CD47 acts as a defense mechanism for tumor cells by sending a “don’t eat me” signal via its bond with SIRPα. With CD47’s overexpression linked to poor cancer outcomes, its pathway has become a target in cancer immunotherapy. Though monoclonal antibodies offer specificity, they have limitations like the large size and production costs. Nanobodies, due to their small size and unique properties, present a promising therapeutic alternative. In our study, a high-affinity anti-CD47 nanobody was engineered from an immunized alpaca. We isolated a specific VHH from the phage library, which has nanomolar affinity to SIRPα, and constructed a streptavidin-based tetramer. The efficacy of the nanobody and its derivative was evaluated using various assays. The new nanobody demonstrated higher affinity than the monoclonal anti-CD47 antibody, B6H12.2. The nanobody and its derivatives also stimulated substantial phagocytosis of tumor cell lines and induced apoptosis in U937 cells, a response confirmed in both in vitro and in vivo settings. Our results underscore the potential of the engineered anti-CD47 nanobody as a promising candidate for cancer immunotherapy. The derived nanobody could offer a more effective, cost-efficient alternative to conventional antibodies in disrupting the CD47–SIRPα axis, opening doors for its standalone or combinatorial therapeutic applications in oncology.

Achieving the precise targeting of lentiviral vectors (LVs) to specific cell populations is crucial for effective gene therapy, particularly in cancer treatment where the modulation of the tumor microenvironment can enhance anti-tumor immunity. Programmed cell death protein 1 (PD-1) is overexpressed on activated tumor-infiltrating T lymphocytes, including regulatory T cells that suppress immune responses via FOXP3 expression. We developed PD1-targeted LVs by incorporating the anti-PD1 nanobody nb102c3 into receptor-blinded measles virus H and VSV-G_mut glycoproteins. We assessed the retargeting potential of nb102c3 and evaluated transduction efficiency in activated T lymphocytes. FOXP3 expression was suppressed using shRNA delivered by these LVs. Our results demonstrate that PD1-targeted LVs exerted pronounced tropism towards PD1⁺ cells, enabling the selective transduction of activated T lymphocytes while sparing naive T cells. The suppression of FOXP3 in Tregs reduced their suppressive activity. PD1-targeted glycoprotein H provided greater specificity, whereas the VSV-G_mut, together with the anti-PD1 pseudoreceptor, achieved higher viral titers but was less selective. Our study demonstrates that PD1-targeted LVs may offer a novel strategy to modulate immune responses within the tumor microenvironment with the potential for developing new therapeutic strategies aimed at enhancing anti-tumor immunity.

Selective gene delivery to defined cell populations remains one of the key challenges in lentiviral vector-based gene therapy. The vesicular stomatitis virus glycoprotein (VSV-G) confers high infectivity but lacks cell-type specificity because of the ubiquitous expression of its receptor, LDLR. To enable modular, receptor-specific targeting while retaining the production efficiency of VSV-G-pseudotyped vectors, we designed a bispecific adapter, 929-B6, comprising a VSV-G-binding nanobody and an ERBB2-binding DARPin 9.29. Anti-VSV-G nanobodies were isolated from an alpaca immune library and screened in cell-based pseudoreceptor assays to identify the optimal binder (VSVG-B6). The resulting adapter was evaluated with receptor-ablated (VSV-G_mut) and wild-type VSV-G-pseudotyped LVs across ERBB2-positive and -negative cell lines and in a mouse xenograft model. 929-B6 enabled efficient, receptor-specific transduction of ERBB2-expressing cells without increasing infection of ERBB2-negative controls. Pre-incubation of VSV-G_mut-pseudotyped LVs with 1–2 µg/mL 929-B6 increased transduction up to eight-fold in ERBB2⁺ cells, with similar but smaller effects for VSV-G and VSV-G_mut + 929R pseudotypes. Across breast cancer lines, transduction enhancement correlated with ERBB2 surface density, and co-culture experiments confirmed selective entry into ERBB2⁺ populations. In vivo imaging of ERBB2⁺ tumors revealed a visible tumor-localized luminescent signal following administration of 929-B6-treated vectors. The 929-B6 adapter provides a rapid, scalable means to retarget standard LV stocks toward chosen receptors without re-engineering the envelope or co-packaging pseudoreceptor plasmids. Its modularity suggests a generalizable platform for both gene therapy and oncolytic applications requiring flexible, receptor-defined tropism.