補体依存性細胞傷害を作用機序とするラット抗Thy-1抗体投与モデルを用いた抗体医薬の薬効及び毒性発現予測要因に関する病理学的研究

• Material Type: 博士論文
• Title: 補体依存性細胞傷害を作用機序とするラット抗Thy-1抗体投与モデルを用いた抗体医薬の薬効及び毒性発現予測要因に関する病理学的研究
• Author/Editor: 加藤, 千恵
• Author Heading: 加藤, 千恵
• Publication Date: 2013-07-22
• Publication Date (W3CDTF): 2013-07-22
• Alternative Title: Pathological study of factors that can predict efficacy and toxicity of therapeutic antibody using rats treated with anti-Thy-1 antibody to induce complement-dependent cytotoxicity
• Degree grantor/type: 麻布大学
• Date Granted: 2013-07-22
• Date Granted (W3CDTF): 2013-07-22
• Dissertation Number: 乙第427号
• Degree Type: 博士(獣医学)
• Conferring No. (Dissertation): 乙第427号
• Text Language Code: jpn
• Target Audience: 一般
• Note (General): 抗体医薬は標的分子に対して高い特異性に基づく治療効果が期待できることや、抗体工学の発展を背景として、近年開発が活発に行われている。抗体医薬品は、標的分子の中和作用、生体内の免疫機構を利用したcomplement-dependent cytotoxicity (CDC)や antibody-dependent cell-mediated cytotoxicity (ADCC)などによる標的分子発現細胞傷害作用、drug delivery carrierとして標的分子発現細胞を傷害する作用などにより、その薬理作用を発揮する。抗体医薬品を含むバイオテクノロジー応用医薬品の開発については、日米EU医薬品規制調和国際会議 (ICH)における合意に基づくガイドラインが定められ、それに基づき非臨床試験における安全性評価を遂行することが定められている。その中でも、モノクローナル抗体を本体とする抗体医薬では、ヒト組織パネルを用いた免疫組織化学的染色（IHC）による組織交差反応性試験によって、抗体と組織中の標的抗原の結合を評価することが求められている。抗体医薬品では標的抗原の生体内分布と投与抗体による傷害臓器が一致すると考えられており、組織交差反応性試験はヒト初回投与臨床試験以前に標的臓器を予測し、安全性を担保する重要な試験と位置付けられている。しかし、抗原の分布や発現量と抗体に誘導される傷害臓器が一致しないとの報告もあり、抗体医薬の有効性・安全性をより正確に予測することが、抗体医薬の開発において緊急課題となっている。標的抗原以外の生体反応を規定する要因の研究は現在までほとんど行われておらず、また、IHCによる抗原分布解析と抗体投与によるin vivoでの生体反応を比較解析することのできる動物モデルの探索も行われていない。そこで本研究では、CDCを作用機序とする抗Thy-1.1抗体投与ラットモデルに着目し、この実験モデルがCDC誘導において標的抗原発現以外の生体反応を規定する要因を解析するために有用である事を示し、投与抗体の分布や膜補体制御因子 (mCRPs)の評価を加えた新たな抗体医薬の有効性・安全性予測方法について新しい知見を提示した。第１章 正常ラットにおけるThy-1.1抗原の分布及び抗Thy-1.1抗体投与ラットにおけるCDCの誘導抗Thy-1.1抗体投与ラットが本研究目的に適していることを評価する目的で、正常ラットにおけるThy-1.1抗原の分布及び抗Thy-1.1抗体投与ラットにおけるCDC誘導臓器を検索した。今モデルにおける標的抗原であるThy-1.1抗原は、IHCによって胸腺及び脾臓リンパ球を含む免疫系、腎糸球体メサンギウム（Mes）細胞を含む泌尿器系、副腎髄質細胞を含む神経内分泌系、間葉系の細胞など全身に広く分布していることが確認された。また、胸腺、副腎、脳組織を用いたRT-PCRおよびWestern blotにおいても、Thy-1.1抗原の発現を確認した。これらの結果より、ラットへ抗Thy-1.1抗体を投与すると、Thy-1.1抗原を発現する様々な臓器に組織傷害が起こると予測されたが、実際には組織傷害は腎臓のみに認められた。病理組織学的には、腎臓において抗体投与後0.5h及び1hより、Mes領域におけるKaryolysis（核融解）及び好中球の浸潤が認められ、続いて抗体投与後8hよりMes細胞の減数及び糸球体毛細血管の拡張が、抗体投与後24h及び48hではMes領域の減少が認められた。IHCでは、CDCの補体反応カスケードの要であるC3の沈着も腎臓メサンギウム領域でのみ認められた。これら腎臓における病理組織学的所見及びC3沈着は、抗Thy-1.1抗体投与に起因するCDCによる変化として報告されている所見と一致しており、Mes細胞の細胞死は抗Thy-1.1抗体投与に起因するCDCにより誘導されたものと判断された。一方、その他の抗原発現臓器では、病理組織学的変化並びにC3沈着は認められず、標的抗原の分布とCDCにより誘導される生体反応が一致しないことが明らかとなり、抗Thy-1.1抗体投与ラットがCDC誘導における生体反応を規定する抗原発現量以外の要因を解析する有用なモデルとなることが明らかにした。第２章 抗Thy-1.1抗体投与ラットにおける投与抗体分布と膜補体制御因子抗Thy-1.1抗体投与ラットにおける標的抗原分布と生体反応の不一致の理由として、1) 投与抗体が標的抗原発現部位に到達・結合していない、2) 抗体と抗原が結合した後、mCRPsがCDC誘導を抑制していること、という2つの要因を想定し、抗Thy-1.1抗体投与ラットにおける投与抗体の分布並びに正常ラットにおけるmCRPs (CrryおよびCD55)の分布を免疫組織学的に解析した。その結果、投与抗体は標的抗原の分布と必ずしも一致せず、腎糸球体Mes細胞、胸腺皮質リンパ球、脾臓赤脾髄リンパ球、副腎髄質細胞のみに分布していた。胸腺皮質リンパ球及び副腎髄質細胞については、胸腺の血管周囲や、副腎の皮髄境界部など、抗原発現細胞のごく一部にのみ投与抗体が限局して分布していた。それ以外の標的抗原発現臓器には投与抗体は分布していなかった。mCRPsについては、胸腺皮質リンパ球にCrryがmoderateに、副腎髄質細胞にはCrryがweak及びCD55がstrongに発現していた。副腎および胸腺についてはこれらmCRPsのタンパク質発現をWestern blotにより確認し、相対的な発現量も免疫組織学的な染色性と一致する事を確認した。これらの臓器においては、投与抗体は到達したが、mCRPsが抗Thy-1.1抗体投与によるC3沈着に抑制的に働き、補体活性化が起こらないものと考えられた。いっぽう、腎糸球体Mes細胞にはCrryがweakに認められた。これらの結果から、抗Thy-1.1抗体投与ラットにおいては、投与抗体の分布、及び一定以上発現したmCRPsによるC3沈着抑制が、CDC生体反応を規定する要因となると考えられた。以上の結果から、標的抗原発現臓器についてCDC誘導の段階を抗体の分布、mCRPsの発現、C3の沈着、及び組織傷害の発現をもとに3つに分類した。すなわち、抗Thy-1.1抗体投与ラットにおいて、標的抗原発現臓器は、A) 抗原抗体反応が起き、CDC活性化が起き、細胞死が誘導されるもの (腎糸球体Mes細胞)、B) 抗原抗体反応が起きるが、mCRPsの抑制作用によりCDCが活性化せず、細胞死も誘導されないもの (胸腺皮質リンパ球及び副腎髄質細胞)、C) 抗原抗体反応が起こらず、CDC活性化も細胞死も誘導されないもの (その他の抗原発現臓器)、の3つに分類された。AとBの間にはC3沈着の有無という明確な差が認められ、C3沈着を抑制するmCRPsがCDC誘導に深く関わっていることが示唆された。第３章 CDCを作用機序とする抗体医薬品の標的臓器予測における補体制御因子解析の有用性第１章，２章において、抗Thy-1.1抗体投与ラットでは、抗原分布のみではなく、1) 投与抗体の抗原部位への到達・結合の有無、2) 抗体と抗原が結合した後CDCを制御するmCRPsの発現が、CDC生体反応を規定する要因となることが明らかとなった。そこで本章では、mCRPs (Crry及びCD55)のラット全身諸臓器における分布を検索し、CDCの予測要因となるか考察したその結果、Crry及びCD55は正常ラット全身諸臓器に広く分布するものの、同一臓器においても別々の細胞に発現しており、両者はC3沈着抑制という共通の機能を持っているが、それぞれの分子が特定の役割を持っていることが示唆された。さらに、Thy-1.1抗原分布、投与抗体の分布、mCRPs分布を合わせ、本ラットモデルにおける抗体投与後の生体反応を予測した。Approach 1として標的抗原の分布する組織を、Approach 2として標的抗原分布に加え投与抗体の分布した組織を、Approach 3としてApproach 2の条件に加えてmCRPsがmoderate以上に分布する臓器を除外した組織を生体反応の起こり得る組織として取り上げ、抗Thy-1.1抗体投与による生体反応の実際の結果と比較した。その結果、抗原分布に加え、投与抗体の分布、mCRPsの発現を加えたApproach 3により生体反応が予測された腎糸球体Mes細胞のみに細胞傷害を認めた実際の生体反応と一致した。本研究によって、従来の抗原分布から予測するApproach 1は最も生体反応の起こる可能性を見積るが、抗原に加え抗体分布並びにmCRPsを合わせたApproach 3はより正確に予測ができることが示された。CDC生体反応規定要因としてのmCRPsの分布解析は生体反応予測に有用であり、CDCを作用機序とする抗体医薬の有効性・安全性をより正確に予測することが可能となった。以上、本研究は抗体に起因するCDC生体反応が、これまでの抗原の分布のみからの予測に比べ、抗原分布、投与抗体の分布、mCRPs分布を合わせた評価がより正確に有効性・安全性を予測することを示唆しており、今後、分子標的医薬である抗体医薬品のより正確な有効性・安全性予測に寄与し、今後の研究開発に貢献するものである。; Disease-related molecules that have been discovered through genomic research can be targeted therapeutically by antibodies. Thanks to the advance of antibody and genetic engineering techniques, research and development of therapeutic antibodies has progressed, and over 30 therapeutic monoclonal antibodies are now on the market in the United States, Europe, and Japan. These monoclonal antibodies exert efficacy as anticancer agents on many kinds of molecules through various natural functions, namely, neutralization to block the physiological function of the target antigens, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or by acting as drug delivery carriers. These recent scientific advances and the shared experience of preclinical safety evaluation of biotechnology-derived pharmaceuticals have been incorporated in a guideline by the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (the ICH S6 R1 guideline), and compliance to this guideline is required for preclinical safety testing. One requirement of the guideline is that, because tissue injury induced by antibody treatment is thought to be consistent with antigen distribution, the binding of therapeutic monoclonal antibodies to antigens within tissues be evaluated in tissue cross-reactivity (TCR) studies using a panel of human tissues. This TCR study with a panel of human tissues can predict target organs of toxicity prior to the initial clinical dosing of these products and is a recommended component of the safety assessment package. On the other hand, because some reports show that the tissue distribution or expression level of the antigen is not consistent with tissue injury, the issue remains of how to exactly predict efficacy and toxicity when developing therapeutic antibodies, and a model suited for studying the factors that predict the biological response of therapeutic antibodies is necessary to address these matters. The anti-Thy-1.1 antibody-treated rat (rat anti-Thy-1 model) is known as an animal model for the involvement of antibody-mediated CDC in the induction of tissue injury. In the present study, we examine how the antigen and membrane complement regulatory proteins (mCRPs) are distributed, what effect an antibody has on the biological response and the factors that predict that effect, and we present novel information on, and methods for predicting, efficacy and toxicity of a therapeutic antibody.In Chapter 1, we examined the distribution of Thy-1.1 antigen in normal rats and the tissue injury induced by CDC in the rat anti-Thy-1 model to evaluate and confirm that the model would be suited for investigating what other factors than antigen expression can predict the activation of CDC. We demonstrated that Thy-1.1 antigen is broadly distributed across several organs and cells, including lymphocytes of the thymus and spleen, mesangial cells of the kidney, medullary cells of the adrenal gland, and stromal cells in several organs. We expected that injecting anti-Thy-1.1 antibody would result in tissue injury in all these Thy-1.1-expressing cells, but when the rat anti-Thy-1 model was histopathologically evaluated in detail, cell death induced by the anti-Thy-1.1 antibody was observed only in mesangial cells. Morphologically, at 0.5 and 1 hour after treatment karyolysis in the mesangial cells and infiltration of neutrophils were found; at 8 hours after injection, the number of mesangial cells had decreased and the capillaries of the glomerulus were dilated; and at 24 and 48 hours after injection, the mesangial area had decreased. Deposition of C3, the key molecule of the CDC cascade, was detected by immunohistochemistry only in the mesangial area from 0.5 hours after treatment. Judging from these results of the histopathological examination and C3 deposition, cell death of mesangial cells was induced by CDC mechanisms, as previously reported, but the other organs and tissues that express Thy-1.1 did not show cell death in this model. This result indicates that the antigen distribution data was not consistent with the organs in which antibody-mediated CDC was induced. This chapter concludes that the rat anti-Thy-1 model is thought to be a suitable model for analyzing the factors other than the expression levels of the target antigen that predict the induction of CDC, based on the following reasons: 1) mesangial cell death due to CDC was induced by external administration of the antibody, 2) although Thy-1.1 antigen was distributed broadly, it was not consistent with cell death induced by treatment with anti-Thy-1.1 antibody. In Chapter 2, to clarify the reason why the Thy-1.1 antigen distribution was not consistent with cell death we next considered two possible causes: 1) the injected antibody was not distributed in organs and tissues expressing Thy-1.1 and 2) mCRPs inhibited complement activation in the CDC reaction after the antigen bound to the antibody. Thus to elucidate the probable cause, the distribution of injected anti-Thy-1 antibody in the rat anti-Thy-1 model and the expression of Crry and CD55 in normal rats were evaluated. The injected anti-Thy-1.1 antibody was distributed in the mesangial cells of the kidney, in the lymphocytes in the perivascular areas of the cortex in the thymus and the red pulp of the spleen, and in medullary cells in the cortico-medullary junction of the adrenal gland. These results indicate that the injected anti-Thy-1.1 antibody did not bind to all of the cells that expressed the antigen but only to those cells that expressed more than a certain level of antigen. The expression of mCRPs was found in glomerular cells of the kidney, lymphocytes of the thymus, and medullary cells of the adrenal gland. In the kidney, weak expression of Crry and no expression of CD55 were observed in the mesangial cell. In the thymus, moderate, diffuse expression of Crry and no expression of CD55 were seen in the lymphocytes. In the adrenal gland, weak expression of Crry and strong expression of CD55 were observed in medullary cells. Thus, Crry or CD55, which inhibit C3 activation, are more than moderately expressed in cells that have a level of antigen-antibody binding that does not induce C3 deposition and cell death. Through our results concerning antigen expression, antibody distribution, and cell death, the relationship between antigen-antibody binding and CDC activation was categorized into the following three types: A) antigen-antibody binding that causes cell death (mesangial cells of the kidney); B) antigen-antibody binding that does not induce cell death (lymphocytes of the thymus and medullary cells in the adrenal gland); C) no antigen-antibody binding and no cell death (the other antigen-expressing cells). There were definite differences in C3 deposition between type A and type B cells. In other words, C3 deposition was observed in mesangial cells, which showed cell death, but was not seen in lymphocytes of the thymus and medullary cells in the adrenal gland, which did not show cell death. These results suggest that mCRPs are related to CDC induction. As a conclusion of this chapter, the factors regulating CDC reaction in the rat anti-Thy-1 model were not only the distribution of antigen but also 1) distribution of the injected antibody and 2) expression of mCRPs that inhibit complement activation after antigen-antibody binding.In Chapters 1 and 2, distribution of the Thy-1.1 antigen was not consistent with cell death induced by treating the rat anti-Thy-1 model with anti-Thy-1.1 antibody; thus, the antigen distribution data alone is not sufficient to predict the induction of antibody-mediated CDC. This conclusion was supported by the following two findings: 1) regulation through the distribution of the injected antibody and 2) inhibition of complement activation after antigen-antibody binding by the expression of mCRPs. Having analyzed the distribution of the injected antibody in Chapter 2, in Chapter 3 the distributions of mCRPs (Crry and CD55) in normal rat were examined and we considered the possibility that mCRPs could be used to predict CDC reaction. Because there were 2 factors other than distribution of antigen that were related to CDC induction, we analyzed the distribution of the injected antibody and expression of mCRPs and discussed how analyzing these factors would contribute to better prediction of the biological reaction induced by treatment with a CDC-type antibody. Crry and CD55 were detected widely in rat organs and tissues. The complement system can be effective in destroying external pathogens but unintended activation of complements can cause unnecessary injury. Thus the distribution of mCRPs may be involved in tight regulation of nonspecific activation in these tissues. Crry and CD55 were co-expressed in the same organs but they were expressed distinctly differently between cells. The two molecules have a common function in inhibiting C3 deposition, but the present results show that they have a separate expression pattern, a fact that indicates specific roles in CDC regulation.We predicted the occurrence of lesion caused by a Thy-1.1 antibody injection according to 3 approaches, in which different tissues were selected as potential targets of biological reaction and were compared with the tissues that actually were affected in the rat anti-Thy-1 model: tissues in which antigen was expressed (Approach 1), tissues in which both the antigen and the injected antibody were distributed (Approach 2), and tissues in which both the antigen and the injected antibody were distributed and which had less than moderate expression of mCRPs (Approach 3). As a result, Approach 3, the approach that considers the distribution of antigen, the distribution of the injected antibody, and the expression of mCRPs, was consistent with the tissues that were actually affected, namely, the mesangial cell in the kidney. In conclusion, combining the analysis of antigen distribution, distribution of the injected antibody and the expression of mCRPs enabled us to predict the efficacy and toxicity of a CDC-type antibody more precisely.The results of TCR studies designated in the Guideline predict the target organs of therapeutic antibodies in human to a certain extent but are not necessarily consistent with the biological response caused by therapeutic antibodies in target organs, because it is difficult to predict efficacy and toxicity of therapeutic antibody in human only from the distribution of the antigen. The main achievement of this study was the discovery that analyzing the distribution of antigen, the distribution of the injected antibody, and the expression of mCRPs makes it possible to predict the efficacy and toxicity of a CDC-type antibody more precisely. These results will contribute to greatly improved prediction of efficacy and toxicity of therapeutic antibodies and will also contribute to the enhanced research and development of novel therapeutic antibodies.
• DOI: 10.14944/00003407; https://doi.org/10.14944/00003407
• Persistent ID (NDL): info:ndljp/pid/8948263; https://dl.ndl.go.jp/pid/8948263
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• Periodical Title (URI): http://dx.doi.org/10.14944/00003407; http://id.nii.ac.jp/1112/00003407/
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