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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.

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

Purpose: Approved anti-EGFR antibodies cetuximab and panitumumab provide significant clinical benefit in patients with metastatic colorectal cancer (MCRC). However, patients ultimately develop disease progression, often driven by acquisition of mutations in the extracellular domain (ECD) of EGFR. Sym004 is a novel 1:1 mixture of two nonoverlapping anti-EGFR mAbs that recently showed promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations.Experimental Design: Functional studies were performed to assess drug–receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab, and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored EGFR mutation in the post-cetuximab tumor sample.Results: Contrary to cetuximab and panitumumab, Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring mutations in EGFR maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As proof-of-principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy.Conclusions: Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Clin Cancer Res; 22(13); 3260–7. ©2016 AACR.

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

Figure S1: EGFR mutants binding to CTX, Sym004, PNM Figure S2: Clonogenic assay in EGFR mutants with CTX, Sym004, PNM

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

Supplementary Figure 1 from Sym004: A Novel Synergistic Anti–Epidermal Growth Factor Receptor Antibody Mixture with Superior Anticancer Efficacy

Epidermal growth factor receptor (EGFR) is a validated therapeutic target in cancer and EGFR antagonists with greater effectiveness than existing clinical agents remain of interest. Here, we report a novel approach based on Sym004, a mixture of two anti-EGFR monoclonal antibodies directed against distinct nonoverlapping epitopes in EGFR extracellular domain III. Like anti-EGFR monoclonal antibodies in current clinical use, Sym004 inhibits cancer cell growth and survival by blocking ligand-binding receptor activation and phosphorylation and downstream receptor signaling. However, unlike the other antibodies, Sym004 induces rapid and efficient removal of the receptor from the cancer cell surface by triggering EGFR internalization and degradation. Compared with reference anti-EGFR monoclonal antibodies, Sym004 exhibited more pronounced growth inhibition in vitro and superior efficacy in vivo. Together, these findings illustrate a strategy to target EGFR more effectively than existing clinical antibodies. Cancer Res; 70(2); 588–97

Supplementary Figure 4 from Sym004: A Novel Synergistic Anti–Epidermal Growth Factor Receptor Antibody Mixture with Superior Anticancer Efficacy

Supplementary Figure and Table Legends from Sym004: A Novel Synergistic Anti–Epidermal Growth Factor Receptor Antibody Mixture with Superior Anticancer Efficacy

Supplementary Figure 3 from Sym004: A Novel Synergistic Anti–Epidermal Growth Factor Receptor Antibody Mixture with Superior Anticancer Efficacy

Supplementary Table 1 from Sym004: A Novel Synergistic Anti–Epidermal Growth Factor Receptor Antibody Mixture with Superior Anticancer Efficacy

Supplementary Figure 2 from Sym004: A Novel Synergistic Anti–Epidermal Growth Factor Receptor Antibody Mixture with Superior Anticancer Efficacy

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.

Discovery of therapeutic antibodies is a field of intense development, where immunization of rodents remains a major source of antibody candidates. However, high orthologue protein sequence homology between human and rodent species disfavors generation of antibodies against functionally conserved binding epitopes. Chickens are phylogenetically distant from mammals. Since chickens generate antibodies from a restricted set of germline genes, the possibility of adapting the Symplex antibody discovery platform to chicken immunoglobulin genes and combining it with high-throughput humanization of antibody frameworks by “mass complementarity-determining region grafting” was explored. Hence, wild type chickens were immunized with an immune checkpoint inhibitor programmed cell death 1 (PD1) antigen, and a repertoire of 144 antibodies was generated. The PD1 antibody repertoire was successfully humanized, and we found that most humanized antibodies retained affinity largely similar to that of the parental chicken antibodies. The lead antibody Sym021 blocked PD-L1 and PD-L2 ligand binding, resulting in elevated T-cell cytokine production in vitro. Detailed epitope mapping showed that the epitope recognized by Sym021 was unique compared to the clinically approved PD1 antibodies pembrolizumab and nivolumab. Moreover, Sym021 bound human PD1 with a stronger affinity (30 pM) compared to nivolumab and pembrolizumab, while also cross-reacting with cynomolgus and mouse PD1. This enabled direct testing of Sym021 in the syngeneic mouse in vivo cancer models and evaluation of preclinical toxicology in cynomolgus monkeys. Preclinical in vivo evaluation in various murine and human tumor models demonstrated a pronounced anti-tumor effect of Sym021, supporting its current evaluation in a Phase 1 clinical trial. Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CD, cluster of differentiation; CDC, complement-dependent cytotoxicity; CDR, complementarity determining region; DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorting; FR, framework region; GM-CSF, granulocyte-macrophage colony-stimulating factor; HRP, horseradish peroxidase; IgG, immunoglobulin G; IL, interleukin; IFN, interferon; mAb, monoclonal antibody; MLR, mixed lymphocyte reaction; NK, natural killer; PBMC, peripheral blood mono-nuclear cell; PD1, programmed cell death 1; PDL1, programmed cell death ligand 1; RT-PCR, reverse transcription polymerase chain reaction; SEB, Staphylococcus Enterotoxin B; SPR, surface Plasmon Resonance; VL, variable part of light chain; VH, variable part of heavy chain

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

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.

Purpose: Approved anti-EGFR antibodies cetuximab and panitumumab provide significant clinical benefit in patients with metastatic colorectal cancer (MCRC). However, patients ultimately develop disease progression, often driven by acquisition of mutations in the extracellular domain (ECD) of EGFR. Sym004 is a novel 1:1 mixture of two nonoverlapping anti-EGFR mAbs that recently showed promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. Experimental Design: Functional studies were performed to assess drug–receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab, and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored EGFR mutation in the post-cetuximab tumor sample. Results: Contrary to cetuximab and panitumumab, Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring mutations in EGFR maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As proof-of-principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. Conclusions: Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Clin Cancer Res; 22(13); 3260–7. ©2016 AACR.

The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.

Inhibition of immunologic checkpoints like Programmed Cell Death 1 (PD-1) has shown clinical efficacy in a broad range of cancers by improving or restoring T-cell activity. Anti-PD-1 antibodies show great promise in treating cancer malignances when administered alone or in combination with other immune activating approaches. However, high protein sequence identity between human and mammalian species used for antibody generation often disfavor generation of antibodies against functionally conserved epitopes, or prevents isolating antibodies cross reacting with ortholog species used for evaluating potential toxicity. Chickens are phylogenetically distant from mammals and are better at generating antibodies against epitopes that are conserved in mammals. Because chickens generate antibodies from a very restricted set of V-gene germline genes that are diversified by “gene conversion”, we envisioned that high throughput humanization of antibody frameworks was achievable by “mass CDR grafting” after recovering antibodies by immunization and B-cell cloning. Wild type chickens were immunized with PD-1 antigen, and a repertoire of 120 antibodies was generated with Symplex™ technology, by combining single B-cell FACS sorting and high throughput RT-PCR cloning of cognate VH and VL chains. The isolated PD-1 repertoire was cloned with an inert Fc backbone and humanized by a combination of in silico CDR grafting and gene synthesis. Humanized antibodies were expressed and screened for retained binding affinity and functionality in T-cell based assays. We successfully generated a humanized PD-1 antibody repertoire and found that most antibodies retained affinity and functionality similar to that of parental chicken antibodies. Furthermore, the antibody repertoire displayed broad binding epitope coverage on PD-1, often with strong pM affinity, and showed biophysical properties acceptable for drug development. Our lead antibody Sym021 blocked PD-L1 and PD-L2 ligand binding and downstream PD-1 signaling, resulting in elevated T-cell cytokine production in vitro. Moreover, Sym021 bound human PD-1 with much stronger affinity of 30 pM compared to clinical PD-1 mAbs nivolumab and pembrolizumab, while also cross reacting to cynomolgous and mouse PD-1. This enabled direct testing of Sym021 in syngenic mouse in vivo models and evaluation of preclinical toxicology in cynomolgus monkeys. Detailed epitope mapping showed that the epitope recognized by Sym021 was unique compared to clinical antibodies pembrolizumab and nivolumab. These results supported entry of PD-1 targeting Sym021 into clinical trials. Citation Format: Torben Gjetting, Monika Gad, Camilla Fröhlich, Maria C. Melander, Gunther Galler, Johan Lantto, Thomas Bouquin, Ivan D. Horak, Michael Kragh, Mikkel W. Pedersen, Klaus Koefoed. Characterization of the first chicken-derived anti-PD-1 clinical stage antibody with a unique epitope and promising anticancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3822.

Immunotherapy has become a major focus of research in oncology and blockade of immune checkpoints such as cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) and programmed cell death protein 1 (PD1) has been some of the most successful immunotherapies. The next wave of immunomodulatory targets that are being explored for cancer therapy include T cell immunoglobulin and mucin domain protein 3 (TIM3). TIM3 is constitutively expressed on cells of myeloid origin whereas the TIM3 expression is induced on T-cells upon activation. The exact function of TIM3 on the different immune cells is not clear and may be context dependent suggesting that TIM3 is not a classical immune check-point. Sym023 is a human anti-TIM3 antibody, which binds human TIM3 and cross-reacts with cynomolgus monkey TIM3. Sym023 blocks binding of phosphatidyl serine but not galectin 9 and stimulates T-cell proliferation in mixed lymphocyte reactions and tumor growth inhibition in vivo. Here, we present data demonstrating that ligation of TIM3 by Sym023 increase cytokine production and T cell proliferation in vitro through a novel mechanism of action. Citation Format: Trine Lindsted, Monika Gad, Michael V. Grandal, Camilla Frölich, Vikram K. Bhatia, Torben Gjetting, Johan Lantto, Ivan D. Horak, Michael Kragh, Klaus Koefoed, Mikkel W. Pedersen. Preclinical characterization of Sym023 a human anti-TIM3 antibody with a novel mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5629.

Importance Acquired resistance to anti-EGFR therapy (epidermal growth factor receptor) is frequently due toRASandEGFRextracellular domain (ECD) mutations in metastatic colorectal cancer (mCRC). Some anti-EGFR–refractory patients retain tumor EGFR dependency potentially targetable by agents such as Sym004, which is a mixture of 2 nonoverlapping monoclonal antibodies targeting EGFR. Objective To determine if continuous blockade of EGFR by Sym004 has survival benefit. Design, Setting, and Participants Multicenter, phase 2, randomized, clinical trial comparing 2 regimens of Sym004 with investigator’s choice from March 6, 2014, through October 15, 2015. Circulating tumor DNA (ctDNA) was analyzed for biomarker and tracking clonal dynamics during treatment. Participants had wild-typeKRASexon 2 mCRC refractory to standard chemotherapy and acquired resistance to anti-EGFR monoclonal antibodies. Interventions Participants were randomly assigned in a 1:1:1 ratio to Sym004, 12 mg/kg/wk (arm A), Sym004, 9 mg/kg loading dose followed by 6 mg/kg/wk (arm B), or investigator’s choice of treatment (arm C). Main Outcomes and Measures Overall survival (OS). Secondary end points included preplanned exploratory biomarker analysis in ctDNA. Results A total of 254 patients were randomized (intent-to-treat [ITT] population) (median age, 63 [range, 34-91] years; 63% male; n = 160). Median OS in the ITT population was 7.9 months (95% CI, 6.5-9.9 months), 10.3 months (95% CI, 9.0-12.9 months), and 9.6 months (95% CI, 8.3-12.2 months) for arms A, B, and C, respectively (hazard ratio [HR], 1.31; 95% CI, 0.92-1.87 for A vs C; and HR, 0.97; 95% CI, 0.68-1.40 for B vs C). The ctDNA revealed high intrapatient genomic heterogeneity following anti-EGFR therapy. Sym004 effectively targetedEGFRECD-mutated cancer cells, and a decrease inEGFRECD ctDNA occurred in Sym004-treated patients. However, this did not translate into clinical benefit in patients withEGFRECD mutations, likely owing to co-occurring resistance mechanisms. A subgroup of patients was defined by ctDNA (RAS/BRAF/EGFRECD-mutation negative) associated with improved OS in Sym004-treated patients in arm B compared with arm C (median OS, 12.8 and 7.3 months, respectively). Conclusions and Relevance Sym004 did not improve OS in an unselected population of patients with mCRC and acquired anti-EGFR resistance. A prospective clinical validation of Sym004 efficacy in a ctDNA molecularly defined subgroup of patients with refractory mCRC is warranted. Trial Registration clinicaltrialsregister.eu Identifier:2013-003829-29

Epidermal growth factor receptor (EGFR) is a validated therapeutic target in cancer and EGFR antagonists with greater effectiveness than existing clinical agents remain of interest. Here, we report a novel approach based on Sym004, a mixture of two anti-EGFR monoclonal antibodies directed against distinct nonoverlapping epitopes in EGFR extracellular domain III. Like anti-EGFR monoclonal antibodies in current clinical use, Sym004 inhibits cancer cell growth and survival by blocking ligand-binding receptor activation and phosphorylation and downstream receptor signaling. However, unlike the other antibodies, Sym004 induces rapid and efficient removal of the receptor from the cancer cell surface by triggering EGFR internalization and degradation. Compared with reference anti-EGFR monoclonal antibodies, Sym004 exhibited more pronounced growth inhibition in vitro and superior efficacy in vivo. Together, these findings illustrate a strategy to target EGFR more effectively than existing clinical antibodies. Cancer Res; 70(2); 588–97

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

The chemokine receptor CCR5 is the major coreceptor for infection by macrophage-tropic R5 HIV-1. A 32-bp deletion in the gene coding for CCR5 (CCR5Δ32) occurs with a frequency of 10% in the Caucasian population and results in a receptor protein that is truncated and not expressed at the cell surface. CCR5Δ32 homozygous individuals are apparently normal but resistant to infection with R5 HIV-1. In two individuals homozygous for CCR5Δ32, who had been repeatedly exposed to CCR5-expressing blood cells through sexual activity, we have identified antibodies to CCR5 that bound specifically to the surface of CCR5-expressing cell lines. Serum from these individuals, in contrast to serum from CCR5 +/+ individuals, competed with radiolabeled RANTES for binding to the CCR5 receptor and inhibited infection of peripheral blood mononuclear cells with R5, but not X4, primary isolates of HIV-1. The identified human antibodies to CCR5 define an alloantigen that may cause allograft rejection in a mismatch situation even in individuals with no history of blood transfusions or i.v. drug abuse.

The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti-CD5 Abs that engage secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) both as single mAbs and combinations of two mAbs against non-overlapping epitopes on human CD5. The results demonstrated that combinations of two mAbs significantly increased the level of CDC compared to the single mAbs, while no enhancement of ADCC was seen with anti-CD5 mAb combinations. High levels of CDC and ADCC correlated with low levels of Ab-induced CD5 internalization and degradation. Importantly, an anti-CD5 mAb combination enhanced CDC of CLL cells when combined with the anti-CD20 mAbs rituximab and ofatumumab as well as with the anti-CD52 mAb alemtuzumab. These results suggest that an anti-CD5 mAb combination inducing CDC and ADCC may be effective alone, in combination with mAbs against other targets or combined with chemotherapy for CLL and other CD5-expressing haematological or lymphoid malignancies.

Characterization of conformational and dynamic changes associated with protein interactions can be done by hydrogen/deuterium exchange mass spectrometry (HDX-MS) by comparing the deuterium uptake in the bound and unbound state of the proteins. Investigation of local hydrogen/deuterium exchange in heteromultimeric protein complexes poses a challenge for the method due to the increased complexity of the mixture of peptides originating from all interaction partners in the complex. Previously, interference of peptides from one interaction partner has been removed by immobilizing the intact protein on beads prior to the HDX-MS experiment. However, when studying protein complexes of more than two proteins, immobilization can possibly introduce steric limitations to the interactions. Here, we present a method based on the high affinity biotin-streptavidin interaction that allows selective capture of biotinylated proteins even under the extreme conditions for hydrogen/deuterium exchange quenching i.e. pH 2.5 and 0 °C. This biotin-streptavidin capture strategy allows hydrogen/deuterium exchange to occur in proteins in solution and enables characterization of specific proteins in heteromultimeric protein complexes without interference of peptides originating from other interaction partners in the complex. The biotin-streptavidin strategy has been successfully implemented in a model system with two recombinant monoclonal antibodies that target nonoverlapping epitopes on the human epidermal growth factor receptor (EGFR). We present a workflow for biotinylation and characterization of recombinant antibodies and demonstrate affinity capture of biotinylated antibodies under hydrogen/deuterium exchange quench conditions by the biotin-streptavidin strategy.

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen., Author Summary Staphylococcus aureus and S. epidermidis are major bacterial pathogens that can cause life-threatening human diseases. Following entry into the circulation, S.aureus can infect virtually any organ. However, it must first counter antibacterial mechanisms of the innate immune system, including those involving macrophages and neutrophils. Important for staphylococcal adhesion to and successful colonization of host tissues, is a family of bacterial surface proteins containing multiple repeats of serine-aspartate repeats (SDR) adjacent to an adhesive A-domain. The biological functions of the SDR-domain of these SDR proteins remain elusive. We found that the SDR-domain of all staphylococcal SDR proteins is heavily glycosylated. We identified two novel glycosylases, SdgA and SdgB, which are responsible for glycosylation in two steps, and found that this glycosylation protects the adhesive SDR proteins against proteolytic attack by human neutrophil cathepin G. Since pathogen binding to human tissues, including the extracellular matrix protein fibrinogen, depends on SDR proteins, this glycosylation may be important for successful colonization of the human host. We also show that the SdgB-mediated glycosylation creates an immunodominant epitope for highly opsonic antibodies in humans. These antibodies account for a significant proportion of the total anti-staphylococcal IgG response.

The receptor tyrosine kinase MET (Hepatocyte Growth Factor Receptor, HGFR) has been associated with development and progression of a range of human tumors due to its regulation of cell proliferation, migration, invasion, and angiogenesis. A subset of human tumors, particularly of lung or gastric origin, appear to have a primary dependency on MET, which is driven by alterations, such as MET-gene amplification, MET-exon 14 deletion, activating mutations, or autocrine HGF production. Furthermore, MET-amplification has been reported as a key mechanism of de novo resistance to EGFR targeting agents. Sym015 is a mixture of two humanized monoclonal antibodies against non-overlapping epitopes on MET. The specific pair of antibodies was identified in a high-throughput cell based screen searching for antibody mixtures with superior growth inhibitory activity against four MET-dependent cell lines. Both antibodies bind the SEMA domain of MET with high affinity. Synergistic activity was confirmed by dose-response curves in several MET-dependent cell lines and cell line and patient-derived xenograft models. Mechanistically, Sym015 blocks HGF binding to MET, induces MET internalization and degradation effectively diminishing MET oncogenic signaling. Sym015 also induces higher levels of antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro compared to individual mAbs. In conclusion, Sym015, a novel monoclonal antibody mixture against MET, shows strong inhibitory activity against MET driven cell lines and xenografts due to a combined effect of MET degradation and secondary effector functions. These data support initiation of clinical trials. Citation Format: Michael M. Grandal, Thomas T. Poulsen, Klaus Koefoed, Karsten W. Eriksen, Anna Dahlman, Paolo Conrotto, Thomas Bouquin, Helle Jacobsen, Ivan D. Horak, Michael Kragh, Johan Lantto, Mikkel W. Pedersen. A novel synergistic antibody pair targeting non-overlapping epitopes of MET effectively inhibits MET-driven cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1218.

The anti-EGFR monoclonal antibodies (MoAbs) cetuximab and panitumumab are used to treat ‘RAS’ wild type metastatic colorectal cancer (MCRC), providing significant clinical benefit in patients. However, all patients ultimately develop disease progression, driven by acquisition of mutations in downstream effectors and in the extracellular domain (ECD) of EGFR, in approximately 25% of the cases. Sym004 is a novel 1:1 mixture of two non-overlapping anti-EGFR MoAbs that has recently shown promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. Functional studies were performed to assess drug-receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored an EGFR mutation in the post-cetuximab tumor sample. Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring EGFR ECD mutations maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As a proof of principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. These data suggest that Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. Overall, EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Citation Format: Francisco J. Sanchez-Martin, Alba Dalmases, Beatriz Bellosillo, Guillem Argiles, Mariona Gelabert, Israel Cañadas, Joana Vidal, Giulia Siravegna, Sabrina Arena, Klaus Koefoed, Laura Visa, Oriol Arpí, Ivan D. Horak, Mar Iglesias, Christopher Stroh, Michael Kragh, Ana Rovira, Joan Albanell, Alberto Bardelli, Josep Tabernero, Clara Montagut. The anti-EGFR antibody mixture Sym004 overcomes acquired resistance to cetuximab in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2149.

HIV-1-associated thrombocytopenia (HIV-1-ITP) is a common complication of HIV-1 infection, frequently caused by increased peripheral platelet destruction mediated by antiplatelet antibodies (Abs) and/or platelet-bound immune complexes. Little is known about the specificity of the antiplatelet Abs at a molecular level. Here, we used immunoglobulin G (IgG) phage-display libraries generated from 3 HIV-1-ITP patients to isolate a large panel of human monoclonal antiplatelet Abs by selection on unfixed platelets. The platelet antigen recognized by all the cloned Abs was identified to be the talin head domain (talin-H), a cleavage product of talin that can be generated by platelet activation or HIV-1 protease. Talin-H was found in HIV-1-ITP-circulating immune complexes, and antitalin Abs were detected in HIV-1-ITP sera but not in controls. The cloned anti-talin-H IgGs were highly somatically mutated, indicative of an antigen-driven, affinity-matured response. These findings suggest that talin-H Ab may be a marker of HIV-1-ITP elicited due to exposure of immunodominant epitopes on talin-H as a result of a disease-related process. Abs to talin-H and related immune complexes (ICs) may contribute to HIV-1-ITP. (Blood. 2004;104:4054-4062)

Human bone marrow is a major repository for maturated antibody-secreting plasma cells, which produce the majority of the antibodies found in serum, making it an attractive source for generating human immune antibody libraries. Unfortunately, bone marrow is not always readily available and, although human immune libraries can be generated from circulating B cells, the low frequency of antigen-specific B cells in the circulation yield few monoclonal antibodies of interest. We used a pre-selection strategy to enrich for antigen-specific B cells prior to library generation, and applied this approach to evaluate, at a molecular level, the nature of the human anti-HIV-1 gp120 repertoire encoded by circulating B cells. IgG antibody phage display libraries were generated from HIV-1 seropositive individuals using either affinity-selected anti-gp120 IgG-bearing circulating B cells, predominantly exhibiting memory/activated B cell phenotype, or unselected PBMCs. These libraries were selected against HIV-1 gp120, resulting in isolation of a panel of gp120-specific antibodies. Whereas only 2 gp120-specific antibodies were retrieved from the non-pre-selected HIV-1 library, 9 gp120-specific antibodies were retrieved from the 10-fold smaller library generated from the pre-selected B cells, demonstrating the feasibility of the approach. The anti-gp120 antibodies derived from the circulating B cells of HIV-1 donors generally resembles those from bone marrow plasma cells with respect to epitope specificity, affinity and neutralization ability. They exhibit high affinity for gp120, are directed against a variety of epitopes, but rarely exhibit the ability to neutralize HIV-1.

Deregulation of HER family members plays an important role in the development and progression of human cancer, and EGFR and HER2 are also clinically validated targets in many cancer indications. Accumulating evidence indicates, however, a high degree of HER family cross-talk and compensatory signaling leading to resistance upon therapeutic intervention with one of the receptors. Simultaneous targeting of more than one HER family receptor in vivo is frequently able to overcome resistance to the initial drug and the combined treatment is often also more efficacious than single-receptor targeting alone. Blocking of signaling from more than one HER family member may thus be necessary to effectively treat cancer and limit drug resistance. We have generated Sym013, a mixture of monoclonal antibodies specifically targeting EGFR, HER2 and HER3. Preclinical testing has shown that Sym013 displays potent growth inhibitory activity in a broad range of cell lines of diverse tissue origin and genetic background, including cell lines with acquired resistance to cetuximab, trastuzumab, or pertuzumab. Sym013 effectively inhibits all three targets simultaneously and thus prevents compensatory receptor up-regulation/activation, a commonly observed cellular response that can lead to drug escape when a single receptor is targeted. Furthermore, Sym013 is highly efficacious in the presence of EGFR and HER3 ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. Overall, simultaneous targeting of three receptors provides broader efficacy than targeting of a single receptor or any combination of two receptors in the HER family. The ability to simultaneously inhibit EGFR, HER2 and HER3 in cancer cells in vitro also translates into broad and efficacious tumor growth suppression in vivo. All three target specificities have been demonstrated to contribute to the efficacy of Sym013 in vivo, and there is a clear benefit of combining HER family target specificities compared to single-receptor targeting. Sym013 is also highly efficacious in hard-to-treat KRAS mutated patient-derived pancreatic cancer models, and is frequently able to overcome resistance to HER family targeted therapeutics. Our data suggest that a mixture of antibodies, which simultaneously inhibits EGFR, HER2 and HER3, is superior to existing targeted therapies in dealing with both primary and acquired resistance due to intra-tumor heterogeneity and plasticity in terms of HER family dependency. Citation Format: Johan Lantto, Mikkel W. Pedersen, Helle J. Jacobsen, Thomas T. Poulsen, Ida Kjær, Klaus Koefoed, Jette W. Sen, Dietmar Weilguny, Bolette Bjerregaard, Christina R. Andersen, Ivan D. Horak, Michael Kragh. Simultaneous inhibition of EGFR, HER2 and HER3 by an antibody mixture provides broad and potent tumor inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4751. doi:10.1158/1538-7445.AM2013-4751