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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.

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 × 108 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.

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.

Shenxia Xie,1,2,* Xiaoqiong Hou,1,3,* Wei Yang,1,3,* Wei Shi,1,3 Xiaomei Yang,1,3 Siliang Duan,3 Fengzhen Mo,3 Aiqun Liu,3 Wu Wang,1,4 Xiaoling Lu1,3,5 1School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi, People’s Republic of China; 2Pharmaceutical College, Guangxi Medical University, Nanning, Guangxi, People’s Republic of China; 3International Nanobody Research Center of Guangxi, Guangxi Medical University, Nanning, Guangxi, People’s Republic of China; 4Laboratory of Tropical Biomedicine and Biotechnology, School of Tropical Medicine and Laboratory Medicine, Hainan Medical University, Haikou, Hainan, 571101, People’s Republic of China; 5College of Stomatology, Guangxi Medical University, Nanning, Guangxi, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaoling LuCollege of Stomatology, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of ChinaEmail luxiaoling@gxmu.edu.cnWu WangLaboratory of Tropical Biomedicine and Biotechnology, School of Tropical Medicine and Laboratory Medicine, Hainan Medical University, Haikou, Hainan, 571101, People’s Republic of ChinaEmail lxd8860@163.comBackground: 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 fun

Shuyang Sun,1,2 Ziqiang Ding,1 Xiaomei Yang,1,3 Xinyue Zhao,1,3 Minlong Zhao,1,2 Li Gao,1,3 Qu Chen,1,2 Shenxia Xie,1,4 Aiqun Liu,1 Shihua Yin,1 Zhiping Xu,5 Xiaoling Lu1,2 1International Nanobody Research Center, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 2School of Stomatology, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 3School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 4Department of Pharmacology, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 5Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD, 4072, AustraliaCorrespondence: Xiaoling LuNanobody Research Center, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of ChinaTel/Fax +86 771-2387 518Email luxiaoling@gxmu.edu.cnZhiping XuAustralian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD, 4072, AustraliaEmail gordonxu@uq.edu.auAbstract: 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

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.

Shenxia Xie,1,2,* Xiaoqiong Hou,1,3,* Wei Yang,1,3,* Wei Shi,1,3 Xiaomei Yang,1,3 Siliang Duan,3 Fengzhen Mo,3 Aiqun Liu,3 Wu Wang,1,4 Xiaoling Lu1,3,5 1School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi, People’s Republic of China; 2Pharmaceutical College, Guangxi Medical University, Nanning, Guangxi, People’s Republic of China; 3International Nanobody Research Center of Guangxi, Guangxi Medical University, Nanning, Guangxi, People’s Republic of China; 4Laboratory of Tropical Biomedicine and Biotechnology, School of Tropical Medicine and Laboratory Medicine, Hainan Medical University, Haikou, Hainan, 571101, People’s Republic of China; 5College of Stomatology, Guangxi Medical University, Nanning, Guangxi, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaoling LuCollege of Stomatology, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of ChinaEmail luxiaoling@gxmu.edu.cnWu WangLaboratory of Tropical Biomedicine and Biotechnology, School of Tropical Medicine and Laboratory Medicine, Hainan Medical University, Haikou, Hainan, 571101, People’s Republic of ChinaEmail lxd8860@163.comBackground: 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 fun

Shuyang Sun,1,2 Ziqiang Ding,1 Xiaomei Yang,1,3 Xinyue Zhao,1,3 Minlong Zhao,1,2 Li Gao,1,3 Qu Chen,1,2 Shenxia Xie,1,4 Aiqun Liu,1 Shihua Yin,1 Zhiping Xu,5 Xiaoling Lu1,2 1International Nanobody Research Center, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 2School of Stomatology, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 3School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 4Department of Pharmacology, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 5Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD, 4072, AustraliaCorrespondence: Xiaoling LuNanobody Research Center, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of ChinaTel/Fax +86 771-2387 518Email luxiaoling@gxmu.edu.cnZhiping XuAustralian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD, 4072, AustraliaEmail gordonxu@uq.edu.auAbstract: 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

Topic evolution modeling has received significant attentions in recent decades. Although various topic evolution models have been proposed, most studies focus on the single document corpus. However in practice, we can easily access data from multiple sources and also observe relationships between them. Then it is of great interest to recognize the relationship between multiple text corpora and further utilize this relationship to improve topic modeling. In this work, we focus on a special type of relationship between two text corpora, which we define as the "lead-lag relationship". This relationship characterizes the phenomenon that one text corpus would influence the topics to be discussed in the other text corpus in the future. To discover the lead-lag relationship, we propose a jointly dynamic topic model and also develop an embedding extension to address the modeling problem of large-scale text corpus. With the recognized lead-lag relationship, the similarities of the two text corpora can be figured out and the quality of topic learning in both corpora can be improved. We numerically investigate the performance of the jointly dynamic topic modeling approach using synthetic data. Finally, we apply the proposed model on two text corpora consisting of statistical papers and the graduation theses. Results show the proposed model can well recognize the lead-lag relationship between the two corpora, and the specific and shared topic patterns in the two corpora are also discovered.

Ziqiang Ding,1,2,* Dujin Wang,1,2,* Wei Shi,2,3,* Xiaomei Yang,2,3 Siliang Duan,2 Fengzhen Mo,2 Xiaoqiong Hou,2,3 Aiqun Liu,2 Xiaoling Lu2,4 1National Center for International Research of Biological Targeting Diagnosis and Therapy, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China; 2International Nanobody Research Center of Guangxi, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China; 3School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China; 4College of Stomatology, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaoling LuInternational Nanobody Research Center of Guangxi, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of ChinaTel/Fax +86 771-2387 518Email luxiaoling@gxmu.edu.cnPurpose: Cancer tissue-specific and nuclei-targeted drug delivery is ideal for the delivery of chemotherapy. However, it has only been achieved in in vitro studies mainly due to low efficiency in vivo. In this study, we aimed to establish an efficient dual-targeted system that targets liver cancer tissue as well as the nuclei of cancer cells in vivo.Methods: We first synthesized TAT peptide (TATp)-mesoporous silica nanoparticle (MSN) complex (TATp-MSN) and generated liposomes that carried liver cancer-specific aptamer TLS11a (TLS11a-LB). We then generated the drug TLS11a-LB@TATp-MSN/doxorubicin (DOX) by mixing TLS11a-LB and DOX-loaded TATp-MSN. After physical and chemical characterization of the nanoparticles, DOX release from these formulations was evaluated at pH 5.0 and 7.4. Furthermore, we also evaluated nuclear localization and cytotoxicity of the drug in H22 cells in vitro and investigated the liver cancer targeting and antitumor activities of the nano-drug in vivo using a H22 tumor-bearing mice model.Results

Ziqiang Ding,1,2,* Dujin Wang,1,2,* Wei Shi,2,3,* Xiaomei Yang,2,3 Siliang Duan,2 Fengzhen Mo,2 Xiaoqiong Hou,2,3 Aiqun Liu,2 Xiaoling Lu2,4 1National Center for International Research of Biological Targeting Diagnosis and Therapy, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China; 2International Nanobody Research Center of Guangxi, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China; 3School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China; 4College of Stomatology, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaoling LuInternational Nanobody Research Center of Guangxi, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of ChinaTel/Fax +86 771-2387 518Email luxiaoling@gxmu.edu.cnPurpose: Cancer tissue-specific and nuclei-targeted drug delivery is ideal for the delivery of chemotherapy. However, it has only been achieved in in vitro studies mainly due to low efficiency in vivo. In this study, we aimed to establish an efficient dual-targeted system that targets liver cancer tissue as well as the nuclei of cancer cells in vivo.Methods: We first synthesized TAT peptide (TATp)-mesoporous silica nanoparticle (MSN) complex (TATp-MSN) and generated liposomes that carried liver cancer-specific aptamer TLS11a (TLS11a-LB). We then generated the drug TLS11a-LB@TATp-MSN/doxorubicin (DOX) by mixing TLS11a-LB and DOX-loaded TATp-MSN. After physical and chemical characterization of the nanoparticles, DOX release from these formulations was evaluated at pH 5.0 and 7.4. Furthermore, we also evaluated nuclear localization and cytotoxicity of the drug in H22 cells in vitro and investigated the liver cancer targeting and antitumor activities of the nano-drug in vivo using a H22 tumor-bearing mice model.Results