Therapeutic Target For Cancer Immunotherapy Research Articles

CD106 in tumor-specific exhausted CD8+ T cells mediates immunosuppression by inhibiting TCR signaling.

T cell exhaustion is a major contributor to immunosuppression in the tumor microenvironment (TME). Blockade of key regulators of T cell exhaustion, such as PD-1, can reinvigorate tumor-specific T cells and activate anti-tumor immunity in various types of cancer. Here, we identified that CD106 was specifically expressed in exhausted CD8+ T cells in the TME using single-cell RNA-sequencing. High CD106 expression in the TME in clinical samples corresponded to improved response to cancer immunotherapy. CD106 in tumor-specific T cells suppressed anti-tumor immunity both in vitro and in vivo, and loss of CD106 in CD8+ T cells suppressed tumor growth and improved response to PD-1 blockade. Mechanistically, CD106 inhibited T-cell receptor (TCR) signaling by interacting with the TCR/CD3 complex and reducing its surface expression. Together, these findings provide insights into the immunosuppressive role of CD106 expressed in tumor-specific exhausted CD8+ T cells, identifying it as a potential biomarker and therapeutic target for cancer immunotherapy.

STING agonist diABZI enhances the cytotoxicity of T cell towards cancer cells

Antigen-specific T cell receptor-engineered T cell (TCR-T) based immunotherapy has proven to be an effective method to combat cancer. In recent years, cross-talk between the innate and adaptive immune systems may be requisite to optimize sustained antigen-specific immunity, and the stimulator of interferon genes (STING) is a promising therapeutic target for cancer immunotherapy. The level of expression or presentation of antigen in tumor cells affects the recognition and killing of tumor cells by TCR-T. This study aimed at investigating the potential of innate immune stimulation of T cells and engineered T cells to enhance immunotherapy for low-expression antigen cancer cells. We systematically investigated the function and mechanism of cross-talk between STING agonist diABZI and adaptive immune systems. We established NY-ESO-1 full knockout Mel526 cells for this research and found that diABZI activated STING media and TCR signaling pathways. In addition, the results of flow cytometry showed that antigens presentation from cancer cells induced by STING agonist diABZI also improved the affinity of TCR-T cells function against tumor cells in vitro and in vivo. Our findings revealed that diABZI enhanced the immunotherapy efficacy of TCR-T by activating STING media and TCR signaling pathways, improving interferon-γ expression, and increasing antigens presentation of tumor cells. This indicates that STING agonist could be used as a strategy to promote TCR-T cancer immunotherapy.

CD155-TIGIT Axis as a Therapeutic Target for Cancer Immunotherapy.

Immune checkpoint inhibitors (ICIs) have shown unprecedented efficacy in treating many advanced cancers. Although FDA-approved ICIs have shown promising efficacy in treating many advanced cancers, their application is greatly limited by the low response rate, immune-related adverse events (irAE), and drug resistance. Developing novel ICIs holds great promise to improve the survival and prognosis of advanced cancer patients. T-Cell immunoglobulin and ITIM domain (TIGIT) is an inhibitory receptor expressed on T cells, natural killer (NK) cells, and T regulatory cells. Increasing reports have shown that the disrupting CD155-TIGIT axis could activate the immune system and restore antitumor immune response. This review briefly summarized the role of TIGIT in tumor immune escape and targeting CD155-TIGIT axis drugs in preclinical and clinical trials for cancer immunotherapy.

MicroRNA-561-3p indirectly regulates the PD-L1 expression by targeting ZEB1, HIF1A, and MYC genes in breast cancer

Globally, breast cancer is the second most common cause of cancer-related deaths among women. In breast cancer, microRNAs (miRNAs) are essential for both the initiation and development of tumors. It has been suggested that the tumor suppressor microRNA-561-3p (miR-561-3p) is crucial in arresting the growth of cancer cells. Further research is necessary to fully understand the role and molecular mechanism of miR-561 in human BC. The aim of this study was to investigate the inhibitory effect of miR-561-3p on ZEB1, HIF1A, and MYC expression as oncogenes that have the most impact on PD-L1 overexpression and cellular processes such as proliferation, apoptosis, and cell cycle in breast cancer (BC) cell lines. The expression of ZEB1, HIF1A, and MYC genes and miR-561-3p were measured in BC clinical samples and cell lines via qRT-PCR. The luciferase assay, MTT, Annexin-PI staining, and cell cycle experiments were used to assess the effect of miR-561-3p on candidate gene expression, proliferation, apoptosis, and cell cycle progression. Flow cytometry was used to investigate the effects of miR-561 on PD-L1 suppression in the BC cell line. The luciferase assay showed that miRNA-561-3p targets the 3′-UTRs of ZEB1, HIF1A and MYC genes significantly. In BC tissues, the qRT-PCR results demonstrated that miR-561-3p expression was downregulated and the expression of ZEB1, HIF1A and MYC genes was up-regulated. It was shown that overexpression of miR-561-3p decreased PD-L1 expression and BC cell proliferation, and induced apoptosis and cell cycle arrest through downregulation of candidate oncogenes. Furthermore, inhibition of candidate genes by miR-561-3p reduced PD-L1 at both mRNA and protein levels. Our research investigated the impact of miR-561-3p on the expression of ZEB1, HIF1A and MYC in breast cancer cells for the first time. Our findings may help clarify the role of miR-561-3p in PD-L1 regulation and point to this miR as a potential biomarker and novel therapeutic target for cancer immunotherapy.

Discovery of Pyrido[2,3-d]pyrimidin-7-one Derivatives as Highly Potent and Efficacious Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) Inhibitors for Cancer Treatment.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an extracellular enzyme responsible for hydrolyzing cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), the endogenous agonist for the stimulator of interferon genes (STING) pathway. Inhibition of ENPP1 can trigger STING and promote antitumor immunity, offering an attractive therapeutic target for cancer immunotherapy. Despite progress in the discovery of ENPP1 inhibitors, the diversity in chemical structures and the efficacy of the agents are far from desirable, emphasizing the demand for novel inhibitors. Herein, we describe the design, synthesis, and biological evaluation of a series of ENPP1 inhibitors based on the pyrido[2,3-d]pyrimidin-7-one scaffold. Optimization efforts led to compound 31 with significant potency in both ENPP1 inhibition and STING pathway stimulation in vitro. Notably, 31 demonstrated in vivo efficacy in a syngeneic 4T1 mouse triple negative breast cancer model. These findings provide a promising lead compound with a novel scaffold for further drug development in cancer immunotherapy.

Unveiling the structural mechanisms of nonpeptide ligand recognition and activation in human chemokine receptor CCR8.

The human CC chemokine receptor 8 (CCR8) is an emerging therapeutic target for cancer immunotherapy and autoimmune diseases. Understanding the molecular recognition of CCR8, particularly with nonpeptide ligands, is valuable for drug development. Here, we report three cryo-electron microscopy structures of human CCR8 complexed with Gi trimers in the ligand-free state or activated by nonpeptide agonists LMD-009 and ZK 756326. A conserved Y1.39Y3.32E7.39 motif in the orthosteric binding pocket is shown to play a crucial role in the chemokine and nonpeptide ligand recognition. Structural and functional analyses indicate that the lack of conservation in Y1143.33 and Y1724.64 among the CC chemokine receptors could potentially contribute to the selectivity of the nonpeptide ligand binding to CCR8. These findings present the characterization of the molecular interaction between a nonpeptide agonist and a chemokine receptor, aiding the development of therapeutics targeting related diseases through a structure-based approach.

NF-κB: Governing Macrophages in Cancer.

Tumor-associated macrophages (TAMs) are the major component of the tumor microenvironment (TME), where they sustain tumor progression and or-tumor immunity. Due to their plasticity, macrophages can exhibit anti- or pro-tumor functions through the expression of different gene sets leading to distinct macrophage phenotypes: M1-like or pro-inflammatory and M2-like or anti-inflammatory. NF-κB transcription factors are central regulators of TAMs in cancers, where they often drive macrophage polarization toward an M2-like phenotype. Therefore, the NF-κB pathway is an attractive therapeutic target for cancer immunotherapy in a wide range of human tumors. Hence, targeting NF-κB pathway in the myeloid compartment is a potential clinical strategy to overcome microenvironment-induced immunosuppression and increase anti-tumor immunity. In this review, we discuss the role of NF-κB as a key driver of macrophage functions in tumors as well as the principal strategies to overcome tumor immunosuppression by targeting the NF-κB pathway.

Pumilio1 regulates NPM3/NPM1 axis to promote PD-L1-mediated immune escape in gastric cancer

Abnormal regulation of RNA binding proteins (RBPs) plays an essential role in tumorigenesis and progression, but their functions and mechanisms remain largely elusive. Previously, we reported that Pumilio 1 (PUM1), a RBP, could regulate glycolysis metabolism and promote the progression of gastric cancer (GC). However, the role of PUM1 in tumor immune regulation remains largely elusive. In this study, we report that PUM1 induces immune escape through posttranscriptional regulation of PD-L1 in GC. We used multiplexed immunohistochemistry to analyze the correlation between PUM1 expression and immune microenvironment in GC. The effect of PUM1 deficiency on tumor killing of T cells was examined in vitro and in vivo. The molecular mechanism of PUM1 was evaluated via RNA immunoprecipitation, chromatin immunoprecipitation, Western blot, co-immunoprecipitation, and RNA stability assays. Clinically, elevated PUM1 expression is associated with high-expression of PD-L1, lack of CD8+ T cell infiltration and poor prognosis in GC patients. PUM1 positively regulates PD-L1 expression and PUM1 reduction enhances T cell killing of tumors. Mechanistically, PUM1 directly binds to nucleophosmin/nucleoplasmin 3 (NPM3) mRNA and stabilizes NPM3. NPM3 interacts with NPM1 to promote NPM1 translocation into the nucleus and increase the transcription of PD-L1. PUM1 inhibits the anti-tumor activity of T cells through the PUM1/NPM3/PD-L1 axis. In summary, this study reveals the critical post-transcriptional effect of PUM1 in the modulation of PD-L1-dependent GC immune escape, thus provides a novel indicator and potential therapeutic target for cancer immunotherapy.

Ablation of ERO1A induces lethal endoplasmic reticulum stress responses and immunogenic cell death to activate anti-tumor immunity

Immunophenotyping of the tumor microenvironment (TME) is essential for enhancing immunotherapy efficacy. However, strategies for characterizing the TME exhibit significant heterogeneity. Here, we show that endoplasmic reticular oxidoreductase-1α (ERO1A) mediates an immune-suppressive TME and attenuates the response to PD-1 blockade. Ablation of ERO1A in tumor cells substantially incites anti-tumor Tcell immunity and promotes the efficacy of aPD-1 in therapeutic models. Single-cell RNA-sequencing analyses confirm that ERO1A correlates with immunosuppression and dysfunction of CD8+ Tcells along anti-PD-1 treatment. In human lung cancer, high ERO1A expression is associated with a higher risk of recurrence following neoadjuvant immunotherapy. Mechanistically, ERO1A ablation impairs the balance between IRE1α and PERK signaling activities and induces lethal unfolded protein responses in tumor cells undergoing endoplasmic reticulum stress, thereby enhancing anti-tumor immunity via immunogenic cell death. These findings reveal how tumor ERO1A induces immunosuppression, highlighting its potential as a therapeutic target for cancer immunotherapy.

USP2 promotes tumor immune evasion via deubiquitination and stabilization of PD-L1.

The abnormal upregulation of programmed death ligand-1 (PD-L1) on tumor cells impedes T-cell mediated cytotoxicity through PD-1 engagement, and further exploring the mechanisms regulation of PD-L1 in cancers may enhance the clinical efficacy of PD-L1 blockade. Here, using single-guide RNAs (sgRNAs) screening system, we identify ubiquitin-specific processing protease 2 (USP2) as a novel regulator of PD-L1 stabilization for tumor immune evasion. USP2 directly interacts with and increases PD-L1 abundance in colorectal and prostate cancer cells. Our results show that Thr288, Arg292 and Asp293 at USP2 control its binding to PD-L1 through deconjugating the K48-linked polyubiquitination at lysine 270 of PD-L1. Depletion of USP2 causes endoplasmic reticulum (ER)-associated degradation of PD-L1, thus attenuates PD-L1/PD-1 interaction and sensitizes cancer cells to T cell-mediated killing. Meanwhile, USP2 ablation-induced PD-L1 clearance enhances antitumor immunity in mice via increasing CD8+ T cells infiltration and reducing immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), whereas PD-L1 overexpression reverses the tumor growth suppression by USP2 silencing. USP2-depletion combination with anti-PD-1 also exhibits a synergistic anti-tumor effect. Furthermore, analysis of clinical tissue samples indicates that USP2 is positively associated with PD-L1 expression in cancer. Collectively, our data reveal a crucial roleof USP2 for controlling PD-L1 stabilization in tumor cells, and highlight USP2 as a potential therapeutic target for cancer immunotherapy.

Comprehensive analysis of PILRΑ’s association with the prognosis, tumor immune infiltration, and immunotherapy in pan-cancer

Paired immunoglobulin-like type 2 receptor alpha (PILRA) plays a vital role in regulating broad immune responses. However, the roles of PILRA in cancer immunity remain unexplored yet. In the current study, we comprehensively analyzed the oncogenic and immunologic roles of PILRA at a pan-cancer level based on the Cancer Genome Atlas and Gene Expression Omnibus datasets. PILRA was significantly dysregulated and frequently mutated in pan-cancer. Its expression and mutation status significantly impacted patient prognosis in several cancers. Besides, PILRA expression was positively correlated with ESTIMATE scores and the abundances of tumor-infiltrating immune cells. Concurrently, PILRA expression was significantly associated with predictive biomarkers of cancer immunotherapy, and positively correlated with the prognostic outcomes of cancer patients receiving immunotherapy. Mechanistically, enrichment analysis implied that PILRA might be involved in the regulation of immune response and metabolic process. This study uncovered the immunological roles of PILRA in cancers and its potential as a novel biomarker and therapeutic target for cancer immunotherapy.

Retracted: NOD1 and NOD2 Are Potential Therapeutic Targets for Cancer Immunotherapy.

[This retracts the article DOI: 10.1155/2022/2271788.].

C1q+ tumor-associated macrophages contribute to immunosuppression through fatty acid metabolic reprogramming in malignant pleural effusion

BackgroundAlthough immune checkpoint blockade (ICB) therapy has shown remarkable benefits in cancers, a subset of patients with cancer exhibits unresponsiveness or develop acquired resistance due to the existence of abundant...

ZIC2 induces pro-tumor macrophage polarization in nasopharyngeal carcinoma by activating the JUNB/MCSF axis

Nasopharyngeal carcinoma (NPC) is a common malignant epithelial tumor of the head and neck that often exhibits local recurrence and distant metastasis. The molecular mechanisms are understudied, and effective therapeutic targets are still lacking. In our study, we found that the transcription factor ZIC2 was highly expressed in NPC. Although ZIC family members play important roles in neural development and carcinogenesis, the specific mechanism and clinical significance of ZIC2 in the tumorigenesis and immune regulation of NPC remain elusive. Here, we first reported that high expression of ZIC2 triggered the secretion of MCSF in NPC cells, induced M2 polarization of tumor-associated macrophages (TAMs), and affected the secretion of TAM-related cytokines. Mechanistically, ChIP-seq and RNA-seq analyses identified JUNB as a downstream target of ZIC2. Furthermore, ZIC2 was significantly enriched in the promoter site of JUNB and activated JUNB promoter activity, as shown by ChIP-qPCR and luciferase assays. In addition, JUNB and MCSF participated in ZIC2-induced M2 TAMs polarization. Thus, blocking JUNB and MCSF could reverse ZIC2-mediated M2 TAMs polarization. Moreover, Kaplan-Meier survival analyses indicated that high expression of ZIC2, JUNB, and CD163 was positively associated with a poor prognosis in NPC. Overexpression of ZIC2 induced tumor growth in vivo, with the increase of JUNB, MCSF secretion, and CD163. In summary, our study implies that ZIC2 induces M2 TAM polarization, at least in part through regulation of JUNB/MCSF and that ZIC2, JUNB, and CD163 can be utilized as prognostic markers for NPC and as therapeutic targets for cancer immunotherapy.

RelB is a potential molecular biomarker for immunotherapy in human pan-cancer.

Introduction: The nuclear factor kB (NF-κB) pathway emerges as a critical regulator of immune responses and is often dysregulated in human cancers. It consists of a family of transcription factors involved in many biological responses. Activated NF-κB subunits results in the nuclear translocation and activation of transcription, and the NF-κB pathway is known to influence the transcription of many genes. Noncanonical NF-κB and its components have been shown to have effects, usually protumorigenic, in many different cancer types. Besides, NF-κB signaling had diverse and complicated roles in cancer with studies that NF-κB could both contribute to tumor promotion and suppression of oncogenesis relying on the cellular context. RelB, a member of noncanonical NF-κB was abnormally regulated in most cancer types, however the molecular features and clinical signature of RelB expression, as well as its role in cancer immunity in human pan-cancer remains to be elucidated. Methods: We used the open databases to explore RelB expression, clinical features and the association with tumor-infiltration cells in human pan-cancer. In this study, we investigated the aberration expression and prognostic significance of RelB, and the correlation with clinicopathological characters and immune cells infiltration in various cancers. The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases were used to analyze the mRNA expression level in different cancer types. Kaplan-Meier analysis and Cox regression were used to explore the prognostic significance of RelB in human pan-cancer. Then we took advantage of the TCGA database to analyze the relationship between RelB expression and DNA methylation, the infiltration of immune cells, immune checkpoint genes, tumor mutation burden (TMB), microsatellite instability (MSI), mismatch repair (MSS). Results: Higher expression of RelB was significantly detected in human cancer tissues and a high level of RelB expression was significantly linked with a worse outcome in LGG, KIPAN, ACC, UVM, LUAD,THYM, GBM, LIHC and TGCT but associated with a favorable overall survival (OS) in SARC, SKCM and BRCA. According to the Human Protein Altas database, RelB was considered as an independent factor in breast cancer and renal cancer prognosis. GSEA results revealed that RelB was involved in many oncogenesisrelated processes and immunity-related pathways. RelB was significantly correlated with DNA methylation in 13 types of cancer. Meanwhile, RelB expression was associated with TMB in 5 types of cancer and MSI in 8 types of cancer. In the final, we analyzed the relationship between RelB expression and immune-infiltration cells in human pan-cancer, which suggested RelB could be a promising therapeutic target for cancer immunotherapy. Discussion: Our study further provided insights into a deeper understanding of RelB as a prognostic biomarker.

The SWI/SNF chromatin remodeling complexes BAF and PBAF differentially regulate epigenetic transitions in exhausted CD8+ T cells

CD8+ Tcell exhaustion (Tex) limits disease control during chronic viral infections and cancer. Here, we investigated the epigenetic factors mediating major chromatin-remodeling events in Tex-cell development. A protein-domain-focused invivo CRISPR screen identified distinct functions for two versions of the SWI/SNF chromatin-remodeling complex in Tex-cell differentiation. Depletion of the canonical SWI/SNF form, BAF, impaired initial CD8+ Tcell responses in acute and chronic infection. In contrast, disruption of PBAF enhanced Tex-cell proliferation and survival. Mechanistically, PBAF regulated the epigenetic and transcriptional transition from TCF-1+ progenitor Tex cells to more differentiated TCF-1- Tex subsets. Whereas PBAF acted to preserve Tex progenitor biology, BAF was required to generate effector-like Tex cells, suggesting that the balance of these factors coordinates Tex-cell subset differentiation. Targeting PBAF improved tumor control both alone and in combination with anti-PD-L1 immunotherapy. Thus, PBAF may present a therapeutic target in cancer immunotherapy.

ISG15 targets glycosylated PD-L1 and promotes its degradation to enhance antitumor immune effects in lung adenocarcinoma

BackgroundImmunocheckpoint inhibitors (ICIs) have been widely used in the clinical treatment of lung cancer. Although clinical studies and trials have shown that patients can benefit significantly after PD-1/PD-L1 blocking therapy, less than 20% of patients can benefit from ICIs therapy due to tumor heterogeneity and the complexity of immune microenvironment. Several recent studies have explored the immunosuppression of PD-L1 expression and activity by post-translational regulation. Our published articles demonstrate that ISG15 inhibits lung adenocarcinoma progression. Whether ISG15 can enhance the efficacy of ICIs by modulating PD-L1 remains unknown.MethodsThe relationship between ISG15 and lymphocyte infiltration was identified by IHC. The effects of ISG15 on tumor cells and T lymphocytes were assessed using RT-qPCR and Western Blot and in vivo experiments. The underlying mechanism of PD-L1 post-translational modification by ISG15 was revealed by Western blot, RT-qPCR, flow cytometry, and Co-IP. Finally, we performed validation in C57 mice as well as in lung adenocarcinoma tissues.ResultsISG15 promotes the infiltration of CD4+ T lymphocytes. In vivo and in vitro experiments demonstrated that ISG15 induces CD4+ T cell proliferation and invalidity and immune responses against tumors. Mechanistically, we demonstrated that the ubiquitination-like modifying effect of ISG15 on PD-L1 increased the modification of K48-linked ubiquitin chains thus increasing the degradation rate of glycosylated PD-L1 targeting proteasomal pathway. The expression of ISG15 and PD-L1 was negatively correlated in NSCLC tissues. In addition, reduced accumulation of PD-L1 by ISG15 in mice also increased splenic lymphocyte infiltration as well as promoted cytotoxic T cell infiltration in the tumor microenvironment, thereby enhancing anti-tumor immunity.ConclusionsThe ubiquitination modification of PD-L1 by ISG15 increases K48-linked ubiquitin chain modification, thereby increasing the degradation rate of glycosylated PD-L1-targeted proteasome pathway. More importantly, ISG15 enhanced the sensitivity to immunosuppressive therapy. Our study shows that ISG15, as a post-translational modifier of PD-L1, reduces the stability of PD-L1 and may be a potential therapeutic target for cancer immunotherapy.

Macrophage CD5L is a target for cancer immunotherapy

Reprogramming of immunosuppressive tumor-associated macrophages (TAMs) presents an attractive therapeutic strategy in cancer. The aim of this study was to explore the role of macrophage CD5L protein in TAM activity and assess its potential as a therapeutic target. Monoclonal antibodies (mAbs) against recombinant CD5L were raised by subcutaneous immunization of BALB/c mice. Peripheral blood monocytes were isolated from healthy donors and stimulated with IFN/LPS, IL4, IL10, and conditioned medium (CM) from different cancer cell lines in the presence of anti-CD5L mAb or controls. Subsequently, phenotypic markers, including CD5L, were quantified by flow cytometry, IF and RT-qPCR. Macrophage CD5L protein expression was studied in 55 human papillary lung adenocarcinoma (PAC) samples by IHC and IF. Anti-CD5L mAb and isotype control were administered intraperitoneally into a syngeneic Lewis Lung Carcinoma mouse model and tumor growth was measured. Tumor microenvironment (TME) changes were determined by flow cytometry, IHC, IF, Luminex, RNAseq and RT-qPCR. Cancer cell lines CM induced an immunosuppressive phenotype (increase in CD163, CD206, MERTK, VEGF and CD5L) in cultured macrophages. Accordingly, high TAM expression of CD5L in PAC was associated with poor patient outcome (Log-rank (Mantel-Cox) test p=0.02). We raised a new anti-CD5L mAb that blocked the immunosuppressive phenotype of macrophages invitro. Its administration invivo inhibited tumor progression of lung cancer by altering the intratumoral myeloid cell population profile and CD4+ T-cell exhaustion phenotype, thereby significantly modifying the TME and increasing the inflammatory milieu. CD5L protein plays a key function in modulating the activity of macrophages and their interactions within the TME, which supports its role as a therapeutic target in cancer immunotherapy. For a full list of funding bodies, please see the Acknowledgements.

Discovery of 7H-Pyrrolo[2,3-d]pyrimidine Derivatives as potent hematopoietic progenitor kinase 1 (HPK1) inhibitors

Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cells and is a negative regulator of T cell receptor (TCR) signaling. Recent studies have demonstrated that HPK1 is a promising therapeutic target for cancer immunotherapy. However, despite significant progress in the development of HPK1 inhibitors, none of them has been approved for cancer therapy. Development of HPK1 inhibitors with a structurally distinct scaffold is still needed. Herein, we describe the design and synthesis of a series of HPK1 inhibitors with a 7H-pyrrolo[2,3-d]pyrimidine scaffold, exemplified by 31. Compound 31 showed potent inhibitory activity against HPK1 with an IC50 value of 3.5 nM and favorable selectivity within a panel of kinases. It also potently inhibited the phosphorylation level of SLP76, a substrate of HPK1, and enhanced the IL-2 secretion in Jurkat cells (human T cell leukemia). Our findings provide new clues for further optimization and development to generate HPK1 inhibitors for cancer immunotherapy.

Abstract 462: A novel and potent Cblb inhibitor demonstrates robust immunological profile and anti-tumor efficacy

Abstract Background: Casitas B-lineage lymphoma b (Cbl-b), a RING finger E3 ligase, is a negative regulator of immune cell activation1. Genetic deletion or pharmacological inhibition of Cbl-b resulted in hyper-reactive and co-stimulation independent T cell activation and cytokine production1. In syngeneic tumor models, CD8 T-cell and NK-cell mediated rejection of tumours were observed1. These findings point to Cbl-b as a therapeutic target in cancer immunotherapy. Inhibition of Cbl-b also demonstrated the potential to enhance the efficacy of check-point blockers like anti-PD-1 antibody, an unmet need in the clinic. Methods: Using intuitive medicinal chemistry design supported by computational approaches, we identified a lead Cbl-b inhibitor. SAR was developed using a battery of biochemical assays, functional read-outs and primary human in vitro T-cell activation and exhaustion assays. In vivo efficacy was demonstrated in syngeneic mouse colon tumor model. Results: Our lead Cbl-b inhibitor demonstrated potent binding to Cbl-b, robust anti-tumor cytokine secretion in human and mouse T cells, whole blood and potent reversal of T cell exhaustion. A strong tumor growth inhibition was demonstrated by the lead compound in a mouse colon tumor model. Compared to single agent, a combination of the lead compound with anti-PD-1 antibody induced enhanced complete tumor rejections. Conclusions: We have identified a novel, potent and orally bioavailable Cbl-b inhibitor that demonstrated robust in vitro and in vivo anti-tumor profiles. Acknowledgements: We thank Sanjib Das, Ajit Patil, Gauri Gawas, Savita Pandita, Priya Yadav, Sneha Pusadkar, Mayura Behere, Subhadip Das, Shravankumar Kolli and Radheshyam Yadav for their contributions to the project