Bispecific CD3-HAC carried by E1A-engineered mesenchymal stromal cells against metastatic breast cancer by blocking PD-L1 and activating T cells.

Background: PD-1/PD-L1 blockade can confer durable benefits in the treatment of metastatic cancers, but the response rate remains modest and potential adverse effects occur sometimes. Concentrating immunotherapeutic agents at the site of disease was believed to break local immune tolerance and reduce systemic toxicity. E1A-engineered mesenchymal stromal cell (MSC.E1A) was an attractive transfer system that preferentially homing and treating cancer metastasis, through which the tumor cells were modified by locally replicated adenoviruses to release CD3-HAC, a bifunctional fusion protein that anti-CD3 scfv linked with high-affinity consensus (HAC) PD-1. Subsequently, CD3-HAC, wbich was bound on PD-L1-positive breast cancer cells, recruited T cells to exhibit a potent antitumor immunity incombination with immune checkpoint blockade.

Methods: We constructed the CD3-HAC gene driven by human telomerase reverse transcriptase (hTERT) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. The homing property of MSCs in vivo was analyzed with firefly luciferase-labeled MSCs (MSC.Luc) by bioluminescent imaging (BLI). The cytotoxicity of T cells induced by CD3-HAC towards PD-L1-positive cells was detected in vitro and in vivo in combination with 5-FU.

Results: Our data suggest that CD3-HAC could specifically bind to PD-L1-positive tumor cells and induce lymphocyte-mediated lysis effectively both in vitro and in vivo. The intervention with HAC diminished the effects of PD-1/PD-L1 axis on T cells exposed to MDA-MB-231 cells and increased lymphocytes activation. MSCs infected by AdCD3-HAC followed by LentiR.E1A could specially migrate to metastasis of breast cancer and produce adenoviruses in the tumor sites. Furthermore, treatment with MSC.CD3-HAC.E1A in combination with 5-FU significantly inhibited the tumor growth in mice.

Conclusions: This adenovirus-loaded MSC.E1A system provides a promising strategy for the identification and elimination of metastasis with locally released immuno-modulator.