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T-cell engager (TCE) molecules provide a targeted immunotherapy approach to treat hematologic malignancies and solid tumors. Since the approval of the CD19-targeted BiTE® (bispecific T-cell engager) molecule blinatumomab, multiple TCE molecules against different targets have been developed in several tumor types, with the approval of three additional TCE molecules in 2022. Some of the initial challenges, such as the need for continuous intravenous administration and low productivity, have been addressed in subsequent iterations of the platform by advancing half-life extended, Fc-based molecules. As clinical data from these molecules emerge, additional optimization of formats and manufacturability will be necessary. Ongoing efforts are focused on further improving TCE efficacy, safety, and convenience of administration., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A comprehensive understanding of structure-reactivity relationships is critical to the design and optimization of cysteine-targeted covalent inhibitors. Herein, we report glutathione (GSH) reaction rates for N -phenyl acrylamides with varied substitutions at the α- and β-positions of the acrylamide moiety. We find that the GSH reaction rates can generally be understood in terms of the electron donating or withdrawing ability of the substituent. When installed at the β-position, aminomethyl substituents with amine p K a 's > 7 accelerate, while those with p K a 's < 7 slow the rate of GSH addition at pH 7.4, relative to a hydrogen substituent. Although a computational model was able to only approximately capture experimental reactivity trends, our calculations do not support a frequently invoked mechanism of concerted amine/thiol proton transfer and C-S bond formation and instead suggest that protonated aminomethyl functions as an electron-withdrawing group to reduce the barrier for thiolate addition to the acrylamide.

The serum level of iron in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing the internalization and degradation of ferroportin 1 . Aberrant ferroportin activity can lead to diseases of iron overload, such as haemochromatosis, or iron limitation anaemias 2 . Here we determine cryogenic electron microscopy structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with hepcidin and the iron mimetic cobalt. These structures and accompanying molecular dynamics simulations identify two metal-binding sites within the N and C domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy terminus of hepcidin directly contacts the divalent metal in the ferroportin C domain. Hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, in which only ferroportin molecules loaded with iron are targeted for degradation. More broadly, our structural and functional insights may enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.

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., (© 2020 by The American Society of Hematology.)

We investigated here the novel immunomodulation and anti-multiple myeloma (MM) function of T cells engaged by the bispecific T-cell engager molecule AMG 701, and further examined the impact of AMG 701 in combination with immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide). AMG 701 potently induced T-cell-dependent cellular cytotoxicity (TDCC) against MM cells expressing B-cell maturation antigen, including autologous cells from patients with relapsed and refractory MM (RRMM) (half maximal effective concentration, <46.6 pM). Besides inducing T-cell proliferation and cytolytic activity, AMG 701 also promoted differentiation of patient T cells to central memory, effector memory, and stem cell-like memory (scm) phenotypes, more so in CD8 vs CD4 T subsets, resulting in increased CD8/CD4 ratios in 7-day ex vivo cocultures. IMiDs and AMG 701 synergistically induced TDCC against MM cell lines and autologous RRMM patient cells, even in the presence of immunosuppressive bone marrow stromal cells or osteoclasts. IMiDs further upregulated AMG 701-induced patient T-cell differentiation toward memory phenotypes, associated with increased CD8/CD4 ratios, increased Tscm, and decreased interleukin 10-positive T and T regulatory cells (CD25highFOXP3high), which may downregulate T effector cells. Importantly, the combination of AMG 701 with lenalidomide induced sustained inhibition of MM cell growth in SCID mice reconstituted with human T cells; tumor regrowth was eventually observed in cohorts treated with either agent alone (P < .001). These results strongly support AMG 701 clinical studies as monotherapy in patients with RRMM (NCT03287908) and the combination with IMiDs to improve patient outcomes in MM., (© 2020 by The American Society of Hematology.)

Feline McDonough Sarcoma-like tyrosine kinase 3 (FLT3), a tyrosine-protein kinase involved in hematopoiesis, is detectable on the cell surface of approximately 80% of leukemia isolates from adult patients with acute myeloid leukemia (AML). AMG 553 is an investigational chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of AML. FLT3 expression analysis and in vitro and in vivo studies were leveraged to evaluate the nonclinical safety of AMG 553. Cynomolgus monkeys administered autologous anti-FLT3 CAR T cells demonstrated no evidence of CAR T-cell-mediated toxicity, expansion, or persistence, likely due to restricted cell surface FLT3 protein expression in healthy animals. This highlights the limited value of such in vivo studies for safety assessment of the CAR T-cell modality when directed against a target with restricted expression. To complement these studies and directly evaluate the potential toxicities of eliciting T-cell-mediated cytotoxicity against cells with surface expression of FLT3 protein in vivo, data from cynomolgus monkey toxicology studies with 2 bispecific T-cell engager molecules targeting FLT3 were leveraged; findings were consistent with the targeted killing of bone marrow cells expressing cell surface FLT3. Potential AMG 553-induced cytotoxicity was assessed against a wide range of normal human primary cells and cell lines; cytotoxicity was observed against FLT3-positive AML cell lines and a percentage of primary bone marrow CD34+ cells. In conclusion, the nonclinical safety data suggest that AMG 553 can target FLT3 protein on AML cells, whereas only affecting a percentage of normal hematopoietic stem and progenitor cells, supporting clinical development., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society of Toxicology.)

Despite advances in the treatment of acute myeloid leukemia (AML), novel therapies are needed to induce deeper and more durable clinical response. Bispecific T-cell Engager (BiTE) molecules, which redirect patient T cells to lyse tumor cells, are a clinically validated modality for hematologic malignancies. Due to broad AML expression and limited normal tissue expression, fms-related tyrosine kinase 3 (FLT3) is proposed to be an optimal BiTE molecule target. Expression profiling of FLT3 was performed in primary AML patient samples and normal hematopoietic cells and nonhematopoietic tissues. Two novel FLT3 BiTE molecules, one with a half-life extending (HLE) Fc moiety and one without, were assessed for T-cell-dependent cellular cytotoxicity (TDCC) of FLT3-positive cell lines in vitro, in vivo , and ex vivo FLT3 protein was detected on the surface of most primary AML bulk and leukemic stem cells but only a fraction of normal hematopoietic stem and progenitor cells. FLT3 protein detected in nonhematopoietic cells was cytoplasmic. FLT3 BiTE molecules induced TDCC of FLT3-positive cells in vitro , reduced tumor growth and increased survival in AML mouse models in vivo Both molecules exhibited reproducible pharmacokinetic and pharmacodynamic profiles in cynomolgus monkeys in vivo , including elimination of FLT3-positive cells in blood and bone marrow. In ex vivo cultures of primary AML samples, patient T cells induced TDCC of FLT3-positive target cells. Combination with PD-1 blockade increased BiTE activity. These data support the clinical development of an FLT3 targeting BiTE molecule for the treatment of AML., (©2020 American Association for Cancer Research.)

Bleeding and altered iron distribution occur in multiple gastrointestinal diseases, but the importance and regulation of these changes remain unclear. We found that hepcidin, the master regulator of systemic iron homeostasis, is required for tissue repair in the mouse intestine after experimental damage. This effect was independent of hepatocyte-derived hepcidin or systemic iron levels. Rather, we identified conventional dendritic cells (cDCs) as a source of hepcidin that is induced by microbial stimulation in mice, prominent in the inflamed intestine of humans, and essential for tissue repair. cDC-derived hepcidin acted on ferroportin-expressing phagocytes to promote local iron sequestration, which regulated the microbiota and consequently facilitated intestinal repair. Collectively, these results identify a pathway whereby cDC-derived hepcidin promotes mucosal healing in the intestine through means of nutritional immunity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours 1,2 . The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity 3-5 . Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRAS G12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRAS G12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

KRAS regulates many cellular processes including proliferation, survival, and differentiation. Point mutants of KRAS have long been known to be molecular drivers of cancer. KRAS p.G12C , which occurs in approximately 14% of lung adenocarcinomas, 3-5% of colorectal cancers, and low levels in other solid tumors, represents an attractive therapeutic target for covalent inhibitors. Herein, we disclose the discovery of a class of novel, potent, and selective covalent inhibitors of KRAS G12C identified through a custom library synthesis and screening platform called Chemotype Evolution and structure-based design. Identification of a hidden surface groove bordered by H95/Y96/Q99 side chains was key to the optimization of this class of molecules. Best-in-series exemplars exhibit a rapid covalent reaction with cysteine 12 of GDP-KRAS G12C with submicromolar inhibition of downstream signaling in a KRAS G12C -specific manner., Competing Interests: The authors declare no competing financial interest.

For targets that are homogenously expressed, such as CD19 on cells of the B lymphocyte lineage, immunotherapies can be highly effective. Targeting CD19 with blinatumomab, a CD19/CD3 bispecific antibody construct (BiTE®), or with chimeric antigen receptor T cells (CAR-T) has shown great promise for treating certain CD19-positive hematological malignancies. In contrast, solid tumors with heterogeneous expression of the tumor-associated antigen (TAA) may present a challenge for targeted therapies. To prevent escape of TAA-negative cancer cells, immunotherapies with a local bystander effect would be beneficial. As a model to investigate BiTE®-mediated bystander killing in the solid tumor setting, we used epidermal growth factor receptor (EGFR) as a target. We measured lysis of EGFR-negative populations in vitro and in vivo when co-cultured with EGFR-positive cells, human T cells and an EGFR/CD3 BiTE® antibody construct. Bystander EGFR-negative cells were efficiently lysed by BiTE®-activated T cells only when proximal to EGFR-positive cells. Our mechanistic analysis suggests that cytokines released by BiTE®-activated T-cells induced upregulation of ICAM-1 and FAS on EGFR-negative bystander cells, contributing to T cell-induced bystander cell lysis.

We have implemented a solid-phase extraction based time-of-flight mass spectrometer system in combination with novel informatics to rapidly screen and characterize the covalent binding of different irreversible inhibitors to intact proteins. This high-throughput screening platform can be used to accurately detect and quantitate the extent of formation of different covalent protein-inhibitor adducts between electrophilic inhibitors and nucleophilic residues such as cysteine or lysine. For a representative 19.5 kDa protein, the analysis time is approximately 20 s per sample, including an efficient sample loading and desalting step. Accurate protein masses are measured (±0.5 amu of the theoretical molecular weight; measured precision of ±0.02 amu). The fraction of protein reacted with an electrophilic compound is determined relative to an unmodified protein control. A key element of the workflow is the automated identification and quantitation of the expected masses of covalent protein-inhibitor adducts using a custom routine that obviates the need to manually inspect each individual spectrum. Parallel screens were performed on a library of approximately 1000 acrylamide containing compounds (different structures and reactivities) using the solid-phase extraction mass spectrometry based assay and a fluorescence based thiol-reactive probe assay enabling comparison of false positives and false negatives between these orthogonal screening approaches., (© 2015 Society for Laboratory Automation and Screening.)

Success in the design of targeted covalent inhibitors depends in part on a knowledge of the factors influencing electrophile reactivity. In an effort to further develop an understanding of structure-reactivity relationships among N-arylacrylamides, we determined glutathione (GSH) reaction rates for a family of N-arylacrylamides independently substituted at ortho-, meta-, and para-positions with 11 different groups common to inhibitor design. We find that substituent effects on reaction rates show a linear Hammett correlation for ortho-, meta-, and para-substitution. In addition, we note a correlation between (1)H and (13)C NMR chemical shifts of the acrylamide with GSH reaction rates, suggesting that NMR chemical shifts may be a convenient surrogate measure of relative acrylamide reactivity. Density functional theory calculations reveal a correlation between computed activation parameters and experimentally determined reaction rates, validating the use of such methodology for the screening of synthetic candidates in a prospective fashion.

Filgrastim, a recombinant methionyl human granulocyte colony-stimulating factor (G-CSF) (r-metHuG-CSF), is efficacious in stimulating neutrophil production and maturation to prevent febrile neutropenia (FN) in response to chemotherapy. Because of its relatively short circulating half-life, daily filgrastim injections are required to stimulate neutrophil recovery. In an effort to develop a long-acting form of filgrastim that was as safe and efficacious as filgrastim but had a longer in vivo residence time, a number of strategies were considered. Ultimately, fusion of filgrastim to polyethylene glycol (PEG) was selected. Following extensive analysis of conjugation chemistries as well as in vitro and in vivo characterization of a panel of PEGylated proteins, a construct containing a 20 kDa PEG moiety covalently conjugated to the N-terminus of filgrastim was chosen for advancement as pegfilgrastim. Pegfilgrastim is primarily cleared by neutrophils and neutrophil precursors (rather than the kidneys), meaning that clearance from the circulation is self-regulating and pegfilgrastim is eliminated only after neutrophils start to recover. Importantly, addition of PEG did not alter the mechanism of action and safety profile compared to filgrastim. Clinical evaluation revealed that a single 6 mg dose effectively reduces the duration of neutropenia and risk of FN in patients receiving chemotherapy. This work demonstrates the benefit of using PEGylation to generate pegfilgrastim, which allows for once-per-chemotherapy cycle administration while maintaining similar safety and efficacy profiles as those for multiple daily administration of filgrastim. Approaches that may provide advances for therapeutic agonists of G-CSF receptor are also discussed.

Hepcidin is a circulating peptide hormone made by the liver that is a central regulator of systemic iron uptake and recycling. Here, we report that prostate epithelial cells also synthesize hepcidin, and that synthesis and secretion of hepcidin are markedly increased in prostate cancer cells and tissue. Prostatic hepcidin functions as an autocrine hormone, decreasing cell surface ferroportin, an iron exporter, increasing intracellular iron retention, and promoting prostate cancer cell survival. Synthesis of hepcidin in prostate cancer is controlled by a unique intersection of pathways that involves BMP4/7, IL6, Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epigenetic silencing of SOSTDC1 through methylation is increased in prostate cancer and is associated with accelerated disease progression in patients with prostate cancer. These results establish a new connection between iron metabolism and prostate cancer, and suggest that prostatic dysregulation of hepcidin contributes to prostate cancer growth and progression., (©2015 American Association for Cancer Research.)

Hepcidin is a four disulfide 25-residue peptide hormone which has a central role in the regulation of iron homeostasis. To support studies on hepcidin we have sought to establish reliable and robust synthetic methods for the preparation of correctly folded materials. While correctly-folded hepcidin has good aqueous solubility, we have found that its direct synthetic precursor, linear (reduced) hepcidin peptide, is resistant to solubilization, prone to precipitation at pH > or = 6, and thus difficult to fold efficiently. Attempts to directly fold either the crude or purified linear hepcidin peptide by air or DMSO oxidation methods under basic conditions were ineffective. However, addition of a glutathione redox pair system improved folding of purified linear hepcidin at mild basic pH (pH 7.5). Under acidic conditions, it was possible to oxidatively fold both crude and purified hepcidin using a polymer-supported oxidizing strategy. Peptide precipitation was also avoided under acidic conditions. Isolated folding yields of human hepcidin under acidic polymer-assisted conditions were superior to yields under basic folding conditions. These studies enabled identification of a reliable synthetic route for correctly-folded hepcidin.

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys(1)-Cys(8), Cys(3)-Cys(6), Cys(2)-Cys(4), and Cys(5)-Cys(7). NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.

HFE2 (hemochromatosis type 2 gene) is highly expressed in skeletal muscle and liver hepatocytes. Its encoded protein, hemojuvelin (HJV), is a co-receptor for the bone morphogenetic proteins 2 and 4 (BMP2 and BMP4) and enhances the BMP-induced hepcidin expression. Hepcidin is a central iron regulatory hormone predominantly secreted from hepatocytes. HJV also binds neogenin, a membrane protein widely expressed in many tissues. Neogenin is required for the processing and release of HJV from cells. The role that neogenin plays in HJV trafficking was investigated, using HepG2 cells, a human hepatoma cell line. Knockdown of endogenous neogenin markedly suppresses HJV release but has no evident effect on HJV trafficking to the plasma membrane. The addition of a soluble neogenin ectodomain to cells markedly inhibits HJV release, indicating that the HJV shedding is not processed before trafficking to the cell surface. At the plasma membrane it undergoes endocytosis in a dynamin-independent but cholesterol-dependent manner. The additional findings that HJV release is coupled to lysosomal degradation of neogenin and that cholesterol depletion by filipin blocks both HJV endocytosis and HJV release suggest that neogenin-mediated HJV release occurs after the HJV-neogenin complex is internalized from the cell surface.