Transforming growth factor-beta (TGF-β), a potent promoter of extracellular matrix deposition and suppressor of infiltrating immunity, has arisen as an attractive target for improving outcomes in tissue fibrosis and cancer immune therapy. Despite the promise of TGF-β inhibitors for attenuating the progression of fibrotic disorders or as adjuncts for cancer immunotherapy, current systemically administered inhibitors that target the ligand or receptors have significant on-target liabilities, including cardiotoxicity and development of pre-malignant cutaneous squamous lesions. Recently, an engineered mini monomer of TGF-β (mmTGF-β), which potently and specifically inhibits TGF-β activity, was shown to strongly synergize with checkpoint inhibitors to suppress cancer progression in an aggressive model of melanoma when genetically delivered using an engineered form of vaccinia virus that preferentially infects cancer cells. Despite these promising results, however, a significant fraction of the mmTGF-β was found to misfold, likely due to mispairing of the cysteines that comprise its cystine knot. Here, we demonstrate that inclusion of a modified form of the TGF-β pro-domain that lacks its dimerization motif, the bowtie knot, dramatically improves both the folding and inhibitory activity upon secretion by mammalian cells, thus overcoming one of the major limitations of genetically delivering mmTGF-β. Furthermore, we show that fusion of mmTGF-β to a CD44 binding domain enhances the inhibitory potential of mmTGF-β on immune cells, and on other cell types which express CD44, by more than 30-fold compared to cells negative for CD44. Together, these modifications provide a framework for further enhancing the efficacy and safety of mmTGF-β for cancer immune therapy, and possibly also tissue fibrosis, when delivered genetically using vaccinia, or other related approaches.

Background and objectives: The PWWP domain of lens epithelium-derived growth factor p75 (LEDGF/p75) mediates chromatin engagement through recognition of histone H3 lysine 36 di- and trimethylation (H3K36me2/3) and nucleosomal DNA. LEDGF/p75 plays a role in multiple human diseases. In particular, its interaction with HIV-1 integrase enables viral genome integration. However, the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets. Here we report the generation of high-affinity nanobodies (Nbs) to investigate the structure and function of this domain. Methods: Camelids were immunized with recombinant LEDGF PWWP domain, and immune phage display libraries were screened for affinity. Selected Nbs were recombinantly expressed in E. coli and purified. Their interaction with the PWWP domain of LEDGF and its close homolog HRP-2 was characterized using size-exclusion chromatography and surface plasmon resonance. Structural characterization of the Nbs was performed by X-ray crystallography. Functional effects on chromatin engagement were evaluated using the AlphaScreen assay. Results: Nine sequence-distinct Nbs were identified, seven of which were confirmed to bind the LEDGF PWWP domain with nanomolar affinities. Five Nbs also bound the HRP-2 domain, consistent with conserved functional surfaces, while two showed reduced affinity. Crystal structures of two Nbs (NbC03 and NbH10) confirmed canonical immunoglobulin folds, while the latter additionally revealed a domain-swapped dimer. Moreover, NbH10 dose-dependently inhibited the interaction between full-length LEDGF/p75 and H3K36me3-modified nucleosomes in vitro. Conclusions: This work establishes a validated panel of Nbs targeting the LEDGF PWWP domain and demonstrates their ability to functionally disrupt the LEDGF-chromatin interaction. These Nbs serve as valuable tools towards functional studies and structure-based drug design.

Background and objectives: The PWWP domain of lens epithelium-derived growth factor p75 (LEDGF/p75) mediates chromatin engagement through recognition of histone H3 lysine 36 di- and trimethylation (H3K36me2/3) and nucleosomal DNA. LEDGF/p75 plays a role in multiple human diseases. In particular, its interaction with HIV-1 integrase enables viral genome integration. However, the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets. Here, we report the generation of high-affinity nanobodies (Nbs) to investigate the structure and function of this domain. Methods: Camelids were immunized with recombinant LEDGF PWWP domain, and immune phage display libraries were screened for affinity. Selected Nbs were recombinantly expressed in E. coli and purified. Their interaction with the PWWP domain of LEDGF and its close homolog HRP-2 was characterized using size-exclusion chromatography and surface plasmon resonance. Structural characterization of the Nbs was performed using X-ray crystallography. Functional effects on chromatin engagement were evaluated using an AlphaScreen assay. Results: Nine sequence-distinct Nbs were identified, seven of which were confirmed to bind the LEDGF PWWP domain with nanomolar affinities. Five Nbs also bound the HRP-2 domain, consistent with conserved functional surfaces, while two showed reduced affinity. The crystal structures of two Nbs (NbC03 and NbH10) confirmed there were canonical immunoglobulin folds, while the latter additionally revealed a domain-swapped dimer. Moreover, NbH10 dose-dependently inhibited the interaction between full-length LEDGF/p75 and H3K36me3-modified nucleosomes in vitro. Conclusions: This work establishes a validated panel of Nbs targeting the LEDGF PWWP domain and identifies one Nb capable of functionally disrupting the LEDGF–chromatin interaction. These Nbs serve as valuable tools for functional studies and structure-based drug design.