Evaluation of a TGN1412 analogue using in vitro assays and two immune humanized mouse models.

Cytokine release syndrome (CRS) is a serious and potentially life-threatening complication typically associated with biological drug products. Pre-clinical testing in vitro and in vivo studies using non-human primates had failed to reliably predict CRS. To determine if bone marrow-thymus-liver (BLT) humanized mice with a fully engrafted human immune system or a CD34-humanized mouse model could predict CRS, we tested an anti-CD28 monoclonal antibody (mAb) similar to TGN1412. This TGN1412 analogue (TGN1412A) was initially tested in vitro and found to produce significant dose-dependent increases in cytokine production. For in vivo studies, adalimumab, an anti-tumor necrosis factor-alpha antibody known not to cause CRS, served as a negative control. We evaluated immune cell activation and cytokine expression in three independent experiments. In BLT humanized mice, significant increases in levels of human cytokines were identified in animals treated with anti-CD28 mAb. As expected, CD28+ cell detection was strongly reduced in the anti-CD28 treated group. Increased T cell activation was also observed. The control group did not show reductions in CD28+ T-cells and did not experience increased cytokine levels. Responses by CD34-humanized mice showed no significant differences between adalimumab and anti-CD28 treatment at doses used to test BLT-humanized mice. These results suggest that the TGN1412A produces similar results in vitro to the original TGN1412 monoclonal antibody. The BLT immune humanized mice but not the CD34 humanized mice produce both robust and specific cytokine responses and may represent a pre-clinical model to identify CRS.

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.

CCL9 Induced by TGFß Signaling in Myeloid Cells Enhances Tumor Cell Survival in the Premetastatic Organ.

Tumor cell survival in the hostile distant organ is a rate-limiting step in cancer metastasis. Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor-promoting microenvironment. However, it is unclear whether these myeloid cells in the premetastatic site have any direct effect on tumor cell survival. Here, we report that chemokine CCL9 was highly induced in Gr-1(+)CD11b(+) immature myeloid cells and in premetastatic lung in tumor-bearing mice. Knockdown of CCL9 in myeloid cells decreased tumor cell survival and metastasis. Importantly, CCL9 overexpression in myeloid cells lacking TGFß signaling rescued the tumor metastasis defect observed in mice with myeloid-specific Tgfbr2 deletion. The expression level of CCL23, the human orthologue for CCL9, in peripheral blood mononuclear cells correlated with progression and survival of cancer patients. Our study demonstrates that CCL9 could serve as a good candidate for anti-metastasis treatment by targeting the rate-limiting step of cancer cell survival. In addition, targeting CCL9 may avoid the adverse effects of TGFß-targeted therapy.