Preclinical Assessment of AMG 596, a Bispecific T-cell Engager (BiTE) Immunotherapy Targeting the Tumor-specific Antigen EGFRvIII.

AMG 596 is a bispecific T-cell engager (BiTE) immuno-oncology therapy in clinical development for treatment of glioblastoma multiforme (GBM), the most common primary brain tumor in adults with limited therapeutic options. AMG 596 is composed of two single-chain variable fragments that simultaneously bind to the tumor-specific antigen, EGFR variant III (EGFRvIII), on GBM cells and to CD3 on T cells, thereby activating T cells to proliferate and secrete cytotoxic substances that induce lysis of the bound tumor cell. T-cell-redirected lysis by AMG 596 is very potent; in vitro studies revealed EC50 values in the low picomolar range, and in vivo studies showed that AMG 596 treatment significantly increased the overall survival of mice bearing EGFRvIII-expressing orthotopic tumors. In addition, AMG 596 activity is highly specific; no AMG 596-induced T-cell activity can be observed in assays with EGFRvIII-negative GBM cells, and no signs of toxicity and activity were observed in cynomolgus monkeys, which lack expression of EGFRvIII on normal tissues. With EGFRvIII-expressing GBM cells, we showed shedding of EGFRvIII-containing membrane vesicles, followed by vesicle uptake and EGFRvIII cell surface presentation by EGFRvIII noncoding GBM cells. Cell membrane presentation of EGFRvIII following microvesicle transfer allows engagement by AMG 596, resulting in T-cell activation and T-cell-dependent lysis of GBM cells. Together, these data show a compelling preclinical efficacy and safety profile of AMG 596, supporting its development as a novel immunotherapy for treatment of GBM.

AMG 701 induces cytotoxicity of multiple myeloma cells and depletes plasma cells in cynomolgus monkeys.

Multiple myeloma (MM) is a hematologic malignancy that is characterized by the accumulation of abnormal plasma cells (PCs) in the bone marrow (BM). Patient outcome may be improved with BiTE (bispecific T-cell engager) molecules, which redirect T cells to lyse tumor cells. B-cell maturation antigen (BCMA) supports PC survival and is highly expressed on MM cells. A half-life extended anti-BCMA BiTE molecule (AMG 701) induced selective cytotoxicity against BCMA-expressing MM cells (average half-maximal effective concentration, 18.8 ± 14.8 pM), T-cell activation, and cytokine release in vitro. In a subcutaneous mouse xenograft model, at all doses tested, AMG 701 completely inhibited tumor formation (P < .001), as well as inhibited growth of established tumors (P ≤ .001) and extended survival in an orthotopic MM model (P ≤ .01). To evaluate AMG 701 bioactivity in cynomolgus monkeys, a PC surface phenotype and specific genes were defined to enable a quantitative digital droplet polymerase chain reaction assay (sensitivity, 0.1%). Dose-dependent pharmacokinetic and pharmacodynamic behavior was observed, with depletion of PC-specific genes reaching 93% in blood and 85% in BM. Combination with a programmed cell death protein 1 (PD-1)-blocking antibody significantly increased AMG 701 potency in vitro. A model of AMG 701 binding to BCMA and CD3 indicates that the distance between the T-cell and target cell membranes (ie, the immunological synapse) is similar to that of the major histocompatibility complex class I molecule binding to a T-cell receptor and suggests that the synapse would not be disrupted by the half-life extending Fc domain. These data support the clinical development of AMG 701.

Molecular Imaging of Radiolabeled Bispecific T-Cell Engager 89Zr-AMG211 Targeting CEA-Positive Tumors.

Purpose: AMG 211, a bispecific T-cell engager (BiTE) antibody construct, targets carcinoembryonic antigen (CEA) and the CD3 epsilon subunit of the human T-cell receptor. AMG 211 was labeled with zirconium-89 (89Zr) or fluorescent dye to evaluate the tumor-targeting properties.Experimental Design: 89Zr-AMG211 was administered to mice bearing CEA-positive xenograft tumors of LS174T colorectal adenocarcinoma or BT474 breast cancer cells, as well as CEA-negative HL-60 promyelocytic leukemia xenografts. Biodistribution studies with 2- to 10-µg 89Zr-AMG211 supplemented with unlabeled AMG 211 up to 500-µg protein dose were performed. A BiTE that does not bind CEA, 89Zr-Mec14, served as a negative control. 89Zr-AMG211 integrity was determined in tumor lysates ex vivo Intratumoral distribution was studied with IRDye800CW-AMG211. Moreover, 89Zr-AMG211 was manufactured according to Good Manufacturing Practice (GMP) guidelines for clinical trial NCT02760199Results: 89Zr-AMG211 demonstrated dose-dependent tumor uptake at 6 hours. The highest tumor uptake was observed with a 2-µg dose, and the lowest tumor uptake was observed with a 500-µg dose. After 24 hours, higher uptake of 10-µg 89Zr-AMG211 occurred in CEA-positive xenografts, compared with CEA-negative xenografts. Although the blood half-life of 89Zr-AMG211 was approximately 1 hour, tumor retention persisted for at least 24 hours. 89Zr-Mec14 showed no tumor accumulation beyond background level. Ex vivo autoradiography revealed time-dependent disintegration of 89Zr-AMG211. 800CW-AMG211 was specifically localized in CEA-expressing viable tumor tissue. GMP-manufactured 89Zr-AMG211 fulfilled release specifications.Conclusions: 89Zr-AMG211 showed dose-dependent CEA-specific tumor targeting and localization in viable tumor tissue. Our data enabled its use to clinically evaluate AMG 211 in vivo behavior. Clin Cancer Res; 24(20); 4988-96. ©2018 AACR.

Bone morphogenetic protein-7 release from endogenous neural precursor cells suppresses the tumourigenicity of stem-like glioblastoma cells.

Glioblastoma cells with stem-like properties control brain tumour growth and recurrence. Here, we show that endogenous neural precursor cells perform an anti-tumour response by specifically targeting stem-like brain tumour cells. In vitro, neural precursor cells predominantly express bone morphogenetic protein-7; bone morphogenetic protein-7 is constitutively released from neurospheres and induces canonical bone morphogenetic protein signalling in stem-like glioblastoma cells. Exposure of human and murine stem-like brain tumour cells to neurosphere-derived bone morphogenetic protein-7 induces tumour stem cell differentiation, attenuates stem-like marker expression and reduces self-renewal and the ability for tumour initiation. Neurosphere-derived or recombinant bone morphogenetic protein-7 reduces glioblastoma expansion from stem-like cells by down-regulating the transcription factor Olig2. In vivo, large numbers of bone morphogenetic protein-7-expressing neural precursors encircle brain tumours in young mice, induce canonical bone morphogenetic protein signalling in stem-like glioblastoma cells and can thereby attenuate tumour formation. This anti-tumour response is strongly reduced in older mice. Our results indicate that endogenous neural precursor cells protect the young brain from glioblastoma by releasing bone morphogenetic protein-7, which acts as a paracrine tumour suppressor that represses proliferation, self-renewal and tumour-initiation of stem-like glioblastoma cells.

A common haplotype of the IL-31 gene influencing gene expression is associated with nonatopic eczema.

BACKGROUND: IL-31 is a novel cytokine that, when overexpressed in transgenic mice, induces severe itching dermatitis resembling human eczema. OBJECTIVE: We aimed to evaluate the importance of polymorphisms in the human IL-31 gene (IL31) in the genetic susceptibility to eczema. METHODS: We sequenced the entire IL-31 gene, including the promoter region, and determined the haplotype structure. Single nucleotide polymorphisms tagging the main haplotypes were genotyped in 3 independent European populations comprising 690 affected families. An association analysis of IL31 gene variants with atopic and nonatopic eczema was performed. RESULTS: We found significant association of a common IL31 haplotype with the nonatopic type of eczema in all 3 study populations (combined P = 4.5 x 10(-5)). Analysis of PBMCs in healthy individuals revealed a strong induction IL31 mRNA expression on stimulation with anti-CD3 and anti-CD28 that was 3.8-fold higher in individuals homozygous for the risk haplotype (AA) in contrast to non-A haplotype carriers, suggesting that altered regulation of IL-31 gene expression is the disease-causing factor. CONCLUSION: Our results lend strong support to an important role of IL-31 in the pathogenesis of nonatopic eczema. CLINICAL IMPLICATIONS: This study presents the first genetic risk factor for the nonatopic type of eczema and indicates a primary role of IL-31-induced pruritus in the initiation of this disease, thus proposing a new target for the prevention and therapy of eczema.

Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression.

In the immune system, extracellular ATP functions as a "natural adjuvant" that exhibits multiple proinflammatory effects. It is released by damaged cells as an indicator of trauma and cell death but can be inactivated by CD39 (nucleoside triphosphate diphosphohydrolase-1 [NTPDase 1]), an ectoenzyme that degrades ATP to AMP. Here, we show that CD39 is expressed primarily by immune-suppressive Foxp3(+) regulatory T (Treg) cells. In mice, the enzyme is present on virtually all CD4(+)CD25(+) cells. CD39 expression is driven by the Treg-specific transcription factor Foxp3 and its catalytic activity is strongly enhanced by T-cell receptor (TCR) ligation. Activated Treg cells are therefore able to abrogate ATP-related effects such as P2 receptor-mediated cell toxicity and ATP-driven maturation of dendritic cells. Also, human Treg cells express CD39. In contrast to mice, CD39 expression in man is restricted to a subset of Foxp3(+) regulatory effector/memory-like T (T(REM)) cells. Notably, patients with the remitting/relapsing form of multiple sclerosis (MS) have strikingly reduced numbers of CD39(+) Treg cells in the blood. Thus, in humans CD39 is a marker of a Treg subset likely involved in the control of the inflammatory autoimmune disease.