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Background: T cell-based immunotherapies are facing great challenges in the recruitment and activation of tumor-specific T cells against solid tumors. Among which, utilizing nanobody (Nb) or nanobodies (Nbs) to construct T cell engager has emerged as a more practical potential for enhancing the anti-tumor effectiveness of T cells. Here, we designed a new Nb-guided multifunctional T cell engager (Nb-MuTE) that not only recruited effector T cells into the tumor tissues, but also efficiently activated T cells anti-tumor immunity when synergies with photothermal effect., Results: The Nb-MuTE, which was constructed based on an indocyanine green (ICG)-containing liposome with surface conjugation of CD105 and CD3 Nbs, and showed excellent targetability to both tumor and T cells, following enhancement of activation, proliferation and cytokine secretion of tumor-specific T cells. Notably, the immunological anti-tumor functions of Nb-MuTE-mediated T cells were further enhanced by the ICG-induced photothermal effect in vitro and in vivo., Conclusions: Such a new platform Nb-MuTE provides a practical and "all-in-one" strategy to potentiate T cell responses for the treatment of solid tumor in clinic., (© 2024. The Author(s).)

Chimeric antigen receptor (CAR) T-cell immunotherapy has become one of the research hotspots in the treatment of malignant tumors nowadays. However, the available tumor surface antigens are limited in number. Most tumor-associated antigens are intracellular molecules that can't be targeted by conventional CAR T cells. As the major histocompatibility complex (MHC)/peptide complex is a presentation form of intracellular proteins on the surface of tumor cells, here, we chose the Glypican-3 (GPC3) oncoprotein and Wilms tumor 1 (WT1) oncoprotein as examples to explore whether nanobody (Nb)-based T cell receptor (TCR)-like CAR T cells could kill tumor cells by targeting the MHC/peptide complexes. Using the immune nanobody phage display library, we developed human leukocyte antigen (HLA)-A2/GPC3- and HLA-A2/WT1-specific nanobodies for the first time and then incorporated these nanobodies in two TCR-like CARs, targeting HLA-A2/GPC3 and HLA-A2/WT1 respectively. These TCR-like Nb CAR-redirected T cells could selectively recognize and lyse MHC/peptide complex-expressing tumor cells in vitro assays and subcutaneous mouse tumor models. This study offers a possible strategy for targeting intracellular antigens and widening the application of CAR T-cell therapy., Competing Interests: Conflict of interest statement The authors declare that there are no conflicts of interest., (Copyright © 2022. Published by Elsevier Masson SAS.)

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an infectious disease that has become a serious burden on global public health. This study screened and yielded specific nanobodies (Nbs) against SARS-CoV-2 spike protein receptor binding domain (RBD), following testing its basic characteristics. A nanobody phage library was established by immunizing a camel with RBD protein. After three rounds of panning, the positive colonies were screened by enzyme-linked immunosorbent assay (ELISA). By sequencing, four different sequences of nanobody gene fragments were selected. The four nanobody fusion proteins were expressed and purified, respectively. The specificity and affinity of the four nanobodies were identified by ELISA. Our results showed that an immune phage display library against SARS-CoV-2 has been successfully constructed with a library capacity of which was 4.7 × 10 8 CFU. The four purified nanobodies showed specific high-affinity binding SARS-CoV-2 S-RBD. Among these, the antigen binding affinity of Nb61 was more comparable to that of commercial rabbit anti-SARS-CoV-2 S-RBD antibodies. In sum, our study has obtained four nanobody strains against SARS-CoV-2 S-RBD with significant affinity and specificity, therefore laying an essential foundation for further research as well as the applications of diagnostic and therapeutic tools of SARS-CoV-2.

Despite the many successes and opportunities presented by PD-1 blockade in cancer therapies, anti-PD-1 monoclonal antibodies still face multiple challenges. Herein we report a strategy based on a nanobody (Nb) to circumvent these obstacles. A new PD-1-blocking Nb (PD-1 Nb20) in combination with tumor-specific dendritic cell (DC)/tumor-fusion cell (FC) vaccine that aims to improve the activation, proliferation, cytokine secretion, and tumor cell cytotoxicity of CD8 + T-cells. This combination was found to effectively enhance the in vitro cytotoxicity of CD8 + T-cells to kill human non-small cell lung cancer (NSCLC) HCC827 cells, hepatocellular carcinoma (HCC) HepG2 cells, and tongue squamous cell carcinoma (TSCC) Tca8113 cells. Moreover, CD8 + T-cells pre-treated with PD-1 Nb20 and tumor-specific DC/tumor-FCs significantly suppressed the growth of NSCLC-, HCC- and TSCC-derived xenograft tumors and prolonged the survival of tumor-bearing mice, through promoting T-cell infiltration to kill tumor cells and inhibiting tumor angiogenesis. These data demonstrate that PD-1 Nb20 in synergy with DC/tumor-FC vaccine augment the broad spectrum of antitumor activity of CD8 + T-cells, providing an alternative and promising immunotherapeutic strategy for tumor patients who are T-cell-dysfunctional or not sensitive to anti-PD-1 therapy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Cytokine-induced killer cells induced with tumor antigen-pulsed dendritic cells (DC-CIK) immunotherapy is a promising strategy for the treatment of malignant tumors. However, itsefficacy isrestricted by the immunosuppression, which is mediated by the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) pathway. In order to overcome the negative co-stimulation from these T cells,we screened a nanobody targeted for CTLA-4 (Nb36) and blocked the CTLA-4 signaling with Nb36., Methods: Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors to beused to induce CIK cells in vitro, after which they were co-cultured with DC cells that had received tumor antigens. In addition, wetested whether blocking CTLA-4 signaling with Nb36 could promote in vitro DC-CIK cells proliferation, pro-inflammatory cytokine production and cytotoxicity,or not. For the in vivo experiments, we constructed a subcutaneously transplanted tumor model and placed it in NOD/SCID mice to verify the anti-tumor effect of this therapy., Results: After stimulation with Nb36, the DC-CIK cells presented enhanced proliferation and production of IFN-γ in vitro, which strengthened the killing effect on the tumor cells. For the in vivo experiments, it was found that Nb36-treated DC-CIK cells significantly inhibited the growth of subcutaneously transplanted livercancer tumors, as well as reduced the tumor weight and prolonged the survival of tumor-bearing NOD/SCID mice., Conclusions: Ourfindings demonstrated that in response to CTLA-4 specific nanobody stimulation, DC-CIK cells exhibited a better anti-tumor effect. In fact, this Nb-based CTLA-4 blocking strategy achieved an anti-tumor efficacy close to that of monoclonal antibodies. Our findings suggest that DC-CIK cells + Nb36 have the potential totreatmalignant tumors through in vivo adoptive therapy., (© 2021. The Author(s).)

The development of monoclonal antibody treatments for successful tumor-targeted therapies took several decades. However, the efficacy of antibody-based therapy is still confined and desperately needs further improvement. Nanobodies are the recombinant variable domains of heavy-chain-only antibodies, with many unique properties such as small size (~15kDa), excellent solubility, superior stability, ease of manufacture, quick clearance from blood, and deep tissue penetration, which gain increasing acceptance as therapeutical tools and are considered also as building blocks for chimeric antigen receptors as well as for targeted drug delivery. Thus, one of the promising novel developments that may address the deficiency of monoclonal antibody-based therapies is the utilization of nanobodies. This article provides readers the significant factors that the structural and biochemical properties of nanobodies and the research progress on nanobodies in the fields of tumor treatment, as well as their application prospect., Competing Interests: The authors declare that they have no conflicts of interest., (© 2021 Sun et al.)

Retargeting the antigen-binding specificity of T cells to intracellular antigens that are degraded and presented on the tumor surface by engineering chimeric antigen receptor (CAR), also named TCR-like antibody CAR-T, remains limited. With the exception of the commercialized CD19 CAR-T for hematological malignancies and other CAR-T therapies aiming mostly at extracellular antigens achieving great success, the rareness and scarcity of TCR-like CAR-T therapies might be due to their current status and limitations. This review provides the probable optimized initiatives for improving TCR-like CAR-T reprogramming and discusses single-domain antibodies administered as an alternative to conventional scFvs and secreted by CAR-T cells, which might be of great value to the development of CAR-T immunotherapies for intracellular antigens., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Lichen Zhu et al.)

Cytotoxic T‑lymphocyte antigen‑4 (CTLA‑4) is a critical negative regulator of immune responses. CTLA‑4 is rapidly upregulated following T‑cell activation, and then binds to B7 molecules with a higher affinity than CD28. CTLA‑4 may abolish the initiation of the responses of T cells by raising the threshold of signals required for full activation of T cells, and it also may terminate ongoing T-cell responses. This regulatory role has led to the development of monoclonal antibodies (mAbs) designed to block CTLA‑4 activity for enhancing immune responses against cancer. mAbs have several disadvantages including high production cost and unstable behavior. Nanobodies (Nbs) are single‑domain antigen‑binding fragments derived from the camelid heavy‑chain antibodies, which are highly attractive in cancer immunotherapy due to their small size, high specificity, and stability. We selected CTLA‑4‑specific Nbs from a high quality dromedary camel immune library by phage display technology. Four positive colonies were sequenced and classified based on the amino acids sequences in the CDR3 region. These Nbs recognized unique epitopes on CTLA‑4 and displayed high binding rates when used on PHA‑stimulated human T cells. Treatment of B16 melanoma‑bearing C57BL/6 mice with anti‑CTLA‑4 nanobody 16 (Nb16) delayed melanoma growth and prolonged the survival time of mice. These data indicate that anti‑CTLA‑4 Nbs selected from a high quality phage display library may be effective for the treatment of patients with tumors.

The unpredicted spread of avian influenza virus subtype H7N2 in the world is threatening animals and humans. Specific and effective diagnosis and supervision are required to control the influenza. However, the existing detecting methods are laborious, are time-consuming, and require appropriate laboratory facilities. To tackle this problem, we isolated VHH antibodies against the H7N2 avian influenza virus (AIV) and performed an enzyme-linked immunosorbent assay (ELISA) to detect the H7N2 virus. To obtain VHH antibodies with high affinity and specificity, a camel was immunized. A VHH antibody library was constructed in a phage display vector pMECS with diversity of 2.8 × 10(9). Based on phage display technology and periplasmic extraction ELISA, H7N2-specific VHH antibodies were successfully isolated. According to a pairing test, two VHH antibodies (Nb79 and Nb95) with good thermal stability and specificity can recognize different epitopes of H7N2 virus. The capture antibody (Nb79) was biotinylated in vivo, and the detection antibody (Nb95) was coupled with horseradish peroxidase (HRP). Based on biotin-streptavidin interaction, a novel sandwich immune ELISA was performed to detect H7N2. The immunoassay exhibited a linear range from 5 to 100 ng/ml. Given the above, the newly developed VHH antibody-based double sandwich ELISA (DAS-ELISA) offers an attractive alternative to other diagnostic approaches for the specific detection of H7N2 virus., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Chimeric antigen receptor-based T-cell immunotherapy is a promising strategy for treatment of hematological malignant tumors; however, its efficacy towards solid cancer remains challenging. We therefore focused on developing nanobody-based CAR-T cells that treat the solid tumor. CD105 expression is upregulated on neoangiogenic endothelial and cancer cells. CD105 has been developed as a drug target. Here we show the generation of a CD105-specific nanobody, an anti-human CD105 CAR-T cells, by inserting the sequences for anti-CD105 nanobody-linked standard cassette genes into AAVS1 site using CRISPR/Cas9 technology. Co-culture with CD105+ target cells led to the activation of anti-CD105 CAR-T cells that displayed the typically activated cytotoxic T-cell characters, ability to proliferate, the production of pro-inflammatory cytokines, and the specific killing efficacy against CD105+ target cells in vitro. The in vivo treatment with anti-CD105 CAR-T cells significantly inhibited the growth of implanted CD105+ tumors, reduced tumor weight, and prolonged the survival time of tumor-bearing NOD/SCID mice. Nanobody-based CAR-T cells can therefore function as an antitumor agent in human tumor xenograft models. Our findings determined that the strategy of nanobody-based CAR-T cells engineered by CRISPR/Cas9 system has a certain potential to treat solid tumor through targeting CD105 antigen.