InVivoMAb anti-mouse CTLA-4 (CD152)

Interleukin-2 (IL2) was among the earliest reagents used for cancer immunotherapy due to its ability to support the survival and function of tumor-reactive T cells. However, treatment with IL2 is accompanied by off-target toxicity and low response rates in patients. In mouse models, these issues are largely overcome when IL2 is administered as a cytokine/antibody complex (IL2c). The complex has a longer serum half-life and can be designed for preferential cytokine delivery to specific cells of interest. Early studies showed IL2c could boost antitumor immunity in mice by activating tumor-reactive CD8+ T cells. But such functional T cells are often limited in the tumor microenvironment, where instead unresponsive tolerant T cells are eventually eliminated by apoptosis, representing a major obstacle to the success of cancer immunotherapy. We found that IL2c treatment rescued tumor-specific CD8+ T cells from a state of established tolerance, providing effective immunotherapy in tumor-bearing mice. Expression of the transcription factor T-bet was necessary to drive intratumoral IFNγ production and effector activity by T cells rescued with IL2c. Furthermore, IL2c promoted T-bet expression in human CD4+ and CD8+ T cells in humanized tumor-bearing mice, but also increased the frequency of Foxp3+ regulatory T cells. Our study reveals a novel role for IL2c as a powerful immunotherapeutic reagent capable of reversing tolerance in tumor-reactive T cells, and provides the first evidence that IL2c influences human T cells in vivo, highlighting the translational potential to modulate human antitumor immune responses. Cancer Immunol Res; 4(12); 1016-26. ©2016 AACR. ©2016 American Association for Cancer Research.

β-adrenergic signaling has been suggested to promote tumor growth, and β-blockers are being evaluated for repurposing for cancer treatment. Here, we identify a β-adrenergic signaling axis involved in metastasis formation. We show that the β-blocker propranolol has strong anti-metastatic activity in multiple murine models, with this effect being completely dependent on CD4 + T cells and independent of NK or CD8 + T cells. We also observe that CD4 + T cells are required for the anti-tumor effect of propranolol in a syngeneic subcutaneous model of colon cancer. Mechanistically, propranolol induces a Th1-polarized and cytotoxic CD4 + T cell response, which requires MHC class II expression by cancer cells for full efficacy. We also report propanolol-driven systemic changes in the monocyte compartment, and upon depletion of monocytes, propranolol loses its anti-tumor effects. Finally, we show that propranolol treatment synergizes with anti-CTLA-4 therapy to further enhance CD4 + T cell infiltration and control metastasis. Thus, we show that β-adrenergic signaling limits CD4 T cell-mediated anti-tumor immunity, highlighting the potential of repurposing β-blockers for cancer treatment.

Colorectal cancer (CRC) remains a formidable threat to health worldwide. Immunotherapy with immune checkpoint inhibitors results in only a minority of CRC patients experiencing long-term progression-free survival, at the expense of significant autoimmune toxicity. Development of new therapeutics to "wake up" the immune system to fight CRC is necessary. Here we investigated for the first time radioimmunotherapy (RIT) directed towards CCR8, a marker of tumor-infiltrating immunosuppressive T-regulatory cells (ti-Tregs) as a method to recover anti-tumor immunity followed by immunotherapy in CRC models.

Oncogenic KRAS mutations underlie some of the deadliest human cancers. Genetic or pharmacological KRAS inactivation produces mixed outcomes and frequent relapse. Mechanisms of tumor resistance to KRAS inhibition remain poorly understood. We present evidence that STAT3 supports tumor growth following KRAS depletion. Using a conceptual framework of pancreatic ductal adenocarcinoma, we show that cancer cells that survive CRISPR-mediated ablation of mutant KRAS are dependent on STAT3 function to maintain tumorigenicity. Mechanistically, the combined loss of mutant KRAS and STAT3 disrupts a core transcriptional program of cancer cells critical to oncogenic competence. This in turn impairs tumor growth in mice and enhances immune rejection, leading to tumor clearance. We propose that the STAT3 transcriptional program operating in cancer cells enforces their malignant identity, rather than providing classical features of transformation, and shapes cancer persistence following KRAS inactivation. Our findings establish STAT3 as a critical enforcer of oncogenic identity in KRAS-ablated tumors, revealing a key vulnerability.

High-grade serous ovarian carcinoma (HGSOC) is associated with high mortality rates due to late-stage diagnosis and limited treatment options. We investigated the role of FSTL3 in ovarian cancer progression both as a prognostic biomarker and as a potential therapeutic target.We measured levels of follistatin (FST) and follistatin-like 3 (FSTL3) in 96 ovarian cancer patient ascites samples and found that FSTL3 overexpression was more predominant than FST and associated with poorer survival outcomes. Mice implanted with an HGSOC syngeneic cell line bearing common alterations in ovarian cancer (KRASG12 V, P53R172H, CCNE1oe, AKT2oe) had increasing levels of FST and FSTL3 in serum during tumor growth. Further alteration of this model to generate a knockout of FST (KPCA.FSTKO) and an overexpression of human FSTL3 (KPCA.FSTKO_hFSTL3) revealed that FSTL3 expression was associated with a more fibrotic tumor microenvironment, correlating with an increased abundance of cancer-associated myofibroblasts (myCAFs), and cancer cells with a more mesenchymal phenotype. Tumors overexpressing FSTL3 also had significantly less immunocyte infiltration, reduced intratumoral T-cell abundance, and increased CD8+ T cell exhaustion. FSTL3 overexpression completely abrogated tumor response to PPC treatment (Prexasertib combined with PD-1 and CTLA-4 blockade) compared to controls, suggesting that FSTL3 may be involved in immunotherapy resistance. In conclusion, this study suggests a role for FSTL3 as a prognostic marker and as therapeutic target in HGSOC, where it may play a role in promoting a mesenchymal tumor phenotype, maintaining an immunosuppressive tumor microenvironment, and driving immunotherapy resistance.

Immune checkpoint blockers (ICBs) have demonstrated substantial efficacy across various malignancies, yet the benefits of ICBs are limited to a subset of patients. Therefore, it is essential to identify novel therapeutic targets. By integrating multi-omics data from cohorts of patients with melanoma treated with ICBs, a positive correlation is observed between tumor CD137L expression and the efficacy of PD-1 blockade. Functionally, CD137L induction in cancer cells significantly enhances anti-tumor immunity by promoting CD8+ T cell survival, both in vivo and in vitro. Mechanistically, helicase-like transcription factor (HLTF) is identified as a pivotal transcriptional regulator of CD137L, controlling its expression through phosphorylation of serine at position 398. Therapeutically, the AMPK agonist AICAR (acadesine) as an inducer of CD137L, exhibiting synergistic effects with PD-1 or CTLA-4 blockade. In summary, our findings elucidate a mechanism controlling CD137L expression and highlight a promising combination therapy to enhance the efficacy of ICBs in melanoma. One Sentence Summary: Inducing co-stimulatory immune checkpoint CD137L expression in melanoma cells enhances T cell-mediated anti-tumor immunity.

A significant percentage of melanomas are refractory to immune checkpoint inhibitor (ICI) monotherapies and combinations. As there are currently no effective second-line therapies available for ICI-resistant patients, we sought to identify novel checkpoint inhibitor combinations for future clinical evaluation.

Resistance to v-raf murine sarcoma viral oncogene homolog B1 (BRAF) plus mitogen-activated protein kinase kinase (MEK) inhibition (BRAFi+MEKi) in BRAFV600E-mutant gliomas drives rebound, progression, and high mortality, yet it remains poorly understood. This study addresses the urgent need to develop treatments for BRAFi+MEKi-resistant glioma using preclinical mouse models and patient-derived materials. BRAFi+MEKi reveals glioma plasticity by heightening cell state transitions along glial differentiation trajectories, giving rise to astrocyte- and immunomodulatory oligodendrocyte (OL)-like states. PD-L1 upregulation in OL-like cells links cell state transitions to immune evasion, possibly orchestrated by Galectin-3. BRAFi+MEKi induces interferon response signatures, tumor infiltration, and suppression of T cells. Combining BRAFi+MEKi with immune checkpoint inhibition enhances survival in a T cell-dependent manner, reinvigorates T cells, and outperforms individual or sequential therapies in mice. Elevated PD-L1 expression in BRAF-mutant versus BRAF-wild-type glioblastoma supports the rationale for PD-1 inhibition in patients. These findings underscore the potential of targeting glioma plasticity and highlight combination strategies to overcome therapy resistance in BRAFV600E-mutant high-grade glioma.

The lack of T cells in tumors is a major hurdle to successful immune checkpoint therapy (ICT). Therefore, therapeutic strategies promoting T cell recruitment into tumors are warranted to improve the treatment efficacy. Here, we report that Fc-optimized anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) antibodies are potent remodelers of tumor vasculature that increase tumor-associated high endothelial venules (TA-HEVs), specialized blood vessels supporting lymphocyte entry into tumors. Mechanistically, this effect is dependent on the Fc domain of anti-CTLA-4 antibodies and CD4+ T cells and involves interferon gamma (IFNγ). Unexpectedly, we find that the human anti-CTLA-4 antibody ipilimumab fails to increase TA-HEVs in a humanized mouse model. However, increasing its Fc effector function rescues the modulation of TA-HEVs, promotes CD4+ and CD8+ T cell infiltration into tumors, and sensitizes recalcitrant tumors to programmed cell death protein 1 (PD-1) blockade. Our findings suggest that Fc-optimized anti-CTLA-4 antibodies could be used to reprogram tumor vasculature in poorly immunogenic cold tumors and improve the efficacy of ICT.

Cellular heterogeneity presents a significant challenge to cancer treatment. Antibody therapies targeting individual tumor-associated antigens can be extremely effective but are not suited for all patients and often fail against tumors with heterogeneous expression as tumor cells with low or no antigen expression escape targeting and develop resistance. Simultaneously targeting multiple tumor-specific proteins with multiple antibodies has the potential to overcome this barrier and improve efficacy, but relatively few widely expressed cancer-specific antigens are known. In contrast, neoepitopes, which arise from mutations unique to tumor cells, are considerably more abundant. However, since neoepitopes are not commonly shared between individuals, a patient-customized approach is necessary and motivates efforts to develop an efficient means to identify suitable target mutations and isolate neoepitope-specific monoclonal antibodies. Here, focusing on the latter goal, we use directed evolution in yeast and phage display systems to engineer antibodies from nonimmune, human antibody fragment libraries that are specific for neoepitopes previously reported in the B16F10 melanoma model. We demonstrate proof-of-concept for a pipeline that supports rapid isolation and functional enhancement of multiple neoepitope peptide-targeted monoclonal antibodies and demonstrate their robust binding to B16F10 cells and potent effector functions in vitro. These antibodies were combined and evaluated in vivo for anticancer activity in tumor-bearing mice, where they suppressed B16F10 tumor growth and prolonged survival. These findings emphasize the potential for clinical application of patient-customized antibody cocktails in the treatment of the many cancers poorly addressed by current therapies.

Breast cancer is a fatal malignancy facing human health, with most patients experiencing recurrence and resistance to chemotherapy. The immunosuppressive tumor microenvironment (TME) greatly limits the actual outcome of immunotherapy. This study aimed to develop a modality of theranostics nanoparticles for breast cancer based on a near-infrared light-triggered nanoparticle for the targeted delivery of ginsenoside Rg3 and immune adjuvants imiquimod (R837) for effective breast cancer immunotherapy. Folate-receptor (FA) targeting IR780-R837/ginsenoside Rg3-perfluorohexane (PFH) @ polyethylene glycol (PEG)-poly (lactide-co-glycolic acid) (PLGA) nanoparticles (FA-NPs) can be activated by near-infrared laser irradiation in tumors, which leads to rapid release of ginsenoside Rg3 and R837 in the regions with high expression of folate receptors and glucose transporter 1 (GLUT1). Meanwhile, the nanoparticles can be used as dual-mode contrast agents for photoacoustic and ultrasound imaging. This strategy provides a strong immune memory effect, which can prevent tumor recurrence after eliminating the initial tumor.

Immune checkpoint blockade (ICB) therapy, targeting programmed cell death ligand-1 (PD-L1)/programmed cell death protein 1 (PD-1) axis and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), has exhibited amazing clinical outcomes in various types of cancers. However, only a small portion of patients benefit from ICB therapy, indicating that the mechanism underlying immune checkpoint is still unclear. Here, it is reported that motor neuron and pancreas homeobox 1 (MNX1), a homeobox domain-containing transcription factor, contributes to the tumor immune escape. MNX1 increases PD-L1 expression in cancer cells by stabilizing PD-L1 mRNA rather than activating transcription. Mechanistically, MNX1 exists in the cytoplasm of cancer cells and interacts with Y-box binding protein 1 (YBX1), a multifunctional DNA/RNA-binding protein, to enhance the binding of YBX1 to PD-L1 mRNA. MNX1 ablation activates cytotoxic T cell-mediated anti-tumor immunity and sensitizes CTLA-4 blockade therapy. Moreover, MNX1 also facilitates tumor progression in an immune-independent manner in cancer cells. In addition, MNX1 is upregulated by its adjacent long non-coding RNA MNX1-AS1 via HECT and RLD domain containing E3 ubiquitin protein ligase 2 (HERC2). Together, these results reveal MNX1 as a novel immune checkpoint regulator with promising therapeutic potential.

Tumors undergo metabolic reprogramming to meet the energetic, synthetic and redox demands essential for malignancy, often characterized by increased glycolysis and lactate production. However, the role of mitochondrial metabolism in tumor immunity remains unclear. The present study integrates spatial transcriptomics, bulk transcriptomics and proteomics, revealing a strong link between the metabolite succinyl-CoA and tumor immunity as well as the efficacy of anti-programmed cell death protein-1 (PD-1) therapy in patients with melanoma. Elevated succinyl-CoA levels, through α-ketoglutarate or succinate supplementation, enhanced T cell-mediated tumor elimination, both in vitro and in vivo. Mechanistically, succinylation of the ligand of PD-1 (PD-L1) at lysine 129 led to its degradation. Increased carnitine palmitoyltransferase 1A (CPT1A), identified as a succinyltransferase for PD-L1, boosted anti-tumor activity. Preclinically, bezafibrate, a hyperlipidemia drug, upregulated CPT1A and synergized with CTLA-4 monoclonal antibody to inhibit tumor growth. Clinically, higher PD-L1 and lower CPT1A levels in tumors correlated with better anti-PD-1 therapy responses, suggesting potential biomarkers for prediction of treatment efficacy.

KRAS mutations are linked to some of the deadliest forms of cancer. Pharmacological studies suggest that co-targeting KRAS with feedback/bypass pathways could lead to enhanced anti-tumor activity. The underlying premise is that cancers display a deep-rooted hypersensitivity to KRAS inactivation. Here, we investigate the role of intratumor heterogeneity in pancreatic ductal adenocarcinoma, focusing on oncogenic KRAS addiction and treatment resistance. Integrated analysis of single-cell and bulk RNA sequencing data reveals that most tumors display a mixture of cells with vastly different degrees of KRAS dependency. We identify distinct cell populations that vary in their gene expression patterns pertaining to the predicted level of KRAS signaling activity, cell growth, and differentiation commitment within each tumor. Selective targeting of mutant KRAS suppresses the growth of tumor cells with high RAS/mitogen-activated protein kinase (MAPK) activity while sparing pre-existing subsets with low RAS signaling activity, necessitating alternative treatments. Combination immunotherapy leads to durable tumor regression in preclinical models.

Glioblastoma (GBM) carries a dismal prognosis, with a median survival of less than 15 months. Temozolomide (TMZ), the standard frontline chemotherapeutic for GBM, is an alkylating agent that generates DNA O6-methylguanine (O6MeG) lesions. Without O6MeG-methyltransferase (MGMT), this lesion triggers the mismatch repair (MMR) pathway and leads to cytotoxicity via futile cycling. TMZ resistance frequently arises via the somatic acquisition of MMR deficiency (MMRd). Moreover, DNA-damaging agents have been shown capable of increasing tumor immunogenicity and improving response to immune checkpoint blockade (ICB), which has had limited success in glioma. The study of how alkylating chemotherapy such as TMZ impacts antitumor immunity in glioma has been hindered by a lack of immunocompetent models that incorporate relevant DNA repair genotypes. Here, we used CRISPR/Cas9 to generate models isogenic for knockout (KO) of Mlh1 in the syngeneic SB28 murine glioma cell line. MMR KO models readily formed intracranial tumors and exhibited in vitro and in vivo resistance to TMZ. In contrast, MMR KO cells maintained sensitivity to KL-50, a newly developed alkylating compound that exerts MGMT-dependent, MMR-independent cytotoxicity. Lastly, MMR KO tumors remained resistant to ICB, mirroring the lack of response seen in patients with somatic MMRd GBM. The development of syngeneic, immunologically cold glioma models with somatic loss of MMR will facilitate future studies on the immunomodulatory effects of alkylating agents in relevant DNA repair contexts, which will be vital for optimizing combinations with ICB. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Validation of mismatch repair knockouts and in vitro sensitivity to alkylating agents Basic Protocol 2: Stereotaxic injection of isogenic SB28 cells in female C57BL/6J mice and in vivo treatment.

Dual Opdivo plus Yervoy immunotherapy, targeting the immune checkpoints PD1 and CTLA-4, is successful in clinical use. However, it is associated with a high incidence of adverse events, and its therapeutic efficacy needs improving. In this study, polymeric multivalent Fc-binding peptides (PLG-Fc-III-4C) are employed to fabricate a bispecific antibody (PD1/CTLA-4 BsAb) to potentiate dual immunotherapy targeting PD1 and CTLA-4. The PD1/CTLA-4 BsAb is prepared by mixing PLG-Fc-III-4C with aPD1 and aCTLA-4 in an aqueous solution for 3 h using the clinically optimal 3:1 proportion of aPD1 to aCTLA-4. PD1/CTLA-4 BsAb significantly inhibits tumors in MC38 colon cancer-bearing mice more effectively than the combination of aPD1 and aCTLA-4, with tumor suppression rates of 96.8% and 77.3%, respectively. It also induces a higher percentage of CD8+ T cells and increases the secretion of effector cytokines while reducing Treg levels in tumors compared to phosphate-buffered saline, indicating significant tumor immunity regulation. Mechanistically, a 6.3-fold increase in PD1/CTLA-4 BsAb accumulation in tumors due to the tumor targeting ability of aPD1, and the PD1/CTLA-4 BsAb significantly reduces the adverse colitis event in healthy mice, compared to aPD1 and aCTLA-4. Thus, these findings provide a novel approach to enhance antitumor therapy using aPD1 and aCTLA-4.

Numerous human cancers have exhibited the ability to elude immune checkpoint blockade (ICB) therapies. This type of resistance can be mediated by immune-suppressive macrophages that limit antitumor immunity in the tumor microenvironment (TME). Here, we elucidate a strategy to shift macrophages into a proinflammatory state that down-regulates V domain immunoglobulin suppressor of T cell activation (VISTA) via inhibiting AhR and IRAK1. We used a high-throughput microfluidic platform combined with a genome-wide CRISPR knockout screen to identify regulators of VISTA levels. Functional characterization showed that the knockdown of these hits diminished VISTA surface levels on macrophages and sustained an antitumor phenotype. Furthermore, targeting of both AhR and IRAK1 in mouse models overcame resistance to ICB treatment. Tumor immunophenotyping indicated that infiltration of cytotoxic CD8+ cells, natural killer cells, and antitumor macrophages was substantially increased in treated mice. Collectively, AhR and IRAK1 are implicated as regulators of VISTA that coordinate a multifaceted barrier to antitumor immune responses.

Calcium signaling plays a crucial role in the activation of T lymphocytes. However, modulating calcium levels to control T cell activation in vivo remains a challenge. In this study, we investigate T cell activation using 12-myristate 13-acetate (PMA)-encapsulated CaCO3 nanoparticles. We find that anti-PD-1 antibody-conjugated CaCO3 nanoparticles can be internalized by T cells via receptor-mediated endocytosis and then gradually release calcium. This results in an increase in cytosolic calcium, which triggers the activation of NFAT and NF-κB pathways, especially when the surface of the CaCO3 nanoparticles is loaded with PMA. Animal studies demonstrate that the PMA-loaded calcium nanoparticles enhance the activation and proliferation of cytotoxic T cells, leading to improved tumor suppression without additional toxicity. When tested in metastatic tumor models, T cells loaded with the calcium nanoparticles prior to adoptive cell transfer control tumor growth better, resulting in prolonged animal survival. Our approach offers an alternative T cell activation strategy to potentiate immunotherapy by targeting a fundamental signaling pathway.