BackgroundThrombocytopenia is a common problem in the management of patients with cancer and other conditions that affect hematopoietic cells. In previous clinical trials, the polyethylene-glycolconjugated recombinant human megakaryocyte growth and development factor increased platelet counts in patients with idiopathic thrombocytopenic purpura and solid tumors undergoing chemotherapy. However, antibodies to polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor develop in healthy volunteers and patients undergoing chemotherapy and cross-react with endogenous thrombopoietin. As a result, clinical development of polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor was discontinued in 1998. The aim of this study was to identify an orally bioavailable human Mpl activator that does not develop autoantibodies against endogenous thrombopoietin.

Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8–. CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.

Mannan-binding protein (MBP) is a C-type mammalian lectin specific for mannose and N-acetylglucosamine. MBP is mainly synthesized in the liver and occurs naturally in two forms, serum MBP (S-MBP) and intracellular MBP (I-MBP). S-MBP activates Mannan-binding protein (MBP),4 also known as mannanbinding lectin, is a Ca 2ϩ -dependent (C-type) mammalian lectin exhibiting primary specificity for mannose, fucose, and N-acetylglucosamine (1). Because its discovery as a vital serum component associated with innate immunity, this lectin has been regarded as a multifunctional protein. MBP occurs naturally in two forms, secretory serum MBP (S-MBP) and intracellular MBP, which is termed I-MBP in this study and was called liver MBP (L-MBP) in our previous studies (2-4). Human S-MBP and I-MBP are mainly synthesized in the liver and translated from a single form of mRNA. Both S-MBP and I-MBP are homooligomers composed of 32-kDa subunits. Each subunit has an NH 2 -terminal region containing cysteines involved in interchain disulfide bond formation, a collagen-like domain containing hydroxyproline and hydroxylysine residues, and a carbohydrate-recognition domain (CRD) with an amino acid sequence highly homologous to those of other C-type lectins (5). The CRD is specific for high-mannose oligosaccharide structures on exogenous and endogenous ligands, whereas the collagen-like domain is believed to be responsible for interactions with other effector proteins involved in host defense. S-MBP activates complement through interaction with three novel MBP-associated serine proteases, which is called the lectin pathway (6). Furthermore, S-MBP was shown to exhibit novel cytotoxic activity toward some in vivo colorectal carcinoma cell experiments, which we proposed to term MBP-dependent cell-mediated cytotoxicity (4, 7). However, little is known about the subcellular localization and physiological significance of I-MBP. Recently, the functions of glycoprotein glycan chains in protein quality control have been attracting more attention, highmannose-type oligosaccharides especially having been shown to play important roles in this process (8,9). Glycosylation with an asparagine-linked (N-linked) oligosaccharides is a co-translational process that occurs in the ER. High-mannose-type oligosaccharides, presumably representing nascent core-glycosylated proteins, are subsequently transferred to the Golgi

No abstract

Background/Aim: Cancer stem cells (CSCs) are considered to be one of the causes of tumor recurrence after chemotherapy. The purpose of our study was to isolate CSCs from human colorectal cancer cell (CRC) lines. Materials and Methods: Nine CRC lines were screened based on the expression level of potential CSC markers to identify putative CSCs. Tumor formation capacity in immunodeficient mice was compared with that of their counterparts. Stemness, differentiation potency and sensitivity to 5-fluorouracil (5-FU), in vitro, were also assessed. Microarray analysis was used to characterize the features of the putative CSCs. Results: COLO 201 cells were separated into two populations based on CD44 expression. CD44 positive (CD44 + ) cells showed significantly higher tumor formation capacity than CD44 − cells in immunodeficient mice. CD44 + cells also possessed stemness properties and lower sensitivity to 5-FU in vitro. Moreover, cancer stemness and chemoresistance-related genes were highly up-regulated in CD44 + cells. Conclusion: CD44 + COLO 201 cells possessed the features of CSCs; therefore, the present CSC model could serve as a valuable tool to accelerate CSC research.

BackgroundRegulatory T cells (Tregs) are suppressive immune cells required for the maintenance of immune homeostasis, but tumor-infiltrating Tregs are known to suppress the antitumor immune system and promote tumor progression. Therefore, selective reduction of tumor-infiltrating Tregs is anticipated to reinvigorate antitumor immunity without inducing autoimmunity. S-531011 is a novel anti-human IgG1 antibody targeting human CCR8 (C-C motif chemokine receptor 8) which is selectively expressed in tumor-infiltrating Tregs, with both in vitro antibody dependent cellular cytotoxicity (ADCC) against CCR8-expressing cells and neutralizing activity against CCL1-CCR8 signaling. Here, to evaluate antitumor activities and safety aspects of S-531011, we conducted non-clinical pharmacology studies of S-531011 using human CCR8 knock-in (KI) mice and human tissues.MethodsS-531011 was administrated to CT26WT tumor-bearing hCCR8-KI mice, and the effect on the presence of tumor-infiltrating CCR8+ Treg and tumor growth were evaluated. We also investigated the antitumor efficacy of S-531011 in combination with anti-mouse PD-1 antibody. Next, human lung cancer tissues and human NK-cells were co-cultured, and the ex vivo ADCC against tumor-infiltrating Tregs by S-531011 was verified. We also incubated human peripheral blood-derived mononuclear cells (PBMC) from healthy individuals with S-531011 to investigate the effects on the proportion of Tregs in human PBMC.ResultsIntravenous administration of S-531011 to CT26WT tumor-bearing hCCR8-KI mice significantly reduced tumor-infiltrating CCR8+ Tregs and markedly suppressed tumor growth. Furthermore, the combined therapy of S-531011 with anti-mouse PD-1 antibody showed greater anti-tumor effect than monotherapy without any apparent side effects. Ex vivo ADCC studies using human lung cancer tissues and FCM analysis of CCR8 expression in tumor-infiltrating Tregs suggested that most of the tumor-infiltrating CCR8+ Tregs were depleted by S-531011. On the other hand, S-531011 didn’t reduce Tregs in human PBMC.ConclusionsS-531011 is a promising drug which has a strong antitumor effect by depleting tumor-infiltrating CCR8+ Tregs, as a not only monotherapy but also combination therapy with other immune checkpoint inhibitors.Ethics ApprovalThe present study was approved by the Institutional Ethics Committee of Osaka University Hospital (approved number: 13266-15) and Shionogi Co., Ltd. (approved number: 021-003). Animal studies were approved by the Institutional Animal Care and Use Committee (approved number: S20093D, S20197D and S20198D).

Butyzamide is a novel non-peptidyl molecule which has agonistic activity to the thrombopoietin (TPO) receptor Mpl. Butyzamide promotes the proliferation of murine pro B cell line Ba/F3 expressing human Mpl (hMpl), and induces phosphorylation of JAK2, STAT3, STAT5 and MAPK. Interestingly, butyzamide does not promote the proliferation of Ba/F3 cells expressing murine Mpl (mMpl). To elucidate the mechanisms for this species specificity, we created stable transfectants such as Ba/F3-hMpl(H499L) and Ba/F3-mMpl(L490H) cells. Butyzamide induced the proliferation of Ba/F3-mMpl(L490H) but not Ba/F3-hMpl(H499L) cells, indicating that a histidine residue in the transmembrane domain of human Mpl is critical for butyzamide-induced signaling. Butyzamide induced the colony-forming unit-megakaryocyte and polyploid megakayocytes from human CD34+ hematopoietic progenitor cells, and its effects were comparable to those of thrombopoietin. When butyzamide was orally administered to immunodeficient NOD/Shi-scid/IL-2Rγcnull (NOG) mice transplanted with human fetal liver-derived CD34+ cells, the human platelet count increased by 6.2- and 22.9-fold at the doses of 10 and 50 mg/kg for 20 days, respectively. Butyzamide also increased the number of reticulated human platelets and human matured megakaryocytes in NOG mice. These results indicate that butyzamide is an orally bioavailable Mpl activator and suggest its potential for clinical development as a therapeutic agent in patients with thrombocytopenia.

Despite the success of immune checkpoint therapy, most non-small cell lung cancer (NSCLC) patients still receive conventional chemotherapy. Overcoming chemotherapy resistance by identifying specific targets should improve cancer treatment and patient survival. In this study, we examined a prospective chemotherapy-resistant subpopulation in NCI-H1975 NSCLC cells and identified a novel target, neurosecretory protein VGF (VGF). Using flow cytometry, we first analyzed the expression profile of cancer stem cell markers in 100 cancer cell lines and found that NCI-H1975 cells could be divided into three subpopulations, CD44-low (CD44-L), CD44-middle (CD44-M) and CD44-high cells based on the expression pattern of CD44. CD44-M cells account for less than 5% of the NCI-H1975 cells under normal conditions, but only this subpopulation can proliferate in vitro and in vivo after treatment with cytotoxic agents, such as paclitaxel, pemetrexed and 5-fluorouracil. Affymetrix microarray analysis revealed that VGF is specifically expressed in CD44-M cells and we hypothesized that VGF would play a critical role in chemotherapy resistance. We next employed patient-derived xenograft (PDX) models of NSCLC to confirm this hypothesis with human clinical samples. NOD/SCID mice were subcutaneously transplanted with PDX and treated with either vehicle or paclitaxel (12 mg/kg, Q3Dx3, i.v.). VGF immunohistochemistry on resected PDX revealed that the number of VGF-expressing cells increased 5.6-fold in the paclitaxel-treated group compared to the vehicle control. We also performed VGF immunohistochemistry on NSCLC surgical specimens which were obtained from 127 patients, consisting of 42 neoadjuvant chemotherapy treated cases and 85 non-treated cases. The frequency of VGF-expressing specimens was higher in neoadjuvant chemotherapy treated cases (50%) than non-treated cases (18%). These results suggest that VGF-expressing tumor subpopulation is paclitaxel-resistant even in clinical settings. We also analyzed the molecular functions of VGF. Knockdown of VGF expression by siRNAs could signifıcantly suppress paclitaxel resistance in CD44-M cells. On the other hand, overexpression of VGF could significantly induce paclitaxel resistance in CD44-L cells. These results suggest that VGF is functionally responsible for chemotherapy resistance in NSCLC patients and combined administration of an anti-VGF drug and chemotherapy could be effective for preventing cancer recurrence and prolonging cancer free survival. Citation Format: Wataru Nogami, Yumi Tona, Soichi Tofukuji, Yoshino Ishioka, Mitsunobu Matsumoto, Hajime Yamada, Kenji Kuwabara, Hidekazu Tanaka, Shigeki Adachi, Yoko Yamamoto, Ryu Kanzaki, Soichiro Funaki, Yasushi Shintani, Meinoshin Okumura, Taisei Nomura. VGF is functionally responsible for chemotherapy resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 372.