InVivoMAb anti-mouse PD-1 (CD279)

Immunotherapy represents a pivotal strategy for triple-negative breast cancer (TNBC), yet its efficacy is constrained by the immunosuppressive tumor microenvironment (TME). In this study, we demonstrate that bone morphogenetic protein-9 (BMP9) inhibits tumor growth and reprograms the immune TME in orthotopic TNBC models, primarily by attenuating regulatory T cells (Tregs) infiltration. Tregs depletion abrogates si-BMP9-mediated tumor promotion. Mechanistically, BMP9 suppresses CCL2 expression in an exocrine-independent manner to restrict Tregs recruitment. We identify DNA-dependent protein kinase catalytic subunit (PRKDC) as a BMP9-binding transcriptional regulator. The interaction between PRKDC and BMP9 directly impedes CCL2 transcriptional activation by suppressing PRKDC phosphorylation and indirectly suppresses CCL2 expression via NF-κB pathway remodeling. Critically, BMP9 modulation and CCL2 targeting potentiates immunotherapy efficacy without observable toxicity. Our study unveils the BMP9-PRKDC-CCL2 axis as a master regulatory node for TNBC-Tregs crosstalk, providing a strategy to overcome immunotherapy resistance in TNBC.

Head and neck squamous cell carcinoma (HNSCC) is a significant cause of morbidity and mortality worldwide, with limited treatment options for patients with locally advanced disease. CD47 immune checkpoint inhibitors have been used to block the CD47/SIRPa interaction that inhibits antigen-presenting cell phagocytosis, thereby enhancing antigen presentation to cytotoxic T-cells, and have shown promise in combination with anti-PD1 immunotherapy in tumors, including recurrent/metastatic HNSCC. We found that CD47 expression is associated with poor prognosis in HNSCC and explored the anti-tumor activity of an anti-CD47 fusion protein in combination with anti-PD1 and lymphatic-sparing radiotherapy in a locally advanced HNSCC model. In the 4MOSC1 syngeneic HPV-negative HNSCC mouse model, ALX301 (an engineered CD47-blocking SIRPα fusion for murine models) induced complete tumor regression when combined with anti-PD-1, and produced a partial tumor response as a monotherapy. An anti-PD1 immune checkpoint inhibitor in a CD47-null tumor background led to complete tumor regression confirming a key role for CD47 in tumor immunity. ALX301 treated mice demonstrated increased MHC-II expression on dendritic cells within the tumor and upregulation of CD86 co-stimulatory molecule on dendritic cells within the tumor, sentinel lymph nodes, and contralateral lymph nodes. Combination ALX301 and anti-PD1 treatment in an anti-PD1 resistant 4MOSC2 model demonstrated significant tumor regression, enhanced survivability, improved response with neoadjuvant radiotherapy, and greater retention of CD8 + T-cells within the tumor microenvironment. Notably, T-cell receptor sequencing revealed increased shared clonality between the tumor and sentinel lymph nodes of ALX301 treated mice. These data demonstrate that a combination of CD47 blockade and anti-PD1 therapy enhances tumor antigen presentation and immune cell infiltration, while further improving anti-tumor responses in combination with tumor-targeted radiotherapy. This study provides support for the rational design of combinatorial immunoradiotherapy, using anti-CD47 inhibitors and anti-PD1 therapy, in a clinical trial targeting locally advanced HPV-negative HNSCC.

Although disruptions in metal ion homeostasis leading to severe cellular damage and regulated cell death are a promising strategy for cancer immunotherapy, challenges in overloading these ions to tumor cells without premature release have limited their therapeutic applications. In this study, we develop binary mineral nanoparticles incorporating both Ca2+ and Na+ ions to enhance the cytotoxic effects of ion interference in cancer immunotherapy. Engineered using a microfluidic system for uniform size distribution and scalability, these nanoparticles exhibit pH-sensitive ion release. Systemically administered, they preferentially accumulate in tumors, elevating intracellular Ca2+ and Na+ levels and inducing immunogenic cell death without calcium channel activators or other small-molecule inducers. Our binary mineral nanoparticles significantly enhance antitumor immunity, especially when combined with an immune checkpoint inhibitor, leading to long-term immunity and inhibition of metastasis. This nanotechnology-enabled synergistic delivery of Ca²⁺ and Na⁺ ions represents a promising adjunct to existing metalloimmunotherapy strategies for cancer eradication.

The combination of chimeric antigen receptor (CAR)-T cell therapy with immune checkpoint blockade (ICB) using anti-PD-1 has been demonstrated to enhance antitumor CAR-T cell responses. Although, in conventional T cells, PD-1 suppresses T cell activation by binding to PD-L1 with recruitment of a phosphatase SHP2, the precise mechanisms underlying this process in CAR-T cells remain unclear. Here we show that, using real-time single-cell imaging, CD19-CAR aggregates to form "CAR microclusters," which transduce activation signals in coordination with key downstream molecules involved in TCR signaling. Concurrently, PD-1 forms inhibitory signalosomes, recruiting SHP2 into CAR microclusters by PD-L1 binding, leading to diminished CD3ζ phosphorylation, as well as reduced in cytokine production, antigen-specific cytotoxicity, and antitumor CAR-T cell effects in vivo. Importantly, anti-PD-1 treatment impairs PD-1 microcluster formation and restores CAR-T cell activities. Thus, these findings suggest that PD-1 microcluster formation can serve as a trigger for immune tolerance in CAR-T cells.

Liposarcoma is the most common soft tissue sarcoma. Well-differentiated liposarcoma can progress to dedifferentiated liposarcoma (DDLPS), a more aggressive form with higher metastatic potential and poor response to existing therapies. Progress in understanding and treating liposarcoma has been limited. To address this, we sought to develop an immunocompetent genetically engineered mouse model of liposarcoma.

Immunomodulatory agents benefit a small percentage of patients with oral cancer (OC), a subset of head and neck cancer. Cathepsin S (CTSS), a lysosomal protease, has been frequently associated with tumor immunity. This study aimed to investigate the mechanism by which tumor CTSS affects anti-tumor immunity through the regulation of interleukin-7 (IL-7) to overcome this obstacle.

Although ACVR2A mutations are prevalent in non-viral hepatocellular carcinomas (HCCs), the underlying mechanism remains unelucidated. Our molecular investigation reveals that ACVR2A impairment induces hyperglycolysis through the inactivation of the SMAD signaling pathway. Using syngeneic transplantation models and human clinical samples, we clarify that ACVR2A-deficient HCC cells produce and secrete lactate via the upregulation of lactate dehydrogenase A (LDHA) and monocarboxylate transporter 4 (MCT4) expression levels, which promotes regulatory T (Treg) cell accumulation and then acquires resistance to immune checkpoint inhibitors. Remarkably, genetic knockdown and pharmacological inhibition of MCT4 ameliorate the high-lactate milieu in ACVR2A-deficient HCC, resulting in the suppression of intratumoral Treg cell recruitment and the restoration of the sensitivity to PD-1 blockade. These findings furnish compelling evidence that lactate attenuates anti-tumor immunity and that therapeutics targeting this pathway present a promising strategy for mitigating immunotherapy resistance in ACVR2A-deficient HCC.

Lung cancer is the leading cause of cancer-related deaths, of which adenocarcinoma is the most common subtype. Despite this, lung adenocarcinoma and its metastasis are poorly understood, due to difficulties in feasibly recapitulating disease progression and predicting clinical benefits of therapy. We outline a methodology to develop immunogenic orthotopic lung adenocarcinoma mouse models, by injecting cell-specific cre viruses into the lung of a genetically engineered mouse, which mirrors cancer progression defined by the International Association for the Study of Lung Cancer. Evaluation of different cre virus/concentrations models demonstrate remarkable consistency in cancer initiation and metastasis, allowing for high throughput, while showing differences in timing and severity, offering greater flexibility when selecting models. Histological and immune profiles reflect clinical observations suggesting similar mechanisms are recapitulated and preliminary data show resultant tumors to be responsive to clinical treatments. We present a clinically relevant, next-generation murine model for studying lung adenocarcinoma.

Mechanistic understanding of the inhibitory immunoreceptor PD-1 is largely based on mouse models, but human and mouse PD-1 share only 59.6% amino acid identity. Here, we found that human PD-1 is more inhibitory than mouse PD-1, owing to stronger interactions with the ligands PD-L1 and PD-L2 and more efficient recruitment of the effector phosphatase Shp2. In a mouse melanoma model with adoptively transferred T cells, humanization of a PD-1 intracellular domain disrupted the antitumor activity of CD8+ T cells and increased the magnitude of anti-PD-1 response. We identified a motif highly conserved across vertebrate PD-1 orthologs, absent in rodents, as a key determinant for differential Shp2 recruitment. Evolutionary analysis suggested that PD-1 underwent a rodent lineage-specific functional attenuation during evolution. Together, our study uncovers species-specific features of the PD-1 pathway, with implications for PD-1 evolution and differential anti-PD-(L)1 responses in mouse models and human patients.

CD8+ T cells are critical mediators of antitumor immunity but differentiate into a dysfunctional state, known as T cell exhaustion, after persistent T cell receptor stimulation in the tumor microenvironment (TME). Exhausted T (Tex) cells are characterized by upregulation of coinhibitory molecules and reduced polyfunctionality. T cells in the TME experience an immunosuppressive metabolic environment via reduced levels of nutrients and oxygen and a buildup of lactic acid. Here we show that terminally Tex cells uniquely upregulate Slc16a11, which encodes monocarboxylate transporter 11 (MCT11). Conditional deletion of MCT11 in T cells reduced lactic acid uptake by Tex cells and improved their effector function. Targeting MCT11 with an antibody reduced lactate uptake specifically in Tex cells, which, when used therapeutically in tumor-bearing mice, resulted in reduced tumor growth. These data support a model in which Tex cells upregulate MCT11, rendering them sensitive to lactic acid present at high levels in the TME.

PD-1 immune checkpoint blockade (ICB) is a key cancer treatment. While blocking PD-1 binding to ligand is known, the role of internalization in enhancing ICB efficacy is less explored. Our study reveals that PD-1 internalization helps unlock ICB's full potential in cancer immunotherapy. Anti-PD-1 induces 50%-60% surface PD-1 internalization from human and mouse cells, leaving low to intermediate levels of resistant receptors. Complexes then appear in early and late endosomes. Both CD4 and CD8 T cells, especially CD8+ effectors, are affected. Nivolumab outperforms pembrolizumab in human T cells, while PD-1 internalization requires crosslinking by bivalent antibody. While mono- and bivalent anti-PD-1 inhibit tumor growth with CD8 tumor-infiltrating cells expressing increased granzyme B, bivalent antibody is more effective where the combination of steric blockade and endocytosis induces greater CD8+ T cell tumor infiltration and the expression of the cytolytic pore protein, perforin. Our findings highlight an ICB mechanism that combines steric blockade and PD-1 endocytosis for optimal checkpoint immunotherapy.

Neoadjuvant immunochemotherapy (nICT) has dramatically changed the treatment landscape of operable esophageal squamous cell carcinoma (ESCC), but factors influencing tumor response to nICT are not well understood. Here, using single-cell RNA sequencing paired with T cell receptor sequencing, we profile tissues from ESCC patients accepting nICT treatment and characterize the tumor microenvironment context. CXCL13+CD8+ Tex cells, a subset of exhausted CD8+ T cells, are revealed to highly infiltrate in pre-treatment tumors and show prominent progenitor exhaustion phenotype in post-treatment samples from responders. We validate CXCL13+CD8+ Tex cells as a predictor of improved response to nICT and reveal CXCL13 to potentiate anti-PD-1 efficacy in vivo. Post-treatment tumors from non-responders are enriched for CXCL13+CD8+ Tex cells with notably remarkable exhaustion phenotype and TNFRSF4+CD4+ Tregs with activated immunosuppressive function and a significant clone expansion. Several critical markers for therapeutic resistance are also identified, including LRRC15+ fibroblasts and SPP1+ macrophages, which may recruit Tregs to form an immunosuppressive landscape. Overall, our findings unravel immune features of distinct therapeutic response to nICT treatment, providing a rationale for optimizing individualized neoadjuvant strategy in ESCC.

The route of oncolytic virotherapy is pivotal for immunotherapeutic efficacy in advanced cancers. In this preclinical study, an oncolytic reovirus (RC402) is orally administered to induce antitumor immunity. Oral reovirus treatment shows no gross toxicities and effectively suppresses multifocal tumor lesions. Orally administered reovirus interacts with the host immune system in the Peyer's patch of the terminal ileum, increases IgA+ antibody-secreting cells in the lamina propria through MAdCAM-1+ blood vessels, and reshapes the gut microbiome. Oral reovirus promotes antigen presentation, type I/II interferons, and T cell activation within distant tumors, but does not reach or directly infect tumor cells beyond the gastrointestinal tract. In contrast to intratumoral reovirus injection, the presence of the gut microbiome, Batf3+ dendritic cells, type I interferons, and CD8+ T cells are indispensable for orally administered reovirus-induced antitumor immunity. Oral reovirus treatment is most effective when combined with αPD-1(L1) and/or αCTLA-4, leading to complete colon tumor regression and protective immune memory. Collectively, oral reovirus virotherapy is a feasible and effective immunotherapeutic strategy in preclinical studies.

Immunotherapy successfully complements traditional cancer treatment. However, primary and acquired resistance might limit efficacy. Reduced antigen presentation by MHC-I has been identified as potential resistance factor. Here we show that the epigenetic regulator ubiquitin-like with PHD and ring finger domains 1 (UHRF1), exhibits altered expression and aberrant cytosolic localization in cancerous tissues, where it promotes MHC-I ubiquitination and degradation. Cytoplasmic translocation of UHRF1 is induced by its phosphorylation on a specific serine in response to signals provided by factors present in the tumor microenvironment (TME), such as TGF-β, enabling UHRF1 to bind MHC-I. Downregulation of MHC-I results in suppression of the antigen presentation pathway to establish an immune hostile TME. UHRF1 inactivation by genetic deletion synergizes with immune checkpoint blockade (ICB) treatment and induces an anti-tumour memory response by evoking low-affinity T cells. Our study adds to the understanding of UHRF1 in cancer immune evasion and provides a potential target to synergize with immunotherapy and overcome immunotherapeutic resistance.

Understanding tumor-host immune interactions and the mechanisms of lung cancer response to immunotherapy is crucial. Current preclinical models used to study this often fall short of capturing the complexities of human lung cancer and lead to inconclusive results. To bridge the gap, we introduce two new murine monoclonal lung cancer cell lines for use in immunocompetent orthotopic models. We demonstrate how our cell lines exhibit immunohistochemical protein expression (TTF-1, NapA, PD-L1) and common driver mutations (KRAS, p53, and p110α) seen in human lung adenocarcinoma patients, and how our orthotopic models respond to combination immunotherapy in vivo in a way that closely mirrors current clinical outcomes. These new lung adenocarcinoma cell lines provide an invaluable, clinically relevant platform for investigating the intricate dynamics between tumor and the immune system, and thus potentially contributes to a deeper understanding of immunotherapeutic approaches to lung cancer treatment.