METHODS. Two different pipelines of neoantigen identification were established in this study: (a) Clinical-grade targeted sequencing was performed in patients with refractory solid tumor, and mutant peptides with high variant allele frequency and predicted high HLA-binding affinity were synthesized de novo. (b) An inventory-shared neoantigen peptide library of common solid tumors was constructed, and patients' hotspot mutations were matched to the neoantigen peptide library. The candidate neoepitopes were identified by recalling memory T cell responses in vitro. Subsequently, neoantigen-loaded dendritic cell vaccines and neoantigen-reactive T cells were generated for personalized immunotherapy in 6 patients. RESULTS. Immunogenic neoepitopes were recognized by autologous T cells in 3 of 4 patients who used the de novo synthesis mode and in 6 of 13 patients who used the shared neoantigen peptide library. A metastatic thymoma patient achieved a complete and durable response beyond 29 months after treatment. Immune-related partial response was observed in another patient with metastatic pancreatic cancer. The remaining 4 patients achieved prolonged stabilization of disease with a median progression-free survival of 8.6 months. CONCLUSION. The current study provides feasible pipelines for neoantigen identification. Implementing these strategies to individually tailor neoantigens could facilitate neoantigen-based translational immunotherapy research.

Poor infiltration of activated lymphocytes into tumors represents a fundamental factor limiting the therapeutic effect of adoptive cell immunotherapy. A tumor-penetrating peptide, iRGD, has been widely used to deliver drugs into tumor tissues. In this study, we demonstrate for the first time that iRGD could also facilitate the infiltration of lymphocytes in both 3D tumor spheroids and several xenograft mouse models. In addition, combining iRGD modification with PD-1 knockout lymphocytes reveals a superior anti-tumor efficiency. Mechanistic studies demonstrate that the binding of iRGD to neuropilin-1 results in tyrosine phosphorylation of the endothelial barrier regulator VE-cadherin, which plays a role in the opening of endothelial cell contacts and the promotion of transendothelial lymphocyte migration. In summary, these results demonstrate that iRGD modification could promote tumor-specific lymphocyte infiltration, and thereby overcome the bottleneck associated with adoptive immune cell therapy in solid tumors.

BackgroundPoor infiltration and limited activation of transferred T cells are fundamental factors impeding the development of adoptive cell immunotherapy in solid tumors. A tumor-penetrating peptide iRGD has been widely used to deliver drugs deep into tumor tissues. CD3-targeting bispecific antibodies represent a promising immunotherapy which recruits and activates T cells.MethodsT-cell penetration was demonstrated in tumor spheroids using confocal microscope, and in xenografted tumors by histology and in vivo real-time fluorescence imaging. Activation and cytotoxicity of T cells were assessed by flow cytometry and confocal microscope. Bioluminescence imaging was used to evaluate in vivo antitumor effects, and transmission electron microscopy was used for mechanistic studies.ResultsWe generated a novel bifunctional agent iRGD-anti-CD3 which could immobilize iRGD on the surface of T cells through CD3 engaging. We found that iRGD-anti-CD3 modification not only facilitated T-cell infiltration in 3D tumor spheroids and xenografted tumor nodules but also induced T-cell activation and cytotoxicity against target cancer cells. T cells modified with iRGD-anti-CD3 significantly inhibited tumor growth and prolonged survival in several xenograft mouse models, which was further enhanced by the combination of programmed cell death protein 1 (PD-1) blockade. Mechanistic studies revealed that iRGD-anti-CD3 initiated a transport pathway called vesiculovacuolar organelles in the endothelial cytoplasm to promote T-cell extravasation.ConclusionAltogether, we show that iRGD-anti-CD3 modification is an innovative and bifunctional strategy to overcome major bottlenecks in adoptive cell therapy. Moreover, we demonstrate that combination with PD-1 blockade can further improve antitumor efficacy of iRGD-anti-CD3-modified T cells.

BackgroundIt was previously reported that targeting vascular epithelial growth factor (VEGF)/VEGFR could modulate the antitumor immunity. VEGFR2 inhibitor YN968D1 is a highly selective VEGFR2 inhibitor and was approved for the treatment of late-stage gastric cancer in 2014, but its role in antitumor immunity remains unknown.Materials and methodsIn this study, we investigated the effects of YN968D1 on the function of T cells in vitro by testing the cytotoxicity and cytokine production. Next, we constructed peritoneal dissemination and subcutaneous gastric cancer mouse model to assess the cytotoxicity of YN968D1-treated T cells in vivo, respectively.ResultsWe found that the use of YN968D1 in CD8+ T cells could reduce the expression levels of inhibitory checkpoints, such as Lag-3, PD-1, and Tim3, escalate the production of IFN-γ and IL-2 and promote the cytotoxicity of T cells dramatically in vitro. The transfer of YN968D1-treated T cells achieved better tumor control compared to DMSO-treated T cells or control in both peritoneal dissemination and subcutaneous gastric cancer mouse models.ConclusionOur results indicate that YN968D1 can enhance the T cell-mediated antitumor immunity.

Background: Cervical cancer stem cells (CCSCs) represent a subpopulation of tumor cells that possess self-renewal capacity and numerous intrinsic mechanisms of resistance to conventional chemotherapy and radiotherapy. These cells play a crucial role in relapse and metastasis of cervical cancer. Therefore, eradication of CCSCs is the primary objective in cervical cancer therapy. Salinomycin (Sal) is an agent used for the elimination of cancer stem cells (CSCs); however, the occurrence of several side effects hinders its application. Nanoscale drug-delivery systems offer great promise for the diagnosis and treatment of tumors. These systems can be used to reduce the side effects of Sal and improve clinical benefit. Methods: Sal-loaded polyethylene glycol-peptide-polycaprolactone nanoparticles (Sal NPs) were fabricated under mild and non-toxic conditions. The real-time biodistribution of Sal NPs was investigated through non-invasive near-infrared fluorescent imaging. The efficacy of tumor growth inhibition by Sal NPs was evaluated using tumor xenografts in nude mice. Flow cytometry, immunohistochemistry, and Western blotting were used to detect the apoptosis of CSCs after treatment with Sal NPs. Immunohistochemistry and Western blotting were used to examine epithelial-mesenchymal transition (epithelial interstitial transformation) signal-related molecules. Results: Sal NPs exhibited antitumor efficacy against cervical cancers by inducing apoptosis of CCSCs and inhibiting the epithelial-mesenchymal transition pathway. Besides, tumor pieces resected from Sal NP-treated mice showed decreased reseeding ability and growth speed, further demonstrating the significant inhibitory ability of Sal NPs against CSCs. Moreover, owing to targeted delivery based on the gelatinase-responsive strategy, Sal NPs was more effective and tolerable than free Sal. Conclusion: To the best of our knowledge, this is the first study to show that CCSC-targeted Sal NPs provide a potential approach to selectively target and efficiently eradicate CCSCs. This renders them a promising strategy to improve the therapeutic effect against cervical cancer.