A study on the effectiveness of combinationing epitope selection and antibody function for the generation of novel monoclonal antibodies

　モノクローナル抗体は、その特異性から分子生物学的ならびに病理組織学的な分子の検出・解析に威力を発揮するとともに、抗体の持つ機能との組み合わせにより分子標的治療にも広く活用されている。本研究では二つのモノクローナル抗体の取得について報告する。一つは幹細胞の分子生物学的・病理組織学的研究への展開を目指したモノクローナル抗体の取得に関する研究であり、もう一つはそこから得られたナレッジを活用した抗体創薬に向けたモノクローナル抗体の取得に関する研究である。Ⅰ. LGR6(Leucine-rich repeat-containing G protein-coupled receptor)に対する　モノクローナル抗体の作製　LGR6はGタンパク質共役受容体（GPCR）の一つで、ロイシン・リッチ・リピートを含む GPCR(LGR)ファミリーのメンバーである。LGR6は、LGR4, 5とともにLGRのサブファミリーを形成し、これらの分子で最も研究が進んでいるのはLGR5である。LGR5は小腸、胃、皮膚の幹細胞、および大腸癌幹細胞のマーカーとして知られており、我々は先の研究でLGR5の発現が大腸癌幹細胞の増殖と静止状態を区別する分子病理学的マーカーであることを報告した。今回研究を進めたLGR6 も、genetic lineage tracingの解析から原始の皮膚の幹細胞マーカーとして報告されている。しかし、LGR6の組織学的な発現情報やLGR6陽性細胞の機能・役割については、特異的な抗体が得られていないためにいまだ詳細に解明されていない。我々が抗LGR5抗体を取得する際に経験した困難さから LGR6特異的抗体が得られていないのは、このサブファミリーに特徴的なタンパクの構造に起因するものと考えられた。すなわち、LGRサブファミリーはN末端に馬蹄形をした500アミノ酸からなるロイシン・リッチ・リピート領域を有し、この複雑な立体構造を維持した免疫抗原を調製することが難しいこと、また、ロイシン・リッチ・リピート領域を含めサブファミリー分子間の相同性が高く、ファミリー分子に交差しない特異的抗体の取得が難しいことが考えられた。　そこで我々はこれらの課題を克服するために、DNA免疫法によるLGR6特異的抗体の作製を試みた。DNA免疫法では、金粒子にコーティングした発現プラスミドをGeneGunによって高圧でマウスの腹部に接種し、発現プラスミドが導入された細胞ではタンパク質が産生され、これらのタンパク質は立体構造を維持した状態で細胞膜上に提示され、免疫抗原としてマウスでの抗体産生を誘導することができる。LGR6発現プラスミドをDNA免疫法でBalb/cマウスに導入した結果、LGR6に対する液性免疫が誘導され、LGR6に対する抗体が産生された。　LGR6に対する抗体価が上昇したマウスに対し、さらにLGR6に対する免疫を亢進させるためにブースト免疫としてLGR6タンパク質を高発現した細胞株の細胞免疫を実施した。LGRファミリーを含むGPCRは一般に高発現株を取得することが難しいとされているが、我々は先の抗LGR5抗体取得の際にLGR5を高発現させる方法としてマウスProB細胞株であるBa/F3株を使用することが有効であることを経験していた。Ba/F3株は目的の遺伝子を発現する細胞株の樹立にあまり広く利用されていない親株であるが、浮遊細胞のためフローサイトメトリー解析において細胞の調整が容易であり、また細胞の増殖が速いことから細胞株の樹立を早期に実現できるメリットがある。また、Ba/F3株は Balb/cマウスに由来する細胞株であり、Balb/cマウスへ免疫する際にはBa/F3株で発現される抗原のみが外来抗原と認識される。そこで、LGR6高発現Ba/F3株を樹立し、細胞免疫を施すことで特異的な免疫増強による抗体価上昇を誘導し、抗LGR6モノクローナル抗体を取得することができた。　取得された抗体については、以下の流れで特性と機能の解析を行った。1. LGR6特異的抗体は、フローサイトメトリーを用いて細胞外領域への結合の有無でエピトープ分類を行い、N末端の細胞外領域（N-ECD）を認識する抗体と7回膜貫通領域（7TM）の細胞外ループを認識する抗体のエピトープを確認した。その結果、N-ECDを認識する抗体2クローンと7TMを認識する抗体1クローンを取得した。2. LGR6特異的抗体によるLGR6とリガンドRSPO-1との結合阻害活性を解析した。RSPO-1の LGR6への結合を検出するため、タグ付きの組換えタンパク質のRSPO-1を準備し、タグに対する抗体で検出するアッセイ系を構築した。反応系に加えた抗LGR6抗体の濃度依存的な結合阻害を評価した。その結果、43A6, 43D10の二つのクローンはLGR6とリガンドRSPO-1との結合を阻害することが明らかとなった。　DNA免疫と細胞免疫の二つの方法を組み合わせることによって、LGR6に特異的であるとともに、リガンドとレセプターの結合を阻害する中和活性のある抗体の取得にも成功した。これらの成果は、LGR6の立体構造を維持したタンパク質を免疫抗原として用いたことと、立体構造の維持によりリガンドの結合部位が保存されたという、免疫手法の選択・工夫がもたらしたものと考える。今回取得した抗体は、LGR6 陽性細胞の役割・機能の解明など新たな幹細胞生物学の進展に貢献できるものと考える。Ⅱ.デスモグレイン３(Desmoglein 3 (DSG3))に対するモノクローナル抗体の作製　近年、癌を標的としたモノクローナル抗体が分子標的治療薬として利用されてきている。抗体をベースとした創薬の特徴は、抗体の特異性と、中和活性、抗体依存的な細胞傷害活性（ADCC）、補体依存的な細胞傷害活性（CDC）といった抗体の機能の組み 合わせにより癌細胞を死滅させることである。　癌に対する抗体創薬の新規標的分子は、遺伝子発現の多寡から候補遺伝子を絞り込む遺伝子発現解析と、その遺伝子から発現されるタンパク質の組織分布や細胞膜に位置しているかといった細胞内分布を解析する病理解析の両面から評価される。これらの解析から、我々はDSG3を重層扁平上皮癌に対する有望な標的分子として見出した。 DSG3 は一回膜貫通タンパク質で他のカドヘリン分子である DSG1 とともにデスモソー ムを形成し、重層扁平上皮組織での細胞間結合に寄与している。　抗DSG3自己抗体は皮膚粘膜の水疱を特徴とする自己免疫疾患の一つである天疱瘡の原因となることが知られている。DSG3を標的とする創薬を進めるためには、天疱瘡様病変の誘発を回避し、かつ、重層扁平上皮癌に対して薬理作用を発揮しなければならない。これまでの研究より天疱瘡を引き起こす病原性の自己抗体は、Ca2+依存的な構造をとるDSG3を認識すること、自己抗体が認識する領域が N末端の接着界面に存在することが報告されている。これらの知見より、Ca2+非依存的DSG3結合抗体の取得により副作用を回避した治療用抗体を取得できるものと仮説を立てた。　そこで、各種スクリーニング系を駆使し、目的とする特徴を有する抗体の取得を試みた。スクリーニング系では、DSG3が生体内で本来形成している構造に近いタンパク質を準備し、エピトープの分類による抗体の選別を行った。抗マウスDSG3抗体のスクリーニング過程を以下に示す。1. マウスDSG3に結合する抗体をフローサイトメトリーによりスクリーニングした。34個のマウス DSG3結合抗体を取得した。2. 癌細胞の死滅誘導方法としてADCCを薬理作用に持つ抗体12クローンを選抜した。マウスのADCC 活性を測定する安定したアッセイ系がないため、ヒトNK92細胞を遺伝子工学的に改変した細胞株を用いたスクリーニング系を考案し、マウスとヒトのFcRγⅢa融合タンパク質を発現させることによってNK細胞によるマウス抗体のADCC活性を測定できる系が構築できた。3. 抗体によってDSG3のCa2+依存的構造が認識されるかどうかを Ca2+のキレート剤 であるEDTA 存在下でフローサイトメトリーを用いて評価した。3クローンがCa2+非依存的にDSG3に結合する抗体であった。そのうち最も結合アフィニティーの高いクローン18-1を選定した。4. 天疱瘡様病変を誘発する抗体は、細胞間の接着を分離する活性を有していることからマウス由来の皮膚細胞シートを用いたスクリーニング方法により抗体が細胞の接着機能を阻害する能力があるかどうかを判断した。クローン18-1は細胞間接着の分離活性は有していなかった。　上記スクリーニング系でマウスDSG3に対する副作用の回避が可能と考えられる抗体18-1を取得できた。次に、in vivoモデルでの解析として、DSG3を発現するマウス肺癌細胞株LC12を移植したマウスによる抗腫瘍効果を評価した。その結果、抗マウスDSG3抗体18-1の投与による天疱瘡様病変はみられず、LC12癌組織の退縮が観察された。副作用を誘導するエピトープを避け、ADCCを誘導するエピトープを選択することによりDSG3に対する創薬抗体の作製が可能であることがマウスモデル系で示された。　次に、同様のスクリーニングフローを用い、ヒトDSG3に対し天疱瘡様病変の誘発作用がなく、高いADCC作用を持つ抗体DF366を取得した。種々のヒト重層扁平上皮癌担癌モデルでの抗ヒトDSG3 抗体DF366の抗腫瘍効果を確認し、抗体医薬としての可能性を示した。Ⅲ. 総括　二つの解析から目的のエピトープを持つ抗体を作製するための要点は次あげる二点となる。一つは、免疫やスクリーニングに用いるタンパク質は機能的に天然の構造を持ったものを利用することであり、もう一つは、エピトープを分類することで適切な抗体の機能を付加できるような抗体の分類をすることである。これらのステップは目的の機能を持った抗体を同定・取得する有用なアプローチを提供するものである。　現在、抗体創薬標的分子は枯渇している。今回、医薬品とするためには不都合な作用のある抗原であってもエピトープの選択次第で新しい創薬への展開が可能となることを示唆した。この研究は、分子標的薬としての抗体創薬の標的分子の選択可能性を広げるものであり、今後の抗体医薬品の研究開発に貢献するものと考える。　Monoclonal antibodies are considered powerful tools for molecular biological or histopathological detection and analysis of a molecule due to their specificity, and they are also widely utilized for molecular targeted therapy by combining their specificity and functionality.　In the current research the process of the generation of two unique monoclonal antibodies was studied. With the first antibody, the process of obtaining a monoclonal antibody aimed for application to molecular biological or histopathological analysis in stem cell research is studied, and for the second, the knowledge obtained in the process for the first antibody is applied for development of an antibody therapy.I.Generation and characterization of monoclonal antibodies against human LGR6 (Leucine-rich repeat (LRR)-containing G protein-coupled receptor)　LGR6 is a G protein-coupled receptor (GPCR) and a member of the LRR containing GPCR (LGR) family. LGR4, 5 and 6 belong to the same subfamily and one of the best studied is LGR5. LGR5 is known as a marker of intestinal, gastric, skin and also colon cancer stem cells. We previously reported that LGR5 expression could be used as a molecular pathological marker to distinguish the states of proliferating and quiescent cancer stem cells. LGR6 is also known as a marker for primitive epidermal stem cells from genetic lineage tracing analysis. However, information concerning its histological expression, cellular functions and physiological roles of is scarce due to the lack of a specific antibody. From our previous experience in obtaining an LGR5 antibody, we judged that we would face similar difficulties with an LGR6 antibody because of the complex structure of LGR proteins. LGRs have about 500 amino acids’ length of LRR region with a horseshoe structure in the N-terminus and this complex tertiary structural feature makes it a challenge to prepare immunogen. In addition, it is difficult to obtain antibodies that do not cross-reactive to other LGRs because of high homology between subfamily molecules with the LRR region.　Thus we attempted to generate a LGR6-specific antibody by DNA immunization to overcome these problems. For DNA immunization gold particles coated with plasmids expressing the molecule of interest are prepared. The particles are injected into the abdominal skin of mice with a high pressured GeneGun inducing the protein encoded in the plasmids. The protein is displayed on the plasma membrane with a native tertiary structure, and was thought to be a robust immunogen for induction of humoral antibody production in mice. By introducing a plasmid expressing the LGR6 gene in Balb/c mice we succeeded in inducing a humoral immune response to obtain antibodies against LGR6.　Furthermore, to enhance immunity against LGR6, we additionally injected a cell line overexpressing LGR6 as a boosting immunogen into the mice that were immunized by the plasmids with high titers of anti-LGR6 antibodies. Although, it is generally a challenge to obtain a stable cell line overexpressing GPCRs including the LGR family proteins, we found in our experience with LGR5 that it is effective to use the mouse proB cell, Ba/F3 cell. Ba/F3 is not frequently used for the establishment of cell lines with specific gene expression. However this cell line is a floating cell that can be analyzed easily by flow cytometry and because of its rapid growth, the establishment of a cell line can be accomplished in a short time. As Ba/F3 is derived from Balb/c mice, human antigens expressed in Ba/F3 cells, can be recognized specifically as a foreign antigen when injected into Balb/c mice. By this method, LGR6-specific immunity was augmented, making it easier to obtain monoclonal antibodies against LGR6.　According to the following flow, the characteristics and functions of the monoclonal antibodies against LGR6 obtained with the above methods were analyzed.1.Epitope classification was carried out by identifying binders to the N-terminal extracellular domain (N-ECD) or 7-pass transmembrane domain (7TM) of LGR6 by flow cytometry. As a result, we obtained three clones, 43A6 and 43D10, with an epitope against N-ECD and 43A25 against 7TM.2.The inhibitory activity of the antibodies against ligand binding to LGR6 was evaluated. It is well known that RSPO1 is a ligand that binds to the N-ECD of LGR6. Recombinant RSPO1 with a myc-His tag was prepared and an assay system for detection of RSPO1-bindig to LGR6 was established with an anti-myc tag antibody. Binding inhibition dependent on antibody concentration was evaluated. As a result, 2 clones, 43A6 and 43D10, were found to inhibit binding of ligand to LGR6. On the other hand, 43A25 did not inhibit RSPO1-binding.　The key to our success with the LGR6 antibodies was thought to be the selection of the immunization method of combining DNA and cell immunization which enabled immunization with an immunogen preserving the native tertiary structure of the ligand binding site of LGR6. 　Utilizing our unique antibodies may lead to understanding the role and function of LRG6-positive cells, and we anticipate that this may contribute to the progress in stem cell research. II.Generation of anti-desmoglein 3 (DSG3) antibody without pathogenic activity of pemphigus vulgaris for therapeutic application to squamous cell carcinoma　In the second report, therapeutic application of an anti-DSG3 monoclonal antibody is studied. Cancer-targeted monoclonal antibodies are frequently utilized in cancer therapy. The advantage of antibody-based therapeutics is their specificity and functionality such as neutralization, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC), by which the antibody can eliminate cancer cells. Many therapeutic antibodies against cancer have been launched and there have been great benefits for the therapeutic modality in the oncology field.　Prior to our second study, candidate genes with high gene expression levels in tumor tissues compared to normal tissues were selected in order to discover a novel drug target for antibody therapy in cancer patients The tissue distribution of the gene product and their subcellular localization to the cell membrane in tumor tissues was studied to confirm the protein expression. By this process we found that DSG3 is a promising target for squamous cell carcinoma. DSG3 is a one-pass transmembrane protein and forms desmosomes along with another desmosomal cadherin, DSG1, and contributes to cell-cell adhesion in stratified squamous tissues. 　It is well known that anti-DSG3 autoantibodies cause PV, an autoimmune disease characterized by cutaneous and mucosal blistering. To successfully target DSG3 for therapy, it is necessary to avoid PV-like effects and exert pharmacological action against squamous carcinoma cells. It is reported that pathogenic autoantibodies which induce PV recognizes a Ca2+-dependent structure of DSG3 and the region bound by DSG3-autoanibodies is located in the N-terminal adhesive interface. From these findings, we hypothesized that a therapeutic antibody with no severe side effects could be generated by obtaining antibodies that bind DSG3 in Ca2+-independent manner.　Thus, we attempted to obtain such an antibody by using several screening systems. In the screening system we prepared proteins mimicking the native conformation of DSG3 and selected antibodies by epitope classification. Our screening process is shown below.1.Antibodies with the ability to bind mouse DSG3 were selected by flow cytometry. 34 clones were found to bind mouse DSG3. 2.We selected ADCC as the pharmacological action of antibodies, and 12 antibodies with ADCC function through the following screening system. To construct a stable assay for ADCC activity, we designed a screening system using genetically engineered NK cells. We established an ADCC assay system with human NK92 cells expressing a chimeric protein with the ECD of mouse FcγRIIIa fused to the human transmembrane and cytoplasmic domain of FcγRIIIa. 3.Recognition of the Ca2+ dependent structure of DSG3 was evaluated by flow cytometry under the presence of an EDTA, a Ca2+ chelator. Three clones were found to bind DSG3 in Ca2+-independent manner. Clone 18-1 that had the highest binding- affinity to mouse DSG3 was selected as the final candidate.4.Screening with keratinocyte sheets is used to judge the ability of interference to adhesive function by antibodies, as the antibody which could induce PV-like lesion has dissociating activity to cell-cell adhesion. We tested clone 18-1 on the keratinocyte sheets and found there was no dissociating activity.　By this screening system an antibody with no severe side effects, 18-1, was obtained. Next, anti-tumor activity was evaluated in mice subcutaneously inoculated with a mouse lung cancer cell line LC12 overexpressing DSG3. Consequently PV-like changes were not observed in mice and the tumor was regressed by anti-mouse DSG3 antibody administration. The results show that it is possible to generate a therapeutic antibody against DSG3 by selecting an ADCC-promoting epitope that can be distinguished from an epitope inducing pathogenic response.　Next, along with the same screening flow used in generating the anti-mouse DSG3 antibody, an anti-human DSG3 antibody with high ADCC activity DF366 that does not induce PV-like lesions, was successfully generated. The potential of the DF366 antibody as a therapeutic was shown by the efficacy in xenograft models of various human squamous cell carcinomas.III.Summary　The essential points for generating antibodies with an intended epitope are 1) the utilization of protein with functional and native conformation for in vivo immunization and screening, and 2) to confirm adequate function by epitope classification. These steps present a useful approach to identify and generate an antibody with intended function.　Currently, promising target molecules for antibody therapeutics are said to be exhausted. It was suggested in the second report that there is a potential for developing a novel therapeutic antibody in the choice of epitope, even if the antigen has a concern of unwanted effects as a therapeutic target. The current study may expand the option for selecting antibody-based drug targets, and is thought to contribute to the research and development for the future antibody therapeutics.博士(学術)麻布大

Estimating causal effects with a non-paranormal method for the design of efficient intervention experiments

BACKGROUND: Knockdown or overexpression of genes is widely used to identify genes that play important roles in many aspects of cellular functions and phenotypes. Because next-generation sequencing generates high-throughput data that allow us to detect genes, it is important to identify genes that drive functional and phenotypic changes of cells. However, conventional methods rely heavily on the assumption of normality and they often give incorrect results when the assumption is not true. To relax the Gaussian assumption in causal inference, we introduce the non-paranormal method to test conditional independence in the PC-algorithm. Then, we present the non-paranormal intervention-calculus when the directed acyclic graph (DAG) is absent (NPN-IDA), which incorporates the cumulative nature of effects through a cascaded pathway via causal inference for ranking causal genes against a phenotype with the non-paranormal method for estimating DAGs. RESULTS: We demonstrate that causal inference with the non-paranormal method significantly improves the performance in estimating DAGs on synthetic data in comparison with the original PC-algorithm. Moreover, we show that NPN-IDA outperforms the conventional methods in exploring regulators of the flowering time in Arabidopsis thaliana and regulators that control the browning of white adipocytes in mice. Our results show that performance improvement in estimating DAGs contributes to an accurate estimation of causal effects. CONCLUSIONS: Although the simplest alternative procedure was used, our proposed method enables us to design efficient intervention experiments and can be applied to a wide range of research purposes, including drug discovery, because of its generality

Identification and Characterization of pvuA, a Gene Encoding the Ferric Vibrioferrin Receptor Protein in Vibrio parahaemolyticus

We previously reported that Vibrio parahaemolyticus expresses two outer membrane proteins of 78 and 83 kDa concomitant with production of siderophore vibrioferrin in response to iron starvation stress and that these proteins are the ferric vibrioferrin receptor and heme receptor, respectively (S. Yamamoto, T. Akiyama, N. Okujo, S. Matsuura, and S. Shinoda, Microbiol. Immunol. 39:759-766, 1995; S. Yamamoto, Y. Hara, K. Tomochika, and S. Shinoda, FEMS Microbiol. Lett. 128:195-200, 1995). In this study, the Fur titration assay (FURTA) system was applied to isolate DNA fragments containing a potential Fur box from a genomic DNA library of V. parahaemolyticus WP1. Sequencing a 3.2-kb DNA insert in one FURTA-positive clone revealed that an amino acid sequence deduced from a partial gene, which was preceded by a full-length gene (psuA) encoding a receptor for a siderophore of unknown origin, was consistent with the N-terminal amino acid sequence of the 78-kDa ferric vibrioferrin receptor. Then, the full-length gene (pvuA) encoding the ferric vibrioferrin receptor was cloned and characterized. The deduced protein encoded by pvuA displayed the highest similarity (31% identity; 48% similarity) to RumA, a ferric rhizoferrin receptor of Morganella morganii. Primer extension and Northern blot analyses indicated that psuA and pvuA constitute an operon which is transcribed from a Fur-repressed promoter upstream of psuA. The product of the pvuA gene and its function were confirmed by generating a pvuA-disrupted mutant, coupled with genetic complementation studies. A mutant with disruption in the upstream psuA gene also displayed a phenotype impaired in the utilization of ferric vibrioferrin

The Native Form and Maturation Process of Hepatitis C Virus Core Protein

The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles

Phylogenetic subtypes of human T-lymphotropic virus type I and their relations to the anthropological background

Isolates of human T-lymphotropic virus type I(HTLV-I) were phylogenetically analyzed from native inhabitants in India and South America (Colombia and Chile) and from Ainu (regarded as pure Japanese descendants from the preagricultural 'Jomon' period). Their genomes were partially sequenced together with isolates from Gabon in central Africa and from Ghana in West Africa. The phylogenetic tree was constructed from the sequence data obtained and those of previously reported HTLV-I isolates and simian T- lymphotropic virus type I (STLV-I) isolates. The heterogeneity of HTLV-I was recently recognized, and one major type, generally called the 'cosmopolitan' type, contained Japanese, Caribbean, and West African isolates. The phylogenetic tree constructed in the present study has shown that this cosmopolitan type can be further grouped into three lineages (subtypes A, B, and C). Subtype A consists of some Caribbean, two South American, and some Japanese isolates, including that from the Ainu