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The lysosome-targeting chimera (LYTAC) strategy provided a very powerful tool for the degradation of membrane proteins. However, the synthesis of LYTACs, antibody-small molecule conjugates, is challenging. The ability of antibody-based LYTACs to penetrate solid tumor is limited as well, especially to cross the blood-brain barrier (BBB). Here, we propose a covalent chimeric peptide-based targeted degradation platform (Pep-TACs) by introducing a long flexible aryl sulfonyl fluoride group, which allows proximity-enabled cross-linking upon binding with the protein of interest. The Pep-TACs platform facilitates the degradation of target proteins through the mechanism of recycling transferrin receptor (TFRC)-mediated lysosomal targeted endocytosis. Biological experiments demonstrate that covalent Pep-TACs can significantly degrade the expression of PD-L1 on tumor cells, dendritic cells and macrophages, especially under acidic conditions, and markedly enhance the function of T cells and tumor phagocytosis by macrophages. Furthermore, both in anti-PD-1-responsive and -resistant tumor models, the Pep-TACs exert significant anti-tumor immune response. It is noteworthy that Pep-TACs can cross the BBB and prolong the survival of mice with in situ brain tumor. As a proof-of-concept, this study introduces a modular TFRC-based covalent peptide degradation platform for the degradation of membrane protein, and especially for the immunotherapy of brain tumors. LYTAC strategies often face challenges in solid tumor penetration and synthesis. Here, the authors introduce Pep-TACs, a modular TFRC-based covalent peptide degradation platform that effectively degrades membrane protein PD-L1. This approach significantly suppresses both anti-PD-1-responsive and -resistant tumor growth, particularly in brain tumors.

Background Esophageal squamous cell carcinoma (ESCC) is a common gastrointestinal malignancy with poor patient prognosis. Current treatment for ESCC, including immunotherapy, is only beneficial for a small subset of patients. Better characterization of the tumor microenvironment (TME) and the development of novel therapeutic targets are urgently needed. Methods In the present study, we hypothesized that integration of single-cell transcriptomic sequencing and large microarray sequencing of ESCC biopsies would reveal the key cell subtypes and therapeutic targets that determine the prognostic and tumorigenesis of ESCC. We characterized the gene expression profiles, gene sets enrichment, and the TME landscape of a microarray cohort including 84 ESCC tumors and their paired peritumor samples. We integrated single-cell transcriptomic sequencing and bulk microarray sequencing of ESCC to reveal key cell subtypes and druggable targets that determine the prognostic and tumorigenesis of ESCC. We then designed and screened a blocking peptide targeting Chemokine C–C motif ligand 18 (CCL18) derived from tumor associated macrophages and validated its potency by MTT assay. The antitumor activity of CCL18 blocking peptide was validated in vivo by using 4-nitroquinoline-1-oxide (4-NQO) induced spontaneous ESCC mouse model. Results Comparative gene expression and cell–cell interaction analyses revealed dysregulated chemokine and cytokine pathways during ESCC carcinogenesis. TME deconvolution and cell interaction analyses allow us to identify the chemokine CCL18 secreted by tumor associated macrophages could promote tumor cell proliferation via JAK2/STAT3 signaling pathway and lead to poor prognosis of ESCC. The peptide Pep3 could inhibit the proliferation of EC-109 cells promoted by CCL18 and significantly restrain the tumor progression in 4-NQO-induced spontaneous ESCC mouse model. Conclusions For the first time, we discovered and validated that CCL18 blockade could significantly prevent ESCC progression. Our study revealed the comprehensive cell–cell interaction network in the TME of ESCC and provided novel therapeutic targets and strategies to ESCC treatment.

Background Metastasis is the leading cause of mortality in human cancers, including esophageal squamous cell carcinoma (ESCC). As a pro-inflammatory cytokine, IL-32 was reported to be a poor prognostic factor in many cancers. However, the role of IL-32 in ESCC metastasis remains unknown. Methods ESCC cells with ectopic expression or knockdown of IL-32 were established and their effects on cell motility were detected. Ultracentrifugation, Transmission electron microscopy and Western blot were used to verify the existence of extracellular vesicle IL-32 (EV-IL-32). Coculture assay, immunofluorescence, flow cytometry, and in vivo lung metastasis model were performed to identify how EV-IL-32 regulated the crosstalk between ESCC cells and macrophages. Results Here, we found that IL-32 was overexpressed and positively correlated to lymph node metastasis of ESCC. IL-32 was significantly higher in the tumor nest compared with the non-cancerous tissue. We found that IL-32β was the main isoform and loaded in EV derived from ESCC cells. The shuttling of EV-IL-32 derived from ESCC cells into macrophages could promote the polarization of M2 macrophages via FAK-STAT3 pathway. IL-32 overexpression facilitated lung metastasis and was positively correlated with the proportion of M2 macrophages in tumor microenvironment. Conclusions Taken together, our results indicated that EV-IL-32 derived from ESCC cell line could be internalized by macrophages and lead to M2 macrophage polarization via FAK-STAT3 pathway, thus promoting the metastasis of ESCC. These findings indicated that IL-32 could serve as a potential therapeutic target in patients with ESCC.

BackgroundImmunotherapy has achieved remarkable advances via a variety of strategies against tumor cells that evade immune surveillance. As important innate immune cells, macrophages play important roles in maintaining homeostasis, preventing pathogen invasion, resisting tumor cells and promoting adaptive immune response. CD47 is found to be overexpressed on tumor cells and act as a don’t eat me’ signal, which contributes to immune evasion. Macrophages mediated phagocytosis via blockade CD47/SIRPα (signal regulatory protein alpha) interaction was proved to induce effective antitumor immune response.MethodsA novel peptide pep-20, specifically targeting CD47 and blocking CD47/SIRPα interaction, was identified via high-throughput phage display library bio-panning. The capability to enhance the macrophage-mediated phagocytosis activities and antitumor effects of pep-20 were investigated. The mechanism of pep-20 to induce T-cell response was explored by ex vivo analysis and confirmed via macrophage depleting strategy. The structure-activity relationship and D-amino acid substitution of pep-20 were also studied. The antitumor effects and mechanism of a proteolysis resistant D-amino acid derivate pep-20-D12 combined with irradiation (IR) were also investigated.ResultsPep-20 showed remarkable enhancement of macrophage-mediated phagocytosis to both solid and hematologic tumor cells in vitro, and inhibited tumor growth in immune-competent tumor-bearing mice. Furthermore, pep-20 promoted macrophages to mobilize the antitumor T-cell response with minimal toxicity. Furthermore, systemic administration of the derivate pep-20-D12 showed robust synergistic antitumor efficacy in combination with IR.ConclusionIn summary, these results demonstrated that CD47/SIRPα blocking peptides, pep-20 and its derivate, could serve as promising candidates to promote macrophages-mediated phagocytosis and immune response in cancer immunotherapy.

BackgroundAside from immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1), intervention of CD47/Sirpα mediated ‘don’t eat me’ signal between macrophage and tumor cell is considered as a promising therapeutic approach for cancer immunotherapy. Compared with CD47, the novel immune checkpoint CD24/Siglec-10 can also deliver ‘don’t eat me’ signal and CD24 shows much lower expression level in normal tissue which might avoid unwanted side effects.MethodsCell-based phage display biopanning and D-amino acid modification strategy were used to identify the CD24/Siglec-10 blocking peptide. Cell-based blocking assay and microscale thermophoresis assay were used to validate the blocking and binding activities of the peptide. Phagocytosis and co-culture assays were used to explore the in vitro function of the peptide. Flow cytometry was performed to assess the immune microenvironment after the peptide treatment in vivo.ResultsA CD24/Siglec-10 blocking peptide (CSBP) with hydrolysis-resistant property was identified. Surprisingly, we found that CSBP could not only block the interaction of CD24/Siglec-10 but also PD-1/PD-L1. CSBP could induce the phagocytosis of tumor cell by both the macrophages and monocytic myeloid-derived suppressor cells (M-MDSCs), which can further activate CD8+ T cells. Besides, combination of radiotherapy and CSBP synergistically reduced tumor growth and altered the tumor microenvironment in both anti-PD-1-responsive MC38 and anti-PD-1-resistant 4T1 tumor models.ConclusionsIn summary, this is the first CD24/Siglec-10 blocking peptide which blocked PD-1/PD-L1 interaction as well, functioned via enhancing the phagocytosis of tumor cells by macrophages and M-MDSCs, and elevating the activity of CD8+ T cells for cancer immunotherapy.

Background Targeting the tumor microenvironment (TME) has emerged as a promising strategy in cancer treatment, particularly through the utilization of immune checkpoint blockade (ICB) agents such as PD-1/PD-L1 inhibitors. Despite partial success, the presence of tumor-associated macrophages (TAMs) contributes to an immunosuppressive TME that fosters tumor progression, and diminishes the therapeutic efficacy of ICB. Blockade of the CD47/SIRPα pathway has proven to be an effective intervention, that restores macrophage phagocytosis and yields substantial antitumor effects, especially when combined with PD-1/PD-L1 blockade. Therefore, the identification of small molecules capable of simultaneously blocking CD47/SIRPα and PD-1/PD-L1 interactions has remained imperative. Methods SMC18, a small molecule with the capacity of targeting both SIRPα and PD-L1 was obtained using MST. The efficiency of SMC18 in interrupting CD47/SIRPα and PD-1/PD-L1 interactions was tested by the blocking assay. The function of SMC18 in enhancing the activity of macrophages and T cells was tested using phagocytosis assay and co-culture assay. The antitumor effects and mechanisms of SMC18 were investigated in the MC38-bearing mouse model. Results SMC18, a small molecule that dual-targets both SIRPα and PD-L1 protein, was identified. SMC18 effectively blocked CD47/SIRPα interaction, thereby restoring macrophage phagocytosis, and disrupted PD-1/PD-L1 interactions, thus activating Jurkat cells, as evidenced by increased secretion of IL-2. SMC18 demonstrated substantial inhibition of MC38 tumor growths through promoting the infiltration of CD8 + T and M1-type macrophages into tumor sites, while also priming the function of CD8 + T cells and macrophages. Moreover, SMC18 in combination with radiotherapy (RT) further improved the therapeutic efficacy. Conclusion Our findings suggested that the small molecule compound SMC18, which dual-targets the CD47/SIRPα and PD-1/PD-L1 pathways, could be a candidate for promoting macrophage- and T-cell-mediated phagocytosis and immune responses in cancer immunotherapy.

Lymph nodes are the most common metastasis sites for tumor cells, which are intimately linked to patient prognosis. It has been reported that cancer cells can upregulate CC Chemokine Receptor 7 (CCR7) expression and hijack its normal functions, enabling them to migrate along the gradient of CCL19 and CCL21 toward the lymph nodes and colonies as the initial stage of distant metastasis. In tumor patients, the metastatic tumor in the lymph nodes exhibited higher expression of CCR7, as well as inhibitory immune checkpoints PD-1, LAG-3, and TIM-3 compared to the primary tumors with the analysis of TCGA and GEO databases. Also, in mouse tumor model, tumor cells with elevated CCR7 expression were more susceptible to develop popliteal lymph node metastasis. Subsequently, we successfully identified a CCR7 binding peptide TC6 by phage display biopanning, which specifically blocks the interaction of CCR7/CCL19 and CCR7/CCL21. Further, the D-amino acids were introduced to substitute the N- and C-terminus of TC6 peptide to obtain the proteolysis-resistant TC6-D3 peptide, which decreased tumor cell migration in vitro via ERK1/2 pathway and inhibited tumor growth and lymph nodes metastasis in vivo, as well as effectively restored T cells cytotoxicity in both primary tumors and lymph nodes. In conclusion, CCR7 promoted tumor cell metastasis to lymph node and inhibited the anti-tumor immune responses in lymph nodes. Specific blockade of the CCR7 pathway with TC6-D3 peptide can significantly reduce lymph node tumor burden, promoting CD8 + T cell infiltration in primary tumors, meanwhile, enhancing anti-tumor immune responses in lymph nodes.

Background Colorectal cancer (CRC) is the third most prevalent cancer globally, and liver metastasis (CRLM) is the primary cause of death. Hence, it is essential to discover novel prognostic biomarkers and therapeutic drugs for CRLM. Methods This study developed two liver metastasis-associated prognostic signatures based on differentially expressed genes (DEGs) in CRLM. Additionally, we employed an interpretable deep learning model utilizing drug sensitivity databases to identify potential therapeutic drugs for high-risk CRLM patients. Subsequently, in vitro and in vivo experiments were performed to verify the efficacy of these compounds. Results These two prognostic models exhibited superior performance compared to previously reported ones. Obatoclax, a BCL-2 inhibitor, showed significant differential responses between high and low risk groups classified by prognostic models, and demonstrated remarkable effectiveness in both Transwell assay and CT26 colorectal liver metastasis mouse model. Conclusions This study highlights the significance of developing specialized prognostication approaches and investigating effective therapeutic drugs for patients with CRLM. The application of a deep learning drug response model provides a new drug discovery strategy for translational medicine in precision oncology.

The response rates of PD-1/PD-L1 blockade in cancer immunotherapy are relatively low, necessitating the development of novel immune checkpoint inhibitors. Compared with other immune checkpoints, VISTA interacts with its ligand PSGL-1 only under acidic conditions in the tumor microenvironment to suppress the function of CD8 + T cells. On the other hand, drug repurposing offers advantages such as time efficiency and high safety. However, the development of VISTA/PSGL-1 inhibitor based on drug repurposing is still infancy. Here, by screening a library of marketed drugs, we identified Chidamide had a strong binding affinity toward VISTA ( K D  = 5 nM) and blocked VISTA/PSGL-1 under acidic conditions, thereby significantly enhancing the function of CD8 + T cells and inhibiting the tumor growth in immunocompetent murine CT26 tumor model. This study represents the first discovery of Chidamide as VISTA/PSGL-1 blocker for cancer immunotherapy.

The interaction of PD-1/PD-L1 allows tumor cells to escape from immune surveillance. Clinical success of the antibody drugs has proven that blockade of PD-1/PD-L1 pathway is a promising strategy for cancer immunotherapy. Here, we developed a cyclic peptide C8 by using Ph.D.-C7C phage display technology. C8 showed high binding affinity with hPD-1 and could effectively interfere the interaction of PD-1/PD-L1. Furthermore, C8 could stimulate CD8 + T cell activation in human peripheral blood mononuclear cells (PBMCs). We also observed that C8 could suppress tumor growth in CT26 and B16-OVA, as well as anti-PD-1 antibody resistant B16 mouse model. CD8 T cells infiltration significantly increased in tumor microenvironment, and IFN-γ secretion by CD8 + T cells in draining lymph nodes also increased. Simultaneously, we exploited T cells depletion models and confirmed that C8 exerted anti-tumor effects via activating CD8 + T cells dependent manner. The interaction model of C8 with hPD-1 was simulated and confirmed by alanine scanning. In conclusion, C8 shows anti-tumor capability by blockade of PD-1/PD-L1 interaction, and C8 may provide an alternative candidate for cancer immunotherapy.