Nanobody-based trispecific T cell engager (Nb-TriTE) enhances therapeutic efficacy by overcoming tumor-mediated immunosuppression.

BACKGROUND: T cell engagers (TCEs) have been established as an emerging modality for hematologic malignancies, but solid tumors remain refractory. However, the upregulation of programmed cell death 1 (PD-1) is correlated with T cell dysfunction that confer tumor-mediated immunosuppression. Developing a novel nanobody-based trispecific T cell engager (Nb-TriTE) would be a potential strategy to improve therapeutic efficacy. METHODS: Given the therapeutic potential of nanobodies (Nbs), we first screened Nb targeting fibroblast activation protein (FAP) and successfully generated a Nb-based bispecific T cell engager (Nb-BiTE) targeting FAP. Then, we developed a Nb-TriTE by fusing an anti-PD-1 Nb to the Nb-BiTE. The biological activity and antitumor efficacy of the Nb-TriTE were evaluated in vitro and in both cell line-derived and patient-derived xenograft mouse models. RESULTS: We had for the first time successfully selected a FAP Nb for the generation of novel Nb-BiTE and Nb-TriTE, which showed good binding ability to their targets. Nb-TriTE not only induced robust tumor antigen-specific killing, potent T cell activation and enhanced T cell function in vitro, but also suppressed tumor growth, improved survival and mediated more T cell infiltration than Nb-BiTE in mouse models of different solid tumors without toxicity. CONCLUSIONS: This novel Nb-TriTE provides a promising and universal platform to overcome tumor-mediated immunosuppression and improve patient outcomes in the future.

A dendritic/tumor fusion cell vaccine enhances efficacy of nanobody-based CAR-T cells against solid tumor.

Background: Chimeric antigen receptor (CAR) T-cell therapy is practical in treating cancers of hematopoietic origin, but of that in solid tumors compromises efficacy for the loss of the antigen recognized by the CAR. However, dendritic cell (DC)/tumor fusion vaccines present a spectrum of known or unknown tumor antigens to stimulate T cell expansion and enhanced T cell response. Developing a new strategy of enhanced nanobody-based CAR-T (Nb-CAR-T) cells antitumor activity by DC/tumor fusion vaccines stimulation would provide guidance for more effective CAR-T cell therapies. Methods: Considering the therapeutic potential of nanobody (Nb), we first screened EGFRvIII Nb, then constructed and verified the function of EGFRvIII Nb-CAR-T cells in vitro and in vivo. We further combined DC/tumor fusion vaccines to boost EGFRvIII Nb-CAR-T cells antitumor effect, which was evaluated in vitro Nb-CAR-T cell function and in the tumor-bearing xenograft mouse models. Results: We had for the first time successfully selected EGFRvIII Nb for the generation of the novel EGFRvIII Nb-CAR-T cells. Importantly, our results suggested that DC/tumor fusion vaccines stimulate Nb-CAR-T cells response not only in improving T cell proliferation, T cell activation, cytokine secretion and tumor-specific cytotoxicity in vitro, but also significantly reducing tumor burden, prolonging survival and improving Nb-CAR-T cells infiltration. Conclusions: We have innovatively shown that DC/tumor fusion vaccines significantly enhance the efficacy of Nb-CAR-T cells against solid tumors. This new strategy has provided a promising therapeutic platform for promoting the clinical treatment of CAR-T cells therapy.

Nanobody-based CAR T cells targeting intracellular tumor antigens.

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.

Endoglin-Aptamer-Functionalized Liposome-Equipped PD-1-Silenced T Cells Enhance Antitumoral Immunotherapeutic Effects.

BACKGROUND: The broader application of adoptive cell therapy (ACT) in cancer immunotherapies (particularly for solid tumors) has always been limited by the immunosuppressive tumor microenvironment (TME) and the insufficient targetability of effector T cells, resulting in unsatisfied therapeutic outcome. Here, we designed a new strategy by using aptamer-based immunoliposomes to modify PD-1-silencing T cells, which were activated by dendritic cell (DC)/tumor fusion cells (FCs) to improve the antitumor potency of cytotoxic T lymphocytes (CTLs/CD8+ T cells). METHODS: PD-1 gene was knocked out from CD8+ T cells using CRISPR/Cas9 system to liberate T cell activity from immunosuppression. The PD-1- T cells were stimulated with DC/tumor FCs, followed by further functional modification of tumor-specific nanoliposomes (hEnd-Apt/CD3-Lipo) to generate FC/PD-1- CTLs. The activation and proliferation and specificity of the modified FC/PD-1- CTLs were measured. The antitumor activity of these CTLs against HepG2-tumors was evaluated in xenograft NOD/SCID mice, and the antitumor mechanism was investigated based on tissue immunohistochemistry and serum ELISA. RESULTS: Our results indicated that the modification of hEnd-Apt/CD3-Lipo nanocomposites on the FC/PD-1- CTLs had a more substantial synergetic effect in inhibiting tumor growth and prolonging animal survival, rather than other control liposomes. Furthermore, the hEnd-Apt/CD3-Lipo-modified FC/PD-1- CTLs showed a stronger antitumor outcome in the tumor-bearing mouse model, through the mechanisms of suppressing tumor cell proliferation, promoting tumor apoptosis, reducing angiogenesis but increasing the infiltration of the FC/PD-1- CTLs in the tumor tissue, as well as upregulating the systemic levels of IFN-γ, IL-2, TNF-α and IL-6 cytokines, by comparison of the control settings. CONCLUSION: In sum, our investigation suggests an enhancement of antitumor effect by the surface modification of endoglin-targeting nanoliposomes upon DC/tumor FC-activated PD-1- CTLs, therefore, provides a new tumoral endoglin-targeted approach as a promising strategy to reduce immunosuppression of tumor microenvironment and improve the immunotherapeutic outcome of anticancer ACT.

A new PD-1-specific nanobody enhances the antitumor activity of T-cells in synergy with dendritic cell vaccine.

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.

A Single-Chain Variable Fragment Antibody/Chemokine Fusion Protein Targeting Human Endoglin to Enhance the Anti-Tumor Activity of Cytokine-Induced Killer Cells.

Cytokine-induced killer cell immunotherapy is an ideal candidate for adoptive cell transfer therapy. However, therapeutic approaches to enhance the anti-tumor activity of cytokine-induced killer cells remain to be explored. Here, we described the successful development of a novel antibody-chemokine fusion protein containing the anti-human Endoglin antibody in the single-chain variable fragment format and human interferon-gamma-induced protein 10 (hENG scFv/hIP-10). Its anti-Endoglin immunoreactivity and chemotactic activity against the cytokine-induced killer cells were characterized in vitro. To evaluate the anti-tumor effect in vivo, cytokine-induced killer cells were intravenously injected into human hepatocellular carcinoma-bearing nude mice, together with intratumoral administration of the fusion protein hENG scFv/hIP-10 as an enhancer. The tumor volume and survival time of the mice were monitored, whilst the tumor-infiltrating cytokine-induced killer cells, serum levels of interferon-gamma, tumor cell proliferation, apoptosis, and angiogenesis were measured. The results demonstrated that hENG scFv/hIP-10 and cytokine-induced killer cells synergistically inhibited tumor growth and prolonged survival of tumor-bearing mice. Moreover, the number of tumor-infiltrating cytokine-induced killer cells, serum levels of interferon-gamma, and tumor cell apoptosis were increased, accompanied with decreased tumor proliferation and angiogenesis. Thus, our study suggests that hENG scFv/hIP-10 could enhance the anti-tumor activity of cytokine-induced killer cells against human hepatocellular carcinoma.

Nanobody: A Small Antibody with Big Implications for Tumor Therapeutic Strategy.

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.

Nanobody-based chimeric antigen receptor T cells designed by CRISPR/Cas9 technology for solid tumor immunotherapy.

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.

Recombinant Endoglin-Single-Chain Variable Fragment/ Induced Protein 10 Fusion Protein Potently Boosts the Anti-Tumor Efficacy of Adoptively Transferred TRP2-Specific CD8+ CD28+ Cytotoxic T Lymphocytes in Mice.

In this research, we studied the therapeutic efficacy of a newly designed fusion protein containing Endoglin single-chain variable fragment and IP10 (Endoglin-scFv/IP10), together with our recently generated TRP2-specific CD8+ CD28+ CTLs (CD8+ CD28+ CTLs) in controlling melanoma growth in mice. The recombinant Endoglin-scFv/IP10 was expressed in E. coli, purified by affinity chromatography, and characterized in vitro for its chemotactic movement and immunoreactivity with endoglin-expressing cells. In vivo, melanoma xenografts were established in mice (C57BL/6) using B16F10 cells. After that, mice were treated with intravenous injections of vehicle (PBS), Endoglin-scFv/IP10 alone, CD8+ CD28+ CTLs alone, or Endoglin-scFv/IP10+ CD8+ CD28+ CTLs. The therapeutic efficacy was assessed by monitoring tumor growth, mouse survival and cellular biomarkers. Endoglin-scFv/IP10 fusion protein combined with CD8+ CD28+ CTLs observed a reduction in tumor growth, resulting in improved survival. On the cellular level, the combination treatment dramatically reduced the number of systemic and tumor associated myeloid-derived suppressor cells or regulatory T cells, increased tumor-responsive interferon-γ-producing lymphocytes and tumor-associated CD8+ CXCR3+ T cells, and inhibited proliferation and angiogenesis but stimulated apoptosis within melanoma tissue. This study demonstrates the therapeutic potential of Endoglin-scFv/IP10 fusion protein in combination with CD8+ CD28+ CTLs in melanoma treatment.

Single-Domain Antibody-Based TCR-Like CAR-T: A Potential Cancer Therapy.

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.

Opportunities and Challenges for Antibodies against Intracellular Antigens.

Therapeutic antibodies are one most significant advances in immunotherapy, the development of antibodies against disease-associated MHC-peptide complexes led to the introduction of TCR-like antibodies. TCR-like antibodies combine the recognition of intracellular proteins with the therapeutic potency and versatility of monoclonal antibodies (mAb), offering an unparalleled opportunity to expand the repertoire of therapeutic antibodies available to treat diseases like cancer. This review details the current state of TCR-like antibodies and describes their production, mechanisms as well as their applications. In addition, it presents an insight on the challenges that they must overcome in order to become commercially and clinically validated.