CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

Open AccessCCS ChemistryRESEARCH ARTICLES14 Jun 2022CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination Jun-Jun Wu†, Fang-Yuan Chen†, Bei-Bei Han†, Hong-Qing Zhang†, Lang Zhao, Zhe-Rui Zhang, Juan-Juan Li, Bo-Dou Zhang, Ya-Nan Zhang, Yu-Xin Yue, Hong-Guo Hu, Wen-Hao Li, Bo Zhang, Yong-Xiang Chen, Dong-Sheng Guo and Yan-Mei Li Jun-Jun Wu† Key Lab of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084 Key Laboratory of Innate Immune Biology of Fujian Province, Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, Biomedical Research Center of South China, College of Life Sciences, Fujian Normal University, Fuzhou 350117 , Fang-Yuan Chen† College of Chemistry, Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071 , Bei-Bei Han† Key Lab of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084 , Hong-Qing Zhang† Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 University of Chinese Academy of Sciences, Beijing 100049 , Lang Zhao Key Lab of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084 , Zhe-Rui Zhang Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 University of Chinese Academy of Sciences, Beijing 100049 , Juan-Juan Li College of Chemistry, Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071 , Bo-Dou Zhang Key Lab of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084 , Ya-Nan Zhang Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 University of Chinese Academy of Sciences, Beijing 100049 , Yu-Xin Yue College of Chemistry, Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071 , Hong-Guo Hu Key Lab of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084 , Wen-Hao Li Key Lab of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084 , Bo Zhang *Corresponding authors: E-mail Address: email protected E-mail Address: email protected E-mail Address: email protected E-mail Address: email protected Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 University of Chinese Academy of Sciences, Beijing 100049 , Yong-Xiang Chen *Corresponding authors: E-mail Address: email protected E-mail Address: email protected E-mail Address: email protected E-mail Address: email protected Key Lab of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084 , Dong-Sheng Guo *Corresponding authors: E-mail Address: email protected E-mail Address: email protected E-mail Address: email protected E-mail Address: email protected College of Chemistry, Key Laboratory of Functional Polymer Materials, Ministry of Education, State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071 and Yan-Mei Li *Corresponding authors: E-mail Address: email protected E-mail Address: email protected E-mail Address: email protected E-mail Address: email protected Key Lab of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084 Beijing Institute for Brain Disorders, Beijing 100069 https://doi.org/10.31635/ccschem.022.202201859 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Stimulator of interferon genes, namely STING, an adaptor protein located in the endoplasmic reticulum, has been recognized as a shining target for cancer and infection research. However, STING agonists cyclic dinucleotides (CDNs) have shown almost zero efficacy in phase I clinical trials as a monotherapy, likely due to poor cellular permeability and rapid diffusion despite intratumoral injection. These deficiencies further affect other applications of CDNs, such as pandemic SARS-CoV-2 prevention and therapy. Here, we rationally design a supramolecular cytosolic delivery system based on controllable recognition of calixarene, namely CASTING (CAlixarene-STING), to improve CDN druggability, including degradation stability, cellular permeability, and tissue retention. CASTING efficiently enhances the immunostimulatory potency of CDGSF a chemically modified cyclic di-GMP (CDG) to generate an immunogenic microenvironment for melanoma regression, anti-PD-1 response rate increase, and durable memory formation against tumor recurrence. More importantly, CASTING displays a superior adjuvant activity on SARS-CoV-2 recombinant spike/receptor binding domain vaccines, inducing robust and coordinated T-cell and antibody responses against SARS-CoV-2 infection in vivo. Collectively, the CASTING design represents an innovative advancement to facilitate the clinical translational capability of STING agonists. Download figure Download PowerPoint Introduction Through linking innate immunity to adaptive immunity, interferons (IFNs) play an indispensable role in boosting robust antigen-specific responses for long-term immune protection against cancers and pathogens such as viruses.1 Therefore, regulating IFN secretion will significantly affect the efficacy of immunotherapy and the protective effect of vaccines. Despite revolutionizing the treatment of diverse cancers to realize complete and durable effects, immune checkpoint blockade (ICB) does not generate a positive response in the majority of patients.2 Clinical studies have revealed that the infiltration of tumor antigen-specific CD8+ T cells in the tumor microenvironment (TME) correlates with patients' response rate to ICB. Moreover, patients responding to ICB also exhibit a signature of genes for IFN production.2 These suggest that IFNs in the TME could lead to a breakthrough that maximizes the efficacy of ICB. Additionally, IFNs (type I and III) are also involved in inducing an antiviral state during SARS-CoV-2 infection; however, they exhibit delayed activation due to immune evasion by the virus.3 Up to now, the COVID-19 pandemic has led to over 500,000,000 cases and over 6,000,000 deaths globally, making vaccine-based global immunization an urgent necessity. Along with the emergence of SARS-CoV-2 variants of concern, the protective efficiency as well as antibody neutralizing activity of currently approved vaccines have shown a substantial diminishment of infection.4 In principle, T-cell responses (multiple T-cell epitopes distributed across the spike protein) are more efficient than neutralizing antibodies (targeting a narrow region in the spike) in preventing variant evasion and disease containment.5,6 Indeed, several studies on COVID-19 patients have identified that the disease severity was limited by a coordinated SARS-CoV-2-specific adaptive immunity (CD4+ and CD8+ T cells).7 Moreover, recent studies have revealed that SARS-CoV-2 variants of concern (B.1.1.7 and B.1.351) can partially escape humoral but not T-cell responses in COVID-19 convalescent donors and BNT162b2 vaccine recipients.6,8 Therefore, enhancing the cellular immunity of COVID-19 vaccines through the IFN pathway (mimicking viral infection) could potentially prevent variant evasion. However, except for mRNA and viral vector vaccines, currently approved inactivated and recombinant protein vaccines dominantly adopt aluminum hydroxide as an adjuvant, which hardly elicits cellular immunity.9 Famous for inducing type I IFN for T-cell cross-priming and activation, the stimulator of interferon genes (STING) pathway provides a potential choice for improving the efficacy of cancer immunotherapy and the SARS-CoV-2 vaccine.10,11 STING is a cytosolic endoplasmic reticulum (ER)-locating adaptor protein which recognizes endogenous and exogenous cyclic dinucleotides (CDNs).12–14 CDN binding results in the induction of type I IFN (IFN-β) and other inflammatory cytokines, which in turn selectively stimulate dendritic cells (DCs) and macrophages to prime antigen-specific T cells and activate antigen-independent natural killer (NK) cells.15 Further, as revealed by Chen et al.,16 STING is essential for the antitumor effect of ICB. In light of these findings, CDNs are being studied in several clinical trials (NCT02675439, NCT03010176, and NCT03956680) by Novartis, Merck, and BMS for advanced solid tumor treatment.13 However, the intratumoral injection of ADU-S100 (a CDN derivative) has shown almost zero activity as a monotherapy and a low response rate when combined with ICB (9% for ADU-S100 + Spartalizumab, 7% for ADU-S100 + Ipilimumab) in phase I clinical trials.17 These results are likely due to CDNs' poor cellular permeability and rapid diffusion, limited drug exposure, and permeability in tumor tissues and/or lymphoid organs to interact with STING in the cytosol.18–21 Thus, enhancing the cellular permeability and tissue retention of CDNs would be a breakthrough. In addition, the degree of agonist molecule accumulation in the lymph nodes (LNs) also critically affects the T-cell and antibody responses of therapeutic or preventive vaccines, meanwhile determining the intensity of off-target inflammation caused by systemic administration (a common challenge for clinical use).22 In this context, we aimed to design a supramolecular calixarene-based STING system, namely CASTING (CAlixarene-STING), as a novel host–guest formulation to address the deficiencies (instability, poor cellular permeability, and rapid diffusion) of CDNs (Figures 1a and 1b). Supramolecular prodrugs are drug delivery systems based on stimuli-responsive recognition,23,24 exhibiting the advantages of easy construction, molecular-level protection, quantitative loading, controlled release, reproducibility, and multidrug adaptability.25–29 Owing to the strong host–guest binding affinity ((1.0 ± 0.2) × 108 M−1) and cationic amphiphilic properties of guanidinium-modified calix5arene pentadodecyl ether (GC5A-12C), CDGSF a chemically modified cyclic di-GMP (CDG) is efficiently encapsulated into the calixarene cavity. Nanovesicles based on GC5A-12C protect CDGSF from enzymatic degradation, promote endosomal escape with surface guanidinium groups,30 and rapidly release CDGSF in response to the high adenosine triphosphate (ATP) concentration in the cytosol. Consequently, CASTING has remarkably improved the immunostimulatory activity of CDGSF in multiple cell types, and further generated an immunogenic T cell-inflamed TME to enhance the therapeutic impact of CDGSF and increase the ICB response rate in a melanoma model (Figure 1b). Moreover, the adjuvant efficacy of CASTING on SARS-CoV-2 recombinant spike/receptor binding domain (RBD) vaccines was validated to induce robust and coordinated T-cell and antibody responses which efficiently protected mice against SARS-CoV-2 infection in vivo (Figure 1b). Therefore, our results prove that CASTING provides an ideal supramolecular platform to improve the druggability of CDNs for cancer immunotherapy and a COVID-19 vaccine adjuvant. Figure 1 | Design and characterization of a calixarene-based supramolecular cytosolic delivery system (CASTING). (a and b) Schematics of CASTING preparation (a) and strategy for improving cytosolic delivery of STING agonist CDGSF for cancer immunotherapy and SARS-CoV-2 vaccine (b). (c and d) Competitive fluorescence titration (λex = 500 nm) (c) of GC5A-12C•Fl (1/0.5 μM) in HEPES buffer with CDGSF (up to 7 μM) and the associated titration curve (d) fitting by a 1:1 competitive binding model with only outer layer binding mode (λem = 512 nm).56 (e) TEM image of CASTING. (f and g) DLS analysis (f) and zeta potential distribution (g) of 12C-NV and CASTING. (h) HPLC analysis of CDGSF release from CASTING (10 μM CDGSF) in response to ATP (100 μM) incubation. Download figure Download PowerPoint Experimental Methods Preparation of CASTING CDGSF, GC5A-12C, and PEG-12C were synthesized according to our previous work ( Supporting Information Figures S1 and S2).28,31 GC5A-12C (1 mM in chloroform) and PEG-12C (1 mM in methanol) were mixed at a molar ratio of 1:1. The solvent was removed under vacuum for 12 h. 12C-NV was obtained by hydrating the residue in 10 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) buffer (pH 7.4), which was then sonicated at 80 °C for 4 h. CDGSF (2 mg/mL in HEPES buffer) was added into the 12C-NV solution at a molar ratio of 1:2 (CDGSF:GC5A-12C) to generate CASTING after mixing it well. The transmission electron microscopy (TEM) sample was prepared by dropping the solution onto a copper grid with a formvar and stained with uranyl acetate. The TEM sample was examined by high-resolution TEM (HITACHI HT7700 Exalens; HITACHI, Tokyo, Japan), equipped with a charge-coupled device camera. Dynamic light scattering (DLS) was performed on a laser light scattering spectrometer (NanoBrook 173plus and Brookhaven ZetaPals/BI-200SM; Brookhaven, New York, United States) equipped with a digital correlator at 659 and 532 nm at a scattering angle of 90°. Binding affinity determination The binding affinity between CDGSF and GC5A-12C was determined via a competitive fluorescence titration method. Fluorescein (Fl) was used as the reporter dye. In a solution of GC5A-12C•Fl (1.0 μM/ 0.5 μM) in HEPES buffer (10 mM, pH 7.4), CDGSF was added up to 7.0 μM. The binding affinity was fitted by a 1:1 competitive binding model (only the outer layer of the binding model) with OriginPro (2021b) software. Cells and animals The RAW264.7 cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Utah, United States). Bone marrow-derived dendritic cells (BMDCs) and B16F10 cell line were cultured in RPMI-1640 medium (HyClone, Utah, United States). Vero-E6 cell line used for SARS-CoV-2 plaque assay was cultured in DMEM. All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, California, United States), 100 U mL−1 penicillin (Sigma, Missouri, United States), and 100 μg mL−1 streptomycin (Sigma). For BMDCs preparation, bone marrow was isolated from female C57BL/6 mice (6–8 weeks old) and cultured in RPMI-1640 media with 20 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF) after the red blood cell (RBC) lysis. At days 3–4, media were replenished, and half of the media were replaced with fresh ones at days 6–7. At days 8–9, BMDCs were harvested, and the purity was evaluated using flow cytometry on CD11c+ cells. The female C57BL/6 and BALB/c mice used were raised at the Laboratory Animal Research Center of Tsinghua University with food and water provided ad libitum. In vitro evaluation of CDGSF cellular uptake and activity As to cellular uptake, CDGSF (2 mg/mL in HEPES) and AlPcS4 (2 mg/mL in HEPES) were simultaneously added into 12C-NV to prepare dye-coloading CASTING. Cells were plated in a 24-well plate (Thermo Fisher Scientific, Waltham, MA, United States)/8-well confocal imaging chamber (In Vitro Scientific, California, United States) and treated with CASTING at the indicated concentrations for 6 h for flow cytometry analysis and confocal laser scanning microscopy (CLSM). More details can be found in the Supporting Information. For stimulating activity assessments, RAW264.7 cells were plated in 24-well plates and treated with CASTING at a concentration of 3 μM CDGSF and 6 μM GC5A-12C for 24 h. Then, the cells were collected and incubated with PE-αCD86 antibody (GL-1, BD Pharmingen, New Jersey, United States) on ice for 1 h. The cells were washed and then analyzed on BD Calibur flow cytometry after washing. BMDCs were plated in a 24-well plate and treated with CASTING at a concentration of 0.5 μM CDGSF and 1 μM GC5A-12C for 24 h. After treatment, the cells and supernatants were collected. And cells were then strained with APC-αCD11c (N418, Biolegend), FITC-αCD40(3/23, Biolegend, California, United States), and PE-αCD86 (GL-1, Biolegend) and analyzed in BD LSRFortessa. IFN-β and CXCL-10 in supernatants were determined with an enzyme linked immunosorbent assay (ELISA) kit (Dakewe Biotech, Shenzhen, China) according to manufacturer's instructions. In vivo B16F10 tumor treatment Female C57BL/6 mice (4–6 weeks) were subcutaneously injected in the right flank with 1.5 × 105 or 2 × 105 B16F10 cells in 100 μL RPMI-1640 serum-free media. The tumor volume was measured every other day with callipers and calculated using an equation of V = L × W × W × 0.5. When the tumor diameter reached 15 mm, the mice were euthanized. Mice with established tumors were injected with CASTING in HEPES containing 6.2 μg or 15.5 μg CDGSF through an intratumoral or subcutaneous route. Anti-PD-1 (RMP1-14, Bio X Cell) was intraperitoneally injected at a dose of 200 μg in 100 μL phosphate-buffered saline (PBS). Tumor-tissue and tumor-draining lymph node imaging Female C57BL/6 mice with established B16F10 tumors were intratumorally injected with CASTING, CDGSF, 12C-NV, and AlPcS4 at a dose of 6.2 μg CDGSF and 1 μg AlPcS4. At 24 and 48 h after injection, tumors and tumor-draining lymph nodes (TDLNs) were harvested and imaged using in vivo imaging system (IVIS) Spectrum (PerkinElmer, Massachusetts, United States) (AlPcS4 was detected in the allophycocyanin (APC) channel). SARS-CoV-2 vaccine immunization The vaccines were prepared by adding proteins into CASTING solution and injected subcutaneously at the tail base of female BALB/c mice (6–8 weeks old) with doses of 5 μg spike proteins/10 μg RBD proteins and 6.2 μg CDGSF. Alhydrogel® adjuvant (cat. no.: vac-alu-250, InvivoGen, California, United States) was mixed with antigens (5 μg spike proteins or 10 μg RBD proteins) according to manufacturer's instructions and injected subcutaneously into the right flank of the mice at a dose of 300 μg. Immunizations were performed thrice biweekly. One week after the last immunization, antisera and spleens were harvested. SARS-CoV-2 antigen-binding ELISA Antibody titers and isotypes were determined according to previous procedures.31 More details can be found in the Supporting Information. IFN-γ ELISpot One week after the last vaccination, the mice spleens were ground, and single splenocytes were isolated through a cell strainer of 40 μm. After removing RBCs, splenocytes were counted and added to an IFN-γ-precoated 96-well plate (Dakewe Biotech, Shenzhen, China) at a density of 500,000/well. Then, SARS-CoV-2 spike/RBD proteins were added to wells (final concentration: 50 μg/mL for spike protein, 25 μg/mL for RBD protein). Splenocytes were stimulated for 36 h, using PMA + ionomycin as a positive control. IFN-γ spots were developed according to the ELISpot Kit instructions and read in the AID EliSpot Reader Systems. SARS-CoV-2 challenge experiment After two immunizations, mice were intranasally challenged with a 2 × 104 PFU mouse-adapted SARS-CoV-2 MP7 strain at day 56 and then monitored and weighed for 14 days. Blood samples from the orbit were collected and analyzed using a ProCyte Dx Hematology Analyzer (IDEXX) for five days after the challenge. Meanwhile, mice were sacrificed at days 1 and 3 postchallenge, and the lungs were collected. The left lungs were ground and centrifuged at 10,000 rpm for 10 min, and then the supernatant was used for viral titration by plaque assay while the right lungs were fixed with 4% paraformaldehyde for histopathological analysis by hematoxylin and eosin (H&E) staining. Flow cytometric analysis Immune cell populations were analyzed according to previous procedures.31 More details of tissue processing, cell separation, and antibody staining can be found in the Supporting Information. and Preparation of CASTING synthesized the molecule GC5A-12C as in our previous As in our previous for CDNs we CDGSF (Figure a of as a CDN model with improved stimulating the between CDNs and GC5A-12C, we evaluated binding affinity using an assay As GC5A-12C efficiently encapsulated CDGSF, by a in the fluorescence (1.0 ± 0.2) × 108 M−1) (Figures and on we GC5A-12C by GC5A-12C with at a molar ratio of 1:1 to protein and the (Figure After a solvent and 12C-NV was obtained with an diameter of 25 nm (Figure the CASTING was prepared by mixing CDGSF with 12C-NV in The of CASTING and 12C-NV was using DLS and TEM analysis (Figures and and Supporting Information Figure Additionally, the from of 12C-NV to of CASTING, a between CDGSF and GC5A-12C (Figure Moreover, such a of 25 nm and of the are for accumulation through and tumor tissue retention via the permeability and retention for improving the therapeutic and adjuvant efficacy of As a in the mM ATP was found to to with an affinity of over 108 the binding affinity of CDGSF and ATP GC5A-12C, we further evaluated the release of CDGSF from CASTING via of the CDGSF was rapidly from in response to 100 μM ATP (Figure the for the in vivo applications of CASTING. CASTING enhances the immunostimulatory activity of CDGSF evaluated the potential of CASTING to address in vitro (Figure As to the CDGSF a enzymatic with after a 6 h = with more than = 1 (Figure At the the calixarene in more than CDGSF after 48 h of = 80 that CASTING further improved the enzymatic of CDGSF. we examined the of CASTING to improve the cellular uptake of CDGSF using a strategy (Figure aluminum a used is to CDGSF in GC5A-12C binding affinity × 108 × 108 and Therefore, AlPcS4 was as a and with CDGSF on the CASTING to the uptake of CDGSF. dendritic cells (BMDCs) and macrophages incubated with (AlPcS4 + CDGSF) a increase in fluorescence intensity with CASTING the uptake by and in BMDCs and (Figure immune tumor cells such as melanoma have also been found to to STING and CASTING the melanoma uptake by over (Figure The uptake results were by confocal microscopy with an distribution of the fluorescence in the (Figure a endosomal escape due to the the of guanidinium and in that AlPcS4 fluorescence was binding to the GC5A-12C, these results an efficient cytosolic delivery and release of CDGSF, in or in tumor cells. Figure 2 | CASTING enhances the immunostimulatory activity of CDGSF by improving and cellular (a) of CDGSF delivery and in analysis of CDGSF, and CASTING 100 after with in CDN was using (c) of AlPcS4 CASTING. (d) Flow cytometric analysis of the cellular uptake of CDGSF with AlPcS4 by RAW264.7 and B16F10 cells. (e) of RAW264.7 and B16F10 cells incubated with CDGSF with AlPcS4. flow cytometry and of by RAW264.7 cells and (h) by BMDCs after with 12C-NV, CDGSF, and CASTING μM CDGSF for RAW264.7 0.5 μM CDGSF for for 24 h = analysis of with and The secretion of IFN-β and CXCL-10 in the supernatant of BMDCs after with 12C-NV, CDGSF, and CASTING μM CDGSF) for 24 h determined by ELISA = with Download figure Download PowerPoint on we further evaluated the effect of cellular uptake on immune cell activation (Figure CASTING treatment in robust RAW264.7 (Figure and (Figures and activation, as by the increase to in and to CDGSF 12C-NV treatment was also to activate RAW264.7 cells by which not The to be Moreover, we measured the secretion of IFN-β and CXCL-10 from BMDCs treated with CDGSF which are for T-cell activation and In line with the CDGSF uptake and BMDCs activation, CASTING the release of IFN-β and CXCL-10 to the CDGSF (Figures and based on the host–guest CASTING improved the potency of CDGSF by the of and poor cellular permeability CASTING an immunogenic tumor microenvironment we the impact of CASTING on the of B16F10 melanoma CASTING was injected intratumorally at and after melanoma (Figure CDGSF and 12C-NV were as 48 h after the injection, the and spleens were harvested for the flow cytometry an activation was evaluated to the T-cell CASTING treatment the of CD8+ T cells to CDGSF 12C-NV and (Figures and Further, in a