e14574 Background: TIGIT plays a crucial role in immune regulation, particularly in tumor environments, by acting as an inhibitory receptor. Leveraging its interaction with ligands like CD155 on tumor cells, we developed an innovative CAR-T cell therapy using a mutated TIGIT co-receptor to overcome inhibitory effect induced by CD155, together with targeting Prostate Stem Cell Antigen (PSCA) CAR, chosen for its high expression in bladder and other cancers versus its minimal presence in normal tissues. Methods: We initiated our approach by inducing strategic mutations in TIGIT, selecting a variant with heightened affinity for CD155. Bio-Layer Interferometry (BLI) analysis confirmed the variant's high binding affinity (KD = 1.635 nM). This mutated TIGIT, combined with CD28's signaling components, was integrated as a co-receptor in our CAR construct. For PSCA targeting, we isolated a high-affinity ScFv against PSCA from a fully human phage display library. The specificity of this ScFv for PSCA was rigorously validated using Cell Membrane Protein Targeting Catcher (CMPTC) technology, covering over 6000 human cell membrane proteins. Results: In our comparative analyses, CAR-T cells featuring a mutated TIGIT co-receptor (T-PSCA-CAR-T) matched the cytotoxicity of traditional second-generation BBz-structured CAR-T cells (PSCA-CAR-T) against PSCA-expressing HT-1376 bladder cancer cells. However, T-PSCA-CAR-T cells distinguished themselves by producing significantly higher IFN-γ levels (3805.683±65.506 vs 1271.712±39.768 pg/mL). Our in vivo tumor model, utilizing luciferase-expressing HT-1376 cells, demonstrated T-PSCA-CAR-T's enhanced anti-tumor efficacy, as evidenced by a more significant reduction in bioluminescence signal intensity (average decreasing ratio: 0.02 for T-PSCA-CAR-T vs 0.35 for PSCA-CAR-T; p < 0.05). These results underscore the efficacy of the mutated TIGIT co-receptor in improving CAR-T cell therapy. Conclusions: Our study signifies a transformative advancement in CAR-T cell therapy by incorporating a mutated TIGIT co-receptor, markedly improving therapeutic outcomes in bladder cancer treatment. Utilizing PSCA as a model target not only validates the efficacy of this novel approach but also paves the way for its application in a broader range of cancers, representing a significant leap in the field of immunotherapy.

Introduction:Chimeric antigen receptor T (CAR-T) cell therapy presents a promising treatment option for various cancers, including solid tumors. Carcinoembryonic antigen (CEA) is an attractive target due to its high expression in many tumors, particularly gastrointestinal cancers, while limited expression in normal adult tissues. In our previous clinical study, we reported a 70% disease control rate with no severe side effects using a humanized CEA-targeting CAR-T cell. However, the selection of the appropriate single-chain variable fragment (scFv) significantly affects the therapeutic efficacy of CAR-T cells by defining their specific behavior towards the target antigen. Therefore, this study aimed to identify the optimal scFv and investigate its biological functions to further optimize the therapeutic potential of CAR-T cells targeting CEA-positive carcinoma. Methods:We screened four reported humanized or fully human anti-CEA antibodies (M5A, hMN-14, BW431/26, and C2-45), and inserted them into a 3rd-generation CAR structure. We purified the scFvs and measured the affinity. We monitored CAR-T cell phenotype and scFv binding stability to CEA antigen through flow cytometry. We performed repeated CEA antigen stimulation assays to compare the proliferation potential and response of the four CAR-T cells, then further evaluated the anti-tumor efficacy of CAR-T cells ex vivo and in vivo. Results:M5A and hMN-14 CARs displayed higher affinity and more stable CEA binding ability than BW431/26 and C2-45 CARs. During CAR-T cell production culture, hMN-14 CAR-T cells exhibit a larger proportion of memory-like T cells, while M5A CAR-T cells showed a more differentiated phenotype, suggesting a greater tonic signal of M5A scFv. M5A, hMN-14, and BW431/26 CAR-T cells exhibited effective tumor cell lysis and IFN-γ release when cocultured with CEA-positive tumor cells in vitro, correlating with the abundance of CEA expression in target cells. While C2-45 resulted in almost no tumor lysis or IFN-γ release. In a repeat CEA antigen stimulation assay, M5A showed the best cell proliferation and cytokine secretion levels. In a mouse xenograft model, M5A CAR-T cells displayed better antitumor efficacy without preconditioning. Discussion:Our findings suggest that scFvs derived from different antibodies have distinctive characteristics, and stable expression and appropriate affinity are critical for robust antitumor efficacy. This study highlights the importance of selecting an optimal scFv in CAR-T cell design for effective CEA-targeted therapy. The identified optimal scFv, M5A, could be potentially applied in future clinical trials of CAR-T cell therapy targeting CEA-positive carcinoma.

e14530 Background: Nowadays, CAR-T cell therapy still faces limited therapeutic efficacy for solid tumors. SIRPγ is known as a ligand for a well-known immune checkpoint molecule CD47. It is distinguished from other members of its family in that it has a very short intracytoplasmic tail and is incapable of transducing signals on its own. Of note, the role of SIRPγ still largely unknown to date. Preliminary data indicated SIRPγ plays a key role in T-cell transendothelial migration and promotes antigen-specific T-cell proliferation. We thus designed a novel SIRPγ-CD28 chimeric receptor comprising the extracellular part of SIRPγ, the transmembrane and intracellular domains of CD28 and used as the co-receptor for a CEA-targeting CAR. Here, we report on the activity of this "armed" CAR-T with the SIRPγ-CD28 co-receptor in colorectal tumor (CRC) and its anti-tumor efficacy in mice xenograft model. Methods: In vitro luciferase-based cytotoxicity assay were used by coculturing CAR-T and DLD1-CEA CRC cells to assess the activities of CAR-T w/wo the co-receptor. The incubation supernatants were collected for detecting the release of cytokines. In vivo evaluation of the anti-tumor efficacy of CAR-T with the SIRPγ-CD28 co-receptor versus control T cell, and control CAR-T was performed in NOG mice CRC xenograft model. DLD1-CEA cells expressing luciferase reporter were s.c. implanted and T cells were i.v. injected. Anti-tumor efficacy was assessed by in vivo system. Results: CAR-T with the SIRPγ-CD28 co-receptor exhibited specific cytotoxicity only to CEA + CRC cells, and the efficacies of CAR-T w/wo the co-receptor were comparable (88.6% vs 87.8%, p > 0.05). However, cytokines were significantly higher in the CAR-T group with the co-receptor. The levels of IFNγ, IL2 and TNFα in groups of CAR-T w/wo the co-receptor were 72,793.33 vs 19,013.33 pg/ml (p < 0.05), 1834.67 vs 25.15 pg/ml (p < 0.05) and 79.65 vs 0 pg/ml (p < 0.05), respectively. In mice CRC xenografts model, the tumors were totally eliminated in the CAR-T group with the co-receptor at 21 days and lasted until the end of experiment, while the tumor growth was suppressed in the control CAR-T group at first and out-of-control in the end (Table). Conclusions: These preclinical studies demonstrated the potential of SIRPγ-CD28 co-receptor as a novel T cellular activation signals for CAR-T. The "armed" CAR-T exhibits a fabulous anti-tumor efficacy in CRC and has great potential for applications in other solid tumors. [Table: see text]

Nature killer (NK) cells, which don't require the human leukocyte antigen (HLA) matching, were considered as a promising immune cell for adoptive immunotherapy. Chimeric antigen receptor (CAR) has been proved as a powerful weapon to enhance antitumor ability of NK cell, which makes CAR-NK therapy an attractive candidate for "off-the-shelf" products. However, to date, CAR-NK development still faces several issues including low transduction efficiency, large-scale preparation, and short-time persistence in vivo. NK cells express a variety of signal-activating molecules, such as NKp44, NKp46, NKG2D, DAP10 and DAP12, which are the significant features of NK distinguish them from T cells. Thus, we speculated that CAR structure may be a critical factor in both tumoricidal function and persistence of CAR-NK. Therefore, we designed multiple CAR structures based on the signal pathways of NK (Figure 1A top). We engaged the transmembrane domain of NKp44, which could make CAR form a tripolymer with NK active molecule DAP12, and intracellular domains of 4-1BB and DAP10, which could either activate NF-κB or MAPK pathway, respectively (Figure 1A bottom). The antitumor efficacy of CAR-NK cells with the different CAR were evaluated in NCG model. Interestingly, CD19 scfv-8h-p44TM-BB-DAP10 (The scfv targeting CD19 was attached to the hinge of CD8, and fused to the NKp44 (transmembrane domain), 4-1BB and DAP10 (cytoplasmic) domains, CAR-4) showed the superior tumoricidal function (Figure 1B, C) and exhibited an outstanding persistence in NCG model without exogenous cytokines (Figure 1D). Our data suggested that natural immune receptor-like CAR structure provides a new perspective for CAR-NK optimization. To improve the transduction efficiency of CAR gene in PBMC derived NK cells (PBNK), we developed a lentiviral based NK.affi-LV vector. Our data showed that the positive rate of CAR could reach above 80% when transduced with NK.affi-LV (MOI=3) (Figure 1E) and kept stable in vitro and in vivo. Moreover, we confirmed that the vector could efficiently delivery a large exogenous gene (up to 4, 300 bp, a polycistron linked by 2A self-cleaving peptides). Apart from PBNK cells, highly efficient transduction of other lymphocytes, including γδ-T and B cells, was also achieved. Over 50% of cells were transduced at MOI of 3. These results indicate that the NK.affi-LV vectors have a broader application potency for adoptive immunotherapy. Additionally, a feeders-free culture procedure was established for producing CAR-NK from PBNK. The proliferation curve of CAR-NK from 3 health donors were concluded at Figure 1F, and the average amplification was 8250 (range, 3,000-15,000) after 21 days of culture. Thus, with this procedure, we are able to produce over 8e12 cells per batch when initialed from 1e9 PBNK, which should be sufficient for hundreds of patients. Together, our data give some new solutions to overcome the barriers of the clinical application of CAR-NK, and bring a new hope for the future "Off-the-shelf" CAR-NK production. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal