{"created":"2023-06-19T07:18:07.357056+00:00","id":3245,"links":{},"metadata":{"_buckets":{"deposit":"ccfcd6e9-2ab0-4323-a496-b04f6c1ec7e6"},"_deposit":{"created_by":4,"id":"3245","owners":[4],"pid":{"revision_id":0,"type":"depid","value":"3245"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:00003245","sets":["370:15:392"]},"author_link":["16320"],"item_10006_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2004-02-25"}]},"item_10006_degree_grantor_9":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"麻布大学"}]}]},"item_10006_degree_name_8":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士(獣医学)"}]},"item_10006_description_22":{"attribute_name":"Abstract","attribute_value_mlt":[{"subitem_description":"Anti-HM1.24 monoclonal antibody (MoAb) is a humanized monoclonal IgG_1 antibody that recognizes the HM1.24 antigen. The antigen is highly expressed in multiple myeloma cells and is being developed as a possible therapeutic agent for the treatment of multiple myeloma. The pharmacological mechanism of anti-HM1.24 MoAb is antibody-dependent cell-mediated cytotoxicity (ADCC) with natural killer (NK) cells as effectors. However, the HM1.24 antigen in human is also distributed not just in myeloma cells but throughout the tissues of the body including normal B and T lymphocytes. It is also known that the expression of this antigen increases when these lymphocytes are activated. In a cross-reactivity study using lymphocytes from various animal species, cross-reactivity with anti-HM1.24 MoAb was detected in cynomolgus monkeys but not present in rats, mice, guinea pigs, rabbits or dogs. In another study using normal tissues, it was confirmed that cynomolgus monkey and human tissues showed the similar cross-reactivity with anti-HM1.24 MoAb. The demonstration of this cross-reactivity supports the use of the cynomolgus monkey for the assessment of the biological activity of this MoAb. The animals used in the toxicity study were treated in accordance with Chugai Pharmaceutical's ethical guidelines of animal care, handling and termination.\n As part of the anti-HM1.24 MoAb safety assessment, single-dose and one-month repeated-dose intravenous toxicity studies in cynomolgus monkeys were performed. The results of a single dose of 2, 20 and 200 mg/kg showed no toxic changes attributable to anti-HM1.24 MoAb administration. However anti- \"anti-HM1.24 MoAb\" antibodies were detected in all animals except for female of the 2 mg/kg group, since 14 or 21 days after administration. The anti- \"anti-HM1.24 MoAb\" antibody level gradually increased over time, reaching a steady state on 57 days. On the other hand, anti-HM1.24 MoAb was administered intravenously twice a week at dosage of 2, 20 and 200 mg/kg for one month. The principal toxic changes attributable to MoAb-treatment were as follows; increases of monocytes and basophils and elevated serum globulin, and histopathological changes in the lymphoid tissue associated with the immune system in the 20 and 200 mg/kg groups; cytotoxic changes (necrosis) in males from the 200 mg/kg group; anemia in females from the 200 mg/kg group. In a one-month study, anti- \"anti-HM1.24 MoAb\" antibodies were detected in males from the 2 mg/kg during the treatment period and in all animals except for female of the 200 mg/kg group during 2 month withdrawal period.\n The anemia detected from day 21 through the end of treatment period in two female monkeys was diagnosed as hemolytic anemia. It was caused by the accelerated destruction of red blood cells and this conclusion was supported by the observation of an increase of reticulocytes in the peripheral blood and an increase of erythroblasts in bone marrow. Since an increase in morphologically peripheral round erythrocytes was observed, and splenic enlargement, tissue congestion and hemosiderin deposition in the spleen were also detected, the primary cause of the hemolytic anemia was postulated to be the promotion of extravascular hemolysis. These anemic monkeys exhibited no detectable abnormality in their general condition throughout the treatment period. However one female monkey developed extreme erythrocytopenia after the treatment period. Therefore this animal was judged to be moribund and was sacrificed in extremis 10 days after the final administration. The lymphocytes subset analysis in this anemic animal revealed the abnormal differentiation of thymic T cells (cellular immunity) and the increase of splenic B cells (humoral immunity) activation by stimulation with concanavalin A accompanied by the suppression of increase in CD25 (IL-2 receptor alpha-subunit) positive cells. These findings suggest the enhancement of humoral immunity due to the induction of the immune response by anti-HM1.24 treatment, causing the pathogenesis of systemic autoimmune diseases.\n Because the hematological, blood chemical and histopathological changes in immunoreactivity were marked for these anemic monkeys, there is a strong possibility of immunoglobulin binding by erythrocytes. Immunoglobulin binding by the erythrocytes could occur not only with anti-erythrocyte antibodies but also with anti-HM1.24 MoAb or anti- \"anti-HM1.24 MoAb\" antibodies or the immune complexes. The hemolytic anemia described in the cynomolgus monkey is particularly similar to the autoimmune hemolytic anemia (AIHA) documented in human.\n As the further investigation study, we investigated the mechanism of hemolytic anemia detected in a toxicity study using cynomolgus monkeys. In order to determine the possibility of direct affect with anti-HM1.24 MoAb against erythrocytes, whether HM1.24 antigen is present on the erythrocyte membranes and the erythrocyte agglutination test following the addition of anti-HM1.24 MoAb was performed. Moreover, to examine the presence and characteristics of anti-erythrocyte antibodies, an indirect Coombs' test, a hemolysis test by complement activation and an anti-single stranded DNA antibodies assay were conducted using test animal serum or plasma.\n If anti-HM1.24 MoAb could bind directly to the erythrocytes, the primary cause of the hemolytic anemia is clear. Therefore the specific binding of anti-HM1.24 MoAb to the human and cynomolgus monkey erythrocytes was investigated using FITC- and ^<125>I -labeled anti-HM1.24 MoAb. However, negative results were obtained using both test methods. Furthermore, ^<125>I-labeled anti-HM1.24 MoAb was judged not to bind specifically to the erythrocytes, either in the presence or absence of the monkey anti- \"anti-HM1.24 MoAb\" idiotype antibody. However the fact that labeled anti-HM1.24 MoAb utilized in both methods may be entirely in the form of a monomer should be taken into consideration, because there might be few heterogeneities in the form of dimers and oligomers and heteromolecules from ion-exchange chromatogram in the anti-HM1.24 MoAb solution, the characteristics of these related substances are unclear.\n When anti-HM1.24 MoAb was added to the erythrocyte suspensions in physiological saline, the high concentrations (more than 713 μg/mL) caused the direct agglutination of normal cynomolgus monkey and human erythrocytes. For the analogous recombinant humanized IgG_1 drug used as the comparative control, agglutination was negative at the maximum concentration of 2,850 μg/mL. When the facts that anti-HM1.24 MoAb does not directly bind to the erythrocytes, that IgG class antibodies cannot normally bridge the gap between erythrocytes and cannot directly cause agglutination are taken into consideration, the direct agglutination effect of anti-HM1.24 MoAb addition is assumed to be caused by non-specific adhesion. The specific reason for the erythrocyte agglutination remains unclear but the participation of the above-mentioned dimers, oligomers and distinct electrically charged heteromolecules cannot be ruled out. However, when using the erythrocyte suspensions in serum, no agglutination was detected. These results obtained under in vitro conditions are difficult to extrapolate to an in vivo reaction, but it suggests that the serum components would be able to avoid non-specific adhesion.\n In an indirect Coombs' test using test serum collected from the one-month study, agglutination was detected with the addition of anti-monkey IgG as secondary antibody in one female monkey from the 20 mg/kg group and in three females, including two anemic monkeys from the 200 mg/kg treatment group. Usually, to avoid false positives attributable to non-self recognition in an indirect Coombs' test, the serum and erythrocyte of identical blood groups must be used. Also a possible non-specific reaction due to the existence of allo-antibodies cannot be dismissed. Cynomolgus monkey blood group classification is, however, difficult and because there was no observation of erythrocyte agglutination for both the male and female sera from the control group, non-self recognition was considered to have no effect on the results obtained in this study. For future examination definitive diagnosis of confirmed agglutination using a direct Coombs' test against autologous erythrocytes is essential.\n From the results of hemolysis assay caused by activation of complement, one anemic monkey was judged to be positive. Generally speaking, extravascular hemolysis is the main cause of hemolytic anemia while intravascular hemolysis is rare. This animal developed sudden erythrocytopenia with concurrent increases in the serum LDH and indirect bilirubin and a decrease in haptoglobin. Therefore it was believed that intravascular hemolysis had occurred in this animal. Complement hemolysis was also observed in another female from the 200 mg/kg group when male erythrocytes were used, but was negative when tested with female erythrocytes. This was thought to be due to the participation of natural antibodies that recognized the antigens from blood group and sex differences, although the specific details are unclear.\n For the ELISA measurement of antibodies against single stranded DNA (ssDNA), all of the male animals were negative. However one female monkey from the 20 mg/kg group and three females from the 200 mg/kg group displayed a gradual increase in their anti-ssDNA titers on the 10th and 28th day of the treatment period. The measurement of anti-ssDNA antibodies is a useful diagnostic method for autoimmune diseases in human and is also helpful in the detection of general autoantibodies following the breakdown of self-tolerance. This test alone does not directly represent anti-erythrocyte antibody production. However, from the positive reaction in the indirect Coombs' test and those that were also positive for anti-ssDNA antibodies, the production of anti-erythrocyte autoantibody is thought to be a valid diagnosis as the primary cause for hemolytic anemia. In the anti-ssDNA antibody-positive animals, one female monkey with an extremely high value prior to the beginning of the treatment period is noteworthy. This animal developed anemia at an early stage and experienced marked changes in immunoreactivity, suggesting the possibility of having a latent risk of autoimmune disease.\n From the results of the present mechanistic investigations and clinical pathology of the anemic animals, it is estimated that AIHA probably appeared according to following processes. By combining with the HM1.24 antigen of the lymphocytes, Anti-HM1.24 causes abnormal differentiation of T cell in addition to activation of B cell. It is made to fall into the condition of the humoral immunity superiority, so that the autoreactive B cells could be activated. Under such situation, it seemed to recognize the erythrocyte membrane by some causes as an autoantigen. It is considered that as one of the this factors, high concentration Anti-HM1.24 caused the erythrocyte agglutination and anti- \"anti-HM1.24 MoAb\" antibodies bind to the anti-HM1.24 MoAb on the erythrocyte. The modified erythrocytes are detained by the reticular endothelial system and the splenic macrophages phagocytize the erythrocytes through the IgG-Fc receptor. The antigen of the erythrocyte membrane is released, and being recognized in the immune system which is activated by MoAb-treatment. Accordingly, the autoimmune response is induced, and the anti-erythrocyte autoantibodies are produced. This hypothesis could be supported by the facts that the manifestation of hemolytic anemia agreed with the time when the specific antibody is generally produced (after 2 week of treatment), and the phagocytosis through Fc receptor for specific antibodies of the self-production is the strong immune response. In addition, remarkable producing of the specific antibodies, since the anti-HM1.24 MoAb had the significant immunogenicity for cynomolgus monkeys, would exacerbate these processes.\n In conclusion, the hemolytic anemia observed in the repeated-dose toxicity study of the anti-HM1.24 MoAb using cynomolgus monkeys was diagnosed to be drug-induced autoimmune hemolytic anemia (AIHA). The primary cause of this AIHA was assumed to be the production of IgG class anti-erythrocyte autoantibodies.\n\n Multiple myeloma as targeted disease of anti-HM1.24 MoAb is an intractable disease that is difficult to cure, therefore if therapeutic action for human will be expected certainly, the benefits of anti-HM1.24 MoAb for patient can be assumed to be very considerable. Although it is possible that an immune response to the humanized antibody as a foreign protein for cynomolgus monkeys may have contributed to the manifestation of autoimmune hemolytic anemia in the present study, close attention must be paid to assuring safety in human clinical trials. This means that patients with previous history of autoimmune diseases such as AIHA or SLE and patients with positive antinuclear or anti-DNA antibodies must be excluded from enrolment. Furthermore detailed immune toxicological parameters include quantitation of immune globulins (IgG, IgM, IgA, IgE, IgD), immune complexes and complement concentrations, lymphocyte subset analysis, determination of antinuclear and anti-DNA antibodies, direct Coombs tests and haptoglobin must be carried out in addition to the usual hematological and blood chemistry tests, as indicators for monitoring autoimmune hemolytic anemia.","subitem_description_type":"Other"}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"緒言\n　抗HM1.24モノクローナル抗体(以下Anti-HM1.24)は骨髄腫細胞に高頻度に発現しているヒトHM1.24抗原を認識するヒト型化抗体(IgG1)であり、多発性骨髄腫の治療薬としての開発が期待されている。Anti-HM1.24は主に骨髄腫細胞に対してナチュラルキラー細胞を介した抗体依存性細胞傷害作用(antibody-dependent cell-mediated cytotoxicity:ADCC)を有しており、補体依存性細胞傷害活性は殆どないとされている。しかし、ヒトではHM1.24抗原は骨髄腫細胞に限らず正常なBリンパ球またはTリンパ球を含む全身の組織に分布しており、標的以外の組織に対する影響が懸念される。\n　マウス、ラット、モルモット、ウサギおよびイヌではAnti-HM1.24に対して殆ど交差反応性を示さないが、カニクイザルはヒト組織と類似した交差反応性を有する動物種である。よって、Anti-HM1.24のヒトにおける毒性を予測するためには、カニクイザルが前臨床試験に用いる唯一の動物種として位置付けられる。本研究は前臨床安全性試験を通して、多発性骨髄腫患者における発現メカニズムを解明することを目的とした。カニクイザルを用いた試験は全て中外製薬(株)動物実験指針に則り、動物福祉に対する倫理的配慮をもって遂行した。\n\n第1章　Anti-HM1.24のカニクイザルにおける前臨床安全性評価\n1. カニクイザルにおける単回投与毒性試験\n　Anti-HM1.24を0、2、20、200mg/kg(各群雌雄各1例)の用量で静脈内単回投与した。\n　高用量の200mg/kgはヒトでの予想最高用量の約40倍に相当した。単回投与試験では200mg/kgの用量まで何ら急性毒性は認められず、また遅発性の毒性も認められなかった。\n\n2.カニクイザルにおける1ヶ月間反復投与毒性試験\n　Anti-HM1.24を0、2、20、200mg/kg(各群雌雄各4例)の用量で1ヶ月間(週2回間欠)静脈内反復投与した。1ヵ月間の反復投与では2mg/kgは無毒性量であったが、20mg/kg以上で免疫反応性の変化として、好塩基球、単球および血清グロブリンの増加や脾臓、リンパ節、扁桃、消化管付属リンパ組織および胸腺におけるリンパ球の増減を伴う増殖性または萎縮性の病理学的変化が観察された。最高用量の200mg/kgでは雄4例中1例でAnti-HM1.24に交差反応性を示す臓器に一致して、限局性または巣状壊死が認められ、ADCC作用の過大発現によるものと推察された。また、200mg/kgの雌4例中2例では貧血が認められ、1例では投与開始後3週以降に赤血球の減少、網赤血球および小型・球形赤血球の増加を示し、脾臓の腫大・ヘモジデリン沈着の増加および骨髄赤芽球の増加が認められた。従って、この貧血は主に網内系における血管外溶血の亢進による溶血性貧血であると考えられた。もう1例は最終投与後10日(休薬開始後6日)に極度の貧血を呈し、乳酸脱水素酵素および間接ビリルビンの上昇並びにハプトグロビンの減少を伴い瀕死状態となったため切迫と殺した。本切迫と殺例は投与開始後21日まで末梢血CD20陽性リンパ球(B細胞)比率の増加がみられた以外に血液学的な異常は認められなかったことから、急激な赤血球減少が生じたと推察され、血管外溶血に加え血管内溶血が起こった可能性が高いと考えられた。また、同例では剖検時に胸腺中のT細胞(細胞性免疫)の分化異常および脾臓中リンパ球のB細胞(体液性免疫)の活性化亢進が認められた。このことから、Anti-HM1.24がリンパ球に結合することによって、リンパ球の分化・増殖に影響を及ぼしたため、機能異常が発現して体液性免疫優位となり、全身性自己免疫疾患が発症しやすい状態に陥ったことが示唆された。さらに、抗Anti-HM1.24抗体(Feb'に対するIgG抗体)が単回および1ヵ月間反復投与ともに2mg/kg以上で検出され、産生量に用量依存性はないものの単回投与においても投与後9ヵ月以上にわたり高濃度に検出された。このことから、Anti-HM1.24はカニクイザルに対して強い免疫原性を有することが明らかとなり、産生された抗Anti-HM1.24抗体は種々の免疫反応性の毒性変化に影響を及ぼしていることが推察された。なお、持続的な免疫複合体の形成に伴うと考えられる明らかな炎症性変化や組織沈着による腎障害などは認められなかった。\n\n第2章　Anti-HM1.24に誘発されたカニクイザルの溶血性貧血の機序に関する検討\n　カニクイザルにおける前臨床安全性評価において見出された溶血性貧血の病態はヒトにおける自己免疫性溶血性貧血(autoimmune hemolytic anemia:AIHA)に酷似していたことから、ヒトにおいて最も懸念される毒性であると考えられた。この溶血性貧血は通常の臨床検査では発症の兆候を把握することが困難であったこと、発症後に急激な病態の増悪を示したことから、別途溶血性貧血の誘発機序を解明することが、本薬の臨床試験における副作用発現の有無を予測し、患者のリスクを回避する手段を講じる上で重要な課題となった。\n　すなわち、Anti-HM1.24が直接的に赤血球に作用して溶血貧血を誘発した可能性を検証するために、標識Anti-HM1.24を用いて赤血球に対する結合性およびAnti-HM1.24添加による赤血球凝集反応の有無を調べた。また、AIHAを発症したことを検証するために、1ヵ月投与試験の被験動物血清または血漿を用いて、抗赤血球抗体の有無を間接Coombs試験で調べ、また抗赤血球抗体が血管内溶血を引き起こした可能性を調べるために、補体依存性の溶血性試験を実施した。また、自己抗体産生の有無を調べるために、抗Single stranded DNA抗体(抗ssDNA抗体)の測定を行った。\n　その結果、fluorescein isothiocyanate(FITC)あるいは125I標識したAnti-HM1.24はカニクイザルおよびヒトの赤血球膜に対して、特異的な結合は認められなかった。\n　また、これらの標識Anti-HM1.24にカニクイザル抗イディオタイプAnti-HM1.24抗体を共存させた条件下でも特異的な結合は認められなかった。従って、Anti-HM1.24が赤皿球に特異的に結合して、溶血性貧血の初期要因となった可能性は否定され、ヒトにおいてもその危険性はないと推察された。カニクイザルおよびヒト赤血球浮遊液(生理食塩液)に高濃度(713μg/mL<)のAnti-HM1.24を添加すると、in vitroで直接的に赤血球を凝集させた。しかし、2次抗体として抗ヒトIgGを添加しても、この赤血球凝集は増強されなかった。Anti-HM1.24は赤血球に対して特異的に結合しないことから、直接的な赤血球凝集の成因は不明であるが、Anti-HM1.24溶液中に混在(3％>)する多量体や電荷の異なる異性体が関与している可能性も考えられた。一方、生理食塩液の代わりに自己血清を用いて作製した赤血球浮遊液ではカニクイザルおよびヒトともAnti-HM1.24添加による赤血球凝集は認められなかったことから、血清成分が直接的な赤血球凝集を回避させ得ることが示唆された。\n　間接Coombs試験において、200mg/kgで溶血性貧血を発症した雌2例に加え、発症はみられなかった20および200mg/kgの雌各1例に陽性反応が認められ、赤血球に特異的に結合するIgG抗体の存在が確認された。また、切迫と殺例では補体の活性化によるin vitro溶血反応も認められ、産生された抗赤血球抗体によって血管内溶血が生じた可能性が示唆された。さらに、間接Coombs陽性例では抗ssDNA抗体が投与後に経時的に増加していた。抗ssDNA抗体の測定は自己に対する免疫寛容の破綻により産生される汎自己抗体を検出しているため、直接的に抗赤血球抗体の増加を示すものではないが、抗ssDNA抗体が増加した個体が全例とも間接Coombsで陽性反応が認められたことから、自己免疫性の抗赤血球抗体が産生されていると判断した。","subitem_description_type":"Abstract"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"乙第399号"}]},"item_10006_version_type_18":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_ab4af688f83e57aa","subitem_version_type":"AM"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"渡部, 一人"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2013-02-20"}],"displaytype":"detail","filename":"diss_dv_otsu0399.pdf","filesize":[{"value":"10.0 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0399","url":"https://az.repo.nii.ac.jp/record/3245/files/diss_dv_otsu0399.pdf"},"version_id":"bc2f2765-1214-434d-a8ea-96cfbd97facf"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2014-08-19"}],"displaytype":"detail","filename":"diss_dv_otsu0399_jab&rev.pdf","filesize":[{"value":"404.6 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0399_jab&rev","url":"https://az.repo.nii.ac.jp/record/3245/files/diss_dv_otsu0399_jab&rev.pdf"},"version_id":"e12516e6-3b5a-43fd-b097-57f4816f7d5f"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis"}]},"item_title":"薬物誘発性の自己免疫性溶血性貧血に関する研究 : カニクイザルにおけるヒト型化抗HM1.24モノクローナル抗体による溶血性貧血の誘発機序","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"薬物誘発性の自己免疫性溶血性貧血に関する研究 : カニクイザルにおけるヒト型化抗HM1.24モノクローナル抗体による溶血性貧血の誘発機序"}]},"item_type_id":"10006","owner":"4","path":["392"],"pubdate":{"attribute_name":"公開日","attribute_value":"2013-02-13"},"publish_date":"2013-02-13","publish_status":"0","recid":"3245","relation_version_is_last":true,"title":["薬物誘発性の自己免疫性溶血性貧血に関する研究 : カニクイザルにおけるヒト型化抗HM1.24モノクローナル抗体による溶血性貧血の誘発機序"],"weko_creator_id":"4","weko_shared_id":4},"updated":"2023-06-19T08:15:20.872874+00:00"}