Engineering Recombinant Chimeric Proteins That Deliver Therapies to the Brain.

To engineer biotherapeutics that can specifically target glioma, a highly lethal and recurrent type of brain tumor, we created a class of recombinant chimeric proteins containing multiple functional modules, such as a blood-brain barrier (BBB)-penetrating domain and an immune checkpoint blockade (ICB) agent, PD-L1 nanobody, through fusion protein engineering. The recombinant proteins that could prolong circulation time, cross the BBB, target brain tumors, penetrate cell membranes, and release PD-L1 nanobody were demonstrated. Further, by covalently linking doxorubicin (DOX), an inducer of immunogenic cell death (ICD), to the recombinant proteins, we endow the recombinant protein-drug conjugates (RPDCs) with a combining capacity to induce ICD and block immune checkpoints in cancer cells, achieving significant inhibition of glioma growth and prolonging the survival time of orthotopic glioma-bearing mice by enhancing intratumoral dendritic cell maturation and T-cell activation. In addition, the RPDC also improves the intratumoral immune-suppressive microenvironment, reduces excess extracellular matrix, and alleviates tumor hypoxia. Our work not only offers a new opportunity for glioma treatment but also establishes the groundwork for the design of multifunctional and multitargeting protein-based therapeutics.