Tissue factor overexpression in triple-negative breast cancer promotes immune evasion by impeding T-cell infiltration and effector function.

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&TGFßR 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.

Tumor-targeting anti-EGFR x anti-PD1 bispecific antibody inhibits EGFR-overexpressing tumor growth by combining EGFR blockade and immune activation with direct tumor cell killing.

We developed a strategy to combine conventional targeted therapy with immune checkpoint blockade using a tumor-targeting bispecific antibody (BsAb) to treat solid tumors. The BsAb was designed to simultaneously engage a tumor-associated antigen, epidermal growth factor receptor (EGFR), and programed cell death protein 1 (PD1). In addition to its direct anti-tumor activity via EGFR inhibition, the BsAb mediated efficient antibody-dependent cellular cytotoxicity (ADCC) and activated T cell antitumor im munity through blockade of PD1 from interacting with its counterpart, programed cell death ligand 1 (PDL1). Further, the BsAb exhibited a potent direct tumor cell killing activity in the presence of PBMC, most likely, via activating and, at the same time, physically engaging T cells with tumor cells. Taken together, we here illustrate a new strategy in the design and production of novel BsAbs with enhanced therapeutic efficacy through both direct tumor growth inhibition and T cell activation via tumor-targeted immune checkpoint blockade.

Dual Mechanisms of Novel CD73-Targeted Antibody and Antibody-Drug Conjugate in Inhibiting Lung Tumor Growth and Promoting Antitumor Immune-Effector Function.

Although tyrosine kinase inhibitor therapy and immunotherapy have significantly improved lung cancer management, many patients do not benefit or become resistant to treatment, highlighting the need for novel treatments. We found elevated CD73 expression to be prevalent in non-small cell lung cancer (NSCLC) including those harboring the RAS- or RTK (EGFR, EML4-ALK) oncogenes. CD73 expression is enriched closely with the transcriptome signature of epithelial-mesenchymal transition and the immune-tolerant tumor microenvironment, which are increasingly relevant for disease progression and therapy resistance. We developed two novel series of CD73 antibody, Ab001/Ab002 and humanized version Hu001/Hu002, which demonstrated high CD73 binding affinity, potent enzyme inhibition, and efficiently protected effector T lymphocyte function from adenosine/cancer-imposed toxicity. Hu001/Hu002 inhibited growth of RAS-mutant NSCLC tumors in mice via enhanced antibody-dependent cell-mediated cytotoxicity and multifaceted remodeling of the tumor immune environment, reflecting diminished levels of tumor-associated macrophages, myeloid-derived suppressor cells, and tumor vasculature. A novel MMAE-conjugated CD73-ADC (Hu001-MMAE) elicited potent cytotoxicity against CD73-high expressing tumor cells (IC50<0.1 nmol/L) and suppressed in vivo growth of multiple NSCLC and glioma tumors, including the RAS-mutant models [minimum effective dose <1 mg/kg]. Treatment with CD73-ADC triggered a robust intratumoral accumulation of proinflammatory macrophages and activated dendritic cells (DC), which were not observed with naked CD73 antibody or standard chemotherapy. Studies with human PBMC-derived systems confirmed CD73-ADC as fully functional in protecting effector T cells and stimulating DCs thus providing dual benefits in killing CD73-high tumors and improving cancer immunity response. These results warrant clinical investigation of CD73-targeted antibody and ADC for treating advanced lung cancer.

mTOR Promotes Tissue Factor Expression and Activity in EGFR-Mutant Cancer.

Mechanistic target of rapamycin (mTOR) signaling pathway mediates the function of oncogenic receptor tyrosine kinases (RTKs). We aimed to elucidate new role of mTOR in EGFR-mutant (EGFR-mut) non-small cell lung cancer (NSCLC) and glioblastoma (GBM) with a focus on tumor microenvironments. Here, we report a novel regulatory link between mTOR complexes (mTORCs) and tissue factor (TF), an initiator of tumor-derived thrombosis. TF is elevated in EGFR-mut NSCLC/GBM cell lines and tumors from patients with poor prognosis. Application of mTORC1/2 inhibitors (AZD8055, WYE-125132, MTI-31, and rapamycin) or genetic mTORC-depletion all reduced TF expression, which appeared to be differentially mediated depending on cellular context. In U87MG and HCC827 cells, mTORC1 exerted a dominant role via promoting TF mRNA transcription. In EGFR-TKI-resistant H1975 and PC9 cells, it was mTORC2 that played a major role in specific repression of lysosomal-targeted TF protein degradation. Successful inhibition of TF expression was demonstrated in AZD8055- or MTI-31-treated H1975 and U87MG tumors in mice, while a TF-targeted antibody antagonized TF activity without reducing TF protein. Both the mTOR- and TF-targeted therapy induced a multifaceted remodeling of tumor microenvironment reflecting not only a diminished hypercoagulopathy state (fibrin level) but also a reduced stromal fibrosis (collagen distribution), compromised vessel density and/or maturity (CD31 and/or α-SMA) as well as a substantially decreased infiltration of immune-suppressive M2-type tumor-associated macrophages (CD206/F4/80 ratio). Thus, our results have identified TF as a functional biomarker of mTOR. Downregulation of mTOR-TF axis activity likely contributes to the therapeutic mechanism of mTORC1/2- and TF-targeted agents in EGFR-mut advanced NSCLC and GBM.

A new anti-HER2 antibody that enhances the anti-tumor efficacy of trastuzumab and pertuzumab with a distinct mechanism of action.

The majority of patients with metastatic breast cancer who are treated with the anti-HER2 monoclonal antibody, trastuzumab, generally develop resistance to the drug within a year after initiation of the treatment. Here we describe a new anti-HER2 humanized monoclonal antibody, 19H6-Hu, which binds to HER2 extracellular domain (ECD) with high affinity and inhibits proliferation of multiple HER2-overexpressing cancer cell lines as a single agent or in combination with trastuzumab. 19H6-Hu binds to the domain III in proximity to the domain IV of HER2 ECD, which differs from trastuzumab and pertuzumab. 19H6-Hu in combination with trastuzumab was more effective at blocking phosphorylation of ERK1/2, AKT(S473)and HER2 (Y1248) in HER2-positive cancer cells compared to trastuzumab alone or in combination with pertuzumab. Combination of three antibodies, 19H6-Hu, inetetamab (a trastuzumab analog) and pertuzumab exhibited much stronger inhibition of large NCI-N87 tumor xenografts (>400mm3) than the current standard of care, inetetamab (trastuzumab) plus Docetaxel (DTX), as well as the combination of 19H6-Hu, inetetamab and DTX. Our results highlight the functional variability of HER2 domains and provide a new insight into the design of HER2-targeting agents.

A Novel mTORC1/2 Inhibitor (MTI-31) Inhibits Tumor Growth, Epithelial-Mesenchymal Transition, Metastases, and Improves Antitumor Immunity in Preclinical Models of Lung Cancer.

PURPOSE: We aimed to investigate efficacy and mechanism of MTI-31 (LXI-15029), a novel mTORC1/mTORC2 inhibitor currently in human trial (NCT03125746), in non-small cell lung cancer (NSCLC) models of multiple driver mutations and tyrosine kinase inhibitor (TKI)-resistance. EXPERIMENTAL DESIGN: Gene depletion, inhibitor treatment, immunological, flow cytometry, cellular, and animal studies were performed to determine in vitro and in vivo efficacy in NSCLC models of driver mutations and elucidate roles by mTOR complexes in regulating migration, epithelial-mesenchymal transition (EMT), metastasis, intracranial tumor growth, and immune-escape. RESULTS: MTI-31 potently inhibited cell proliferation (IC50 <1 µmol/L) and in vivo tumor growth in multiple NSCLC models of EGFR/T790M, EML4-ALK, c-Met, or KRAS (MED <10 mg/kg). In EGFR-mutant and/or EML4-ALK-driven NSCLC, MTI-31 or disruption of mTORC2 reduced cell migration, hematogenous metastasis to the lung, and abrogated morphological and functional traits of EMT. Disruption of mTORC2 inhibited EGFR/T790M-positive tumor growth in mouse brain and prolonged animal survival correlating a diminished tumor angiogenesis and recruitment of IBA1+ microglia/macrophages in tumor microenvironment. MTI-31 also suppressed programmed death ligand 1 (PD-L1) in EGFR- and ALK-driven NSCLC, mediated in part by mTORC2/AKT/GSK3ß-dependent proteasomal degradation. Depletion of mTOR protein or disruption of mTOR complexes profoundly downregulated PD-L1 and alleviated apoptosis in Jurkat T and primary human T cells in a tumor-T cell coculture system. CONCLUSIONS: Our results highlight mTOR as a multifaceted regulator of tumor growth, metastasis, and immune-escape in EGFR/ALK-mutant and TKI-resistant NSCLC cells. The newly characterized mechanisms mediated by the rapamycin-resistant mTORC2 warrant clinical investigation of mTORC1/mTORC2 inhibitors in patients with lung cancer.

Novel-smoothened inhibitors for therapeutic targeting of naïve and drug-resistant hedgehog pathway-driven cancers.

The G protein-coupled receptor (GPCR) smoothened (SMO) is a key signaling component of the sonic hedgehog (Hh) pathway and a clinically validated target for cancer treatment. The FDA-approved SMO inhibitors GDC-0449/Vismodegib and LDE225/Sonidegib demonstrated clinical antitumor efficacy. Nevertheless, relatively high percentage of treated patients would eventually develop acquired cross resistance to both drugs. Here, based on published structure and activity of GDC-0449 inhibitor class, we replaced its amide core with benzimidazole which retained bulk of the SMO-targeting activity as measured in our Hh/SMO/Gli1-reporter system. Synthesis and screening of multiple series of benzimidazole derivatives identified HH-1, HH-13, and HH-20 with potent target suppression (IC50: <0.1 µmol/L) in the reporter assays. In NIH3T3 cells stimulated with a secreted Hh (SHH), these inhibitors dose dependently reduced mRNA and protein expression of the endogenous pathway components PTCH-1, Gli1, and cyclin D1 resulting in growth inhibition via G0/G1 arrest. Mechanistically, the SMO-targeted growth inhibition involved downregulation of mTOR signaling inputs and readouts consistent with diminished mTORC1/mTORC2 functions and apoptosis. In mice, as with GDC-0449, orally administered HH inhibitors blocked paracrine activation of stromal Hh pathway in Calu-6 tumor microenvironment and attenuated growth of PTCH+/-/P53-/- medulloblastoma allograft tumors. Furthermore, HH-13 and HH-20 potently targeted the drug-resistant smoothened SMO-D473H (IC50: <0.2 µmol/L) compared to the poor inhibition by GDC-0449 (IC50: >60 µmol/L). These results identify HH-13 and HH-20 as potent inhibitors capable of targeting naïve and drug-resistant Hh/SMO-driven cancers. The current leads may be optimized to improve pharmaceutical property for potential development of new therapy for treatment of Hh pathway-driven cancers.

Pathological expression of tissue factor confers promising antitumor response to a novel therapeutic antibody SC1 in triple negative breast cancer and pancreatic adenocarcinoma.

The pathological presence of tissue factor (TF) in cancer cells promotes tumor-initiated thrombosis and cancer metastasis. We found that TF is aberrantly present in large percentage of aggressive triple negative breast cancer (TNBC) and pancreatic adenocarcinoma (PaC), two most lethal forms of malignancy that urgently need effective treatment. TF expression in TNBC clustered with higher levels of vimentin, basal-type keratins KRT5/14 and caveolin-1 but lower levels of luminal-type biomarkers. We developed a novel and specific anti-TF therapeutic antibody SC1, which displayed an exceedingly high potency against TF extracellular domain (EC50: 0.019 nM), TF-positive TNBC- or PaC cells (EC50: 2.5 nM), intracellular protease activated receptor 2 (PAR2) signaling (IC50: 2-3 nM) and tumor-initiated coagulation (IC50: <10 nM). Depletion of TF or SC1-treatment in TNBC or PaC cells inhibited TF-induced cell migration, lung metastasis and tumor growth in vivo, accompanied by diminished levels of tumor angiogenesis and stromal fibrosis. We further propose TF as a promising target for antibody-drug conjugate (ADC) development based on its rapid and efficient internalization of SC1-drug conjugate. Both SC1-DM1 and SC1-MMAE elicited exquisite cytotoxicity in TF-positive TNBC and PaC cells (IC50: 0.02-0.1 nM) but not in TF-negative cells (>100 nM) achieving >5000 fold target selectivity. Following a weekly intravenous administration, SC1-MMAE and its humanized hSC1-MMAE inhibited TNBC- and PaC tumor growth achieving MED of 0.3-1 mg/kg and were both well tolerated. Thus, the prevalent TF expression in TNBC and PaC renders these challenging tumors highly susceptible to TF-targeted treatment and may offer new opportunity in cancer patients.

Stapled RGD Peptide Enables Glioma-Targeted Drug Delivery by Overcoming Multiple Barriers.

Malignant glioma, the most frequent and aggressive central nervous system (CNS) tumor, severely threatens human health. One reason for its poor prognosis and short survival is the presence of the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB), which restrict the penetration of therapeutics into the brain at different stages of glioma. Herein, inspired by the peptide stapling technique, we designed a cyclic RGD ligand via an all-hydrocarbon staple (stapled RGD, sRGD) to facilitate BBB penetration while retaining the capacity of BBTB penetration and targeting ability to glioma cells. As expected, sRGD-modified micelles were able to penetrate the in vitro BBB model while retaining the glioma targeted capability. The results of the in vivo imaging studies further revealed that this nanocarrier could not only efficiently transverse the intact BBB of normal mice, but also could specifically target glioma cells of intracranial glioma-bearing nude mice. Furthermore, Paclitaxel-loaded sRGD-modified micelles exhibited improved antiglioma efficacy in vitro and significantly prolonged survival time of glioma-bearing nude mice. Overall, this sRGD peptide showed potency for glioma-targeted drug delivery by overcoming multiple barriers.

Targeting TNFα Ameliorated Cationic PAMAM Dendrimer-Induced Hepatotoxicity via Regulating NLRP3 Inflammasomes Pathway.

Hepatotoxicity of cationic poly amidoamine (PAMAM) dendrimers is one of the most urgent challenges to their medicinal application. Recent studies have indicated that proinflammatory cytokines were critical in nanomaterials-induced toxicity. However, little is known about the roles and underlying regulatory mechanisms of proinflammatory cytokines in cationic PAMAM dendrimer-induced hepatotoxicity. Thus, the aim of the current study was to explore the role of proinflammatory cytokine tumor necrosis factor alpha (TNFα) in cationic PAMAM dendrimer-induced liver injury and its underlying mechanism and develop novel strategies to reduce hepatotoxicity of cationic PAMAM dendrimers through regulating TNFα. In this study, we verified the significant overexpression of TNFα in cationic PAMAM dendrimer-induced hepatotoxicity in mice and found that targeting TNFα by etanercept could protect against cationic PAMAM dendrimer-induced liver injury. Interestingly, etanercept suppressed cationic PAMAM dendrimer-induced inflammasome signaling as demonstrated by reduced activation of NALP3, cleavage of Caspase-1, and maturation of interleukin (IL)-1ß. Moreover, suppression of NLRP3 inflammasomes by belnacasan could also protect against cationic PAMAM dendrimer-induced hepatotoxicity and TNFα-induced acute hepatotoxicity. Notably, targeting either TNFα or inflammasomes reduced autophagy activation in hepatotoxicity triggered by cationic PAMAM dendrimers. In general, these findings revealed that targeting TNFα could ameliorate cationic PAMAM dendrimer-induced hepatotoxicity via regulating NLRP3 inflammasome pathway, underscoring that TNFα antagonism by etanercept could be used as an effective pharmacological approach to control hepatotoxicity of cationic PAMAM dendrimers and thus providing novel therapeutic strategies for managing liver toxicity of nanomaterials via regulating inflammatory mediators.

mTOR complex-2 stimulates acetyl-CoA and de novo lipogenesis through ATP citrate lyase in HER2/PIK3CA-hyperactive breast cancer.

The mechanistic target of rapamycin (mTOR) is a major regulator of cell growth and is frequently dysregulated in cancer. While mTOR complex-1 (mTORC1) is a validated cancer target, the role of mTOR complex-2 (mTORC2) remains less defined. Here, we reveal mTORC2 as a critical regulator of breast cancer metabolism. We showed that hyperphosphorylation in ATP citrate lyase (ACL) occurs frequently in human breast tumors and correlates well with HER2+ and/or PIK3CA-mutant (HER2+/PIK3CAmut) status in breast tumor cell lines. In HER2+/PIK3CAmut cells, mTORC2 controls Ser-455 phosphorylation of ACL thereby promoting acetyl-CoA production, de novo lipogenesis and mitochondrial physiology, all of which were inhibited by an mTORC1/mTORC2 kinase inhibitor (mTOR-KI) or cellular depletion of mTORC2 or ACL. mTOR-KI but not rapamycin blocked the IGF-1-induced ACL phosphorylation and glucose to lipid conversion. Depletion of mTORC2 but not mTORC1 specifically inhibited the ACL-dependent acetyl-CoA production. In the HER2+/PIK3CAmut MDA361, MDA453, BT-474 and T47D cells, depletion of mTORC2 or ACL led to growth inhibition and mitochondrial hyperpolarization, which were partially rescued by an alternate source of acetyl-CoA. These same changes were not apparent in mTORC2- or ACL-depleted HER2-/PIK3CAwt MDA231 and HCC1806 cells, highlighting a differential dependence of mTORC2-ACL for survival in these two cell types. Moreover, ACL Ser-455 mutants S455E (phosphomimetic) and S455A (non-phosphorylatable) each increased or decreased, respectively, the acetyl-CoA production, mitochondrial homeostasis and survival in ACL-depleted MDA453 cells. These studies define a new and rapamycin-resistant mechanism of mTORC2-ACL in lipogenesis and acetyl-CoA biology and provide a rationale for targeting of mTORC1 and mTORC2 in HER2+/PIK3CAmut breast cancer.

Interplay of Oxidative Stress and Autophagy in PAMAM Dendrimers-Induced Neuronal Cell Death.

Poly-amidoamine (PAMAM) dendrimers are proposed to be one of the most promising drug-delivery nanomaterials. However, the toxicity of PAMAM dendrimers on the central nervous system seriously hinders their medical applications. The relationship between oxidative stress and autophagy induced by PAMAM dendrimers, and its underlying mechanism remain confusing. In this study, we reported that PAMAM dendrimers induced both reactive oxygen species and autophagy flux in neuronal cells. Interestingly, autophagy might be triggered by the formation of reactive oxygen species induced by PAMAM dendrimers. Suppression of reactive oxygen species could not only impair PAMAM dendrimers-induced autophagic effects, but also reduce PAMAM dendrimers-induced neuronal cell death. Moreover, inhibition of autophagy could protect against PAMAM dendrimers-induced neuronal cell death. These findings systematically elucidated the interplay between oxidative stress and autophagy in the neurotoxicity of PAMAM dendrimers, which might encourage the application of antioxidants and autophagy inhibitors to ameliorate the neurotoxicity of PAMAM dendrimers in clinic.