Bone marrow-liver-thymus (BLT) immune humanized mice as a model to predict cytokine release syndrome.

Cytokine release syndrome (CRS) is a serious and potentially life-threatening complication that can be associated with biological drug products. In vitro assays or in vivo tests using nonhuman primates may fail to predict CRS due to species differences and the complexity of immune system. Therefore, model species that have human-specific immune components may improve the ability to identify CRS and enhance product safety. In this study we used bone marrow-liver-thymus (BLT) humanized mice to test muromonab (OKT3), an anti-CD3 antibody with a black box warning for CRS. Initially, we completed pilot and dose escalation studies with muromonab and showed that when the dose was increased sufficiently, BLT-humanized mice experienced serious adverse outcomes including moribundity. Full studies compared muromonab treatment with adalimumab, saline, and a group pretreated with methylprednisolone prior to muromonab. We evaluated immune cell activation using flow cytometry and cytokine expression using a custom 10-plex cytokine assay to assess levels of human TNF-α, IFN-γ, IL-2, IL-6, IL-8, IL-10, IL-13, IL-17A, IL12/23p40, and GM-CSF. Muromonab treated mice had significant increases in all cytokines tested with T-cell depletion and T-cell activation noted. Adalimumab (active) and saline (inactive) control groups did not demonstrate cytokine expression changes or alterations in T-cell numbers or activation. Further, pretreatment with methylprednisolone blunted or abrogated cytokine increases. This study demonstrates that BLT-humanized mice are capable of experiencing CRS, and could be used to screen biologics for this adverse event to enhance patient safety.

BLT-Immune Humanized Mice as a Model for Nivolumab-Induced Immune-Mediated Adverse Events: Comparison of the NOG and NOG-EXL Strains.

Checkpoint inhibitors represent a new class of therapeutics in the treatment of cancer that has demonstrated remarkable clinical effectiveness. However, some patients have experienced serious immune-mediated adverse effects including pneumonitis, hepatitis, colitis, nephritis, dermatitis, encephalitis, and adrenal or pituitary insufficiency. These adverse events were not predicted by nonclinical studies. To determine if bone marrow-liver-thymus (BLT) immune humanized mice could demonstrate these adverse effects, we studied the effect of nivolumab on 2 strains of BLT-humanized mice, NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) and NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(SV40/HTLV-IL3, CSF2)10-7Jic/JicTac (NOG-EXL). Mice were treated with 2.5, 5.0, or 10.0 mg/kg nivolumab or saline twice weekly for 28 days. BLT-NOG mice had significantly reduced survival compared with BLT-NOG-EXL mice. In spite of the difference in survival, both BLT-humanized strains showed adverse reactions similar to those reported in humans, including pneumonitis and hepatitis, with nephritis, dermatitis and adrenalitis also noted in some individuals. Additional histopathologic findings included pancreatic atrophy, myositis, and osteomyelitis in some animals. T-cell activation increased with concomitant loss of PD-1 detection. These findings show that BLT immune humanized mice can demonstrate immune-mediated adverse effects of antiPD1 therapy, and may represent a model that can be used to better understand toxicity of this class of drugs.

Evaluation of the Ability of Immune Humanized Mice to Demonstrate CD20-Specific Cytotoxicity Induced by Ofatumumab.

CD20 monoclonal antibodies are well-established therapeutics for the treatment of B-cell malignancies. Several mechanisms of target cell killing occur from anti-CD20 therapy, including complement-dependent cytotoxicity (CDC) cell lysis and antibody-dependent cell-mediated cytotoxicity. Human Fc receptors (FcRs) are required to mediate these functions and are either not present or not cross-reactive in mice and most animal species. In contrast, some nonhuman primates have cross-reactive FcR; however, their cellular expression and function may differ from humans. Therefore, we tested bone marrow-liver-thymus (BLT) humanized mice to determine if they could recapitulate the pharmacokinetics (PKs), pharmacodynamics, and potential toxicities of ofatumumab, for which CDC is the predominant mechanism of action. Ofatumumab-treated BLT mice depleted B cells in a dose-dependent manner in all tissues sampled and recapitulated the PKs observed in humans, suggesting that BLT mice can mediate the CDC effector mechanism associated with biological drug products.