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Aside from antibodies, peptides show great potential as immune checkpoint inhibitors (ICIs) due to several advantages, such as better tumor penetration and lower cost. Lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint which can induce T cell dysfunction through interaction with its soluble ligand fibrinogen like protein-1 (FGL1). Here, we found that LAG-3 expression was higher than programmed cell death protein 1 (PD-1) in multiple human cancers by TCGA databases, and successfully identified a LAG-3 binding peptide LFP-6 by phage display bio-panning, which specifically blocks the interaction of LAG-3/FGL1 but not LAG-3/MHC-II. Subsequently, d -amino acids were introduced to substitute the N- and C-terminus of LFP-6 to obtain the proteolysis-resistant peptide LFP-D1, which restores T cell function in vitro and inhibits tumor growth in vivo. Further, a bispecific peptide LFOP targeting both PD-1/PD-L1 and LAG-3/FGL1 was designed by conjugating LFP-D1 with PD-1/PD-L1 blocking peptide OPBP-1(8–12), which activates T cell with enhanced proliferation and IFN- γ production. More importantly, LFOP combined with radiotherapy significantly improve the T cell infiltration in tumor and elevate systemic antitumor immune response. In conclusion, we developed a novel peptide blocking LAG-3/FGL1 which can restore T cell function, and the bispecific peptide synergizes with radiotherapy to further enhance the antitumor immune response. The LFP-D1 peptide specifically blocks the interaction between LAG-3 and FGL1, and LFOP peptide provides a biological rationale for co-blockade LAG-3/FGL1 and PD-1/PD-L1 pathway as an effective cancer immunotherapeutic strategy when combined with radiotherapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Developing new therapeutic agents for cancer immunotherapy is highly demanding due to the low response ratio of PD-1/PD-L1 blockade in cancer patients. Here, we discovered that the novel immune checkpoint VISTA is highly expressed on a variety of tumor-infiltrating immune cells, especially myeloid derived suppressor cells (MDSCs) and CD8+ T cells. Then, peptide C1 with binding affinity to VISTA was developed by phage displayed bio-panning technique, and its mutant peptide VS3 was obtained by molecular docking based mutation. Peptide VS3 could bind VISTA with high affinity and block its interaction with ligand PSGL-1 under acidic condition, and elicit anti-tumor activity in vivo. The peptide DVS3-Pal was further designed by d -amino acid substitution and fatty acid modification, which exhibited strong proteolytic stability and significant anti-tumor activity through enhancing CD8+ T cell function and decreasing MDSCs infiltration. This is the first study to develop peptides to block VISTA/PSGL-1 interaction, which could act as promising candidates for cancer immunotherapy. DVS3-Pal was designed to block VISTA/PSGL-1 interaction effectively under acidic conditions by phage display peptide library, virtual mutation and hydrolysis-resistant modification. DVS3-Pal effectively activated anti-tumor immune response and inhibited tumor growth by activating CD8+ T cells. [Display omitted] [ABSTRACT FROM AUTHOR]

The low response rate and adaptive resistance of PD‐1/PD‐L1 blockade demands the studies on novel therapeutic targets for cancer immunotherapy. We discovered that a novel immune checkpoint TIGIT expressed higher than PD‐1 in many tumors especially anti‐PD‐1 resistant tumors. Here, mirror‐image phage display bio‐panning was performed using the d‐enantiomer of TIGIT synthesized by hydrazide‐based native chemical ligation. d‐peptide DTBP‐3 was identified, which could occupy the binding interface and effectively block the interaction of TIGIT with its ligand PVR. DTBP‐3 showed proteolytic resistance, tumor tissue penetrating ability, and significant tumor suppressing effects in a CD8+ T cell dependent manner. More importantly, DTBP‐3 could inhibit tumor growth and metastasis in anti‐PD‐1 resistant tumor model. This is the first d‐peptide targeting TIGIT, which could serve as a potential candidate for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

[Display omitted] Immune checkpoint inhibitors have unveiled promising clinical prospects in cancer treatment. Nonetheless, their effectiveness remains restricted, marked by consistently low response rates and affecting only a subset of patients. The co-blockade of TIGIT with PD-1 has exhibited substantial anti-tumor effects. Notably, there is a dearth of reports on small-molecule inhibitors concurrently targeting both TIGIT and PD-1. In this study, we employed Microscale Thermophoresis (MST) to screen our laboratory's existing repository of small molecules. Our findings illuminated Gln(TrT) 's affinity for both TIGIT and PD-1, affirming its potential to effectively inhibit TIGIT/PVR and PD-1/PD-L1 pathways. In vitro co-culture experiments substantiated Gln(TrT)'s proficiency in restoring Jurkat T-cell functionality by blocking both TIGIT/PVR and PD-1/PD-L1 interactions. In the MC38 murine tumor model, Gln(TrT) emerges as a pivotal modulator, promoting the intratumoral infiltration and functional competence of CD8+ T cells. Furthermore, whether used as a monotherapy or in conjunction with radiotherapy, Gln(TrT) substantially impedes MC38 tumor progression, significantly extending the survival of murine subjects. [ABSTRACT FROM AUTHOR]

[Display omitted] PD-1/PD-L1 blockade has achieved substantial clinical results in cancer treatment. However, the expression of other immune checkpoints leads to resistance and hinders the efficacy of PD-1/PD-L1 blockade. T cell immunoglobulin and mucin domain 3 (TIM-3), a non-redundant immune checkpoint, synergizes with PD-1 to mediate T cell dysfunction in tumor microenvironment. Development of small molecules targeting TIM-3 is a promising strategy for cancer immunotherapy. Here, to identify small molecule inhibitors targeting TIM-3, the docking pocket in TIM-3 was analyzed by Molecular Operating Environment (MOE) and the Chemdiv compound database was screened. The small molecule SMI402 could bind to TIM-3 with high affinity and prevent the ligation of PtdSer, HMGB1, and CEACAM1. SMI402 reinvigorated T cell function in vitro. In the MC38-bearing mouse model, SMI402 inhibited tumor growth by increasing CD8+ T and natural killing (NK) cells infiltration at the tumor site, as well as restoring the function of CD8+ T and NK cells. In conclusions, the small molecule SMI402 shows promise as a leading compound which targets TIM-3 for cancer immunotherapy. [ABSTRACT FROM AUTHOR]