can be obtained either as a syringe-type or as a filter-type. While the syringe-type can be used straight away the filter-type has to be mounted in a sampler. However, the latter design outperformed the former regarding sampling as well as elution procedure. Conclusion The novel design of SPE-discs works well for sampling of ITZ down to 1 nmol/m3 (0.15 µg/m3) when sampling for 8 hours. It is thereby possible to assess workers’ exposure to air concentrations of ITZ previously reported using impinger flasks.

After observing several clinical patients with respiratory symptoms, we initiated a questionnaire survey to assess prevalence of and predictors for asthma and work-related symptoms among workers in fish processing plants. A questionnaire with items on work conditions, work-related symptoms, and respiratory symptoms/diseases was sent to 916 fish processing workers, the 1836 licenced fishermen in Sweden, and 1965 controls; of those, 43%, 57%, and 53%, respectively, responded. Risks, hazard ratios (HRs), and prevalence ratios (PRs) were calculated with Cox regression, and 95% confidence intervals (CIs) were computed. The risk of asthma among fish filleting workers was increased during the years working in the fish processing industry when compared to the other fish processing workers and controls (HR 3.6, 95% CI 1.6-8.1, adjusted for atopy, gender, and ever smoking). The filleters had an increased PR for most of the work-related respiratory symptoms investigated. All fish processing workers had a higher PR for flu-like symptoms. Use of a pressure sprayer was identified as a risk for asthma and respiratory symptoms among both fish processing workers and controls. Filleters had changed work tasks because of respiratory symptoms more often (Fisher's exact test, p = 0.02) than other fish processing workers. In conclusion the fish filleters and pressure sprayer users reported more adult asthma and cough with phlegm compared to the other fish processing workers and controls. The use of pressure sprayers must be reduced and machinery should be completely encased to reduce workers' exposure to bioaerosols and its effects on the respiratory tract.

Objectives The aim of this study was to characterize personal occupational exposure to endotoxin in size-separated airborne particles of MWF aerosol, using a Sioutas cascade impactor (SCI). Methods Exposure to inhalable fractions of MWF aerosol and endotoxin was measured by personal sampling of 52 individuals over an 8-h work shift using a PAS-6 sampler in parallel with a SCI ( Results A total of 117 measurements of inhalable MWF aerosols were made among 52 workers. The geometric mean of inhalable MWF aerosol was 0.16 mg m−3 air. The geometric mean of endotoxin concentration on the inhalable sampler was 0.15 EU m−3. Airborne endotoxin was found on all size fractions from the impactor, with the major part seen in the fraction (2.5–10 µm). There was a correlation between the inhalable fraction of endotoxin measured by the PAS-6 sampler and on the SCI sampler (2.5–10 µm), estimated to be 0.51 for all samples (P < 0.0001). The concentration of endotoxin varied between the MWFs, as did the proportion of Gram-negative bacteria among the culturable bacteria (>80% in one MWF and Conclusions The personal exposure to inhalable fractions of endotoxin contained in the MWF aerosol were low, where most of the endotoxin were found in fraction (2.5–10 µm), measured by SCI. There are differences between factories and MWF systems regarding the distribution of endotoxin and so results from one context should not be generalized to other plants and systems. Compressed air was used for less than 10 min shift−1. The mixed-effect model showed that working with open machines and grinding as cutting task were important determinants of exposure to inhalable aerosol. It is important to keep occupational exposure to aerosols low with the help of good ventilation systems, enclosed machines, and organization of work.

ObjectivesOccupational exposures to metal fumes have been associated with increased pneumonia risk, but the risk of invasive pneumococcal disease (IPD) has not been characterised previously.MethodsWe studied 4438 cases aged 20–65 from a Swedish registry of invasive infection caused by Streptococcus pneumoniae. The case index date was the date the infection was diagnosed. Six controls for each case, matched for gender, age and region of residency, were selected from the Swedish population registry. Each control was assigned the index date of their corresponding case to define the study observation period. We linked cases and controls to the Swedish registries for socioeconomic status (SES), occupational history and hospital discharge. We applied a job–exposure matrix to characterise occupational exposures. We used conditional logistic analyses, adjusted for comorbidities and SES, to estimate the OR of IPD and the subgroup pneumonia–IPD, associated with selected occupations and exposures in the year preceding the index date.ResultsWelders manifested increased risk of IPD (OR 2.99, 95% CI 2.09 to 4.30). Occupational exposures to fumes and silica dust were associated with elevated odds of IPD (OR 1.11, 95% CI 1.01 to 1.21 and OR 1.33, 95% CI 1.11 to 1.58, respectively). Risk associated with IPD with pneumonia followed a similar pattern with the highest occupational odds observed among welders and among silica dust exposed.ConclusionWork specifically as a welder, but also occupational exposures more broadly, increase the odds for IPD. Welders, and potentially others with relevant exposures, should be offered pneumococcal vaccination.

Increased MET activity is linked with poor prognosis and outcome in several human cancers currently lacking targeted therapies. Here, we report on the characterization of Sym015, an antibody mixture composed of two humanized IgG1 antibodies against nonoverlapping epitopes of MET. Sym015 was selected by high-throughput screening searching for antibody mixtures with superior growth-inhibitory activity against MET-dependent cell lines. Synergistic inhibitory activity of the antibodies comprising Sym015 was observed in several cancer cell lines harboring amplified MET locus and was confirmed in vivo. Sym015 was found to exert its activity via multiple mechanisms. It disrupted interaction of MET with the HGF ligand and prompted activity-independent internalization and degradation of the receptor. In addition, Sym015 induced high levels of CDC and ADCC in vitro. The importance of these effector functions was confirmed in vivo using an Fc-effector function–attenuated version of Sym015. The enhanced effect of the two antibodies in Sym015 on both MET degradation and CDC and ADCC is predicted to render Sym015 superior to single antibodies targeting MET. Our results demonstrate strong potential for use of Sym015 as a therapeutic antibody mixture for treatment of MET-driven tumors. Sym015 is currently being tested in a phase I dose escalation clinical trial (NCT02648724). Mol Cancer Ther; 16(12); 2780–91. ©2017 AACR.

Purpose: Activation of the receptor tyrosine kinase MET is associated with poor clinical outcome in certain cancers. To target MET more effectively, we developed an antagonistic antibody mixture, Sym015, consisting of two humanized mAbs directed against nonoverlapping epitopes of MET. Experimental Design/Results: We screened a large panel of well-annotated human cancer cell lines and identified a subset with highly elevated MET expression. In particular, cell lines of lung cancer and gastric cancer origin demonstrated high MET expression and activation, and Sym015 triggered degradation of MET and significantly inhibited growth of these cell lines. Next, we tested Sym015 in patient- and cell line–derived xenograft models with high MET expression and/or MET exon 14 skipping alterations, and in models harboring MET amplification as a mechanism of resistance to EGFR-targeting agents. Sym015 effectively inhibited tumor growth in all these models and was superior to an analogue of emibetuzumab, a monoclonal IgG4 antibody against MET currently in clinical development. Sym015 also induced antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggesting that secondary effector functions contribute to the efficacy of Sym015. Retrospectively, all responsive, high MET-expressing models were scored as highly MET-amplified by in situ hybridization, pointing to MET amplification as a predictive biomarker for efficacy. Preclinical toxicology studies in monkeys showed that Sym015 was well tolerated, with a pharmacokinetic profile supporting administration of Sym015 every second or third week in humans. Conclusions: The preclinical efficacy and safety data provide a clear rationale for the ongoing clinical studies of Sym015 in patients with MET-amplified tumors. Clin Cancer Res; 23(19); 5923–35. ©2017 AACR.

Occupational hazards exist in the processing of seafood both in land-based facilities as well as on board vessels. Recent findings on occupational injury and respiratory health risks among seafood processing workers were presented and discussed at the IFISH5 conference. Particular emphasis was put on the challenges that im/migrant workers encounter, the greater risks onboard factory vessels, especially where processing machinery are retrofitted to older vessels not primarily designed for this purpose, and the difficulties in assessing and preventing bioaerosol exposures and associated respiratory health risks despite recent advances in characterising agents responsible for allergic and non-allergic reactions. Based on appraisal of existing knowledge in the published literature and new findings presented at the conference, recommendations for immediate actions as well as for future research have been proposed. Among these include the importance of improving extraction ventilation systems, optimising machinery performance, enclosure of bioaerosol sources, improved work organization, and making special efforts to identify and support the needs of im/migrant workers to ensure they also benefit from such improvements. There is a need for studies that incorporate longitudinal study designs, have improved exposure and diagnostic methods, and that address seafood processing in countries with high seafood processing activities such as Asia and those that involve im/migrant workers worldwide. The medical and scientific community has an important role to play in prevention but cannot do this in isolation and should cooperate closely with hygienists, engineers, and national and international agencies to obtain better health outcomes for workers in the seafood industry.

Background The CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile. Methods ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8+ T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry. Results ATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8+ T cells. Conclusions By targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467). Electronic supplementary material The online version of this article (10.1186/s40425-019-0570-8) contains supplementary material, which is available to authorized users.

Background: Occupational exposures to metal fumes and inorganic dust have been associated with increased pneumonia risk, but the risk of invasive pneumococcal disease (IPD) has not been characterized previously. Methods: We studied 4 438 cases 20 to 65 years of age from a Swedish national registry of invasive infection caused by Streptococcus pneumoniae. The case index date was the day the infection was diagnosed. Six controls for each case, matched for gender and age, were selected from the Swedish population registry. Each control was assigned the index date of their corresponding case to define the study observation period. We linked cases and controls to the Swedish registries for socioeconomic status (SES), occupational history, hospital discharge and mortality. We applied a job-exposure matrix (JEM) to characterize occupational exposures. We used conditional logistic analyses, adjusted for co-morbidities and SES, to estimate the odds ratio (OR) of IPD and the subgroup with pneumonia, associated with selected occupations and exposures the year before index date. Results: Welders manifested increased risk of IPD (OR 2.99, 95% confidence interval (CI) 2.09 - 4.30). Occupations likely to have exposure to fumes based on a JEM-assignment were also associated with elevated odds of IPD (OR 2.40, 95% CI 1.72-3.36). Risk associated with IPD with pneumonia followed a similar pattern with the highest occupational odds observed among welders (OR 3.28, 95% CI 2.22-4.84). Conclusions: Work as welder and occupational exposure to fumes increase the odds for IPD. Welders and potentially others with relevant exposures should be offered pneumococcal vaccination. Funding Statement: Swedish Heart and Lung Foundation, Swedish Council for Working Life, Health, and Welfare (FORTE) and by grants from the Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement, 74570. Declaration of Interests: All authors have completed the Unified Competing Interest form at www.icmje/coi_disclosure.pdf (available in request from the corresponding author): KT, IQ, OA, NM, RN, ADH, PDB and LS do not have any conflict of interest to report. Ethical Approval Statement: The Gothenburg Committee of Ethics approved the study (Dnr 729-16).

Objective: Hairdressers have an increased risk for airway symptoms especially when using hair-bleaching powder containing persulfate. To minimize exposure, dust-free bleaching powder (DFP) has been made available. We studied the effects of regular powder (RP) or DFP on the airway symptoms of hairdressers with hair-bleaching associated rhinitis. Methods: Twelve hairdressers each performed three hair-bleachings on a wig in an exposure chamber. Half of the subjects used RP and half used DFP. Exposure to persulfate and ammonia was measured. Before and after each bleaching, the participants stated their degree of airway symptoms on a visual analogue scale. Nasal lavage and blood were sampled before exposure, after the last bleaching, and in the morning after exposure to measure inflammatory markers. Results: Exposure to persulfate was higher when using RP compared to DFP, 22 (11-55) vs. 12 (8-13) μg/m3; median (min-max). Exposure to ammonia did not differ between the groups. Both groups reported an increase in asthma-like symptoms and this increase was significant. Neutrophils, lymphocytes, and monocytes increased after exposure in both groups; monocytes decreased the day after. In nasal lavage, IL-8 was increased the morning after for both types of powder, and the increase was significant in the total group. IL-6 increased immediately after exposure and the day after only in the group using RP. Conclusions: Although DFP powder emits lower levels of persulfate, effects are still elicited in symptomatic hairdressers.

Patients with airway symptoms working in metal working industries are increasing, despite efforts to improve the environmental air surrounding the machines. Our aim was to analyse the amount of endotoxin in size-separated airborne particles of metal working fluid (MWF) aerosol, by using the personal sampler Sioutas cascade impactor, to compare filter types, and to compare the concentration of airborne endotoxin to that of the corresponding MWFs. In a pilot field study, aerosols were collected in two separate machine halls on totally 10 occasions, using glass fibre and polytetrafluoroethylene (PTFE) filters in parallel at each station. Airborne endotoxin was distributed over all size fractions. While a major part was found in the largest size fraction (72%, 2.5-10 µm), up to 8% of the airborne endotoxin was detected in the smallest size fraction (

// Carsten H. Nielsen 1,2,* , Mette M. Jensen 1,2,* , Lotte K. Kristensen 1,2 , Anna Dahlman 3 , Camilla Frohlich 3 , Helle J. Jacobsen 3 , Thomas T. Poulsen 3 , Johan Lantto 3 , Ivan D. Horak 3 , Michael Kragh 3 and Andreas Kjaer 1,2 1 Minerva Imaging ApS, Copenhagen, Denmark 2 Department of Clinical Physiology, Nuclear Medicine and PET and Cluster for Molecular Imaging, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark 3 Symphogen A/S, Ballerup, Denmark * These authors have contributed equally to this work Correspondence to: Andreas Kjaer, email: // Keywords : PET/CT, FDG and FLT, HER family, antibody therapy, pancreatic cancer Received : July 28, 2015 Accepted : September 24, 2015 Published : October 09, 2015 Abstract Purpose: Overexpression of the human epidermal growth factor receptor (HER) family and their ligands plays an important role in many cancers. Targeting multiple members of the HER family simultaneously may increase the therapeutic efficacy. Here, we report the ability to image the therapeutic response obtained by targeting HER family members individually or simultaneously using the novel monoclonal antibody (mAb) mixture Pan-HER. Experimental design and results: Mice with subcutaneous BxPC-3 pancreatic adenocarcinomas were divided into five groups receiving vehicle or mAb mixtures directed against either EGFR (HER1), HER2, HER3 or all three receptors combined by Pan-HER. Small animal positron emission tomography/computed tomography (PET/CT) with 2’-deoxy-2’-[ 18 F]fluoro-D-glucose (FDG) and 3’-deoxy-3’-[ 18 F]fluorothymidine (FLT) was performed at baseline and at day 1 or 2 after initiation of therapy. Changes in tumor uptake of tracers were quantified and compared to reduction in tumor size. Imaging results were further validated by immunohistochemistry and qPCR. Mean FDG and FLT uptake in the Pan-HER treated group decreased by 19±4.3% and 24±3.1%, respectively. The early change in FDG and FLT uptake correlated with tumor growth at day 23 relative to day 0. Ex vivo molecular analyses of markers associated with the mechanisms of FDG and FLT uptake confirmed the in vivo imaging results. Conclusions: Taken together, the study supports the use of FDG and FLT as imaging biomarkers of early response to Pan-HER therapy. FDG and FLT PET/CT imaging should be considered as imaging biomarkers in clinical evaluation of the Pan-HER mAb mixture.

HER2 plays an important role in the development and maintenance of the malignant phenotype of several human cancers. As such, it is a frequently pursued therapeutic target and two antibodies targeting HER2 have been clinically approved, trastuzumab and pertuzumab. It has been suggested that optimal inhibition of HER2 is achieved when utilizing two or more antibodies targeting nonoverlapping epitopes. Superior clinical activity of the trastuzumab plus pertuzumab combination in metastatic breast cancer supports this hypothesis. Because trastuzumab and pertuzumab were not codeveloped, there may be potential for further optimizing HER2 targeting. The study herein evaluated functional activity of anti-HER2 antibody combinations identifying optimal epitope combinations that provide efficacious HER2 inhibition. High-affinity antibodies to all four extracellular domains on HER2 were identified and tested for ability to inhibit growth of different HER2-dependent tumor cell lines. An antibody mixture targeting three HER2 subdomains proved to be superior to trastuzumab, pertuzumab, or a combination in vitro and to trastuzumab in two in vivo models. Specifically, the tripartite antibody mixture induced efficient HER2 internalization and degradation demonstrating increased sensitivity in cell lines with HER2 amplification and high EGFR levels. When compared with individual and clinically approved mAbs, the synergistic tripartite antibody targeting HER2 subdomains I, II, and IV demonstrates superior anticancer activity. Mol Cancer Ther; 14(3); 669–80. ©2015 AACR.

WORK-RELATED ASTHMA comprises occupational asthma (OA) that is caused by conditions at work and work-exacerbated asthma (WEA), in which existing asthma is made worse by workplace conditions.1 Work-related asthma is common, with work accounting for an average 16.9% of all new adult-onset asthma cases,2 and with WEA detected in an estimated 21.5% of working adults with asthma.3 While substantial costs and lost work time are associated with both WEA and OA,4,5 WEA has received less attention in terms of research and prevention efforts.1 WEA is potentially preventable by minimizing harmful workplace exposures,3 but a better understanding of which agents pose a risk is needed to guide prevention efforts. While surveillance in the United States indicates that WEA is frequently caused by irritants, low molecular weight (e.g., acids, bases, aldehydes) and high molecular weight (e.g., latex) agents are also implicated.6 The absence of denominators (i.e., the number of workers exposed) prohibits using surveillance data to estimate agent-specific risk. We used data from a previous study to identify occupational risk factors for exacerbation of asthma.7 In previous analyses of data from this study, we determined WEA status on a case-by-case basis. However, for the current investigation, we modeled severe exacerbation of asthma and determined which occupational exposures were associated with it, while simultaneously controlling for potential confounders. We assessed occupational exposures using a new asthma-specific job-exposure matrix (JEM).8

The ability to induce potent anti-tumor activity by stimulating 4-1BB (CD137), a key co-stimulatory receptor, makes 4-1BB an attractive immunotherapeutic target. However, a clinically tested, 4-1BB targeting monospecific antibody has been hampered by dose-limiting hepatic toxicities. To improve safety of 4-1BB targeting therapies we have developed a 4-1BB x 5T4 bispecific antibody designed to direct tumor-specific T cell responses to the tumor by stimulating 4-1BB only when co-engaged with 5T4, a tumor-associated antigen. The preclinical dataset presented here provides an overview of the mechanism of action and the efficacy and safety profile of ALG.APV-527, supporting its advancement into the clinic. ALG.APV-527 was built using the ADAPTIR™ platform with binding domains from the ALLIGATOR-GOLD® human scFv library. Its 5T4-dependent agonistic function was assessed using primary CD8+ T cells or NK cells in the presence of 5T4-expressing cells. Secretion of IFN-γ or granzyme B was measured at 72 hrs using ELISA. To measure proliferation, PBMCs were labelled with Cell Trace™ and gated CD8+ T cells were analyzed using flow cytometry. For tumor inhibition studies, the human 5T4-expressing colon carcinoma HCT116 xenograft model was used. 5T4 expression was evaluated in normal human tissues and different human tumors by immunohistochemistry. The preclinical safety profile of ALG.APV-527 was evaluated in a single and repeated dose, dose-range finding toxicology study in non-human primates (NHP). The study design included all the standard repeated dose toxicity parameters and in addition, pharmacokinetics, immunogenicity, and pharmacodynamic end-points. ALG.APV-527 induces a 5T4-dependent increase in IFN-γ and granzyme B production and enhances proliferation of T cells and NK cells. Furthermore, ALG.APV-527 inhibits tumor growth in a human 5T4-expressing colon carcinoma xenograft model. 5T4 is overexpressed in multiple solid tumors, potentially directing the activity of 4-1BB induced by ALG.APV-527 to 5T4-expressing tumors, improving the risk/benefit profile. Four doses (administered once weekly) did not cause any adverse events in the NHP toxicity study. In conclusion, ALG.APV-527 induces potent CD8+ T cell and NK co-stimulation but only in the presence of 5T4. Based on its efficacy and preclinical safety profile, ALG.APV-527 is a promising anti-cancer therapeutic for the treatment of multiple 5T4-expressing solid tumors. Citation Format: Anna Dahlman, Michelle Nelson, Jeannette Bannink, Starrla Johnson, Doreen Werchau, Anneli Nilsson, Lill Ljung, Gabriele Blahnik-Fagan, Robert Bader, Adnan Deronic, Peter Ellmark, Maria Askmyr, Gabriela Hernandez-Hoyos, Cathy McMahan, Sara Fritzell. Preclinical safety and efficacy of a tumor-directed T cell activating 4-1BB x 5T4 ADAPTIR™ bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2380.

TPS2653 Background: ATOR-1015 is a human bispecific IgG1 antibody targeting cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and the tumor necrosis factor receptor superfamily member 4, OX40 (also known as CD134). Both in vitro and in vivo, ATOR-1015 induces activation of cytotoxic T cells and depletion of regulatory T cells (1). In syngeneic tumor models, using human OX40 transgenic mice cross-reacting with both targets, ATOR-1015 is demonstrated to localize to the tumor. Further, the effects of ATOR-1015 are shown to occur in the tumor area and not in the spleen (1). Treatment with ATOR-1015 also reduces tumor growth and improves survival in several tumor models in mice, including bladder, colon and pancreatic cancer (1). The non-clinical safety profile and the pharmacokinetics were established in cynomolgus monkeys and the data were used for the dosing schedule. Methods: This is a multicenter, open-label, dose escalation study enrolling patients with advanced and/or refractory solid malignancies (NCT03782467). The primary objective of the study is to determine the maximum tolerated dose (MTD) or the recommended phase 2 dose (RP2D) and to establish the safety profile of ATOR-1015. ATOR-1015 is administered intravenously biweekly as a single agent until confirmed progressive disease, unacceptable toxicity or withdrawal of consent. The study will start with single patient cohorts until grade 2 toxicities are observed, thereafter the study follows a modified 3+3 design. At the MTD/RP2D, or a lower dose, up to 20 patients are planned for additional safety and efficacy evaluation. Study enrollment was initiated in January 2019. A total of up to 53 patients are estimated to be enrolled in the study. (1) Månsson Kvarnhammar et al. Journal for ImmunoTherapy of Cancer 2018; 6(Suppl 1):115. Abstract P683. Clinical trial information: NCT03782467.

4-1BB (CD137) is an inducible co-stimulatory receptor expressed on activated T-cells and natural killer (NK) cells. Activation of 4-1BB induces proliferation, cytokine production, cytotoxic function and survival of T and NK cells. Most importantly, it has been shown to result in tumor eradication and long-term tumor immunity in numerous experimental tumor models. Thus, 4-1BB agonists may have great potential in cancer immunotherapy. However, the 4-1BB antibodies in clinical development are associated with toxic side effects and/or poor efficacy, potentially limiting their clinical use. ATOR-1017 is an anti-4-1BB IgG4 antibody designed to overcome the limitations observed with other 4-1BB antibodies. It binds to a distinct 4-1BB epitope located in domain 2 and blocks 4-1BB ligand binding. Furthermore, its agonistic effect is dependent on crosslinking by Fcγ Receptor (FcγR) I and IIb. A potentially advantageous safety profile was supported by the lack of observed immunotoxicity in a cytokine release assay based on human PBMC using a panel of proinflammatory cytokines as readout. Furthermore, expression profiling of human tumor and normal tissue demonstrates that 4-1BB and FcγRs are highly expressed in the tumor environment. In contrast, co-expression of 4-1BB and FcγRs in non-tumor tissues, such as the liver, is low. Therefore, ATOR-1017 is expected to result in tumor-localized immune activation, thereby avoiding systemic adverse events, including hepatotoxicity, which has been observed by another, FcγR-independent 4-1BB antibody. Finally, to further investigate the safety profile of ATOR-1017, a pilot toxicology study was conducted. ATOR-1017 binds with similar affinity of approximately 0.2 nM to human and cynomolgus monkey 4-1BB, whereas no detectable binding to mouse 4-1BB is observed. Thus, cynomolgus monkeys were administered four weekly doses of 5, 15 or 50 mg/kg ATOR-1017. No clinical signs were observed at any dose, indicating a favorable safety profile. In conclusion, ATOR-1017 was designed to be highly active in the tumor environment, and to have minimal systemic effects, thereby enabling a superior safety/efficacy profile. This is supported by a preclinical data package, demonstrating dependency on FcγR-mediated crosslinking, a clean profile in cytokine release assay, 4-1BB and FcγRs co-expression in the tumor and no clinical adverse events observed in a repeat dosing pilot toxicology study. ATOR-1017 is in preclinical development with initiation of phase I anticipated in 2019. Citation Format: Eva Dahlén, Anna Rosén, Karin Barchan, Anna Dahlman, Peter Ellmark, Tina Furebring, Karin Enell Smith. ATOR-1017: A 4-1BB antibody designed for superior safety/efficacy profile in cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A183.

OBJECTIVES: • To investigate whether cysteine-rich secretory protein 3 (CRISP-3) and/or β-microseminoprotein (β-MSP) expression in diagnostic prostate needle biopsies have predictive value for prostate cancer (PC) on radical prostatecomy (RP). • To evaluate their potential clinical implementation in a preoperative setting. PATIENTS AND METHODS: • In total, 174 participants from the European Randomized Study of Screening for Prostate Cancer, Rotterdam section, treated by RP for PC were included in the present study. • CRISP-3 and β-MSP immunohistochemistry was performed on corresponding diagnostic needle biopsies. • Outcome was correlated with clinicopathological parameters (prostate-specific-antigen, PSA; number of positive biopsies; Gleason score, GS; pT-stage; surgical margins at RP) and significant PC at RP (pT3/4, or GS > 6, or tumour volume ≥0.5 mL) in the total cohort (n= 174) and in a subgroup with low-risk features at biopsy (PSA ≤ 10 ng/ml, cT ≤ 2, PSA density

BACKGROUND: Signal transducer and activator of transcription 3 (Stat3) is constitutively active (phosphorylated) in several forms of cancer, including prostate cancer (PCa). Stat3 signaling may be an interesting target for cancer therapy since inhibition of this pathway mediates growth inhibition and apoptosis of these cells. In this study we investigated the in vitro and in vivo effects of the fungal metabolite galiellalactone, a direct inhibitor of Stat3, on PCa cells. METHODS: The human PCa cell lines DU145, PC-3, and LNCaP were used. Nude mice with subcutaneous PCa cell xenografts were subjected to daily intraperitoneal injections of galiellalactone for 3 weeks. The effect of galiellalactone on the induction of apoptosis of cultured PCa cells was investigated by Western blot analysis, immunocytochemistry, and annexin V staining. Effects of galiellalactone on Stat3 signaling were investigated by a luciferase reporter gene assay. Expression of Stat3 associated proteins and mRNA was investigated by Western blot and real-time quantitative PCR analysis. RESULTS: Galiellalactone induced apoptosis of p-Stat3 positive PCa cells (androgen-insensitive DU145 and PC-3) but not in cells lacking p-Stat3 (androgen-sensitive LNCaP). Galiellalactone inhibited Stat3-mediated luciferase activity (IC(50) approximately 5 microM) and reduced the expression of Bcl-2, Bcl-x(L), c-myc, and cyclin D1. Furthermore, galiellalactone significantly suppressed DU145 xenograft growth in vivo (42% growth reduction; P < 0.002) and reduced the relative mRNA expression of Bcl-x(L) and Mcl-1. CONCLUSIONS: Galiellalactone induced growth inhibition and apoptosis in androgen-insensitive PCa cells expressing p-Stat3. We suggest that galiellalactone is a potential anti-tumor lead against hormone-refractory PCa with constitutively active Stat3. Prostate 68: 269-280, 2008. (c) 2007 Wiley-Liss, Inc.

Background Exposure to allergen may induce a modified T H 2 response characterized by high IgG 4 levels, absence of IgE sensitization, and a decreased risk of allergic respiratory symptoms. Objective To assess the association of IgG 4 level with allergic respiratory symptoms in a community-based sample of adults. Methods Information on exposure to cats, respiratory symptoms, and mattress allergen levels was obtained from 2780 adults. Levels of cat and house dust mite (HDM) specific IgE, IgG, and IgG 4 were measured. The association of exposure to allergen with IgG 4 and of IgG 4 with symptoms was assessed. Results Geometric mean (GM) cat specific IgG and IgG 4 was higher in subjects who had a cat that was allowed in the bedroom than in subjects without a cat (adjusted ratio of GM IgG 4 , 1.41; 95% CI, 1.25-1.57). Levels of HDM specific IgG and IgG 4 were similar in subjects with undetectable and high (>20.22 μg/g) mattress Der 1 levels (adjusted ratio of GM IgG 4 , 1.02; 95% CI, 0.89-1.17). There was no evidence that high cat or HDM specific IgG 4 levels were associated with less IgE sensitization or with fewer symptoms. Conclusion In this community-based sample of adults, high IgG 4 levels to cat or HDM were not associated with a lower risk of allergic respiratory symptoms. Clinical implications In adults, high cat allergen exposure does not protect against respiratory symptoms.

Background: Several studies in European homes have described allergen levels from the house dust mite species Dermatophagoides pteronyssinus and to a lesser extent Dermatophagoides farinae, but geographic comparisons of exposure levels and risk factors have been hampered by a lack of standardized methods. Objective: To study the distribution and determinants of the major house dust mite allergens Der p 1 and Der f 1 in 10 European countries using a common protocol. Methods: During home visits with 3580 participants of the European Community Respiratory Health Survey II from 22 study centers, mattress dust was sampled and analyzed for Der p 1, Der f 1, and Der 2 allergen. Information on housing characteristics was obtained by both observations and interview. Results: Der 1 and Der 2 allergens were detectable (≥0.1 μg/g) in 68% and 53% of the samples, respectively. Large differences in allergen levels between study centers were observed, and geographic patterns for Der p 1 and Der f 1 were different. Low winter temperatures reduced Der p 1 rather than Der f 1. Important risk factors for high allergen levels included an older mattress, a lower floor level of the bedroom, limited ventilation of the bedroom, and dampness for Der p 1 but not for Der f 1. Conclusion: There are large qualitative and quantitative differences of house dust mite allergen levels in Europe, which can partly be explained by geographic and housing characteristics. Clinical implications: Mite allergen exposure may be reduced by replacing the mattress regularly and increasing ventilation of the bedroom, particularly in winter. © 2006 American Academy of Allergy, Asthma and Immunology.

An association between indoor dampness and respiratory symptoms has been reported, but dampness as a risk factor for the onset or remission of respiratory symptoms and asthma is not well documented.This follow up study included 16 190 subjects from Iceland, Norway, Sweden, Denmark, and Estonia who had participated in the European Community Respiratory Health Survey (ECRHS I). Eight years later the same subjects answered a postal questionnaire that included questions on respiratory symptoms and indicators of indoor dampness.Subjects living in damp housing (18%) had a significantly (p0.001) higher prevalence of wheeze (19.1% v 26.0%), nocturnal breathlessness (4.4% v 8.4%), nocturnal cough (27.2% v 36.5%), productive cough (16.6% v 22.3%) and asthma (6.0% v 7.7%). These associations remained significant after adjusting for possible confounders. Indoor dampness was a risk factor for onset of respiratory symptoms but not for asthma onset in the longitudinal analysis (OR 1.13, 95% CI 0.92 to 1.40). Remission of nocturnal symptoms was less common in damp homes (OR 0.84, 95% CI 0.73 to 0.97).Subjects living in damp housing had a higher prevalence of respiratory symptoms and asthma. Onset of respiratory symptoms was more common and remission of nocturnal respiratory symptoms was less common in subjects living in damp housing.

Purpose: Accumulating evidence indicates a high degree of plasticity and compensatory signaling within the human epidermal growth factor receptor (HER) family, leading to resistance upon therapeutic intervention with HER family members. Experimental Design/Results: We have generated Pan-HER, a mixture of six antibodies targeting each of the HER family members EGFR, HER2, and HER3 with synergistic pairs of antibodies, which simultaneously remove all three targets, thereby preventing compensatory tumor promoting mechanisms within the HER family. Pan-HER induces potent growth inhibition in a range of cancer cell lines and xenograft models, including cell lines with acquired resistance to therapeutic antibodies. Pan-HER is also highly efficacious in the presence of HER family ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. All three target specificities contribute to the enhanced efficacy, demonstrating a distinct benefit of combined HER family targeting when compared with single-receptor targeting. Conclusions: Our data show that simultaneous targeting of three receptors provides broader efficacy than targeting a single receptor or any combination of two receptors in the HER family, especially in the presence of HER family ligands. Pan-HER represents a novel strategy to deal with primary and acquired resistance due to tumor heterogeneity and plasticity in terms of HER family dependency and as such may be a viable alternative in the clinic. Clin Cancer Res; 21(18); 4110–22. ©2015 AACR. See related commentary by Yarden and Sela, p. 4030