thromboplastin and Triple-Negative-Breast-Neoplasms

Triple-negative breast cancer (TNBC) remains a most deadly human malignancy with limited response to chemotherapy, targeted therapy and immunotherapy. Tumor immunoenvironment plays an increasingly important role in therapy outcome. Tissue factor (TF) is the target of the FDA-approved ADC Tivdak. HuSC1-39 is the parent antibody of MRG004A, a clinical stage TF-ADC (NCT04843709). Here, we employed HuSC1-39 (termed "anti-TF") to investigate the role of TF in regulating immune-tolerance in TNBC. We found that patients with aberrant TF expression had a poor prognosis and low immune effector cell infiltration, characterizing as "cold tumor". In the 4T1 TNBC syngeneic mouse model, knockout of tumor cell TF inhibited tumor growth and increased tumor infiltration of effector T cell, which was not dependent on the clotting inhibition. In an immune-reconstituted M-NSG mouse model of TNBC, anti-TF inhibited tumor growth, which was further enhanced by a dual-targeting anti-TF&TGFβR fusion protein. There were diminished P-AKT and P-ERK signaling and profound tumor cell death in treated tumors. Transcriptome analyses and immunohistochemistry revealed a dramatically improved tumor immunoenvironment including the increase of effector T cells, decrease of Treg cells and the transformation of tumor into "hot tumor". Moreover, employing qPCR analysis and T cell culture, we further demonstrated that TF expression in tumor cells is sufficient to block the synthesis and secretion of T cell-recruiting chemokine CXCL9/10/11. Treatment of TF-high TNBC cells with anti-TF or TF-knockout all stimulated CXCL9/10/11 production, promoted T cell migration and effector function. Thus, we have identified a new mechanism of TF in TNBC tumor progression and therapy resistance.

Topics: Animals; Cell Line, Tumor; Humans; Immune Evasion; Immunotherapy; Mice; Signal Transduction; Thromboplastin; Triple Negative Breast Neoplasms

Triple-negative breast cancer (TNBC), representing ~15% of globally diagnosed breast cancer, is typically an incurable malignancy due to the lack of targetable surface targets for development of effective therapy. To address the unmet need for TNBC treatment, we recently determined that tissue factor (TF) is a useful surface target in 50-85% of patients with TNBC and developed a second-generation TF-targeting antibody-like immunoconjugate (called L-ICON) for preclinical treatment of TNBC. Using the chimeric antigen receptor (CAR) approach, here we develop and test TF-targeting CAR-engineered natural killer (TF-CAR-NK) cells that co-express CD16, the Fc receptor (FcγIII) to mediate antibody-dependent cellular toxicity (ADCC), for a preclinical assessment of immunotherapy of TNBC using TF-CAR-NK cell as single agent therapy and in combination with L-ICON. Our preclinical results demonstrate that TF-CAR-NK cells alone could kill TNBC cells and its efficacy was enhanced with L-ICON ADCC in vitro. Moreover, TF-CAR-NK cells were effective in vivo for the treatment of TNBC in cell line- and patient's tumor-derived xenograft mouse models. Thus, this study established the proof of concept of targeting TF as a new target in CAR-NK immunotherapy for effective treatment of TNBC and may warrant further preclinical study and potentially future investigation in TNBC patients.

Topics: Animals; Cell Line, Tumor; Cytotoxicity, Immunologic; Female; Humans; Immunotherapy; Killer Cells, Natural; Mice; Receptors, Chimeric Antigen; Thromboplastin; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Triple-negative breast cancer (TNBC) is a leading cause of breast cancer death and is often associated with

Topics: Animals; Antineoplastic Agents, Immunological; Biomarkers, Tumor; Cell Line, Tumor; CHO Cells; Cricetulus; Disease Models, Animal; Female; Gene Expression; Humans; Immunoconjugates; Immunohistochemistry; Molecular Targeted Therapy; Mutation; Thromboplastin; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Targeting cancer stem cell (CSC) represents a promising therapeutic approach as it can potentially fight cancer at its root. The challenge is to identify a surface therapeutic oncotarget on CSC. Tissue factor (TF) is known as a common yet specific surface target for cancer cells and tumor neovasculature in several solid cancers. However, it is unknown if TF is expressed by CSCs. Here we demonstrate that TF is constitutively expressed on CD133 positive (CD133+) or CD24-CD44+ CSCs isolated from human cancer cell lines, tumor xenografts from mice and breast tumor tissues from patients. TF-targeted agents, i.e., a factor VII (fVII)-conjugated photosensitizer (fVII-PS for targeted photodynamic therapy) and fVII-IgG1Fc (Immunoconjugate or ICON for immunotherapy), can eradicate CSC via the induction of apoptosis and necrosis and via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, respectively. In conclusion, these results demonstrate that TF is a novel surface therapeutic oncotarget for CSC, in addition to cancer cell TF and tumor angiogenic vascular endothelial TF. Moreover, this research highlights that TF-targeting therapeutics can effectively eradicate CSCs, without drug resistance, isolated from breast, lung and ovarian cancer with potential to translate into other most commonly diagnosed solid cancer, in which TF is also highly expressed.

Topics: A549 Cells; Animals; Antibody-Dependent Cell Cytotoxicity; Cell Line, Tumor; Female; Humans; Lung Neoplasms; Male; Metalloporphyrins; Mice; Mice, SCID; Molecular Targeted Therapy; Neoplastic Stem Cells; Ovarian Neoplasms; Photochemotherapy; Thromboplastin; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy.. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and (64)Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of (64)Cu-NOTA-ALT-836-Fab.. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of (64)Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR.. (64)Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management.

Topics: Animals; Cell Line, Tumor; Copper Radioisotopes; Female; Humans; Immunoglobulin Fab Fragments; Mammary Neoplasms, Experimental; Mice; Organometallic Compounds; Positron-Emission Tomography; Radiopharmaceuticals; Thromboplastin; Tissue Distribution; Triple Negative Breast Neoplasms