XCL1-secreting CEA CAR-T cells enhance endogenous CD8(+) T cell responses to tumor neoantigens to confer a long-term antitumor immunity.

Therapeutic efficacy of carcinoembryonic antigen (CEA)-specific chimeric antigen receptor (CAR) T cells against colorectal cancer (CRC) remains limited due to the unique characteristics and distinct microenvironments of tumor tissues. We modified CEA-specific CAR-T cells, aiming to stimulate endogenous CD8+ T cell responses against neoantigens that were derived from CEA-positive tumors destroyed by the CAR T cells. In a conventional CEA CAR (reg-CAR), we modified it to express lymphotactin XCL1 and interleukin (IL)-7 genes, constructing a modified 7XCL1-CAR. By generating the CEA-specific 7XCL1-CAR T cells, we assessed their antitumor efficacy against CRC cells with varying levels of CEA expression, both in cell-cultures and in two strains of tumor-bearing syngeneic mice. Following retroviral transduction, 7XCL1-CAR T cells and reg-CAR T cells exhibited similar positive proportions of CEA-CAR and CD4:CD8 ratios. In co-culture system with CEA-negative CT26 cells, no differences in cytotoxicity were observed between 7XCL1-CAR and reg-CAR T cells. However, in co-culture with CT26.CEAhigh and CT26.CEAint cells, 7XCL1-CAR T cells displayed higher cytotoxicity than that reg-CAR T cells after 60 hours. On interaction with CT26.CEA-positive cells, 7XCL1-CAR T cells secreted higher levels of XCL1 and IL-7, effectively recruited the most potent cross-presenting cDC1s (type-I conventional dendritic cells), and sustained the antitumor activity of CAR-T cells. In treating mice that carried tumors derived from universally CEA-positive cells, 7XCL1-CAR T cells exhibited no difference compared with reg-CAR T cells. However, in treating mice with tumors containing both CEA-positive and CEA-negative cells, 7XCL1-CAR T cells displayed greater inhibition than that of reg-CAR-T cells. After treatment of 7XCL1-CAR T cells, tumor-bearing mice exhibited enhanced infiltration of cDC1s, maintained CAR-T activity, and generation of endogenous neoantigen-specific T cells. Consequently, 7XCL1-CAR T cell-treated mice demonstrated resistance to challenge with CEA-negative CT26 cells. Treatment with CEA-specific, XCL1-secreting CAR-T cells for CEA-positive tumors promoted the generation of CD8+ T cells against tumor neoantigens, mediating a long-term antitumor immunity against heterogeneous CRCs.

Li XN ，Wang F ，Chen K ，Wu Z ，Zhang R ，Xiao C ，Zhao F ，Wang D ，Zhao H ，Ran Y ，Qu C ... -  《Journal for ImmunoTherapy of Cancer》

Targeting CEA in metastatic triple negative breast cancer with image-guided radiation followed by Fab-mediated chimeric antigen receptor (CAR) T-cell therapy.

Although CAR-T cell therapy has limited efficacy against solid tumors, it has been hypothesized that prior treatment with Image-Guided Radiation Therapy (IGRT) would increase CAR-T cell tumor infiltration, leading to improved antigen specific expansion of CAR-T cells. To test this hypothesis in a metastatic triple negative breast cancer (TNBC) model, we engineered two anti-CEA single-chain Fab (scFab) CAR-T cells with signaling domains from CD28zeta and 4-1BBzeta, and tested them in vitro and in vivo. The anti-CEA scFab CAR-T cells generated from three different human donors demonstrated robust in vitro expression, expansion, and lysis of only CEA-positive TNBC cells, with the CD28z-CAR-T cells showing the highest cytotoxicity. IFN-γ and granzyme B release assays revealed significantly higher IFN-γ production at a 4:1 effector-to-target (E:T) ratio in CD28z-CAR-T cells compared to 4-1BBz-CAR-T cells. Treatment of CEA-positive TNBC MDA-MB231 xenografts in the mammary fat pads of NSG mice, that produced spontaneous lung metastases over time, resulted in significant tumor growth reduction compared to either therapy alone (p<0.01). Immunohistochemical (IHC) analysis revealed that only combined IGRT and CAR-T therapy resulted in the elimination of lung metastases. These findings demonstrate that the combination of IGRT and anti-CEA scFab CAR-T therapy induces a strong antitumor response, effectively targeting both the primary tumor and distant metastatic lesions in the lungs, thus demonstrating that IGRT enhances CAR-T cell infiltration, persistence, and overall efficacy within both primary and metastatic lesions.

Aniogo E ，Kujawski M ，Awuah D ，Cha SE ，Espinosa R ，Hui S ，Ghimire H ，Yazaki PJ ，Brown CE ，Wang X ，Shively JE ... -  《Frontiers in Immunology》

[Construction of CD138-targeted chimeric antigen receptor- modified T cells and their effect in multiple myeloma therapy].

Guo CC ，Lu Y ，Tang KJ ，Xing HY ，Tian Z ，Rao Q ，Wang M ，Xiong DS ，Wang JX ... -  《-》

Anti-cancer immune effect of human colorectal cancer neoantigen peptide based on MHC class I molecular affinity screening.

Tumor antigen peptide vaccines have shown remarkable efficacy, safety, and reliability in recent studies. However, the screening process for immunopotent antigenic peptides is cumbersome, limiting their widespread application. Identifying neoantigen peptides that can effectively trigger an immune response is crucial for personalized cancer treatment. Whole exome sequencing was performed on patient-derived colon cancer cells to predict 9-amino-acid (9aa) neoantigen peptides. In vitro simulation of endogenous antigen presentation by antigen-presenting cells (dendritic cells) to CD8+ T cells was conducted, aiming to activate the CD8+ immune response to the predicted antigens. The immunological effects of each neoantigen were assessed using flow cytometry and ELISpot assays, while the relationship between neoantigen immunogenicity and MHC molecular affinity was examined. 1. Next-generation sequencing (NGS) predicted 9-amino acid (9aa) neoantigen peptides for subsequent immunological analysis.2. Higher mDC Levels in Experimental Group: CD11c+CD83+ mature dendritic cells (mDCs) were 96.6% in the experimental group, compared to 0.051% in the control group. CD80 fluorescence intensity was also significantly higher in the experimental group, confirming a greater mDC presence.3. Neoantigen Peptides Promote CD4+, CD8+ T, and NK Cell Proliferation: After 14 days, flow cytometry showed higher percentages of CD4+ T (37.41% vs 7.8%), CD8+ T (16.67% vs 14.63%), and NK cells (33.09% vs 7.81%) in the experimental group, indicating that the neoantigen peptides induced proliferation of CD4+, CD8+ T cells, and NK cells. 4. The results, analyzed using two-way ANOVA, showed that the standardized T-value for HLA molecular affinity variation in the 1-4 range (Group B) was significantly higher than for ≤1 (Group A, p < 0.0001) and >4 (Group C, p < 0.05). Regarding HLA-allele genotypes, HLA-Type 1 had a significantly higher standardized T-value than HLA-Type 2 (p < 0.05) and HLA-Type 3 (p < 0.0001). HLA-Type 1 was identified as the allele associated with the highest T-value. 1. The most immunogenic neoantigens typically exhibit an MHC molecular affinity variation between 1 and 4, indicating that stronger immunogenicity correlates with higher MHC molecular affinity variation. 2. Each patient's HLA molecules were classified into Types 1, 2, and 3, with Type 1 showing the highest binding capacity for neoantigens. Our findings indicate that the most immunogenic neoantigens were associated with HLA Type 1. 3. Neoantigen peptides were shown to activate the proliferation of both CD8+ T-cells and induce proliferation of CD4+ T-cells and NK cells. 4. Variation in MHC molecular affinity and HLA neoantigen genotype are anticipated to serve as valuable variables for screening highly immunogenic neoantigens, facilitating more efficient preparation of effective polypeptide tumor vaccines.

Zhang S ，Huang C ，Li Y ，Li Z ，Zhu Y ，Yang L ，Hu H ，Sun Q ，Liu M ，Cao S ... -  《Frontiers in Immunology》

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

《Jove-Journal of Visualized Experiments》