Novel OX40 and 4-1BB derived spacers enhance CD30 CAR activity and safety in CD30 positive lymphoma models.

The chimeric antigen receptor (CAR) derived from the CD30 specific murine antibody, HRS-3, has produced promising clinical efficacy with a favorable safety profile in the treatment of relapsed or refractory CD30-positive lymphomas. However, persistence of the autologous CAR-T cells was brief, and many patients relapsed a year after treatment. The lack of persistence may be attributed to the use of a wild-type immunoglobulin (Ig)G1 spacer that can associate with Fc receptors. We first identified the cysteine-rich domain (CRD) 5 of CD30 as the primary binding epitope of HRS-3 and armed with this insight, attempted to improve the HRS-3 CAR functionality with a panel of novel spacer designs. We demonstrate that HRS-3 CARs with OX40 and 4-1BB derived spacers exhibited similar anti-tumor efficacy, circumvented interactions with Fc receptors, and secreted lower levels of cytokines in vitro than a CAR employing the IgG1 spacer. Humanization of the HRS-3 scFv coupled with the 4-1BB spacer preserved potent on-target, on-tumor efficacy, and on-target, off-tumor safety. In a lymphoma mouse model of high tumor burden, T cells expressing humanized HRS-3 CD30.CARs with the 4-1BB spacer potently killed tumors with low levels of circulating inflammatory cytokines, providing a promising candidate for future clinical development in the treatment of CD30-positive malignancies.

Overexpression of an Engineered SerpinB9 Enhances Allogeneic T-Cell Persistence and Efficacy.

Allogeneic chimeric antigen receptor (CAR)-expressing T cells offer many advantages over autologous therapies, but their benefits are curtailed by graft-versus-host disease (GvHD) and elimination by recipient immune cells. Moreover, just as with autologous therapies, allogeneic CAR T cells are susceptible to activation-induced cell death (AICD) caused by chronic antigen exposure (CAE). Granzyme B (GzmB) and Fas/FasL-initiated, caspase-mediated apoptosis are key mechanisms of T-cell death caused by T/NK cell-mediated allorejection or CAE. We explored a protective strategy of engineering CAR T cells to overexpress variants of the GzmB-specific serine protease inhibitor, SerpinB9 (SB9), to improve allogeneic T-cell persistence and antitumor efficacy. We showed that the overexpression of an SB9 variant with broadened caspase specificity, SB9(CAS), not only significantly reduced rejection of allogeneic CAR T cells, but also increased their resistance to AICD and enabled them to thrive better under CAE, thus improving allogeneic T-cell persistence and antitumor activity in vitro and in vivo. In addition, while SB9(CAS)-overexpression improved the efficacy of allogeneic CAR T-cell therapy by conferring protection to cell death, we did not observe any autonomous growth and the engineered CAR T cells were still susceptible to an inducible suicide switch. Hence, SB9(CAS)-overexpression is a promising strategy that can strengthen current development of cell therapies, broadening their applications to address unmet medical needs.

B7-H3-Targeting Chimeric Antigen Receptors Epstein-Barr Virus-specific T Cells Provides a Tumor Agnostic Off-The-Shelf Therapy Against B7-H3-positive Solid Tumors.

Encouraged by the observations of significant B7-H3 protein overexpression in many human solid tumors compared to healthy tissues, we directed our focus towards targeting B7-H3 using chimeric antigen receptor (CAR) T cells. We utilized a nanobody as the B7-H3-targeting domain in our CAR construct to circumvent the stability issues associated with single-chain variable fragment-based domains. In efforts to expand patient access to CAR T-cell therapy, we engineered our nanobody-based CAR into human Epstein-Barr virus-specific T cells (EBVST), offering a readily available off-the-shelf treatment. B7H3.CAR-armored EBVSTs demonstrated potent in vitro and in vivo activities against multiple B7-H3-positive human tumor cell lines and patient-derived xenograft models. Murine T cells expressing a murine equivalent of our B7H3.CAR exhibited no life-threatening toxicities in immunocompetent mice bearing syngeneic tumors. Further in vitro evaluation revealed that while human T, B, and natural killer cells were unaffected by B7H3.CAR EBVSTs, monocytes were targeted because of upregulation of B7-H3. Such targeting of myeloid cells, which are key mediators of cytokine release syndrome (CRS), contributed to a low incidence of CRS in humanized mice after B7H3.CAR EBVST treatment. Notably, we showed that B7H3.CAR EBVSTs can target B7-H3-expressing myeloid-derived suppressor cells (MDSC), thereby mitigating MDSC-driven immune suppression. In summary, our data demonstrate that our nanobody-based B7H3.CAR EBVSTs are effective as an off-the-shelf therapy for B7-H3-positive solid tumors. These cells also offer an avenue to modulate the immunosuppressive tumor microenvironment, highlighting their promising clinical potential in targeting solid tumors. SIGNIFICANCE: Clinical application of EBVSTs armored with B7-H3-targeting CARs offer an attractive solution to translate off-the-shelf CAR T cells as therapy for solid tumors.

First-in-human trial exploring safety, antitumor activity, and pharmacokinetics of Sym013, a recombinant pan-HER antibody mixture, in advanced epithelial malignancies.

PURPOSE: Sym013 contains six humanized monoclonal antibodies that bind to non-overlapping epitopes on three human epidermal growth factor receptors (HER1-3). Preclinical studies suggested Sym013 strongly suppresses growth of multiple epithelial tumors. This is a first-in-human study exploring safety and efficacy of Sym013 in patients with advanced epithelial malignancies. METHODS: Dose escalation used single-patient cohorts until the stopping rule was met, followed by 3 + 3 design. Dose levels planned were: 1, 2, 4, 6, 9, 12, 15, and 18 mg/kg. Treatment cycles were 28 days with imaging every eight weeks. Serum samples were collected at multiple time points for assessment of pharmacokinetics and development of anti-drug antibodies. RESULTS: Thirty-two patients were enrolled with multiple solid tumors, most common being colorectal cancer (CRC; 10/32, 31%). Due to mucositis, rash, and diarrhea at 4 mg/kg once-weekly, dosing was changed to biweekly (Q2W). Mandatory prophylaxis was added due to Grade 3 infusion-related reaction and oral mucositis at 9 mg/kg Q2W. The 15 mg/kg Q2W cohort was enrolling when the study was terminated for business reasons. Most common adverse events were skin (81%) and gastrointestinal (75%) disorders, including dermatitis/rash, stomatitis, and diarrhea. One patient with CRC achieved a partial response; 12 patients with varied malignancies had stable disease. CONCLUSION: During the conduct of the study, management of frequent infusion-related reactions, skin toxicities, and mucosal disorders, which are indicative of HER inhibition, necessitated multiple protocol amendments. The investigators, in concert with the Sponsor, agreed that achieving a tolerated regimen with acceptable target saturation was unlikely. TRIAL REGISTRY: www. CLINICALTRIALS: gov ; NCT02906670 (September 20, 2016).

Paediatric Strategy Forum for medicinal product development of chimeric antigen receptor T-cells in children and adolescents with cancer: ACCELERATE in collaboration with the European Medicines Agency with participation of the Food and Drug Administration.

The seventh multi-stakeholder Paediatric Strategy Forum focused on chimeric antigen receptor (CAR) T-cells for children and adolescents with cancer. The development of CAR T-cells for patients with haematological malignancies, especially B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), has been spectacular. However, currently, there are scientific, clinical and logistical challenges for use of CAR T-cells in BCP-ALL and other paediatric malignancies, particularly in acute myeloid leukaemia (AML), lymphomas and solid tumours. The aims of the Forum were to summarise the current landscape of CAR T-cell therapy development in paediatrics, too identify current challenges and future directions, with consideration of other immune effector modalities and ascertain the best strategies to accelerate their development and availability to children. Although the effect is of limited duration in about half of the patients, anti-CD19 CAR T-cells produce high response rates in relapsed/refractory BCP-ALL and this has highlighted previously unknown mechanisms of relapse. CAR T-cell treatment as first- or second-line therapy could also potentially benefit patients whose disease has high-risk features associated with relapse and failure of conventional therapies. Identifying patients with very early and early relapse in whom CAR T-cell therapy may replace haematopoietic stem cell transplantation and be definitive therapy versus those in whom it provides a more effective bridge to haematopoietic stem cell transplantation is a very high priority. Development of approaches to improve persistence, either by improving T cell fitness or using more humanised/fully humanised products and co-targeting of multiple antigens to prevent antigen escape, could potentially further optimise therapy. Many differences exist between paediatric B-cell non-Hodgkin lymphomas (B-NHL) and BCP-ALL. In view of the very small patient numbers with relapsed lymphoma, careful prioritisation is needed to evaluate CAR T-cells in children with Burkitt lymphoma, primary mediastinal B cell lymphoma and other NHL subtypes. Combination trials of alternative targets to CD19 (CD20 or CD22) should also be explored as a priority to improve efficacy in this population. Development of CD30 CAR T-cell immunotherapy strategies in patients with relapsed/refractory Hodgkin lymphoma will likely be most efficiently accomplished by joint paediatric and adult trials. CAR T-cell approaches are early in development for AML and T-ALL, given the unique challenges of successful immunotherapy actualisation in these diseases. At this time, CD33 and CD123 appear to be the most universal targets in AML and CD7 in T-ALL. The results of ongoing or planned first-in-human studies are required to facilitate further understanding. There are promising early results in solid tumours, particularly with GD2 targeting cell therapies in neuroblastoma and central nervous system gliomas that represent significant unmet clinical needs. Further understanding of biology is critical to success. The comparative benefits of autologous versus allogeneic CAR T-cells, T-cells engineered with T cell receptors T-cells engineered with T cell receptor fusion constructs, CAR Natural Killer (NK)-cell products, bispecific T-cell engager antibodies and antibody-drug conjugates require evaluation in paediatric malignancies. Early and proactive academia and multi-company engagement are mandatory to advance cellular immunotherapies in paediatric oncology. Regulatory advice should be sought very early in the design and preparation of clinical trials of innovative medicines, for which regulatory approval may ultimately be sought. Aligning strategic, scientific, regulatory, health technology and funding requirements from the inception of a clinical trial is especially important as these are very expensive therapies. The model for drug development for cell therapy in paediatric oncology could also involve a 'later stage handoff' to industry after early development in academic hands. Finally, and very importantly, strategies must evolve to ensure appropriate ease of access for children who need and could potentially benefit from these therapies.

Deep neural networks identify signaling mechanisms of ErbB-family drug resistance from a continuous cell morphology space.

It is well known that the development of drug resistance in cancer cells can lead to changes in cell morphology. Here, we describe the use of deep neural networks to analyze this relationship, demonstrating that complex cell morphologies can encode states of signaling networks and unravel cellular mechanisms hidden to conventional approaches. We perform high-content screening of 17 cancer cell lines, generating more than 500 billion data points from ∼850 million cells. We analyze these data using a deep learning model, resulting in the identification of a continuous 27-dimension space describing all of the observed cell morphologies. From its morphology alone, we could thus predict whether a cell was resistant to ErbB-family drugs, with an accuracy of 74%, and predict the potential mechanism of resistance, subsequently validating the role of MET and insulin-like growth factor 1 receptor (IGF1R) as drivers of cetuximab resistance in in vitro models of lung and head/neck cancer.

Colorectal cancer residual disease at maximal response to EGFR blockade displays a druggable Paneth cell-like phenotype.

Blockade of epidermal growth factor receptor (EGFR) causes tumor regression in some patients with metastatic colorectal cancer (mCRC). However, residual disease reservoirs typically remain even after maximal response to therapy, leading to relapse. Using patient-derived xenografts (PDXs), we observed that mCRC cells surviving EGFR inhibition exhibited gene expression patterns similar to those of a quiescent subpopulation of normal intestinal secretory precursors with Paneth cell characteristics. Compared with untreated tumors, these pseudodifferentiated tumor remnants had reduced expression of genes encoding EGFR-activating ligands, enhanced activity of human epidermal growth factor receptor 2 (HER2) and HER3, and persistent signaling along the phosphatidylinositol 3-kinase (PI3K) pathway. Clinically, properties of residual disease cells from the PDX models were detected in lingering tumors of responsive patients and in tumors of individuals who had experienced early recurrence. Mechanistically, residual tumor reprogramming after EGFR neutralization was mediated by inactivation of Yes-associated protein (YAP), a master regulator of intestinal epithelium recovery from injury. In preclinical trials, Pan-HER antibodies minimized residual disease, blunted PI3K signaling, and induced long-term tumor control after treatment discontinuation. We found that tolerance to EGFR inhibition is characterized by inactivation of an intrinsic lineage program that drives both regenerative signaling during intestinal repair and EGFR-dependent tumorigenesis. Thus, our results shed light on CRC lineage plasticity as an adaptive escape mechanism from EGFR-targeted therapy and suggest opportunities to preemptively target residual disease.

Simultaneous targeting of HER family pro-survival signaling with Pan-HER antibody mixture is highly effective in TNBC: a preclinical trial with PDXs.

BACKGROUND: The human epidermal growth factor receptor (HER) family, notably EGFR, is overexpressed in most triple-negative breast cancer (TNBC) cases and provides cancer cells with compensatory signals that greatly contribute to the survival and development of resistance in response to therapy. This study investigated the effects of Pan-HER (Symphogen, Ballerup, Denmark), a novel mixture of six monoclonal antibodies directed against members of the HER family EGFR, HER2, and HER3, in a preclinical trial of TNBC patient-derived xenografts (PDXs). METHODS: Fifteen low passage TNBC PDX tumor samples were transferred into the right mammary fat pad of mice for engraftment. When tumors reached an average size of 100-200 mm3, mice were randomized (n ≥ 6 per group) and treated following three 1-week cycles consisting of three times/week intraperitoneal (IP) injection of either formulation buffer (vehicle control) or Pan-HER (50 mg/kg). At the end of treatment, tumors were collected for Western blot, RNA, and immunohistochemistry analyses. RESULTS: All 15 TNBC PDXs were responsive to Pan-HER treatment, showing significant reductions in tumor growth consistent with Pan-HER-mediated tumor downmodulation of EGFR and HER3 protein levels and significantly decreased activation of associated HER family signaling pathways AKT and ERK. Tumor regression was observed in five of the models, which corresponded to those PDX tumor models with the highest level of HER family activation. CONCLUSIONS: The marked effect of Pan-HER in numerous HER family-dependent TNBC PDX models justifies further studies of Pan-HER in TNBC clinical trials as a potential therapeutic option.

CD4+ and CD8a+ PET imaging predicts response to novel PD-1 checkpoint inhibitor: studies of Sym021 in syngeneic mouse cancer models.

Predicting the outcome of immunotherapy is essential for efficient treatment. The recent clinical success of immunotherapy is increasingly changing the paradigm of cancer treatment. Accordingly, the development of immune-based agents is accelerating and the number of agents in the global immuno-oncology pipeline has grown 60-70% over the past year. However, despite remarkable clinical efficacy in some patients, only few achieve a lasting clinical response. Treatment failure can be attributed to poorly immunogenic tumors that do not attract tumor infiltrating lymphocytes (TILs). Therefore, we developed positron emission tomography (PET) radiotracers for non-invasive detection of CD4+ and CD8a+ TILs in syngeneic mouse tumor models for preclinical studies. Methods: Seven syngeneic mouse tumor models (B16F10, P815, CT26, MC38, Renca, 4T1, Sa1N) were quantified for CD4+ and CD8a+ TILs using flow cytometry and immunohistochemistry (IHC), as well as for tumor growth response to Sym021, a humanized PD-1 antibody cross-reactive with mouse PD-1. Radiotracers were generated from F(ab)'2 fragments of rat-anti-mouse CD4 and CD8a antibodies conjugated to the p-SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator and radiolabeled with Zirconium-89 (89Zr-DFO-CD4/89Zr-DFO-CD8a). Tracers were optimized for in vivo PET/CT imaging in CT26 tumor-bearing mice and specificity was evaluated by depletion studies and isotype control imaging. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET/CT imaging was conducted in the panel of syngeneic mouse models prior to immunotherapy with Sym021. Results: Syngeneic tumor models were characterized as "hot" or "cold" according to number of TILs determined by flow cytometry and IHC. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a were successfully generated with a radiochemical purity >99% and immunoreactivity >85%. The optimal imaging time-point was 24 hours post-injection of ~1 MBq tracer with 30 µg non-labeled co-dose. Reduced tumor and spleen uptake of 89Zr-DFO-CD8a was observed in CD8a+ depleted mice and the uptake was comparable with that of isotype control (89Zr-DFO-IgG2b) confirming specificity. PET imaging in syngeneic tumor models revealed a varying maximum tumor-to-heart ratio of 89Zr-DFO-CD4 and 89Zr-DFO-CD8a across tumor types and in-between subjects that correlated with individual response to Sym021 at day 10 relative to start of therapy (p=0.0002 and p=0.0354, respectively). The maximum 89Zr-DFO-CD4 tumor-to-heart ratio could be used to stratify mice according to Sym021 therapy response and overall survival was improved in mice with a 89Zr-DFO-CD4 ratio >9 (p=0.0018). Conclusion: We developed 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET radiotracers for specific detection and whole-body assessment of CD4+ and CD8a+ status. These radiotracers can be used to phenotype preclinical syngeneic mouse tumor models and to predict response to an immune checkpoint inhibitor. We foresee development of such non-invasive in vivo biomarkers for prediction and evaluation of clinical efficacy of immunotherapeutic agents, such as Sym021.

Sym021, a promising anti-PD1 clinical candidate antibody derived from a new chicken antibody discovery platform.

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.

Sym004-induced EGFR elimination is associated with profound anti-tumor activity in EGFRvIII patient-derived glioblastoma models.

BACKGROUND: Sym004 is a mixture of two monoclonal antibodies (mAbs), futuximab and modotuximab, targeting non-overlapping epitopes on the epidermal growth factor receptor (EGFR). Previous studies have shown that Sym004 is more efficient at inducing internalization and degradation of EGFR than individual components, which translates into superior cancer cell inhibition. We investigated whether Sym004 induces removal of EGFRvIII and if this removal translates into tumor growth inhibition in hard-to-treat glioblastomas (GBMs) harboring the mutated, constitutively active EGFR variant III (EGFRvIII). METHODS: To address this question, we tested the effect of Sym004 versus cetuximab in eight patient-derived GBM xenograft models expressing either wild-type EGFR (EGFRwt) and/or mutant EGFRvIII. All models were tested as both subcutaneous and orthotopic intracranial xenograft models. RESULTS: In vitro studies demonstrated that Sym004 internalized and removed EGFRvIII more efficiently than mAbs, futuximab, modotuximab, and cetuximab. Removal of EGFRvIII by Sym004 translated into significant in vivo anti-tumor activity in all six EGFRvIII xenograft models. Furthermore, the anti-tumor activity of Sym004 in vivo was superior to that of its individual components, futuximab and modotuximab, suggesting a clear synergistic effect of the mAbs in the mixture. CONCLUSION: These results demonstrate the broad activity of Sym004 in patient-derived EGFRvIII-expressing GBM xenograft models and provide a clear rationale for clinical evaluation of Sym004 in EGFRvIII-positive adult GBM patients.

Acquired Resistance to a MET Antibody In Vivo Can Be Overcome by the MET Antibody Mixture Sym015.

Failure of clinical trials due to development of resistance to MET-targeting therapeutic agents is an emerging problem. Mechanisms of acquired resistance to MET tyrosine kinase inhibitors are well described, whereas characterization of mechanisms of resistance toward MET-targeting antibodies is limited. This study investigated mechanisms underlying in vivo resistance to two antibody therapeutics currently in clinical development: an analogue of the MET-targeting antibody emibetuzumab and Sym015, a mixture of two antibodies targeting nonoverlapping epitopes of MET. Upon long-term in vivo treatment of a MET-amplified gastric cancer xenograft model (SNU-5), emibetuzumab-resistant, but not Sym015-resistant, tumors emerged. Resistant tumors were isolated and used to establish resistant cell lines. Characterization of both tumors and cell lines using extensive protein and signaling pathway activation mapping along with next-generation sequencing revealed two distinct resistance profiles, one involving PTEN loss and the other involving activation of the PI3K pathway, likely via MYC and ERBB3 copy number gains. PTEN loss left one model unaffected by PI3K/AKT targeting but sensitive to mTOR targeting, while the PI3K pathway-activated model was partly sensitive to targeting of multiple PI3K pathway proteins. Importantly, both resistant models were sensitive to treatment with Sym015 in vivo due to antibody-dependent cellular cytotoxicity-mediated tumor growth inhibition, MET degradation, and signaling inhibition. Taken together, our data provide key insights into potential mechanisms of resistance to a single MET-targeting antibody, demonstrate superiority of Sym015 in preventing acquired resistance, and confirm Sym015 antitumor activity in tumors resistant to a single MET antibody. Mol Cancer Ther; 17(6); 1259-70. ©2018 AACR.

Efficacy of Sym004 in Patients With Metastatic Colorectal Cancer With Acquired Resistance to Anti-EGFR Therapy and Molecularly Selected by Circulating Tumor DNA Analyses: A Phase 2 Randomized Clinical Trial.

IMPORTANCE: Acquired resistance to anti-EGFR therapy (epidermal growth factor receptor) is frequently due to RAS and EGFR extracellular 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-type KRAS exon 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 targeted EGFR ECD-mutated cancer cells, and a decrease in EGFR ECD ctDNA occurred in Sym004-treated patients. However, this did not translate into clinical benefit in patients with EGFR ECD mutations, likely owing to co-occurring resistance mechanisms. A subgroup of patients was defined by ctDNA (RAS/BRAF/EGFR ECD-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.

Forty-nine gastric cancer cell lines with integrative genomic profiling for development of c-MET inhibitor.

Receptor tyrosine kinase MET (c-MET) has received considerable attention as a potential target for gastric cancer (GC) therapy and a number of c-MET inhibitors have been developed. For successful drug development, proper preclinical studies especially using patient derived cancer cell lines are very important. We profiled MET and MET-related characteristics in 49 GC cell lines to utilize them as models in preclinical studies of GC. Forty-nine cell lines were analyzed for genetic, biological, and molecular status to characterize MET and MET-related molecules. Four c-MET inhibitors were tested to elucidate the dependency on MET pathway in the 49 GC cell lines. Six of 49 cell lines were MET amplified with overexpression of c-MET and p-MET. The variants of MET were not associated with c-MET expression or amplification. Hs746T showed an exon 14 deletion in conjunction with MET amplification. The cell lines were divided into 6 MET amplified, 2 c-MET overexpressed, 2 hepatocyte growth factor (HGF) overexpressed, and 39 MET-negative subgroups. Except tivantinib, the c-MET inhibitors showed higher inhibition (%) in MET amplified than in MET nonamplified cell lines that MET amplified cell lines showed MET pathway dependency. However, the c-MET overexpressed and HGF overexpressed cell lines showed moderate dependency on MET pathway. Well-characterized cell lines are very important in studying drug development. Our 49 GC cell lines had various characteristics of MET and MET-related molecules and MET pathway dependency. These provide a promising platform for development of various RTK inhibitors including c-MET inhibitors.

An ERBB1-3 Neutralizing Antibody Mixture With High Activity Against Drug-Resistant HER2+ Breast Cancers With ERBB Ligand Overexpression.

Background: Plasticity of the ERBB receptor network has been suggested to cause acquired resistance to anti-human epidermal growth factor receptor 2 (HER2) therapies. Thus, we studied whether a novel approach using an ERBB1-3-neutralizing antibody mixture can block these compensatory mechanisms of resistance. Methods: HER2+ cell lines and xenografts (n ≥ 6 mice per group) were treated with the ERBB1-3 antibody mixture Pan-HER, trastuzumab/lapatinib (TL), trastuzumab/pertuzumab (TP), or T-DM1. Downregulation of ERBB receptors was assessed by immunoblot analysis and immunohistochemistry. Paired pre- and post-T-DM1 tumor biopsies from patients (n = 11) with HER2-amplified breast cancer were evaluated for HER2 and P-HER3 expression by immunohistochemistry and/or fluorescence in situ hybridization. ERBB ligands were measured by quantitative reverse transcription polymerase chain reaction. Drug-resistant cells were generated by chronic treatment with T-DM1. All statistical tests were two-sided. Results: Treatment with Pan-HER inhibited growth and promoted degradation of ERBB1-3 receptors in a panel of HER2+ breast cancer cells. Compared with TL, TP, and T-DM1, Pan-HER induced a similar antitumor effect against established BT474 and HCC1954 tumors, but was superior to TL against MDA-361 xenografts (TL mean = 2026 mm 3 , SD = 924 mm 3 , vs Pan-HER mean = 565 mm 3 , SD = 499 mm 3 , P = .04). Pan-HER-treated BT474 xenografts did not recur after treatment discontinuation, whereas tumors treated with TL, TP, and T-DM1 did. Post-TP and post-T-DM1 recurrent tumors expressed higher levels of neuregulin-1 (NRG1), HER3 and P-HER3 (all P < .05). Higher levels of P-HER3 protein and NRG1 mRNA were also observed in HER2+ breast cancers progressing after T-DM1 and trastuzumab (NRG1 transcript fold change ± SD; pretreatment = 2, SD = 1.9, vs post-treatment = 11.4, SD = 10.3, P = .04). The HER3-neutralizing antibody LJM716 resensitized the drug-resistant cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Finally, Pan-HER treatment inhibited growth of HR6 trastuzumab- and T-DM1-resistant xenografts. Conclusions: These data suggest that upregulation of a NRG1-HER3 axis can mediate escape from anti-HER2 therapies. Further, multitargeted antibody mixtures, such as Pan-HER, can simultaneously remove and/or block targeted ERBB receptor and ligands, thus representing an effective approach against drug-sensitive and -resistant HER2+ cancers.

Simultaneous Targeting of Two Distinct Epitopes on MET Effectively Inhibits MET- and HGF-Driven Tumor Growth by Multiple Mechanisms.

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.

Targeting the HER Family with Pan-HER Effectively Overcomes Resistance to Cetuximab.

Cetuximab, an antibody against the EGFR, has shown efficacy in treating head and neck squamous cell carcinoma (HNSCC), metastatic colorectal cancer, and non-small cell lung cancer (NSCLC). Despite the clinical success of cetuximab, many patients do not respond to cetuximab. Furthermore, virtually all patients who do initially respond become refractory, highlighting both intrinsic and acquired resistance to cetuximab as significant clinical problems. To understand mechanistically how cancerous cells acquire resistance, we previously developed models of acquired resistance using the H226 NSCLC and UM-SCC1 HNSCC cell lines. Cetuximab-resistant clones showed a robust upregulation and dependency on the HER family receptors EGFR, HER2, and HER3. Here, we examined pan-HER, a mixture of six antibodies targeting these receptors on cetuximab-resistant clones. In cells exhibiting acquired or intrinsic resistance to cetuximab, pan-HER treatment decreased all three receptors' protein levels and downstream activation of AKT and MAPK. This correlated with decreased cell proliferation in cetuximab-resistant clones. To determine whether pan-HER had a therapeutic benefit in vivo, we established de novo cetuximab-resistant mouse xenografts and treated resistant tumors with pan-HER. This regimen resulted in a superior growth delay of cetuximab-resistant xenografts compared with mice continued on cetuximab. Furthermore, intrinsically cetuximab-resistant HNSCC patient-derived xenograft tumors treated with pan-HER exhibited significant growth delay compared with vehicle/cetuximab controls. These results suggest that targeting multiple HER family receptors simultaneously with pan-HER is a promising treatment strategy for tumors displaying intrinsic or acquired resistance to cetuximab. Mol Cancer Ther; 15(9); 2175-86. ©2016 AACR.

Pan-HER-An antibody mixture targeting EGFR, HER2 and HER3 abrogates preformed and ligand-induced EGFR homo- and heterodimers.

The human epidermal growth factor receptor (HER)-family is involved in development of many epithelial cancers. Therefore, HER-family members constitute important targets for anti-cancer therapeutics such as monoclonal antibodies (mAbs). A limitation to the success of single HER-targeting mAbs is development of acquired resistance through mechanisms such as alterted receptor dimerization patterns and dependencies. Pan-HER is a mixture of six mAbs simultaneously targeting epidermal growth factor receptor (EGFR), HER2 and HER3 with two mAbs against each receptor. Pan-HER has previously demonstrated broader efficacy than targeting single or dual receptor combinations also in resistant settings. In light of this broad efficacy, we decided to investigate the effect of Pan-HER compared with single HER-targeting with single and dual mAbs on HER-family cross-talk and dimerization focusing on EGFR. The effect of Pan-HER on cell proliferation and HER-family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non-overlapping antibodies. Furthermore, changes in EGFR-dimerization patterns after treatment with Pan-HER were investigated by in situ proximity ligation assay and co-immunoprecipitation, demonstrating that Pan-HER and the EGFR-targeting mAb mixture efficiently down-regulate basal EGFR homo- and heterodimerization in two tested cell lines, whereas single mAbs had limited effects. Pan-HER and the EGFR-targeting mAb mixture also blocked EGF-binding and thereby ligand-induced changes in EGFR-dimerization levels. These results suggest that Pan-HER reduces the cellular capability to switch HER-dependency and dimerization pattern in response to treatment and thus hold promise for future clinical development of Pan-HER in resistant settings.

In vivo imaging of therapy response to a novel pan-HER antibody mixture using FDG and FLT positron emission tomography.

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.

Safety and Activity of the First-in-Class Sym004 Anti-EGFR Antibody Mixture in Patients with Refractory Colorectal Cancer.

UNLABELLED: Tumor growth in the context of EGFR inhibitor resistance may remain EGFR-dependent and is mediated by mechanisms including compensatory ligand upregulation and de novo gene alterations. Sym004 is a two-antibody mixture targeting nonoverlapping EGFR epitopes. In preclinical models, Sym004 causes significant EGFR internalization and degradation, which translates into superior growth inhibition in the presence of ligands. In this phase I trial, we observed grade 3 skin toxicity and hypomagnesemia as mechanism-based dose-limiting events during dose escalation. In dose-expansion cohorts of 9 and 12 mg/kg of Sym004 weekly, patients with metastatic colorectal cancer and acquired EGFR inhibitor resistance were enrolled; 17 of 39 patients (44%) had tumor shrinkage, with 5 patients (13%) achieving partial response. Pharmacodynamic studies confirmed marked Sym004-induced EGFR downmodulation. MET gene amplification emerged in 1 patient during Sym004 treatment, and a partial response was seen in a patient with EGFR(S492R) mutation that is predictive of cetuximab resistance. SIGNIFICANCE: Potent EGFR downmodulation with Sym004 in patients with metastatic colorectal cancer and acquired resistance to cetuximab/panitumumab translates into significant antitumor activity and validates the preclinical hypothesis that a proportion of tumors remains dependent on EGFR signaling. Further clinical development and expanded correlative analyses of response patterns with secondary RAS/EGFR mutations are warranted.