The Use of a Humanized NSG-β2m−/− Model for Investigation of Immune and Anti-tumor Effects Mediated by the Bifunctional Immunotherapeutic Bintrafusp Alfa

Abstract

The lack of serial biopsies in patients with a range of carcinomas has been one obstacle in our understanding of the mechanism of action of immuno-oncology agents as well as the elucidation of mechanisms of resistance to these novel therapeutics. While much information can be obtained from studies conducted with syngeneic mouse models, these models have limitations, including that both tumor and immune cells being targeted are murine and that many of the immuno-oncology agents being evaluated are human proteins, and thus multiple administrations are hampered by host xenogeneic responses. Some of these limitations are being overcome by the use of humanized mouse models where human peripheral blood mononuclear cells (PBMC) are engrafted into immunosuppressed mouse strains. Bintrafusp alfa (M7824) is an innovative first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII to function as a TGF-β "trap" fused to a human IgG1 antibody blocking PD-L1. A phase I clinical trial of bintrafusp alfa showed promising anti-tumor efficacy in heavily pretreated advanced solid tumors, and multiple clinical studies are currently ongoing. There is still much to learn regarding the mechanism of action of bintrafusp alfa, including its effects on both human immune cells in the periphery and in the tumor microenvironment (TME), and any temporal effects upon multiple administrations. By using the NSG-β2m −/− mouse strain humanized with PBMC, we demonstrate here for the first time: (a) the effects of bintrafusp alfa administration on human immune cells in the periphery vs. the TME using three different human xenograft models; (b) temporal effects upon multiple administrations of bintrafusp alfa; (c) phenotypic changes induced in the TME, and (d) variations observed in the use of multiple different PBMC donors. Also discussed are the similarities and differences in the data thus far obtained employing murine syngeneic models, from clinical trials, and in the use of this humanized mouse model. The results described here may guide the future use of this agent or similar immunotherapy agents as monotherapies or in combination therapy studies.

The Development of Next-generation PBMC Humanized Mice for Preclinical Investigation of Cancer Immunotherapeutic Agents

“…Varying degrees of immune activity are observed in humanized mice within the immuno-oncology setting. Data support a model where varied antitumor activity is observed in response to immunomodulatory drugs that are dependent on the immune cell donor (28,33). This varied response is consistent with results obtained in the clinic where most recipients of a therapeutic respond to varying degrees.…”

“…PBMCs may be injected after freezing or injected fresh from human donors. While differences may be observed in some myeloid and B-cell lineages at the time of injection, appropriate freeze/thawing of adoptively transferred cells prior to injection does not appear to change survival or phenotypes of T-cells post engraftment (28,30).…”

“…The number of injected PBMCs varies based on the study; however, most groups report successful engraftment utilizing 5×10 6 -2×10 7 PBMCs per recipient mouse. Although functional differences have been reported when utilizing too few PBMCs (28), it is as yet unclear if there is an advantage or if acceleration of GvHD occurs when injecting larger numbers. Recipient animals may be irradiated or not, and while a phenotypic difference has been noted based on radiation status, a definitive advantage is either uncertain or likely contextual.…”

“…One notable strength of the PBMC model is the ease and speed at which mice can be created and utilized. Circulating human immune cells are detected in recipient animals within 2 weeks post-PBMC engraftment, with an adequate peripheral human immune compartment being observed within 3 weeks (28). Some myeloid, NK, and NK T-cells remain for days to weeks but largely disappear as they lack appropriate survival signals, although exogenous or transgenic expression of various cytokines can aid in their survival (31).…”

“…Human PBMCs injected into recipient mice rapidly expand due to a likely combination of homeostatic proliferation (34) and a xenogeneic response to murine antigens (35), which results in mice harboring physiologically relevant numbers of immune cells within 3 weeks post injection of human PBMCs (28,35,36). In contrast, the HSC model requires months to develop equivalent levels of human immune engraftment.…”