Non-IL-2-blocking anti-CD25 antibody inhibits tumor growth by depleting Tregs and has synergistic effects with anti-CTLA-4 therapy.

CD25, also known as the interleukin-2 receptor α chain (IL-2Rα), is highly expressed on regulatory T cells (Tregs), but relatively lower on effector T cells (Teffs). This makes it a potential target for Treg depletion, which can be used in tumor immunotherapy. However, marketed anti-CD25 antibodies (Basiliximab and Daclizumab) were originally developed as immunosuppressive drugs to prevent graft rejection, because these antibodies can block IL-2 binding to CD25 on Teffs, which in turn destroys the function of Teffs. Recent studies have shown that non-IL-2-blocking anti-CD25 antibodies have displayed exciting antitumor effects. Here, we screened out a non-IL-2-blocking anti-CD25 monoclonal antibody (mAb) 7B7 by hybridoma technology, and confirmed its antitumor activity via depleting Tregs in a CD25 humanized mouse model. Subsequently, we verified that the humanized 7B7, named as h7B7-15S, has comparable activities to 7B7, and that its Treg depletion is further increased when combined with anti-CTLA-4, leading to enhanced remodeling of the tumor immune microenvironment. Moreover, our findings reveal that the Fab form of h7B7-15S has the ability to deplete Tregs, independent of the Fc region. Taken together, our studies expand the application of anti-CD25 in tumor immunotherapy and provide insight into the underlying mechanism.

Combination Foretinib and Anti-PD-1 Antibody Immunotherapy for Colorectal Carcinoma.

Immune checkpoint inhibitors have achieved unprecedented success in cancer immunotherapy. However, the overall response rate to immune checkpoint inhibitor therapy for many cancers is only between 20 and 40%, and even less for colorectal cancer (CRC) patients. Thus, there is an urgent need to develop an efficient immunotherapeutic strategy for CRC. Here, we developed a novel CRC combination therapy consisting of a multiple receptor tyrosine kinase inhibitor (Foretinib) and anti-PD-1 antibody. The combination therapy significantly inhibited tumor growth in mice, led to improved tumor regression without relapse (83% for CT26 tumors and 50% for MC38 tumors) and prolonged overall survival. Mechanistically, Foretinib caused increased levels of PD-L1 via activating the JAK2-STAT1 pathway, which could improve the effectiveness of the immune checkpoint inhibitor. Moreover, the combination therapy remodeled the tumor microenvironment and enhanced anti-tumor immunity by further increasing the infiltration and improving the function of T cells, decreasing the percentage of tumor-associated macrophages (TAMs) and inhibiting their polarization toward the M2 phenotype. Furthermore, the combination therapy inhibited the metastasis of CT26-Luc tumors to the lung in BALB/c mouse by reducing proportions of regulatory T-cells, TAMs and M2 phenotype TAMs in their lungs. This study suggests that a novel combination therapy utilizing both Foretinib and anti-PD-1 antibody could be an effective combination strategy for CRC immunotherapy.

Chlorin e6 and CRISPR-Cas9 dual-loading system with deep penetration for a synergistic tumoral photodynamic-immunotherapy.

Photodynamic therapy (PDT) is a relatively safe and clinically promising treatment to combat primary tumors, especially epidermal carcinoma, while has negligible effects on distant metastasis. Therefore, this work reports a multifunctional nanosystem (HPR@CCP) exerting a combined photodynamic and immunotherapy to amplify the therapeutic effect on primary tumors and distant metastasis. Specifically, this nanosystem was obtained by electrostatic adsorption of a negatively charged hyaluronic acid "shell" with a positively charged "core" consisting of the CRISPR-Cas9 system targeting the Ptpn2 gene (Cas9-Ptpn2) and a modified mitochondria-targeting chlorin e6 (TPP-PEI-Ce6). Cell experiments demonstrated that the HPR@CCP nanoparticles possessed very high transfection efficiency on B16F10 cells, and TPP-PEI-Ce6 in the nanoparticles resulted in a significant PDT efficacy due to the efficient singlet oxygen generation in mitochondria under laser-irradiation. The accumulation of the nanoparticles in the tumor by active and passive tumor-targeting in vivo led to the disruption of the Ptpn2 gene by the Cas9-Ptpn2 plasmids in the nanocarriers, thus sensitizing tumors to immunotherapy by the increase of the IFN-γ and TNF-α signaling and the promotion of the proliferation of CD8+ T cells. In addition, Hyaluronidase was administered in advance to destroy the hyaluronic acid in the condensed extracellular matrix and to remove the hyaluronic acid "shell" from the nanosystem, subsequently leading to an enhanced penetration of oxygen and therapeutic agents. Fortunately, the primary and distant tumors in the experimental animals were remarkably inhibited after the combination of PDT-immunotherapy, thus, this easy-to-built nanomedicine could be used as a potential combination therapy against tumors.

Cryptophycin-55/52 based antibody-drug conjugates: Synthesis, efficacy, and mode of action studies.

Cryptophycin-52 (CR52), a tubulin inhibitor, exhibits promising antitumor activity in vitro (picomolar level) and in mouse xenograft models. However, the narrow therapeutic window in clinical trials limits its further development. Antibody-drug conjugate (ADC), formed by coupling cytotoxic compound (payload) to an antibody via a linker, can deliver drug to tumor locations in a targeted manner by antibody, enhancing the therapeutic effects and reducing toxic and side effects. In this study, we aim to explore the possibility of CR52-based ADC for tumor targeted therapy. Due to the lack of a coupling site in CR52, its prodrug cryptophycin-55 (CR55) containing a free hydroxyl was synthesized and conjugated to the model antibody trastuzumab (anti-HER2 antibody drug approved by FDA for breast cancer therapy) via the linkers based on Mc-NHS and Mc-Val-Cit-PAB-PNP. The average drug-to-antibody ratios (DARs) of trastuzumab-CR55 conjugates (named T-L1-CR55, T-L2-CR55, and T-L3-CR55) were 3.50, 3.29, and 3.35, respectively. These conjugates exhibited potent cytotoxicity in HER2-positive tumor cell lines with IC50 values at low nanomolar levels (0.58-1.19 nM). Further, they displayed significant antitumor activities at the doses of 10 mg/kg in established ovarian cancer (SKOV3) and gastric cancer (NCI-N87) xenograft models without overt toxicities. Finally, the drug releases were analyzed and the results indicated that T-L3-CR55 was able to effectively release CR55 and further epoxidized to CR52, which may be responsible for its best performance in antitumor activities. In conclusion, our results demonstrated that these conjugates have the potential for tumor targeted therapy, which provides insights to further research the CR55/CR52-based ADC for tumor therapy.

Effective antitumor activity of 5T4-specific CAR-T cells against ovarian cancer cells in vitro and xenotransplanted tumors in vivo.

Ovarian cancer is considered to be the most lethal gynecologic malignancy, and despite the development of conventional therapies and new therapeutic approaches, the patient's survival time remains short because of tumor recurrence and metastasis. Therefore, effective methods to control tumor progression are urgently needed. The oncofetal tumor-associated antigen 5T4 (trophoblast glycoprotein, TPBG) represents an appealing target for adoptive T-cell immunotherapy as it is highly expressed on the surface of various tumor cells, has very limited expression in normal tissues, and spreads widely in malignant tumors throughout their development. In this study, we generated second-generation human chimeric antigen receptor (CAR) T cells with redirected specificity to 5T4 (5T4 CAR-T) and demonstrated that these CAR-T cells can elicit lytic cytotoxicity in targeted tumor cells, in addition to the secretion of cytotoxic cytokines, including IFN-γ, IL-2, and GM-CSF. Furthermore, adoptive transfer of 5T4 CAR-T cells significantly delayed tumor formation in xenografts of peritoneal and subcutaneous animal models. These results demonstrate the potential efficacy and feasibility of 5T4 CAR-T cell immunotherapy and provide a theoretical basis for the clinical study of future immunotherapies targeting 5T4 for ovarian cancer.

Targeting of DDR1 with antibody-drug conjugates has antitumor effects in a mouse model of colon carcinoma.

DDR1 has been identified as a cancer-associated receptor tyrosine kinase that is highly expressed in several malignancies relative to normal tissues. Clinically approved multi-kinase inhibitors, such as nilotinib, inhibit DDR1-mediated tumor growth in xenograft models, suggesting DDR1 might be a potential target for cancer treatments. Here, we employed an antibody-based strategy with a novel anti-DDR1 antibody-drug conjugate (ADC) for colon carcinoma treatment. We developed T4 H11 -DM4, an ADC targeting DDR1 which carries the tubulin inhibitor payload DM4. Immunohistochemical analysis of a tissue microarray containing 100 colon cancer specimens revealed that DDR1 was highly expressed in 81% of tumor tissues. Meanwhile, high expression of DDR1 was associated with poor survival in patients. In vitro, T4 H11 -DM4 exhibited potent anti-proliferative activity with half maximal inhibitory concentration (IC50 ) values in the nanomolar range in a panel of colon cancer cell lines. In vivo, the antitumor efficacy of T4 H11 -DM4 was evaluated in three colon cancer cell lines expressing different levels of DDR1. T4 H11 -DM4 achieved complete tumor regression at doses of 5 and 10 mg·kg-1 in HT-29 and HCT116 tumor models. Moreover, a correlation between in vivo efficacy of T4 H11 -DM4 and the levels of DDR1 expression on the cell surface was observed. Tumor cell proliferation was caused by the induction of mitotic arrest, indicating that the antitumor effect in vivo was mediated by DM4. In addition, T4 H11 -DM4 was efficacious in oxaliplatin-resistant colon cancer models. In exploratory safety studies, T4 H11 -DM4 exhibited no overt toxicities when multi-doses were administered at 10 mg·kg-1 into BALB/c nude mice or when a single dose up to 50 mg·kg-1 was administered into BALB/c mice. Overall, our findings highlight the potential of DDR1-targeted ADC and may facilitate the development of a new effective therapeutic strategy for colon cancer.

A novel 5T4-targeting antibody-drug conjugate H6-DM4 exhibits potent therapeutic efficacy in gastrointestinal tumor xenograft models.

5T4, also named as trophoblast glycoprotein, is often upregulated in some cancer cells. Here, we demonstrated that 5T4 was highly expressed in gastric, colorectal, and pancreatic cancer, associated with significantly poor prognosis of gastrointestinal (GI) cancer patients. To search for new targeting drugs for GI cancer, we developed a novel anti-5T4 monoclonal antibody with high affinity and robust internalization ability and conjugated it to the potent microtubule inhibitor DM4 to produce conjugate H6-DM4. This antibody-drug conjugate (ADC) displayed significant cytotoxicity in a panel of GI cancer cell lines with IC50 values in the nanomolar range. H6-DM4 eradicated established GI tumor xenograft models at 2.5 mg/kg or 10 mg/kg without observable toxicity. Further, 5T4 was highly expressed in cancer-initiating cells (CICs) compared with non-CICs in colorectal cancer. In vitro and in vivo, treatment with H6-DM4 exhibited a powerful efficacy on colorectal CICs. Additionally, colorectal cancer cells resistant to platinum were effectively eliminated by H6-DM4. Taken together, our results showed 5T4-positive GI cancer cells, colorectal cancer-initiating cells, and platinum-resistant colorectal cancer cells were potently eliminated by H6-DM4, indicating H6-DM4 may be a potential candidate drug for GI cancer treatment.

Promiximab-duocarmycin, a new CD56 antibody-drug conjugates, is highly efficacious in small cell lung cancer xenograft models.

Small cell lung cancer (SCLC) is of a highly invasive and metastatic lung cancer subtype and there had not been effective targeted therapies. CD56, a cell surface marker highly expressed on most SCLC, is a promising therapeutic target for treatment of this aggressive cancer. In this study, we generated a novel anti-CD56 antibody named promiximab, characterized by high affinity, internalization and tumor specificity. Then, the promiximab was conjugated with a potent DNA alkylating agent duocarmycin via reduced interchain disulfides to yield the promiximab-Duocarmycin (promiximab-DUBA) conjugates. Mass spectrometry analysis showed promiximab-DUBA had an average DAR (Drug-to-Antibody Ratio) of about 2.04. In vitro, promiximab-DUBA exerted strong inhibitory effects on SCLC cell lines NCI-H526, NCI-H524 and NCI-H69, with IC50 values of 0.07 nmol/L, 0.18 nmol/L and 0.29 nmol/L, respectively. In vivo antitumor activity, promiximab-DUBA at the dose of 5 mg/kg and 10 mg/kg every three days with a total of three times were sufficient to induce sustained regression of NCI-H526 tumors over control treatment with promiximab. Mostly, no recurrence was observed until 65 days post treatment with promiximab-DUBA. In the NCI-H69 subcutaneous xenograft model, significant inhibition of tumor growth was also observed following administration of promiximab-DUBA at the dose of 5 mg/kg or 10 mg/kg. Moreover, body weight and histopathology of major organs (liver, spleen, heart, lung and kidney) showed no significant changes after treatment of promiximab-DUBA. In conclusion, promiximab-DUBA is highly efficacious in small cell lung cancer xenograft models, and provides a new immunotherapy approach for SCLC.

Therapeutic potential of an anti-HER2 single chain antibody-DM1 conjugates for the treatment of HER2-positive cancer.

Antibody-drug conjugates (ADCs) take the advantage of monoclonal antibodies to selectively deliver highly potent cytotoxic drugs to tumor cells, which have become a powerful measure for cancer treatment in recent years. To develop a more effective therapy for human epidermal growth factor receptor 2 (HER2)-positive cancer, we explored a novel ADCs composed of anti-HER2 scFv-HSA fusion antibodies conjugates with a potent cytotoxic drug DM1. The resulting ADCs, T-SA1-DM1 and T-SA2-DM1 (drug-to-antibody ratio in the range of 3.2-3.5) displayed efficient inhibition in the growth of HER2-positive tumor cell lines and the half-maximal inhibitory concentration on SKBR-3 and SKOV3 cells were both at the nanomolar levels in vitro. In HER2-positive human ovarian cancer xenograft models, T-SA1-DM1 and T-SA2-DM1 also showed remarkable antitumor activity. Importantly, three out of six mice exhibited complete remission without regrowth in the high-dose group of T-SA1-DM1. On the basis of the analysis of luminescence imaging, anti-HER2 scFv-HSA fusion antibodies, especially T-SA1, showed strong and rapid tumor tissue penetrability and distribution compared with trastuzumab. Collectively, the novel type of ADCs is effective and selective targeting to HER2-positive cancer, and may be a promising antitumor drug candidate for further studies.

Analysis of transcription profile to reveal altered signaling pathways following the overexpression of human desumoylating isopeptidase 2 in pancreatic cancer cells.

Human desumoylating isopeptidase 2 (DESI-2) is a member of the DESI family and contains a conserved PPPDE1 domain. Previous studies have demonstrated that DESI-2 overexpression may induce cell apoptosis. In the present study, differentially expressed genes were analyzed using a transcription microarray in DESI-2 overexpressing PANC-1 pancreatic cancer cells. A total of 45,033 genes were examined by microarray, which identified 1,766 upregulated and 1,643 downregulated genes. A series of altered signaling pathways were analyzed, in which certain essential signaling factors, including retinoid X receptor (RXR), BH3 interacting-domain death agonist, Ras homolog gene family member A (RhoA) and Rho-associated protein kinase, were further investigated at the protein level. The release of cytochrome c and the activation of caspase-3 were also detected by western blot analysis. Immunohistochemistry further revealed the expression features of RXR and RhoA in pancreatic ductal adenocarcinoma tissues with various DESI-2 expression levels. The results serve as a valuable reference for the further elucidation of the functions of DESI-2 in pancreatic cancer.

WNT16B from ovarian fibroblasts induces differentiation of regulatory T cells through ß-catenin signal in dendritic cells.

Treatment for cancer can induce a series of secreted factors into the tumor microenvironment, which can affect cancer progression. Wingless-type MMTV (mouse mammary tumor virus) integration site 16B (WNT16B) is a new member of the WNT family and has been reported to play growth-related roles in previous studies. In this study, we found WNT16B could be expressed and secreted into the microenvironment by human ovarian fibroblasts after DNA damage-associated treatment, including chemotherapy drugs and radiation. We also demonstrated that fibroblast-derived WNT16B could result in accumulation of ß-catenin in dendritic cells and secretion of interleukin-10 (IL-10) and transforming growth factor beta (TGF-ß), which contributed to the differentiation of regulatory T cells in a co-culture environment. These results shed light on the roles of WNT16B in immune regulation, especially in regard to cancer treatment.