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CC-90002 is an anti-CD47 antibody that inhibits CD47-SIRPα interaction and enables macrophage-mediated killing of tumor cells in hematological cancer cell lines. In this first clinical, phase 1, dose-escalation and -expansion study (CC-90002-AML-001; NCT02641002), we evaluated CC-90002 in patients with relapsed/refractory acute myeloid leukemia (AML) or high-risk myelodysplastic syndromes (MDS). CC-90002 was administered in escalating doses of 0.1-4.0 mg/kg, using a modified 3 + 3 design. Primary endpoints included dose-limiting toxicities (DLTs), non-tolerated dose (NTD), maximum tolerated dose (MTD), and recommended phase 2 dose. Secondary endpoints included preliminary efficacy, pharmacokinetics, and presence/frequency of anti-drug antibodies (ADAs). Between March 2016 and July 2018, 28 patients were enrolled (24 with AML and 4 with MDS) at 6 sites across the USA. As of July 18, 2018, all patients had discontinued, mainly due to death or progressive disease. The most common treatment-emergent adverse events were diarrhea (46.4%), thrombocytopenia (39.3%), febrile neutropenia (35.7%), and aspartate aminotransferase increase (35.7%). Four patients experienced DLTs (1 patient had grade 4 disseminated intravascular coagulation and grade 5 cerebral hemorrhage, 1 had grade 3 purpura, 1 had grade 4 congestive cardiac failure and grade 5 acute respiratory failure, and another had grade 5 sepsis). The NTD and MTD were not reached. No objective responses occurred. CC-90002 serum exposure was dose-dependent. ADAs were present across all doses, and the proportion of ADA-positive patients in cycle 1 increased over time. Despite no unexpected safety findings, the CC-90002-AML-001 study was discontinued in dose escalation for lack of monotherapy activity and evidence of ADAs. However, as other anti-CD47 agents in clinical trials are showing promising early results for AML and MDS, understanding preclinical and clinical differences between individual agents in this class will be of high importance., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cluster of differentiation 47 (CD47) is a transmembrane protein ubiquitously expressed on human cells but overexpressed on many different tumor cells. The interaction of CD47 with signal-regulatory protein alpha (SIRPα) triggers a "don't eat me" signal to the macrophage, inhibiting phagocytosis. Thus, overexpression of CD47 enables tumor cells to escape from immune surveillance via the blockade of phagocytic mechanisms. We report here the development and characterization of CC-90002, a humanized anti-CD47 antibody. CC-90002 is unique among previously reported anti-CD47 bivalent antibodies that it does not promote hemagglutination while maintaining high-affinity binding to CD47 and inhibition of the CD47-SIRPα interaction. Studies in a panel of hematological cancer cell lines showed concentration-dependent CC-90002-mediated phagocytosis in acute lymphoblastic leukemia, acute myeloid leukemia (AML), lenalidomide-resistant multiple myeloma (MM) cell lines and AML cells from patients. In vivo studies with MM cell line-derived xenograft models established in immunodeficient mice demonstrated significant dose-dependent antitumor activity of CC-90002. Treatment with CC-90002 significantly prolonged survival in an HL-60-disseminated AML model. Mechanistic studies confirmed the binding of CC-90002 to tumor cells and concomitant recruitment of F4-80 positive macrophages into the tumor and an increase in expression of select chemokines and cytokines of murine origin. Furthermore, the role of macrophages in the CC-90002-mediated antitumor activity was demonstrated by transient depletion of macrophages with liposome-clodronate treatment. In non-human primates, CC-90002 displayed acceptable pharmacokinetic properties and a favorable toxicity profile. These data demonstrate the potential activity of CC-90002 across hematological malignancies and provided basis for clinical studies CC-90002-ST-001 (NCT02367196) and CC-90002-AML-001 (NCT02641002)., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

An international expert group which includes 30 organisations (pharmaceutical companies, contract research organisations, academic institutions and regulatory bodies) has shared data on the use of recovery animals in the assessment of pharmaceutical safety for early development. These data have been used as an evidence-base to make recommendations on the inclusion of recovery animals in toxicology studies to achieve scientific objectives, while reducing animal use. Recovery animals are used in pharmaceutical development to provide information on the potential for a toxic effect to translate into long-term human risk. They are included on toxicology studies to assess whether effects observed during dosing persist or reverse once treatment ends. The group devised a questionnaire to collect information on the use of recovery animals in general regulatory toxicology studies to support first-in-human studies. Questions focused on study design, the rationale behind inclusion or exclusion and the impact this had on internal and regulatory decisions. Data on 137 compounds (including 53 biologicals and 78 small molecules) from 259 studies showed wide variation in where, when and why recovery animals were included. An analysis of individual study and programme design shows that there are opportunities to reduce the use of recovery animals without impacting drug development., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

This study was conducted to assess the safety and tolerability of the alternative formulation vehicles polysorbate 80 (PS80), propylene glycol (PG), and hydroxypropyl-beta-cyclodextrin (HPβCD) in general toxicology studies in the mouse, rat, dog, and monkey. Twenty (20) mg/kg of hydroxypropyl methylcellulose (MC, control), 10 mg/kg PS80, 1000 mg/kg PG, 500 mg/kg HPβCD, or 1000 mg/kg HPβCD were administered by oral gavage to mice, rats, dogs, and cynomolgus monkeys for approximately 90 days. The effects of these formulations on clinical observations, body weight and food consumption parameters, clinical pathology, and histopathology were evaluated across all species. The suitability of formulations containing up to 20 mg/kg MC, 10 mg/kg PS80, and 1000 mg/kg PG for use in preclinical safety studies was confirmed by a lack of effects on all parameters examined. However, formulations containing HPβCD produced elevated transaminase (aspartate and alanine aminotransferase) levels in rats and mice and fecal changes (loose and soft stool) in large animals. Although the etiology and toxicological significance of the transaminase elevations in rats and mice is uncertain, this finding could represent a significant liability for a preclinical formulation because of the critical importance of these biomarkers in the risk assessment of novel therapeutic agents. Based on these data, PS80 and PG are considered to be practical alternatives to MC in preclinical toxicology studies. However, formulations containing HPβCD should be used with caution because of the elevations in rodent transaminase levels.

Many laboratory strains of Escherichia coli are resistant to methotrexate (MTX), a folate analogue that binds dihydrofolate reductase (DHFR). Mutations that inactivate either tolC or acrA confer MTX sensitivity. Further, overexpression of a fusion protein with DHFR activity reverses this sensitivity by titrating out intracellular MTX. These results suggest that MTX accumulates in cells where mutations in acrA or tolC have inactivated the TolC-dependent AcrAB multidrug resistance efflux pump.

Background: Chemically induced dimerization (CID) can be used to manipulate cellular regulatory pathways from signal transduction to transcription, and to create model systems for study of the specific interactions between proteins and small-molecule chemical ligands. However, few CID systems are currently available. The properties of, and interactions between, Escherichia coli dihydrofolate reductase (DHFR) and the ligand methotrexate (MTX) meet many of the desired criteria for the development of a new CID system., Results: BisMTX, a homobifunctional version of MTX, was synthesized and tested for its ability to induce dimerization of DHFR. Gel-filtration analysis of purified DHFR confirmed that, in vitro, the protein was a monomer in the absence of dimerizer drug; in the presence of bisMTX, a complex of twice the monomeric molecular weight was observed. Furthermore, the off-rate was found to be 0.0002 s(-1), approximately 100 times slower than that reported for DHFR-MTX. Interestingly, the addition of excess bisMTX did not result in formation of the binary complex (1 protein:1 dimerizer) over the ternary complex (2 proteins:1 dimerizer), which suggests cooperative binding interactions (affinity modulation) between the two DHFR molecules in the bisMTX:DHFR(2) ternary complex., Conclusions: The combination of DHFR and bisMTX provides a new CID system with properties that could be useful for applications in vivo. Formation of the bisMTX:DHFR(2) ternary complex in vitro is promoted over a wide range of dimerizer concentrations, consistent with the idea that formation of the ternary complex recruits energetically favorable interactions between the DHFR monomers in the complex.

Transcription initiation by RNA polymerase II is a complex, multistep process that minimally involves transcription complex assembly, open complex formation, and promoter clearance. Hydrolysis of the beta--gamma phosphoanhydride bond of ATP has previously been shown to be required for open complex formation, as well as for the phosphorylation of the carboxy-terminal domain of the largest subunit of RNA polymerase II. The observation that ATP-dependent activation of transcription complexes can be blocked by ATP analogues that contain nonhydrolyzable beta--gamma phosphoanhydride bonds (such as ATPgammaS) was exploited to develop a functional kinetic assay for ATP-activated transcription complexes. Activated complexes on the promoter present in the long terminal repeat of the proviral DNA of mouse mammary tumor virus were defined as those that could productively initiate transcription in the presence of excess ATPgammaS. Activation is dependent on treatment of assembled preinitiation complexes with ATP (or dATP) prior to addition of ATPgammaS. At least 15-35% of the total number of preinitiation complexes present become activated within 2 min in the presence of (d)ATP, and activation appears to be rapidly reversible. The time course of formation of activated complexes in the presence of dATP is characterized by two kinetic phases: a rapid formation followed by a relatively slow decay. Activated complexes were estimated to form with a half-time of less than 1 min.