Discovery of amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR and MET.

A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non-small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF ß-chain-Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling.

Rapid COVID-19 Prognostic Blood Test for Disease Severity Using Epigenetic Immune System Biomarkers.

OBJECTIVE: To develop a novel whole-blood epigenetic biomarker of immune system status, or EpiMarker, that would indicate whether a person with a recent COVID-19 diagnosis is at risk for severe symptoms including Acute Respiratory Distress Syndrome. METHODS: Using a novel methyl-sensitive restriction endonuclease approach to measure site-specific DNA methylation profiles, immune system phentoype EpiMarkers are identified using a machine-learning computational bioinformatics platform. The result is a diagnostic network of 20 to 40 immuno DNA methylation sites having the greatest predictive power for identifying patients whose COVID-19 disease will likely progress to ARDS requiring ICU/intubation care. RESULTS: Immune system status in peripheral whole blood provides a sensitive and responsive sentinel signal reflecting how different functional pathways are currently being regulated in a subject. Deciphering this signal status of how immune cells are set to respond provides deep functional information regarding patient health and potential disease phenotypes resulting from a cytokine storm characteristic of a hyper immune inflammatory response to COVID-19 infection. CONCLUSIONS: The ability to identify future potential changes in patient health using this novel EpiMarker technology opens new avenues for defending populations from severe disease risks of Acute Respiratory Distress Syndrome. POLICY IMPLICATIONS: A successful EpiMarker Assay for COVID-19 disease severity risk would allow for two important applications: (1) patients could be triaged early in the course of infection to allow for critical decisions for allocating resources, both in terms of hospital infrastructure (ICU beds, ventilators) and therapeutic drug treatments; and (2) pre-infection, individuals could be screened to identify personnel at low-risk for mission critical assignments (first responders, doctors, nurses, military personnel, etc.) during future pandemics and ongoing battles with viral pathogens like influenza.

Inhibition of tissue factor signaling in breast tumour xenografts induces widespread changes in the microRNA expression profile.

Tissue factor (TF) is a transmembrane receptor for coagulation factor VII/VIIa and is frequently overexpressed by cancer cells. The TF/VIIa complex acts as the main initiator of the clotting cascade in blood and a trigger of intracellular signaling that changes gene expression and the cellular phenotype. However, pathways mediating these changes are still poorly characterized and especially the impact of TF signals on regulatory microRNA (miR) networks in cancer remains unknown. We show that the monoclonal antibody that selectively neutralises the signaling (but not coagulant) function of human TF (CNTO 2559) inhibits progression of MDA-MB-231 breast cancer xenografts in mice and prolongs animal survival. CNTO 2559 blocks FVIIa-induced expression of interleukin 8 (IL-8) by cancer cells without impacting factor Xa (FXa) generation. Notably, acute exposure of MDA-MB-231 tumour xenografts to CNTO 2559 systemic injections triggers wide spread changes in the tumour miR profile including alterations in 75 miRs (55 downregulated) and impacting several miR-regulated and cancer-related pathways. These results suggest that TF signaling in the tumour microenvironment may provoke vast changes in the miR profile of cancer cells, affect disease biology, and reflect tumour interaction with the coagulation system, thereby presenting itself as a possible biomarker.

Crystal structure of tissue factor in complex with antibody 10H10 reveals the signaling epitope.

Tissue factor (TF) initiates the extrinsic pathway of blood coagulation through sequential binding and activation of coagulation factors VII (FVII) and X (FX). In addition, through activation of G-protein-coupled protease activated receptors (PARs) TF induces cell signaling that is related to cancer, angiogenesis and inflammation. Monoclonal antibodies (mAbs) proved to be a useful tool for studying the interplay between TF signaling and coagulation. MAb 10H10 is unique in that it blocks the signaling pathway and thus inhibits angiogenesis and tumor growth without interfering with coagulation. It was also presumed that mAb 10H10 recognizes the cryptic pool of TF devoid of procoagulant activity. The crystal structure of the 10H10 Fab was determined in the absence and in the presence of the TF extracellular domain (ECD). The structures show that the antibody operates by the key-and-lock mechanism causing no conformational changes in either Fab or TF. The TF:10H10 interface is extensive and includes five segments of TF in both the N-terminal and C-terminal domains of the ECD. Neither the known epitope of FVII, nor the putative epitope of FX overlaps with the 10H10 binding site. The 10H10 epitope points to the likely location of the PAR2 exosite. It is also the hypothetical site of TF interaction with integrins that may play a major role in the encryption-decryption process.

A Novel Bispecific Antibody Targeting EGFR and cMet Is Effective against EGFR Inhibitor-Resistant Lung Tumors.

Non-small cell lung cancers (NSCLC) with activating EGFR mutations become resistant to tyrosine kinase inhibitors (TKI), often through second-site mutations in EGFR (T790M) and/or activation of the cMet pathway. We engineered a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action to inhibit primary/secondary EGFR mutations and the cMet pathway. JNJ-61186372 blocked ligand-induced phosphorylation of EGFR and cMet and inhibited phospho-ERK and phospho-AKT more potently than the combination of single receptor-binding antibodies. In NSCLC tumor models driven by EGFR and/or cMet, JNJ-61186372 treatment resulted in tumor regression through inhibition of signaling/receptor downmodulation and Fc-driven effector interactions. Complete and durable regression of human lung xenograft tumors was observed with the combination of JNJ-61186372 and a third-generation EGFR TKI. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372 resulted in no major toxicities, including absence of skin rash observed with other EGFR-directed agents. These results highlight the differentiated potential of JNJ-61186372 to inhibit the spectrum of mutations driving EGFR TKI resistance in NSCLC. Cancer Res; 76(13); 3942-53. ©2016 AACR.

Endothelial protein C receptor function in murine and human breast cancer development.

Several markers identify cancer stem cell-like populations, but little is known about the functional roles of stem cell surface receptors in tumor progression. Here, we show that the endothelial protein C receptor (EPCR), a stem cell marker in hematopoietic, neuronal and epithelial cells, is crucial for breast cancer growth in the orthotopic microenvironment of the mammary gland. Mice with a hypomorphic allele of EPCR show reduced tumor growth in the PyMT-model of spontaneous breast cancer development and deletion of EPCR in established PyMT tumor cells significantly attenuates transplanted tumor take and growth. We find expansion of EPCR(+) cancer stem cell-like populations in aggressive, mammary fat pad-enhanced human triple negative breast cancer cells. In this model, EPCR-expressing cells have markedly increased mammosphere- and tumor-cell initiating activity compared to another stable progenitor-like subpopulation present at comparable frequency. We show that receptor blocking antibodies to EPCR specifically attenuate in vivo tumor growth initiated by either EPCR(+) cells or the heterogenous mixture of EPCR(+) and EPCR(-) cells. Furthermore, we have identified tumor associated macrophages as a major source for recognized ligands of EPCR, suggesting a novel mechanism by which cancer stem cell-like populations are regulated by innate immune cells in the tumor microenvironment.

Tumor-associated macrophages promote invasion while retaining Fc-dependent anti-tumor function.

Tumor-associated macrophages (TAMs) have been shown to promote tumor progression, and increased TAM infiltration often correlates with poor prognosis. However, questions remain regarding the phenotype of macrophages within the tumor and their role in mAb-dependent cytotoxicity. This study demonstrates that whereas TAMs have protumor properties, they maintain Fc-dependent anti-tumor function. CD11b(+)CD14(+) TAMs isolated from primary human breast tumors expressed activating FcγRs. To model breast cancer TAMs in vitro, conditioned medium from breast cancer cells was used to drive human peripheral monocyte differentiation into macrophages. Tumor-conditioned macrophages were compared with in vitro derived M1 and M2a macrophages and were found to promote tumor cell invasion and express M2a markers, confirming their protumor potential. However, unlike M2a macrophages, tumor-conditioned macrophages expressed FcγRs and phagocytosed tumor cells in the presence of a tumor Ag-targeting mAb, unmasking an underappreciated tumoricidal capacity of TAMs. In vivo macrophage depletion reduced the efficacy of anti-CD142 against MDA-MB-231 xenograft growth and metastasis in SCID/beige mice, implicating a critical role for macrophages in Fc-dependent cell killing. M-CSF was identified in tumor-conditioned media and shown to be capable of differentiating macrophages with both pro- and anti-tumor properties. These results highlight the plasticity of TAMs, which are capable of promoting tumor progression and invasion while still retaining tumoricidal function in the presence of tumor-targeting mAbs.

Tissue factor regulation by epidermal growth factor receptor and epithelial-to-mesenchymal transitions: effect on tumor initiation and angiogenesis.

ErbB oncogenes drive the progression of several human cancers. Our study shows that in human carcinoma (A431) and glioma (U373) cells, the oncogenic forms of epidermal growth factor receptor (EGFR; including EGFRvIII) trigger the up-regulation of tissue factor (TF), the transmembrane protein responsible for initiating blood coagulation and signaling through interaction with coagulation factor VIIa. We show that A431 cancer cells in culture exhibit a uniform TF expression profile; however, these same cells in vivo exhibit a heterogeneous TF expression and show signs of E-cadherin inactivation, which is coupled with multilineage (epithelial and mesenchymal) differentiation. Blockade of E-cadherin in vitro, leads to the acquisition of spindle morphology and de novo expression of vimentin, features consistent with epithelial-to-mesenchymal transition. These changes were associated with an increase in EGFR-dependent TF expression, and with enhanced stimulation of vascular endothelial growth factor production, particularly following cancer cell treatment with coagulation factor VIIa. In vivo, cells undergoing epithelial-to-mesenchymal transition exhibited an increased metastatic potential. Furthermore, injections of the TF-blocking antibody (CNTO 859) delayed the initiation of A431 tumors in immunodeficient mice, and reduced tumor growth, vascularization, and vascular endothelial growth factor expression. Collectively, our data suggest that TF is regulated by both oncogenic and differentiation pathways, and that it functions in tumor initiation, tumor growth, angiogenesis, and metastasis. Thus, TF could serve as a therapeutic target in EGFR-dependent malignancies.

Contribution of host-derived tissue factor to tumor neovascularization.

OBJECTIVE: The role of host-derived tissue factor (TF) in tumor growth, angiogenesis, and metastasis has hitherto been unclear and was investigated in this study. METHODS AND RESULTS: We compared tumor growth, vascularity, and responses to cyclophosphamide (CTX) of tumors in wild-type (wt) mice, or in animals with TF levels reduced by 99% (low-TF mice). Global growth rate of 3 different types of transplantable tumors (LLC, B16F1, and ES teratoma) or metastasis were unchanged in low-TF mice. However, several unexpected tumor/context-specific alterations were observed in these mice, including: (1) reduced tumor blood vessel size in B16F1 tumors; (2) larger spleen size and greater tolerance to CTX toxicity in the LLC model; (3) aborted tumor growth after inoculation of TF-deficient tumor cells (ES TF(-/-)) in low-TF mice. TF-deficient tumor cells grew readily in mice with normal TF levels and attracted exclusively host-related blood vessels (without vasculogenic mimicry). We postulate that this complementarity may result from tumor-vascular transfer of TF-containing microvesicles, as we observed such transfer using human cancer cells (A431) and mouse endothelial cells, both in vitro and in vivo. CONCLUSIONS: Our study points to an important but context-dependent role of host TF in tumor formation, angiogenesis and therapy.

CNTO 95, a fully human anti alphav integrin antibody, inhibits cell signaling, migration, invasion, and spontaneous metastasis of human breast cancer cells.

CNTO 95 is a fully human monoclonal antibody that recognizes alphav integrins. Previous studies have shown that CNTO 95 exhibits both anti-tumor and anti-angiogenic activities (Trikha M et al., Int J Cancer 110:326-335, 2004). In this study we investigated the biological activities of CNTO 95 on breast tumor cells both in vitro and in vivo. In vitro treatment with CNTO 95 decreased the viability of breast tumor cells adhering to vitronectin. CNTO 95 inhibited tumor cell adhesion, migration, and invasion in vitro. CNTO 95 treatment also induced tyrosine dephosphorylation of focal adhesion kinase (FAK), and the docking protein paxillin that recruits both structural and signaling molecules to focal adhesions (Turner CE, Int J Biochem Cell Biol 30:955-959, 1998; O'Neil GM et al., Trends Cell Biol 10:111-119, 2000). These results suggest that CNTO 95 inhibits breast tumor cell growth, migration and invasion by interruption of alphav integrin mediated focal adhesions and cell motility signals. In vivo studies of CNTO 95 were conducted in an orthotopic breast tumor xenograft model. Treatment with CNTO 95 resulted in significant inhibition of both tumor growth and spontaneous metastasis of MDA-MB-231 cells to the lungs. CNTO 95 also inhibited lung metastasis in a separate experimental (tail vein injection) model of metastasis. The results presented here demonstrate the anti-tumor and anti-metastatic activities of CNTO 95 in breast cancer models and provide insight into the cellular and molecular mechanisms mediating its inhibitory effects on metastasis.

Alphav integrin-targeted immunoconjugates regress established human tumors in xenograft models.

PURPOSE: Targeted delivery of cytotoxic agents to solid tumors through cell surface antigens can potentially reduce systemic toxicity and increase the efficacy of the targeted compounds. The purpose of this study was to show the feasibility of treating solid tumors by targeting alpha(v) integrins with antibody-maytansinoid conjugates and to test the relative in vivo activities of several linker-maytansinoid chemistries. EXPERIMENTAL DESIGN: CNTO 364, CNTO 365, and CNTO 366 are targeted cytotoxic agents created by conjugating the CNTO 95 anti-alpha(v) integrin antibody with three distinct maytansinoid-linker structures. These structures were designed to have varying degrees of chemical substitution surrounding the disulfide bond linking the cytotoxic agent to the antibody. A model conjugate was shown to be specifically cytotoxic in vitro and highly active against established human tumor xenografts in immunocompromised rats. The in vivo antitumor activities of CNTO 364, CNTO 365, and CNTO 366 were compared in rat xenograft models. RESULTS: CNTO 365, with a linker chemistry of expected intermediate stability, was shown to be substantially more active than the other two conjugates with lesser or greater substitution around the disulfide linkage. CONCLUSION: CNTO 95-maytansinoid immunoconjugates are potent antitumor agents against alpha(v) integrin-expressing human carcinomas. These studies show for the first time the feasibility of targeting alpha(v) integrins on solid tumors with tumor-activated prodrugs. The DM4 linker-maytansinoid configuration of CNTO 365 was substantially more active in the models tested here when compared with alternative configurations with greater or lesser chemical substitution surrounding the linker.

CNTO 859, a humanized anti-tissue factor monoclonal antibody, is a potent inhibitor of breast cancer metastasis and tumor growth in xenograft models.

Thromboembolic complications are frequently associated with advanced cancer. Interestingly, one of the major initiators of blood coagulation, tissue factor (TF), is reported to be overexpressed in several tumor types and can be found on both tumor cells and tumor vasculature. Although the exact mechanisms have yet to be elucidated, TF expressed on tumor cells can trigger intracellular signaling events through various pathways that can lead to tumor angiogenesis, proliferation, and metastasis. There exists preclinical evidence that disruption of TF dependent signaling can effectively inhibit tumor cell migration, metastasis, and angiogenesis. Here, we report for the first time that an antibody to tissue factor can also prevent tumor growth in vivo. Prophylactic administration of CNTO 859, a humanized anti-human TF antibody, was shown to inhibit experimental lung metastasis of MDA-MB-231 human breast carcinoma cells by over 99% compared to a control antibody. Furthermore, therapeutic doses of CNTO 859 were shown to reduce tumor incidence and growth of orthotopically implanted MDA-MB-231 cells.

Therapeutic potential of cytokine and chemokine antagonists in cancer therapy.

A new paradigm is becoming widely accepted, that chronic inflammation, driven in part by chemokines and cytokines at the site of a tumour, may facilitate tumour progression instead of promoting anti-tumour immunity. Tumours and activated stromal cells secrete pro-inflammatory chemokines and cytokines that act either directly or indirectly through stimulation of the vascular endothelium to recruit leukocytes to the tumour. After activation, these tumour-associated leukocytes release angiogenic factors, mitogens, proteolytic enzymes, and chemotactic factors, recruiting more inflammatory cells and stimulating angiogenesis to sustain tumour growth and facilitate tumour metastasis. Breaking this cycle by inhibiting targets such as cytokines, chemokines and other inflammatory mediators, either alone, or more realistically, in combination with other therapies, such as anti-angiogenic or cytotoxic agents, may provide highly efficacious therapeutic regimens for the treatment of malignancies. This article reviews anti-cytokine and anti-chemokine therapies being pursued in cancer, and discusses in more detail anti-tumour necrosis factor-alpha (TNF) approaches.

Tumor necrosis factor-alpha in the pathogenesis and treatment of cancer.

The critical pathogenic role of tumor necrosis factor (TNF)alpha in inflammatory disorders such as rheumatoid arthritis and inflammatory bowel disease is well established. The role played by TNFalpha in both the treatment and pathogenesis of cancer remains less understood. Recent advances help to create a framework for understanding seemingly paradoxical effects of TNFalpha as both an anti-tumour agent and a mediator of tumour growth. High pharmacological doses of TNFalpha combined with chemotherapy can regress otherwise intractable tumours, and efforts continue to optimize delivery to avoid severe toxicities. Mounting evidence demonstrates that pathophysiological concentrations of endogenous TNFalpha act to promote tumour genesis and growth. The cellular and molecular pathways mediating these phenomena are starting to be clarified. Current data support the continued development of both TNFalpha and anti-TNFalpha therapy for clinical treatment of cancers in distinct settings.