Construction of Switch Modules for CAR-T Cell Treatment Using a Site-Specific Conjugation System.

Chimeric antigen receptor T-cell (CAR-T cell) therapy has become a promising treatment option for B-cell hematological tumors. However, few optional target antigens and disease relapse due to loss of target antigens limit the broad clinical applicability of CAR-T cells. Here, we conjugated an antibody (Ab) fusion protein, consisting of an Ab domain and a SpyCatcher domain, with the FITC-SpyTag (FITC-ST) peptide to form a bispecific safety switch module using a site-specific conjugation system. We applied the safety switch module to target CD19, PDL1, or Her2-expressing tumor cells by constructing FMC63 (anti-CD19), antiPDL1, or ZHER (anti-Her2)-FITC-ST, respectively. Those switch modules significantly improved the cytotoxic effects of anti-FITC CAR-T cells on tumor cells. Additionally, we obtained the purified CD8+ T cells by optimizing a shorter version of the CD8-binding aptamer to generate anti-FITC CD8-CAR-T cells, which combined with the CD4-FITC-ST switch module (anti-CD4) to eliminate the CD4-positive tumor cells in vitro and in vivo. Overall, we established a novel safety switch module by site-specific conjugation to enhance the antitumor function of universal CAR-T cells, thereby expanding the application scope of CAR-T therapy and improving its safety and efficacy.

Anti-CD79b/CD3 bispecific antibody combined with CAR19-T cells for B-cell lymphoma treatment.

CD19 CAR-T (chimeric antigen receptor-T) cell immunotherapy achieves a remission rate of approximately 70% in recurrent and refractory lymphoma treatment. However, the loss or reduction of CD19 antigen on the surface of lymphoma cells results in the escape of tumor cells from the immune killing of CD19 CAR-T cells (CAR19-T). Therefore, novel therapeutic strategies are urgently required. In this study, an anti-CD79b/CD3 bispecific antibody (BV28-OKT3) was constructed and combined with CAR19-T cells for B-cell lymphoma treatment. When the CD19 antigen was lost or reduced, BV28-OKT3 redirected CAR19-T cells to CD79b+ CD19- lymphoma cells; therefore, BV28-OKT3 overcomes the escape of CD79b+ CD19- lymphoma cells by the killing action of CAR19-T cells in vitro and in vivo. Furthermore, BV28-OKT3 triggered the antitumor function of CAR- T cells in the infusion product and boosted the antitumor immune response of bystander T cells, markedly improving the cytotoxicity of CAR19-T cells to lymphoma cells in vitro and in vivo. In addition, BV28-OKT3 elicited the cytotoxicity of donor-derived T cells toward lymphoma cells in vitro, which depended on the presence of tumor cells. Therefore, our findings provide a new clinical treatment strategy for recurrent and refractory B-cell lymphoma by combining CD79b/CD3 BsAb with CAR19-T cells.

Bromodomains and Extra-Terminal (BET) Inhibitor JQ1 Suppresses Proliferation of Acute Lymphocytic Leukemia by Inhibiting c-Myc-Mediated Glycolysis.

BACKGROUND Acute lymphocytic leukemia (ALL) is a common blood cancer which induces high mortality in children. Bromodomains and extra-terminal (BET) protein inhibitors, such as JQ1 and ARV-825, are promising cancer therapeutic agents that can be used by targeting c-Myc. A recent work reported that JQ1 effectively attenuates ALL in vitro by suppressing cell proliferation and accelerating apoptosis. The purpose of this research was to probe into the potential mechanism of how JQ1 inhibits ALL cell proliferation in vitro. MATERIAL AND METHODS Cell viability of ALL cells were measured by CTG after treatment by JQ1. Cell cycle analysis was done by EdU and PI staining. Cell apoptosis was assessed by Annexin V/PI staining. Glycolysis was detected using Seahorse and LC-MS kits. The expression of glycolytic rate-limiting enzymes was assessed by RNA-seq, qRT-PCR, and Western blot. RESULTS JQ1 suppressed cell proliferation by arresting the cell cycle and inducing the apoptosis of acute lymphocytic leukemia cells. JQ1 inhibited cell proliferation of B-ALL cells by restraining glycolysis. Conversely, the cell cycle block of B-ALL cells induced by JQ1 was partially abolished after pretreatment with 2-Deoxy-D-glucose (2-DG), an inhibitor of glycolysis. Furthermore, JQ1 restrained the glycolysis of B-ALL cell lines by remarkably downregulating the rate-limiting enzymes of glycolysis, such as hexokinase 2, phosphofructokinase, and lactate dehydrogenase A. Moreover, the cell cycle arrest was reversed in B-ALL cells with overexpressed c-Myc treated by JQ1, which is involved in the enhancement of glycolysis. CONCLUSIONS The BET inhibitor JQ1 suppresses the proliferation of ALL by inhibiting c-Myc-mediated glycolysis, thus providing a new strategy for the treatment of ALL.

CAR-Aptamers Enable Traceless Enrichment and Monitoring of CAR-Positive Cells and Overcome Tumor Immune Escape.

Chimeric antigen receptor (CAR)-positive cell therapy, specifically with anti-CD19 CAR-T (CAR19-T) cells, achieves a high complete response during tumor treatment for hematological malignancies. Large-scale production and application of CAR-T therapy can be achieved by developing efficient and low-cost enrichment methods for CAR-T cells, expansion monitoring in vivo, and overcoming tumor escape. Here, novel CAR-specific binding aptamers (CAR-ap) to traceless sort CAR-positive cells and obtain a high positive rate of CAR19-T cells is identified. Additionally, CAR-ap-enriched CAR19-T cells exhibit similar antitumor capacity as CAR-ab (anti-CAR antibody)-enriched CAR-T cells. Moreover, CAR-ap accurately monitors the expansion of CAR19-T cells in vivo and predicts the prognosis of CAR-T treatment. Essentially, a novel class of stable CAR-ap-based bispecific circular aptamers (CAR-bc-ap) is constructed by linking CAR-ap with a tumor surface antigen (TSA): protein tyrosine kinase 7 (PTK7) binding aptamer Sgc8. These CAR-bc-aps significantly enhance antitumor cytotoxicity with a loss of target antigens by retargeting CAR-T cells to the tumor in vitro and in vivo. Overall, novel CAR-aptamers are screened for traceless enrichment, monitoring of CAR-positive cells, and overcoming tumor cell immune escape. This provides a low-cost and high-throughput approach for CAR-positive cell-based immunotherapy.

Construction of a versatile fusion protein for targeted therapy and immunotherapy.

Antibody (Ab)-based drugs have been widely used in targeted therapies and immunotherapies, leading to significant improvements in tumor therapy. However, the failure of Ab therapy due to the loss of target antigens or Ab modifications that affect its function limits its application. In this study, we expanded the application of antibodies (Abs) by constructing a fusion protein as a versatile tool for Ab-based target cell detection, delivery, and therapy. We first constructed a SpaC Catcher (SpaCC for short) fusion protein that included the C domains of Staphylococcal protein A (SpaC) and the SpyCatcher. SpaCC conjugated with SpyTag-X (S-X) to form the SpaCC-S-X complex, which binds non-covalently to an Ab to form the Ab-SpaCC-S-X protein complex. The "X" can be a variety of small molecules such as fluoresceins, cell-penetrating peptide TAT, Monomethyl auristatin E (MMAE), and DNA. We found that Ab-SpaCC-S-FITC(-TAT) could be used for target cell detection and delivery. Besides, we synthesized the Ab-SpaCC-SN3-MMAE complex by linking Ab with MMAE by SpaCC, which improved the cytotoxicity of small molecule toxins. Moreover, we constructed an Ab-DNA complex by conjugating SpaCC with the aptamer (Ap) and found that Ab-SpaCC-SN3-Ap boosted the tumor-killing function of T-cells by retargeting tumor cells. Thus, we developed a multifunctional tool that could be used for targeted therapies and immunotherapies, providing a cheap and convenient novel drug development strategy.

A novel His-tag-binding aptamer for recombinant protein detection and T cell-based immunotherapy.

Screening novel aptamers for recombinant protein detection is of great significance in industrial mass production of antibody drugs. In addition, construction of structurally stable bispecific circular aptamers (bc-apts) may provide a tumor-targeted treatment strategy by simultaneously binding two different cell types. In this study, we obtained a high-affinity hexahistidine tag (His-tag)-binding aptamer 20S and explored its application in recombinant protein detection and T cell-based immunotherapy. We developed a new molecular beacon (MB) 20S-MB to detect His-tagged proteins in vitro and in vivo with high sensitivity and specificity, and the results showed high consistency with the enzyme-linked immunosorbent assay (ELISA). Moreover, we constructed two kinds of bc-apts by cyclizing 20S or another His-tag-binding aptamer, 6H5-MU, with Sgc8, which specifically recognizes protein tyrosine kinase 7 (PTK7) on tumor cells. After forming a complex with His-tagged OKT3, an anti-CD3 antibody for T cell activation, we utilized these aptamer-antibody complexes (ap-ab complex) to enhance cytotoxicity of T cells by linking T cells and target cells together, and 20S-sgc8 exhibited antitumor efficacy superior to that of 6H5-sgc8. In conclusion, we screened a novel His-tag-binding aptamer and used it to construct a new type of MB for rapid detection of recombinant proteins, as well as establish a feasible approach for T cell-based immunotherapy.

CDK7 Inhibitor THZ1 Induces the Cell Apoptosis of B-Cell Acute Lymphocytic Leukemia by Perturbing Cellular Metabolism.

B-cell acute lymphocytic leukemia (B-ALL) is a malignant blood cancer that develops in children and adults and leads to high mortality. THZ1, a covalent cyclin-dependent kinase 7 (CDK7) inhibitor, shows anti-tumor effects in various cancers by inhibiting cell proliferation and inducing apoptosis. However, whether THZ1 has an inhibitory effect on B-ALL cells and the underlying mechanism remains obscure. In this study, we showed that THZ1 arrested the cell cycle of B-ALL cells in vitro in a low concentration, while inducing the apoptosis of B-ALL cells in vitro in a high concentration by activating the apoptotic pathways. In addition, RNA-SEQ results revealed that THZ1 disrupted the cellular metabolic pathways of B-ALL cells. Moreover, THZ1 suppressed the cellular metabolism and blocked the production of cellular metabolic intermediates in B-ALL cells. Mechanistically, THZ1 inhibited the cellular metabolism of B-ALL by downregulating the expression of c-MYC-mediated metabolic enzymes. However, THZ1 treatment enhanced cell apoptosis in over-expressed c-MYC B-ALL cells, which was involved in the upregulation of p53 expression. Collectively, our data demonstrated that CDK7 inhibitor THZ1 induced the apoptosis of B-ALL cells by perturbing c-MYC-mediated cellular metabolism, thereby providing a novel treatment option for B-ALL.

CDK9 Inhibitor Induces the Apoptosis of B-Cell Acute Lymphocytic Leukemia by Inhibiting c-Myc-Mediated Glycolytic Metabolism.

B-cell acute lymphocytic leukemia (B-ALL), a common blood cancer in children, leads to high mortality. Cyclin-dependent kinase 9 inhibitor (CDK9i) effectively attenuates acute myeloid leukemia and chronic lymphoblastic leukemia by inducing apoptosis and inhibiting cell proliferation. However, the effect of CDK9i on B-ALL cells and the underlying mechanisms remain unclear. In this study, we showed that CDK9i induced the apoptosis of B-ALL cells in vitro by activating the apoptotic pathways. In addition, CDK9i restrained the glycolytic metabolism of B-ALL cells, and CDK9i-induced apoptosis was enhanced by co-treatment with glycolysis inhibitors. Furthermore, CDK9i restained the glycolysis of B-ALL cell lines by markedly downregulating the expression of glucose transporter type 1 (GLUT1) and the key rate-limiting enzymes of glycolysis, such as hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA). Moreover, cell apoptosis was rescued in B-ALL cells with over-expressed c-Myc after treatment with CDK9i, which is involved in the enhancement of glycolytic metabolism. In summary, our findings suggest that CDK9 inhibitors induce the apoptosis of B-ALL cells by inhibiting c-Myc-mediated glycolytic metabolism, thus providing a new strategy for the treatment of B-ALL.