Integrated Pharmacokinetic/Pharmacodynamic Analysis for Determining the Minimal Anticipated Biological Effect Level of a Novel Anti-CD28 Receptor Antagonist BMS-931699.

BMS-931699 (lulizumab pegol), a domain antibody (dAb) conjugated with 40-kDa branched polyethylene glycol, is a human anti-CD28 receptor antagonist under development for the treatment of inflammatory and autoimmune diseases. In the present work, the minimal anticipated biologic effect level (MABEL) was determined for BMS-931699 by integrating all the available preclinical data. The relevance of the in vitro mixed lymphocyte reaction (MLR) assay to a whole blood CD28 receptor occupancy (RO) assessment, as well as the relationship between the CD28 RO and the inhibition of T-cell-dependent antibody response to keyhole limpet hemocyanin in vivo, was demonstrated through an integrated pharmacokinetic/pharmacodynamic analysis using anti-hCD28 dAb-001 (differing from BMS-931699 by two additional amino acids at the N-terminus) and a mouse surrogate. Based on this analysis, the EC10 value (0.32 nM) from the human MLR assay and the human plasma volume (0.04 l/kg) were employed to calculate the MABEL (0.01 mg) of BMS-931699 in humans, with a CD28 RO predicted to be ≤10%. The estimated MABEL dose was threefold higher than the value derived from the binding constant and twofold less than the MABEL converted from animal efficacy studies based on the body surface area. Furthermore, it was 2900-fold lower than the human equivalent dose derived from the no observed adverse effect level in monkeys (15 mg/kg/week for 5 doses, intravenous dosing) with a 10-fold safety factor applied. Therefore, the MABEL dose represented a sound approach to mitigate any potential risk in targeting CD28 and was successfully used as the first-in-human starting dose for BMS-931699.

PEGylated Biopharmaceuticals: Current Experience and Considerations for Nonclinical Development.

PEGylation (the covalent binding of one or more polyethylene glycol molecules to another molecule) is a technology frequently used to improve the half-life and other pharmaceutical or pharmacological properties of proteins, peptides, and aptamers. To date, 11 PEGylated biopharmaceuticals have been approved and there is indication that many more are in nonclinical or clinical development. Adverse effects seen with those in toxicology studies are mostly related to the active part of the drug molecule and not to polyethylene glycol (PEG). In 5 of the 11 approved and 10 of the 17 PEGylated biopharmaceuticals in a 2013 industry survey presented here, cellular vacuolation is histologically observed in toxicology studies in certain organs and tissues. No other effects attributed to PEG alone have been reported. Importantly, vacuolation, which occurs mainly in phagocytes, has not been linked with changes in organ function in these toxicology studies. This article was authored through collaborative efforts of industry toxicologists/nonclinical scientists to address the nonclinical safety of large PEG molecules (>10 kilo Dalton) in PEGylated biopharmaceuticals. The impact of the PEG molecule on overall nonclinical safety assessments of PEGylated biopharmaceuticals is discussed, and toxicological information from a 2013 industry survey on PEGylated biopharmaceuticals under development is summarized. Results will contribute to the database of toxicological information publicly available for PEG and PEGylated biopharmaceuticals.

A monovalent anti-human CD28 domain antibody antagonist: preclinical efficacy and safety.

Targeting the CD28-CD80/86 pathway with an anti-CD28 antagonist is a promising alternative to current therapies for autoimmunity. However, attempts at generating conventional anti-CD28 mAbs lacking stimulatory activity has been challenging. In this study, we describe anti-human CD28 receptor antagonist domain Abs (dAbs) that are specific for human CD28. These dAbs are potent inhibitors of T cell activation, with an EC50 of 35 ± 14 ng/ml for inhibition of proliferation. The EC50 of 53 ± 11 ng/ml in an ex vivo CD28 receptor occupancy assay corresponds with in vitro functional activity, suggesting a direct correlation. The anti-CD28 dAb is equipotent in the inhibition of CD80- and CD86-mediated T cell proliferation and does not interfere with CTLA-4-mediated downmodulation of CD86 expression on APCs. The anti-CD28 dAbs are monomeric and do not demonstrate any evidence of agonism or costimulatory activity. In cynomolgus monkeys, the anti-CD28 dAb demonstrated pharmacodynamic activity, as measured by the inhibition of a T cell-dependent Ab response, without evidence of T cell depletion or cytokine release. Furthermore, there was a strong correlation between systemic exposure, duration, and extent of CD28 receptor occupancy, and pharmacodynamic activity. Taken together, these data support clinical evaluation of this novel anti-CD28 dAb for the treatment of autoimmune diseases.

D-α-tocopheryl polyethylene glycol 1000 succinate-containing vehicles provide no detectable chemoprotection from oxidative damage.

The objective of this study was to evaluate potential protective effects of vehicles containing d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), which may impact nonclinical safety assessments of oxidative processes. This was achieved by evaluating plasma, liver and adrenal gland concentrations of d-α-tocopheryl succinate (TS) and d-α-tocopherol as well as oxidative status of plasma following oral dosing of TPGS-containing vehicles, intraperitoneal (IP) dosing of TS or ex vivo treatment of blood with H2O2. Male and female rats were dosed orally with formulations containing 5% or 40% TPGS (70 or 550 mg kg(-1) day(-1) TS, respectively) for 1 week. A control group was dosed orally with polyethylene glycol-400 (PEG-400; no vitamin E) and positive control animals received a single 100 mg kg(-1) day(-1) IP injection of TS. Whole blood from untreated animals was treated ex vivo with 5 or 50 mm H(2)O(2), with or without TS (0.5, 5, 50 or 500 µm) or ascorbate (1 mm), for 1 h. Oral TPGS treatments did not affect d-α-tocopherol concentrations in plasma or adrenal glands and caused only transient increases in liver. Concentrations of TS in plasma, liver and adrenal glands were undetectable in control animals, but increased in all other groups. Oral administration of TPGS did not reduce plasma lipid peroxidation in vivo. Substantially greater TS concentrations used ex vivo (100× greater than in vivo) were also unable to reduce lipid peroxidation in H2O2 -treated whole blood. These results provide evidence that administration of oral TPGS vehicles is unlikely to impact nonclinical safety assessments of pharmaceuticals.

Cytokines: The Good, the Bad, and the Deadly.

Over the past 30 years, the world of pharmaceutical toxicology has seen an explosion in the area of cytokines. An overview of the many aspects of cytokine safety evaluation currently in progress and evolving strategies for evaluating these important entities was presented at this symposium. Cytokines play a broad role to help the immune system respond to diseases, and drugs which modulate their effect have led to some amazing therapies. Cytokines may be "good" when stimulating the immune system to fight a foreign pathogen or attack tumors. Other "good" cytokine effects include reduction of an immune response, for example interferon ß reduction of neuron inflammation in patients with multiple sclerosis. They may be "bad" when their expression causes inflammatory diseases, such as the role of tumor necrosis factor α in rheumatoid arthritis or asthma and Crohn's disease. Therapeutic modulation of cytokine expression can help the "good" cytokines to generate or quench the immune system and block the "bad" cytokines to prevent damaging inflammatory events. However, care must be exercised, as some antibody therapeutics can cause "ugly" cytokine release which can be deadly. Well-designed toxicology studies should incorporate careful assessment of cytokine modulation that will allow effective therapies to treat unmet needs. This symposium discussed lessons learned in cytokine toxicology using case studies and suggested future directions.

Semimechanistic Physiologically-Based Pharmacokinetic/Pharmacodynamic Model Informing Epcoritamab Dose Selection for Patients With B-Cell Lymphomas.

Epcoritamab is a CD3xCD20 bispecific antibody (bsAb) that induces T-cell-mediated cytotoxicity against CD20-positive B cells. Target engagement and crosslinking of CD3 and CD20 (trimer formation) leads to activation and expansion of T cells and killing of malignant B cells. The primary objective of the dose-escalation part of the phase I/II trial of epcoritamab was to determine the maximum tolerated dose, recommended phase II dose (RP2D), or both. For bsAbs, high target saturation can negatively affect trimer formation. The unique properties and mechanisms of action of bsAbs require novel pharmacokinetic (PK) modeling methods to predict clinical activity and inform RP2D selection. Traditional PK/pharmacodynamic (PD) modeling approaches are inappropriate because they may not adequately predict exposure-response relationships. We developed a semimechanistic, physiologically-based PK/PD model to quantitatively describe biodistribution, trimer formation, and tumor response using preclinical, clinical PK, biomarker, tumor, and response data from the dose-escalation part of the phase I/II trial. Clinical trial simulations were performed to predict trimer formation and tumor response in patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL). Model-predicted trimer formation plateaued at doses of 48 to 96 mg. Simulation results suggest that the 48-mg dose may achieve optimal response rates in DLBCL and FL. Exposure-safety analyses showed a flat relationship between epcoritamab exposure and risk of cytokine release syndrome in the dose range evaluated. This novel PK/PD modeling approach guided selection of 48 mg as the RP2D and provides a framework that may be applied to other CD3 bsAbs.

An Fc-inert PD-L1×4-1BB bispecific antibody mediates potent anti-tumor immunity in mice by combining checkpoint inhibition and conditional 4-1BB co-stimulation.

Immune checkpoint inhibitors (ICI) targeting the PD-1/PD-L1 axis have changed the treatment paradigm for advanced solid tumors; however, many patients experience treatment resistance. In preclinical models 4-1BB co-stimulation synergizes with ICI by activating cytotoxic T- and NK-cell-mediated anti-tumor immunity. Here we characterize the mechanism of action of a mouse-reactive Fc-inert PD-L1×4-1BB bispecific antibody (mbsAb-PD-L1×4-1BB) and provide proof-of-concept for enhanced anti-tumor activity. In reporter assays mbsAb-PD-L1×4-1BB exhibited conditional 4-1BB agonist activity that was dependent on simultaneous binding to PD-L1. mbsAb-PD-L1×4-1BB further blocked the PD-L1/PD-1 interaction independently of 4-1BB binding. By combining both mechanisms, mbsAb-PD-L1×4-1BB strongly enhanced T-cell proliferation, cytokine production and antigen-specific cytotoxicity using primary mouse cells in vitro. Furthermore, mbsAb-PD-L1×4-1BB exhibited potent anti-tumor activity in the CT26 and MC38 models in vivo, leading to the rejection of CT26 tumors that were unresponsive to PD-L1 blockade alone. Anti-tumor activity was associated with increased tumor-specific CD8+ T cells and reduced regulatory T cells within the tumor microenvironment and tumor-draining lymph nodes. In immunocompetent tumor-free mice, mbsAb-PD-L1×4-1BB treatment neither induced T-cell infiltration into the liver nor elevated liver enzymes in the blood. Dual targeting of PD-L1 and 4-1BB with a bispecific antibody may therefore address key limitations of first generation 4-1BB-agonistic antibodies, and may provide a novel approach to improve PD-1/PD-L1 checkpoint blockade.

Preclinical Characterization and Phase I Trial Results of a Bispecific Antibody Targeting PD-L1 and 4-1BB (GEN1046) in Patients with Advanced Refractory Solid Tumors.

Checkpoint inhibitors (CPI) have revolutionized the treatment paradigm for advanced solid tumors; however, there remains an opportunity to improve response rates and outcomes. In preclinical models, 4-1BB costimulation synergizes with CPIs targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis by activating cytotoxic T-cell-mediated antitumor immunity. DuoBody-PD-L1×4-1BB (GEN1046) is an investigational, first-in-class bispecific immunotherapy agent designed to act on both pathways by combining simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation in one molecule. GEN1046 induced T-cell proliferation, cytokine production, and antigen-specific T-cell-mediated cytotoxicity superior to clinically approved PD-(L)1 antibodies in human T-cell cultures and exerted potent antitumor activity in transplantable mouse tumor models. In dose escalation of the ongoing first-in-human study in heavily pretreated patients with advanced refractory solid tumors (NCT03917381), GEN1046 demonstrated pharmacodynamic immune effects in peripheral blood consistent with its mechanism of action, manageable safety, and early clinical activity [disease control rate: 65.6% (40/61)], including patients resistant to prior PD-(L)1 immunotherapy. SIGNIFICANCE: DuoBody-PD-L1×4-1BB (GEN1046) is a first-in-class bispecific immunotherapy with a manageable safety profile and encouraging preclinical and early clinical activity. With its ability to confer clinical benefit in tumors typically less sensitive to CPIs, GEN1046 may fill a clinical gap in CPI-relapsed or refractory disease or as a combination therapy with CPIs. See related commentary by Li et al., p. 1184. This article is highlighted in the In This Issue feature, p. 1171.

Incisor degeneration in rats induced by vascular endothelial growth factor/fibroblast growth factor receptor tyrosine kinase inhibition.

BMS-645737, an inhibitor of vascular endothelial growth factor (VEGF) receptor-2 and fibroblast growth factor (FGF) receptor-1, has anti-angiogenic activity and was evaluated in nonclinical studies as a treatment for cancer. This article characterizes the BMS-645737-induced clinical, gross, and histologic lesions of incisor teeth in Sprague-Dawley (SD) rats. Rats received 0 800 mg/kg BMS-645737 in a single-dose study or consecutive daily doses of 0 20 mg/kg/day in a 1-month study. The reversibility of these effects was assessed in the 1-month study. White discoloration and fracture of incisors were observed clinically and grossly in the 1-month study. In both studies, dose-dependent histopathologic lesions of incisors were degeneration and/or necrosis of odontoblasts and ameloblasts; decreased mineralization of dentin; inflammation and necrosis of the dental pulp; and edema, congestion, and hemorrhage in the pulp and periodontal tissue adjacent to the enamel organ. Partial recovery was observed at lower doses after a two-week dose-free period in the one-month study. Drug-induced incisor lesions were considered to be related to the pharmacologic inhibitory effects on VEGF and FGF signaling, that is, inhibition of growth and maintenance of small-diameter vessels that support the formation of dentin and enamel in growing teeth and/or to perturbances of function of odontoblasts and ameloblasts or their precursors.

DuoBody-CD3xCD20 induces potent T-cell-mediated killing of malignant B cells in preclinical models and provides opportunities for subcutaneous dosing.

BACKGROUND: DuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A. METHODS: T-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys. FINDINGS: DuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies. FUNDING: Genmab.

DcR3/TR6 effectively prevents islet primary nonfunction after transplantation.

Islet primary nonfunction (PNF) is defined as the loss of islet function after transplantation for reasons other than graft rejection. It is a major obstacle to successful and efficient islet transplantation. DcR3/TR6 is a soluble death decoy receptor belonging to the tumor necrosis factor (TNF) receptor family, and it can block apoptosis mediated by several TNF receptor family members such as Fas and LT beta R. In this study, we used TR6 to protect islets from PNF after transplantation. Untreated isogeneic or allogeneic islet transplantation had PNF incidence of 25 and 26.5%, respectively. Administration of TR6 totally prevented PNF in allogeneic islet transplantation. In vitro experiments showed an increased apoptosis among islets that were treated with FasL and gamma-interferon (IFN-gamma) in combination. TR6 significantly reduced such apoptosis. Functional study showed that insulin release was compromised after FasL and IFN-gamma treatment, and the compromise could be prevented with TR6-Fc. This indicates that TR6 indeed protected beta-cells from damage caused by FasL and IFN-gamma. Further in vivo experiments showed that syngeneic islet transplantation between lpr/lpr and gld/gld mice was significantly more efficacious than that conducted between wild-type mice. These results suggest that Fas-mediated apoptosis plays an important role in PNF, and use of TR6 may be a novel strategy to prevent PNF in clinical islet transplantation.

A rugged and accurate liquid chromatography-tandem mass spectrometry method for quantitative determination of BMS-790052 in plasma.

To support toxicokinetic assessments, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of BMS-790052 in rat, dog, monkey, rabbit and mouse K(2)EDTA plasma. The drug was isolated from buffered samples using ISOLUTE C8 96-well solid phase extraction (SPE) plates. Chromatographic separation was achieved on a Waters Atlantis dC18 analytical column (2.1 mm × 50 mm, 5 µm) with detection accomplished using an API 4000 tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 5.00 to 2000 ng/mL for BMS-790052, were fitted to a 1/x(2) weighted linear regression model. The intra-assay precision (%CV) and inter-assay precision (%CV) were within 8.5%, and the assay accuracy (%Dev) was within ±7.1 for rat, dog, monkey, rabbit and mouse K(2)EDTA plasma. This accurate, precise, and selective SPE/LC-MS/MS method has been successfully applied to analyze several thousands of non-clinical study samples.

Mouse T cells receive costimulatory signals from LIGHT, a TNF family member.

LIGHT is a tumor necrosis factor (TNF) family member and is expressed on activated T cells. Its known receptors are TR2 and LTbetaR on the cell surface, and TR6/DcR3 in solution. TR6/DcR3 is a secreted protein belonging to the TNF receptor family. It binds to Fas ligand (FasL), LIGHT, and TL1A, all of which are TNF family members. In the present study, we report that solid-phase TR6-Fc costimulated proliferation, lymphokine production, and cytotoxicity of mouse T cells upon T-cell receptor (TCR) ligation. A monoclonal antibody against LIGHT similarly costimulated mouse T cells in their proliferation response to TCR ligation. These data suggest LIGHT, although a ligand, can receive costimulation when expressed on the T-cell surface. Mechanistically, when T cells were activated by TCR and CD28 co-cross-linking, TCR and rafts rapidly formed caps where they colocalized. LIGHT rapidly congregated and colocalized with the aggregated rafts. This provided a molecular base for the signaling machinery of LIGHT to interact with that of TCR. Indeed, LIGHT cross-linking enhanced p44/42 mitogen-activated protein kinase activation after TCR ligation. This study reveals a new function and signaling event of LIGHT.