第 8 回がん新薬開発合同シンポジウム
Matching event●Establishment of 137 patient-derived cancer cell lines from the asciteswith omics information and their high utility in translational research
Hiroki Sasaki
Chief, Division of Biomarker Discovery
Exploratory Oncology Research & Clinical Trial Center
Chief, Department of Translational Oncology
Fundamental Innovative Oncology Core Center
National Cancer Center
Genome-wide genetic information in about 1,000 cancer cell lines is available on COSMIC DB(Sanger Center, UK); however, among them, only 28 cell lines are derived from gastric cancer (GC).
Since driver gene mutation frequency in a certain cancer is often less than 5%, the establishment ofcell lines from each patient to be analyzed is desired for functional selection of driver genemutations. Furthermore, almost all of the 28 GC cell lines were established many years ago, thereby,
the clinical and pathological information is insufficient. The wait is on for the establishment of new
GC cell lines, especially from metastatic sites after therapy. Peritoneal metastasis is most frequent inGCs, especially diffuse-type GCs.
In 2010-2018, from the ascites, we successfully established 90 diffuse-type GC cell lines(National Cancer Center Stomach Cancer (NSC) series) from 53 patients, and also established
34 pancreatic and 9 ovarian cancer cell lines, and more. We are conducting omics analyses forgene expression and copy number variation, and hot spot- and genome wide-gene alteration in these cell lines. Moreover, for in vivo preclinical study, their tumorigenicity and histopathological characteristics in the xenograft, such as fibroblast rich-, hypovascular-, and dormant-state, were evaluated. Through collaboration with some pharmaceutical industries, in vitro and in vivo
preclinical studies have been conducted to derivate clinical trials in our hospital.
Here I will present the characteristics of the patientʼs derived cancer cell lines and will
underline their utility in translational research.

第 8 回がん新薬開発合同シンポジウム
Matching event●Establishment of gastric cancer Patient-Derived-Xenograft (PDX)
models and cell lines for new drug development

Takeshi Kuwata
Chief
Department of Pathology and Clinical Laboratories
National Cancer Center Hospital East
[Background] It is important to have an appropriate pre-clinical model for developing new drugs.
From May 2015, we have been conducting patient derived xenograft models as well as cell lines
establishment from gastric cancer (GC) patients in NCC.
[Result] Total 250 GC patients, including 233 patients underwent surgery and 17 patients underwent
Cell-free and Concentrated Ascites Reinfusion Therapy (CART), were enrolled.
resected GC specimen, 36 PDXs and 23 cell lines have been established.
PDXs and 8 cell lines have been established.

From 233 surgically

From 17 CART specimen, 1

Totally we have 37 PDX GC models and 24 GC cell lines,

and among these 20 cases have both PDX and cell lines. All the GC PDX tumors have been confirmed
pathologically

andmorethan80%ofPDXtumors

showedanintestinal

Immunohistochemical examination identified HER2 positive GC tumors as well.

phenotype.

NGS-based genemutation analysis revealed repetitive so-called driver mutations (SNV/Indel) in several genes including
KRAS, PIK3CA and PTEN.

In some cases, gene mutation profiles were compared between primarytumor, PDX and cell lines, and most of drive mutations were observed in all specimens.
[Conclusion and future perspective] We have established new GC PDXs and/or cell lines, supplemented
with clinicopathological information, for accelerating new drug development.

This bio-resource may

help pre-clinical studies as well as finding new biomarkers for treating GC patients.

第 8 回がん新薬開発合同シンポジウム
Matching event●Chemotherapy payload of anti-insoluble fibrin antibody drug conjugate isreleased exclusively on the fibrin clot within aggressive solid tumors.
Yasuhiro Matsumura
Director
Division of Developmental Therapeutics
Exploratory Oncology Research & Clinical Trial Center
National Cancer Center
The first study of the enhanced permeability and retention (EPR) effect was published in 1986. Since
then, various formulations of drug delivery system (DDS) based on the EPR effect have been developed
and applied in clinical settings. Meanwhile, we reported that tissue factor (TF) on cancer cell surfaces
and hemorrhage caused by cancer erosion can induce fibrin clot formation, as well as tumor vascularpermeability. Accordingly, common cancers possess a fibrin-rich stroma that hinders the distributionof macromolecular DDS, including monoclonal antibodies (mAbs) and antibody–drug conjugates(ADCs). Recently, we developed an anti-insoluble fibrin (IF) mAb that does not react with fibrinogenor soluble fibrin. We subsequently created an ADC consisting of the anti-IF mAb conjugated with an
ACA via a Val-Leu-Lys linker, which is severed specifically by the fibrinolytic protease plasmin. The antiIF mAb ADC (IF-ADC) can selectively extravasate through leaky tumor vessels, bind to specific pits infibrin clots that are uncovered only when a fibrin clot forms, and create a scaffold from which effective
sustained release of the free ACA occurs. Because plasmin is only active on insoluble fibrin and isneutralized by the innate α2-plasmin inhibitor circulating in the blood, free ACA is only released whenthe IF-ADC is bound to the previously hidden epitopes in the pits on insoluble fibrin. This free ACA caneasily reach cancer cells by diffusion through the stromal barrier. Another benefit of this approach isthat ACA released from IF-ADC can also attack vascular endothelial cells. In this project, we will develop
ADCs using mAbs recognizing IF produced in tumor tissues, and optimize these ADCs during future
clinical development with the goal of achieving cancer stromal targeting (CAST) therapy.
(References)
1. Prince R, Matsumura Y, Angelillo-Scherrer A. et al Targeting anticoagulant protein S to improve hemostasis in hemophilia. Blood 131, 1360-1371 (2018).
2. Fujiwara Y, Furuta M, Manabe S, Koga Y, Yasunaga M, Matsumura Y. Imaging mass spectrometry for the exquisite design of antibody-drug conjugates. Sci. Rep. 6,24954 (2016).
3. Obonai T, Fuchigami H, Furuya F, Kozuka N, Yasunaga M, Matsumura Y. Tumour imaging by detecting fibrin clots in tumour stroma with anti-fibrin Fab fragment. Sci.
Rep. 6, 23613 (2016).
4. Hisada Y, Yasunaga M, Hanaoka S, Saijou S, Sugino T, Tsuji A, Saga T, Tsumoto K, Manabe S, Kuroda J, Kuratsu J, Matsumura Y. Discovery of an uncovered region infibrin clots and its clinical significance. Sci. Rep. 3, 2604 (2013)
5. Matsumura Y. Cancer stromal targeting (CAST) therapy Adv. Drug Deliv. Rev. 64, 710-719 (2012) .
6. Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of tumoritropic accumulation of proteins and the antitumor
agent smancs. Cancer Res. 46, 6387-6392 (1986).

第 8 回がん新薬開発合同シンポジウム
Matching event●Development of novel transcription-based cancer therapies
Susumu S. Kobayashi
ChiefDivision of Translational Genomics
Exploratory Oncology Research & Clinical Trial Center
National Cancer Center
Recent progress of genetic studies has dramatically unveiled pathogenesis of cancer. In addition toconventional cytotoxic agents, targeted therapies and immunotherapies have revolutionized the way
to treat cancers such as chronic myeloid leukemia, melanoma, and non small cell lung cancer. However,overall survival of cancer, particularly advanced cancer, remain unsatisfactory due to primary andacquired resistance to current therapies. Therefore, development of novel therapeutics is required. It
has been shown that CCAAT/Enhancer Binding Protein α (C/EBPα) is one of crucial transcription factorsthat induce differentiation and growth arrest in many types of cells. Mice deficient in C/EBPαdemonstrate a block of differentiation in myeloid cells, hepatocytes, and alveolar cell. Furthermore,
C/EBPα are perturbed in many cancers through multiple mechanisms, such as transcriptional silencing,translational inhibition, posttranslational modification, decrease in DNA binding, or point mutations.
We have previously demonstrated that transfection of C/EBPα can induce differentiation and growtharrest of leukemia cells and lung cancer cells. Moreover, C/EBPα is expressed in many tissues

such as skin, pancreas, adipocytes, prostate, and liver. Therefore, we hypothesize that an increase in
expression/activity of C/EBPα may induce differentiation and subsequent apoptosis of cancer cells,
leading to elimination of tumors. To answer this question, we have established a cell-based highthroughput screening using cell lines in which C/EBPα activity is detected by luciferase assay. Among
severalhits

identified,

2-[( )-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-

quinazolinone (ICCB280), induced myeloid differentiation of leukemia cell lines accompanied byincreased expression of C/EBPα and its downstream target genes. Primary blast cells isolated fromhuman AML patients treated with ICCB280 demonstrated evidence of differentiation and massiveapoptosis. To further characterize chemical and biological nature of ICCB280, we are aiming to: (1)and (2). We believe that the approach describedhere will lead to development of novel differentiation therapies and significantly impact care of patientswith various cancers with unfavorable prognosis in the near future.

第 8 回がん新薬開発合同シンポジウム
Matching event●A Novel method for screening peptide-hormone receptors
Shosei Kishida
Professor, Kagoshima University

Akihiro Asakawa
Professor, Kagoshima University

Yasuhito Uezono
Chief, Department of Supportive Care Research
Exploratory Oncology Research & Clinical Trial Center
National Cancer Center
After identification of human whole genome was completed in 2003, the clarification about the function of each gene have been become more important in the post genome era.

Taken that

membrane proteins consists more than 30 % of human 25000 genes and 60 % of the commerc
ially available drugs target membrane/membrane-associated proteins (ligands, receptors, RTK, etc), analyses of interaction of membrane proteins are still considered to be an important method
for the drug discovery.
However, it is not so easy to handle with membrane proteins.

They are assumed to have physi

ological structures embedded in the lipid-layers. Expression and analyses of recombinant membra
ne proteins are often difficult due to their hydro-phobic properties.
We developed a novel modified assay system using yeasts for intermolecular interactions of me
mbrane-associated proteins (ligands, receptors, transmembrane proteins, etc), whereas original t
wo-hybrid method could not handle with membranous proteins or ligands.

It enables rapid andefficient screening of several interactions such as receptor-ligand interaction, dimerization of me
mbrane proteins etc.

Novel receptors for peptide hormones, neuropeptides could be identified theoretically by our new method, which might lead to the understanding of its signal transduction, the discovery of novel agonists and antagonists for interesting ligands.

Actually, we found thr

ee novel interaction of ligand-membranous proteins which are not reported before.
Recently, we are proceeding the project for identifying new receptors for neuropeptides including
ghrelin and trying to extend our method for detection of interaction of non-peptide ligands andtheir receptors.
We would like to find collaborators/companies which are interested in the molecular interactions
we have found or interested in our method to seek out unknown counterparts of ligands or receptors or chemicals.