The short half-life and use of recombinant bone morphogenetic protein (BMP)2 in large doses poses major limitations in the clinic. Events regulating posttranslational processing and degradation of BMP2 in situ, linked to its secretion, have not been understood. Toward identifying mechanisms regulating intracellular BMP2 stability, we first discovered that inhibiting proteasomal degradation enhances both intracellular BMP2 level and its extracellular secretion. Next, we identified BMP2 degradation occurs through an ubiquitin-mediated mechanism. Since ubiquitination precedes proteasomal turnover and mainly occurs on lysine residues of nascent proteins, we systematically mutated individual lysine residues within BMP2 and tested them for enhanced stability. Results revealed that substitutions on four lysine residues within the pro-BMP2 region and three in the mature region increased both BMP2 stability and extracellular secretion. Structural modeling revealed key lysine residues involved in proteasomal degradation occupy a lysine cluster near proprotein convertase cleavage site. Interestingly, mutations within these residues did not affect biological activity of BMP2. These data suggest that preventing intracellular proteasomal loss of BMP2 through genetic modifications can overcome limitations related to its short half-life. K E Y W O R D SBMP2, proteasomal degradation, protein turnover, ubiquitination | 181 KHATTAR eT Al.
Immune checkpoint inhibitors (ICIs) are promising in adjuvant settings for solid tumors and hematologic malignancies. They are currently used in the treatment as monoclonal antibodies in high concentrations, raising concerns of toxicity and adverse side effects. Among various checkpoint molecules, targeting the program cell death protein-1 (PD-1)-programmed death-ligand 1 (PD-L1) axis has garnered more clinical utility than others have. In order to develop a physiologically relevant and systemically stable level of ICIs from a one-time application by genetic antibody engineering, we endeavored using a non-pathogenic, replication-deficient recombinant adeno-associated vector (rAAV), expressing single-chain variable fragments (scFv) of PD-L1 antibody and tested in syngeneic mouse therapy models of MC38 colorectal and EMT6 breast tumors. Results of this study indicated a significant protection against PD-L1-mediated inhibition of CD8+ T cell function, against the growth of primary and secondary tumors, and durable antitumor cytotoxic T lymphocytes activity by adoptive CD8+ T cell transfer. Stable maintenance of PD-L1 scFv in vivo resulted in an increase in PD-1- CD8+ T cells, and a concomitant decrease in regulatory T cells, M2 macrophages and myeloid-derived suppressor cells in the tumor microenvironment. Overall, these data demonstrate the potential of rAAV-PD-L1-scFv as an alternative to mAb targeting of PD-L1 for tumor therapy.
<div>Abstract<p>Immune checkpoint inhibitors (ICI) are promising in adjuvant settings for solid tumors and hematologic malignancies. They are currently used in the treatment as mAbs in high concentrations, raising concerns of toxicity and adverse side effects. Among various checkpoint molecules, targeting the programmed cell death protein-1 (PD-1)–programmed death-ligand 1 (PD-L1) axis has garnered more clinical utility than others have. To develop a physiologically relevant and systemically stable level of ICIs from a one-time application by genetic antibody engineering, we endeavored using a nonpathogenic, replication-deficient recombinant adeno-associated vector (rAAV) expressing single-chain variable fragments (scFv) of PD-L1 antibody and tested in syngeneic mouse therapy models of MC38 colorectal and EMT6 breast tumors. Results of this study indicated a significant protection against PD-L1–mediated inhibition of CD8<sup>+</sup> T-cell function, against the growth of primary and secondary tumors, and durable antitumor CTLs activity by adoptive CD8<sup>+</sup> T-cell transfer. Stable maintenance of PD-L1 scFv <i>in vivo</i> resulted in an increase in PD-1<sup>−</sup> CD8<sup>+</sup> T cells and a concomitant decrease in regulatory T cells, M2 macrophages, and myeloid-derived suppressor cells in the tumor microenvironment. Overall, these data demonstrate the potential of rAAV-PD-L1-scFv as an alternative to mAb targeting of PD-L1 for tumor therapy.</p></div>
Supplementary Figure from Systemic Checkpoint Blockade by PD-L1 Single-Chain Antibody Confers Potent Antitumor Immunity and Long-term Survival
<div>Abstract<p>Immune checkpoint inhibitors (ICI) are promising in adjuvant settings for solid tumors and hematologic malignancies. They are currently used in the treatment as mAbs in high concentrations, raising concerns of toxicity and adverse side effects. Among various checkpoint molecules, targeting the programmed cell death protein-1 (PD-1)–programmed death-ligand 1 (PD-L1) axis has garnered more clinical utility than others have. To develop a physiologically relevant and systemically stable level of ICIs from a one-time application by genetic antibody engineering, we endeavored using a nonpathogenic, replication-deficient recombinant adeno-associated vector (rAAV) expressing single-chain variable fragments (scFv) of PD-L1 antibody and tested in syngeneic mouse therapy models of MC38 colorectal and EMT6 breast tumors. Results of this study indicated a significant protection against PD-L1–mediated inhibition of CD8<sup>+</sup> T-cell function, against the growth of primary and secondary tumors, and durable antitumor CTLs activity by adoptive CD8<sup>+</sup> T-cell transfer. Stable maintenance of PD-L1 scFv <i>in vivo</i> resulted in an increase in PD-1<sup>−</sup> CD8<sup>+</sup> T cells and a concomitant decrease in regulatory T cells, M2 macrophages, and myeloid-derived suppressor cells in the tumor microenvironment. Overall, these data demonstrate the potential of rAAV-PD-L1-scFv as an alternative to mAb targeting of PD-L1 for tumor therapy.</p></div>
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