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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 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.