201011046 六、發明說明： 具 更 〇 康 0 之 人 是 特 域 領 之 ]術 域技 領物 術生 技於 之關 屬明 所發 明本 發 體地說，本發明關於能辨識表皮生長因子受體（EGFR ) 之胞外區域並阻斷該受體之天然配位體之結合但不完全抑 制該受體二聚化之新穎抗體。 © 【先前技術】 隨著對腫瘤生物學及導致腫瘤形成之機制上之理解的 進展，已經導致鑑別數個腫瘤治療（特別是女性及男性首 要死因之一之肺癌）之標靶。在該等標靶中，EGFR (又 名HER 1 )已成爲肺癌治療之注意焦點。EGFR是一種主要 在上皮來源之細胞中發現之跨膜受體。該受體之胞內區域 之自體磷酸化觸發能導致細胞增生之事件的·級聯。EGFR 經常過度表現於多種固體腫瘤中，並參與細胞存活、增生 G 、轉移及血管形成之控制。 因此，不同策略已經被發展以抑制異常之EGFR相關 性信號傳遞級聯。最重要的治療方式包括小分子酪胺酸激 酶抑制劑和單株抗體，該等抗體與該受體之胞外區域專一 性結合且作爲該受體之天然配位體之拮抗劑。目前一些抗 EGFR抗體正在進行臨床評估，例如西妥昔單抗（ Cetuximab )、帕尼單抗（Panitumumab )及馬突兹單抗（ Matuzumab )。 西妥昔單抗（爾必得舒（Erbitux))是一種能專一性辨 201011046 識EGFR之胞外區域之嵌合單株抗體，該抗體已通過FDA 核准用於治療結直腸癌及晚期頭頸腫瘤。硏究顯示，西妥 昔單抗係A43 1細胞（源自表皮樣癌）增生之強效抑制劑 ，不論於活體外或將該細胞移植至無胸腺小鼠。硏究亦顯 示，該抗體與細胞毒性藥物或放射線治療之組合具有協同 效應。根據這些結果，不同的臨床試驗接著被進行。 第二期臨床試驗之結果顯示，西妥昔單抗不論是單獨 或與愛萊諾迪肯（irinotecan )及草酸銷（oxaliplatin )組 ❹ 合使用，均可作爲晚期轉移性結直腸癌病患之第一線治療 ，使反應率增加10.至20%。 在另一個包含424名晚期局部區域疾病病患之國際性 、多中心臨床試驗中，西妥昔單抗與放射線治療之組合幾 乎延長一倍的存活時間（從28至54個月）。另外，該組 合使病患之存活時間從僅接受放射線治療2及3年後分別 爲55%及44%，增加至組合治療之62%及57%。整體來說 ，來自臨床之結果顯示，使用西妥昔單抗（不論以單一治 _ 療投予或與細胞毒性藥物組合投予）具有顯著之治療效應 。然而，也觀察到習知治療所產生之毒性反應之顯著增加 〇 帕尼單抗（能辨識EGFR之全人抗體）係另一已通過 FDA核准（2006年）之用於單一治療化學治療後疾病惡 化之轉移性結直腸癌病患之抗體。帕尼單抗獲得之臨床結 果類似西妥昔單抗所獲得者，包括不良反應。 許多不同的EGFR拮抗劑（包括若干單株抗體）已經 -6- 201011046 在臨床上進行評估。對大部分該等藥物而言，所獲得之目 標反應是短期的，然而最常見之毒性效應在於嚴重皮疹， 該皮疹在許多病例中導致治療中斷。 大部分EGFR拮抗劑之臨床效用與皮虜毒性之直接相 關性已被證實。因此，有文獻指出皮疹之發生係抗EGFR 作用劑之抗腫瘤反應之預測指標[Jonker et al，2007;201011046 VI. Description of the invention: The person with a more sturdy 0 is the domain of the domain. The domain technology is based on the invention. The present invention relates to the recognition of the epidermal growth factor receptor. A novel antibody that binds to the extracellular region of (EGFR) and blocks the binding of the natural ligand of the receptor but does not completely inhibit the dimerization of the receptor. © [Prior Art] Advances in the understanding of tumor biology and the mechanisms that lead to tumor formation have led to the identification of targets for several cancer treatments, particularly lung cancer, one of the leading causes of death in women and men. Among these targets, EGFR (also known as HER 1) has become the focus of attention in the treatment of lung cancer. EGFR is a transmembrane receptor found primarily in epithelial-derived cells. Autophosphorylation of the intracellular region of the receptor triggers a cascade of events that lead to cell proliferation. EGFR is often overexpressed in a variety of solid tumors and is involved in cell survival, proliferation G, metastasis, and angiogenesis control. Therefore, different strategies have been developed to suppress abnormal EGFR-related signaling cascades. The most important treatment modalities include small molecule tyrosine kinase inhibitors and monoclonal antibodies that specifically bind to the extracellular domain of the receptor and act as antagonists of the natural ligand of the receptor. Some anti-EGFR antibodies are currently undergoing clinical evaluation, such as cetuximab, panitumumab, and matuzumab. Cetuximab (Erbitux) is a chimeric monoclonal antibody that specifically recognizes the extracellular region of EGFR in 201011046. The antibody has been approved by the FDA for the treatment of colorectal cancer and advanced head and neck cancer. Studies have shown that cetuximab is a potent inhibitor of proliferation of A43 1 cells (derived from epidermal-like carcinoma), either in vitro or transplanted into athymic mice. Studies have also shown that this antibody has a synergistic effect with a combination of cytotoxic drugs or radiation therapy. Based on these results, different clinical trials were subsequently conducted. The results of the second phase of clinical trials showed that cetuximab, either alone or in combination with irinotecan and oxaliplatin, can be used as a disease in patients with advanced metastatic colorectal cancer. First-line treatment increases the response rate by 10. to 20%. In another international, multicenter clinical trial involving 424 patients with advanced localized disease, the combination of cetuximab and radiation therapy almost doubled the survival time (from 28 to 54 months). In addition, the combination resulted in a survival time of 55% and 44%, respectively, from 25% to 3 years after radiation therapy alone, and increased to 62% and 57% of combination therapy. Overall, clinical results show that the use of cetuximab (whether administered in a single treatment or in combination with a cytotoxic drug) has a significant therapeutic effect. However, a significant increase in the toxic response from conventional treatments has also been observed. Panibizumab (a fully human antibody that recognizes EGFR) is another disease that has been approved by the FDA (2006) for monotherapy after chemotherapy. Antibodies to patients with exacerbated metastatic colorectal cancer. The clinical results obtained with panitumumab were similar to those obtained with cetuximab, including adverse events. Many different EGFR antagonists (including several monoclonal antibodies) have been clinically evaluated -6- 201011046. For most of these drugs, the target response is short-lived, however the most common toxic effect is a severe rash, which in many cases leads to treatment interruption. The direct relevance of the clinical utility of most EGFR antagonists to cutaneous toxicity has been demonstrated. Therefore, it has been documented that the occurrence of rash is a predictor of anti-tumor response of anti-EGFR agents [Jonker et al, 2007;
Berlin et al，2007](Peedicayil J. y col., en o Correspondence 2004，doi ' 10.016) 〇 格里德利等人（Gridelli et al.，in Results of an Experts Panel Metting, Crit. Rev. Oncol. Hematol. 2007, doi: 10.1016)回顧來自不同臨床試驗之結果，發現在該 等臨床試驗中皮膚皮疹與治療反應及/或病患存活性之間 具有正向相關性。在該文獻中，作者之結論爲皮虜皮疹目 前被認爲是抗EGFR藥物之拮抗活性之間接標記。因此， 專家間之共識認爲皮虜皮疹係抗腫瘤活性及治療效用之重 〇要替代指標。另外，他們建議可將皮虜反應用來識別最可 能得益於該治療之病患。 然而，使用抗EGFR單株抗體h-R3之臨床試驗（EP 07 1 2863B1和US 5,89 1,996 )的結果顯示，病患對該抗體 之耐受性良好。在接受重複劑量之h-R3之病患中未偵測 到皮膚皮疼（Cormbet et al. in Cancer Biology & Therapy 5;4, 375-379, 2006)，與在以其他EGFR阻斷藥物治療之 病患中所觀察到約8 0 %出現皮膚皮疹者不同（ Perez-Soler et al. in Oncologist 2005; 1 0:345-56/ and Thomas et al. in 201011046Berlin et al, 2007] (Peedicayil J. y col., en o Correspondence 2004, doi ' 10.016) Gridelli et al., in Results of an Experts Panel Metting, Crit. Rev. Oncol. Hematol. 2007, doi: 10.1016) reviewing the results from different clinical trials and found a positive correlation between skin rash and treatment response and/or patient viability in these clinical trials. In this document, the authors conclude that the skin rash is currently considered to be an indirect marker of antagonistic activity against EGFR drugs. Therefore, the consensus among experts is that the skin rash is an important substitute for the anti-tumor activity and therapeutic efficacy. In addition, they suggest that the skin reaction can be used to identify patients who are most likely to benefit from the treatment. However, the results of a clinical trial using anti-EGFR monoclonal antibody h-R3 (EP 07 1 2863 B1 and US 5,89 1,996) showed that the patient was well tolerated by the antibody. No skin pain was detected in patients receiving repeated doses of h-R3 (Cormbet et al. in Cancer Biology & Therapy 5; 4, 375-379, 2006), and in the treatment of other EGFR-blocking drugs About 80% of patients with skin rashes are observed (Perez-Soler et al. in Oncologist 2005; 1 0:345-56/ and Thomas et al. in 201011046)
Clin J. Oncol N 2005 ; 9:3 3 2-8 )。 雖然現有技藝以皮虜皮疹與EGFR阻斷之間之直接相 關性爲共識，本發明之作者認爲皮膚皮疹反而是與該特定 藥物有關而非與該標靶本身有關之缺點，因此需要繼續尋 找新穎之抗EGFR作用劑。 【發明內容】 本發明關於表皮生長因子受體（EGFR )之抑制劑類 @ ，該等抑制劑類對表現該受體之細胞具有細胞生長抑制活 性而非細胞毒性作用。 細胞生長抑制劑係任何能抑制細胞增生，使細胞停在 細胞週期中之一期之劑，而細胞毒性劑係指任何能引起細 胞死亡之劑。EGFR抑制劑是一種與該受體結合且因此抑 制由該受體之天然配位體所引發之細胞分裂信號之分子。 意外的是，本發明之作者發現一群EGFR抑制劑，由 於該群抑制劑以特殊方式與EGFR結合，而使該受體之活 ® 化構型和不活化構型之間達到生理平衡，因此能保持基礎 程度之受體信號傳遞。該基礎程度之受體自體磷酸化使該 細胞停在細胞週期之一期。本發現之臨床應用在於我們能 設計以EGFR爲標靶之抗腫瘤藥物，該藥物並非引起腫瘤 細胞死亡而是展現對腫瘤生長之生物控制。此外’該類型 之抗腫瘤藥物具有不產生嚴重不良反應之優點’諸如對腫 瘤具有細胞毒性作用之EGFR抑制劑所報告之皮膚皮疹。 本發明描述具有細胞生長抑制功效之EGFR抑制劑類 -8- 201011046 ，其特徵爲該等抑制劑類辨識EGFR之胞外區域之結構域 I或結構域III (較佳爲結構域III )，且抑制該受體之天 然配位體之結合，因此抑制由該等配位體所誘發之細胞分 裂信號。該等與EGFR胞外區域之結構域I或III結合藉 以競爭天然配位體之結合但仍允許該受體採取其活化構型 且形成能維持基礎磷酸化程度之同型二聚體或雜二聚體之 分子，係對表現EGFR之細胞具有細胞生長抑制活性之分 〇 子，因此該等分子可作爲表現該膜受體之腫瘤之潛在治療 劑。 本發明之作者亦發現以該等能抑制由EGF及其他配位 體所觸發之細胞分裂信號又能維持基礎程度之受體自體磷 酸化之分子（較佳爲單株抗體）作爲惡性腫瘤之治療，對 腫瘤具有阻止該腫瘤生長之細胞生長抑制功效。 目前以EGFR爲標靶之治療劑的功效有限，包括細胞 分裂配位體之拮抗劑及抑制該受體自體磷酸化之小分子， 〇因爲彼等治療劑在許多病例引起嚴重之不良反應而必須中 斷該治療。本發明之作者已經發現以EGFR爲治療標靶之 分子，該等分子能抑制腫瘤生長，但不產生目前爲止在臨 床上測試之EGFR信號傳遞抑制劑所發生之嚴重不良反應 〇 本發明之基礎爲抑制由EGFR天然配位體所誘發之細 胞分裂活性，但不完全抑制該EGFR之自體磷酸化。如前 所述，EGFR在細胞膜上有二種呈現平衡之構型，活化構 型和不活化構型。當該受體採取其活化構型時，彼之結構 -9- 201011046 域II能與其他EGFR分子中之相同結構域（或該EG FR家 族之其他成員）交互作用’藉以形成同型二聚體或雜二聚 體，且因此活化該細胞內結構域之自體磷酸化。此自體磷 酸化能觸發維持該細胞存活所需之事件的級聯。 根據估計，當細胞分裂配位體不存在時’超過90%之 EGFR分子採取較爲節省能源之不活化構型，然而有5至 10%之受體分子採取其活化構型且因此維持磷酸化。該磷 酸化之程度不足以引起細胞分裂，但能讓細胞存活。根據 φ 本發明之作者，使用完全抑制受體自體磷酸化之藥物會造 成大量細胞死亡，包括表現EGFR之腫瘤及正常組織，這 說明嚴重不良反應發生之原因。然而，使用本發明之目標 抑制劑造成抑制細胞分裂活性及因此之腫瘤生長，但維持 能讓細胞存活之基礎程度之受體磷酸化。第一種藥物將產 生伴隨顯著腫瘤消退之細胞毒性作用，但是也會產生嚴重 之不良反應及早期腫瘤復發。本發明之目標藥物不可能產 生伴隨驚人腫瘤消退之大量細胞死亡；相反的因爲彼等對 〇 腫瘤之細胞生長抑制功效而產生疾病穩定狀態，且對正常 組織並不造成顯著之不良反應。此外，本發明之作者亦發 現該等具有細胞生長抑制活性之EGFR抑制劑保有放射線 敏感劑及化學敏感劑之性質，該性質在具有細胞毒性活性 之EGFR抑制劑中已被描述。 根據實驗結果，本發明之作者主張以EGFR爲標靶之 分子爲申請專利範圍，該分子之活性在於對腫瘤生長之生 物控制，不論彼等係以單一治療或與化學或放射線治療組 -10- 201011046 合之方式投予。 因此’本發明之目的係該等天然或合成之EGFR配位 體’較佳爲天然的，且甚至更佳爲單株抗體，該EGF拮抗 劑能抑制EGF與該受體之胞外結構域結合但不干擾受體二 聚化，因此不影響基礎程度之自體磷酸化。 本發明之另一目的係一種用於治療癌症病患之方法， 該方法包含投予含有EGFR配位體之醫藥化合物，該 〇 EGFR配位體較佳係屬於EGF拮抗劑但不抑制基礎受體自 體磷酸化之單株抗體。 本發明之另一目的係一種設計及選擇配位體之方法， 該配位體較佳係具有細胞生長抑制作用以防止惡性腫瘤生 長之單株抗體。 本發明之詳細說明 本發明之目標之EGFR抑制劑包含一天然或合成之分 ® 子，該分子與表皮生長因子受體之胞外區域之結構域I或 結構域III結合，較佳係與結構域III結合，該分子之特 徵在於抑制天然配位體之結合之性質，且另外允許該受體 採取其活化構型。意外的是，以此方式辨識受體之抑制劑 對表現該受體之細胞具有細胞生長抑制作用，這與過去在 本發明之前所描述之對該細胞具有細胞毒性作用之抑制劑 類不同。 用於設計本發明之目標治療劑之方法係根據本發明之 作者所得到之結果，該結果支持該劑與EGFR受體之交互 -11 - 201011046 作用之模型。該等EGF拮抗劑藥物對癌症具有治療效應， 且具有超越先前所描述之EGF拮抗劑藥物之優點。在較佳 之實施態樣中’該等治療劑可爲以EGFR爲標靶之單株抗 體’較佳爲單株抗體hR3。該人化抗體係詳細描述於先前 提到之專利申請案EP 0712863B1及美國專利5,891,996以 及—些科學發表中，例如 Mateo et al.，Immunotechnology 3; 71-81，1997。獲得該抗體之詳細方法亦描述於該些文件 中〇 發明人所描述之交互作用模型亦能解釋來自癌症病患 之臨床試驗之結果，在該試驗中評估本發明目標劑之治療 效果。 已知的是，EGF與EGFR胞外區域之交互作用誘發信 號傳遞級聯，藉以觸發該生長因子之細胞分裂作用。 EGFR胞內結構域之自體磷酸化係此信號傳遞級聯中之第 一生化事件之一。爲了使自體磷酸化得以發生，該受體之 胞外區域必須先發生二聚化。 亦爲已知的是，EGF同時與EGFR胞外區域之結構域 I及III結合藉以穩定該活化受體構型，其中結構域II係 經備妥以與第二單體結合以形成二聚體。 所有已在臨床上測試且已經證實對腫瘤具有治療效應 之以EGFR爲標靶之單株抗體，係藉由抑制受體二聚化及 因此後續之磷酸化以作爲EGF拮抗劑之分子。大部分該等 抗體藉由產生阻止該活化受體構型形成之立體阻礙以抑制 該受體之二聚化。其他抗體直接與EGFR之結構域II結合 201011046 ，因此直接抑制該二聚化。 意外的是本發明之作者發現，阻斷EGF與其受體結合 因此抑制由此配位體產生之細胞分裂信號，但同時允許該 受體採取其可二聚化之活化構型是可能的，因此允許基礎 程度之受體磷酸化。 現有技藝已知的是，在細胞膜上之EGFR分子被發現 呈現活化構型與不活化構型之平衡。有硏究指出介於5% φ 至10%之膜受體呈現活化構型。因此，該部分之受體分子 可形成二聚體並進行後續之磷酸化。該基礎程度之受體磷 酸化不足以觸發細胞增生，但係維持靜止狀態之細胞存活 所必須。 因此，使用具有本發明所描述之特徵之分子將在表現 EGFR之腫瘤之治療上有極大效用。治療上使用本發明之 目標分子將抑制由EGFR所誘發之腫瘤增生，同時能防止 目前臨床上使用之EGF拮抗劑所產生之不良反應。 〇 在具有此處所描述之特性之治療劑中，以EGFR之胞 外區域爲標靶之單株抗體係本發明之較佳實施態樣，以先 前描述於US 5，89 1,996及EP 07 1 2863中之人化單株抗體 hR3特別爲佳。 即使現有技藝中有能辨識該相同標靶之治療劑，本發 明之治療劑所具有之結構及功能特徵提供超越先前描述者 之治療優點。 本發明亦關於一種抑制EGF依賴性腫瘤生長之方法， 其特徵爲投予治療劑量之治療劑所產生之效應，該效應誘 -13- 201011046 發疾病穩定狀態而非驚人之腫瘤消退。本發明之其他目標 係包含至少一種該等細胞生長抑制性抑制劑之含水溶液之 治療組成物，該組成物可用於治療腫瘤或其他與EGFR調 節異常有關之疾病。在本發明之醫藥組成物中，該抑制劑 之濃度（特別是該抗體）係介於10至200毫克/毫升之範 圍內，更具體地介於50至150毫克/毫升之範圍內。 此外，本發明包含該等EGFR抑制劑（特別是該人化 單株抗體hR3 (尼妥珠單抗（Nimotuzumab))與放射線治 ❻ 療或其他治療劑諸如化學治療劑組合以用於治療惡性腫瘤 上之用途。根據本發明，本發明之目標抑制劑之投予可爲 經口、非經腸（經靜脈內或肌肉內）、局部、經皮或經吸 入。 本發明之另一目標係一種用於選擇具有細胞生長抑制 功效之EGFR抑制劑之方法。該方法主要係根據如本發明 實施例ό所述之細胞中DNA含量以碘化丙啶之納入測定 ，該細胞先前經通透性處理及固定。 φ 【實施方式】 實施例 實施例1 : hR3 Fab片段之晶體結構 hR3 Fab片段之晶體結構係以2.5A解析度決定，且具 有經精準化至結晶學R因子/自由r因子分別爲21.5 %及 28.8%之良好之立體化學。 hR3 Fab之整體晶體結構係與其他Fab片段之結構類 ，14 - 201011046 似，但二個在該重鏈之變異結構域中之特殊特徵除外。一 明顯之特徵係關於該重鏈之CDR1，該CDR1不對應任何 被描述之該CDR之基本構型，相反卻在該環之底部呈現 短的α螺旋。圖1顯示hR3 Fab片段之代表性晶體結構， 其中該重鏈之CDR1特徵被強調。 利用電腦程式，比較hR3變異區之晶體結構與所有到 2007年5月爲止寄存於蛋白質資料庫中之類似片段之結構 。在此資料庫中，只有二種抗體具有在CDR H1底部之類 似特徵（PDB 代號 Ib2w([lb2w])及 2cmr( [2cmr])) 。該二個結構之一對應以CDR植入人化之抗體。 實施例2:抗體一受體之交互作用試驗 利用Biacore設備測定，hR3 Fab片段係以2.1xl〇·8 Μ 結合之解離常數（KD)與表皮生長因子受體（EGFR)之 胞外結構域結合。在此試驗中，使用商用抗體西妥昔單抗 © ( Cetuximab )作爲對照。西妥昔單抗Fab片段所得到之 KD値爲1·8χ1〇·9 Μ，與先前報告之値類似。尼妥珠單抗之 KD高出一個數量級，主要是因爲其&。„過低，而其幻7/僅 稍爲較低（表1 )。 這二種抗體間之親和性差異可能解釋彼等在生物效應 上之差異，特別是當該二種抗體被用於系統性治療癌症病 患之不良反應上之差異。 -15- 201011046 表 1 :____Clin J. Oncol N 2005; 9:3 3 2-8 ). Although the prior art is based on the direct correlation between skin rash and EGFR blockade, the authors of the present invention believe that skin rash is a disadvantage associated with the particular drug and not with the target itself, so it is necessary to continue searching A novel anti-EGFR agent. SUMMARY OF THE INVENTION The present invention relates to inhibitors of epidermal growth factor receptor (EGFR), which have cytostatic activity rather than cytotoxic effects on cells expressing the receptor. A cytostatic agent is any agent that inhibits cell proliferation and stops cells in the cell cycle, and a cytotoxic agent refers to any agent that causes cell death. An EGFR inhibitor is a molecule that binds to the receptor and thereby inhibits cell division signals elicited by the natural ligand of the receptor. Surprisingly, the authors of the present invention have discovered a population of EGFR inhibitors that, because of their specific binding to EGFR, result in a physiological balance between the active and inactive configurations of the receptor. Maintain a basic level of receptor signaling. This basal level of autophosphorylation of the receptor stops the cell in one phase of the cell cycle. The clinical application of this discovery is that we can design an anti-tumor drug targeting EGFR that does not cause tumor cell death but exhibits biological control of tumor growth. Furthermore, this type of antitumor drug has the advantage of not causing serious adverse reactions such as skin rashes reported by EGFR inhibitors having cytotoxic effects on tumors. The present invention describes an EGFR inhibitor class-8-201011046 having cytostatic activity, characterized in that the inhibitor class recognizes domain I or domain III (preferably domain III) of the extracellular region of EGFR, and The binding of the natural ligand of the receptor is inhibited, thereby inhibiting cell division signals induced by the ligands. These bind to domain I or III of the extracellular region of EGFR to compete for binding to the natural ligand but still allow the receptor to adopt its activated configuration and form homodimers or heterodimers that maintain the degree of basal phosphorylation. The molecule is a scorpion that has cytostatic activity against cells expressing EGFR, and thus these molecules can be used as potential therapeutic agents for tumors expressing the membrane receptor. The authors of the present invention have also found that molecules capable of inhibiting cell division signals triggered by EGF and other ligands and maintaining a basal level of autophosphorylation of the receptor (preferably monoclonal antibodies) are used as malignant tumors. The treatment has a cell growth inhibiting effect on the tumor to prevent the growth of the tumor. Currently, EGFR-targeted therapeutic agents have limited efficacy, including antagonists of cell division ligands and small molecules that inhibit autophosphorylation of the receptor, because their therapeutic agents cause serious adverse reactions in many cases. This treatment must be discontinued. The authors of the present invention have discovered molecules that target EGFR as a therapeutic target that inhibits tumor growth but does not produce serious adverse reactions that have occurred in clinically tested EGFR signaling inhibitors. The cell division activity induced by the natural ligand of EGFR is inhibited, but autophosphorylation of the EGFR is not completely inhibited. As mentioned previously, EGFR has two equilibrium configurations, an activated configuration and an inactive configuration on the cell membrane. When the receptor adopts its activated configuration, its structure-9- 201011046 domain II can interact with the same domain in other EGFR molecules (or other members of the EG FR family) to form a homodimer or A heterodimer, and thus an autophosphorylation of the intracellular domain. This autophosphorylation triggers a cascade of events required to maintain the survival of the cell. It is estimated that more than 90% of EGFR molecules adopt a more energy-efficient, inactive configuration when the cell division ligand is absent, whereas 5 to 10% of the receptor molecules adopt their activated configuration and thus maintain phosphorylation . The degree of phosphorylation is not sufficient to cause cell division, but it allows cells to survive. According to φ, the authors of the present invention, using drugs that completely inhibit autophosphorylation of the receptor, cause a large number of cell deaths, including tumors and normal tissues exhibiting EGFR, indicating the cause of serious adverse reactions. However, the use of the target inhibitor of the present invention results in inhibition of cell division activity and thus tumor growth, but maintenance of receptor phosphorylation which is essential for cell survival. The first drug will produce a cytotoxic effect with significant tumor regression, but it can also cause serious adverse reactions and early tumor recurrence. The target drug of the present invention is unlikely to produce a large amount of cell death accompanying the abrupt tumor regression; on the contrary, it has a disease-stable state due to its cell growth inhibitory effect on sputum tumors, and does not cause a significant adverse reaction to normal tissues. Furthermore, the authors of the present invention have also found that these EGFR inhibitors having cytostatic activity retain the properties of radiosensitizers and chemosensitizers which have been described in EGFR inhibitors having cytotoxic activity. Based on the experimental results, the authors of the present invention claim that the molecule targeting EGFR is the scope of the patent application, and the activity of the molecule lies in the biological control of tumor growth, whether they are treated with a single treatment or with a chemical or radiotherapy group -10- 201011046 Combined method of investment. Thus, 'the object of the invention is that such natural or synthetic EGFR ligands' are preferably natural, and even more preferably monoclonal antibodies, which inhibit the binding of EGF to the extracellular domain of the receptor. However, it does not interfere with receptor dimerization, and thus does not affect the underlying degree of autophosphorylation. Another object of the invention is a method for treating a cancer patient, the method comprising administering a pharmaceutical compound comprising an EGFR ligand, preferably the EGF antagonist but not the basal receptor Autologous phosphorylated monoclonal antibodies. Another object of the present invention is a method for designing and selecting a ligand which is preferably a monoclonal antibody having a cell growth inhibiting action to prevent malignant growth. DETAILED DESCRIPTION OF THE INVENTION The EGFR inhibitor of the present invention comprises a natural or synthetic moiety which binds to domain I or domain III of the extracellular domain of the epidermal growth factor receptor, preferably a structure and structure. In domain III binding, the molecule is characterized by inhibition of the nature of the binding of the natural ligand and additionally allows the receptor to adopt its activated configuration. Surprisingly, the inhibitor recognizing the receptor in this manner has a cytostatic effect on the cells expressing the receptor, which is different from the inhibitors which have been cytotoxic to the cells described in the prior art. The method for designing the therapeutic agent of the present invention is based on the results obtained by the author of the present invention, which supports the interaction of the agent with the EGFR receptor -11 - 201011046 Model of action. These EGF antagonist drugs have a therapeutic effect on cancer and have advantages over the previously described EGF antagonist drugs. In a preferred embodiment, the therapeutic agents may be EGFR-targeted monoclonal antibodies, preferably monoclonal antibody hR3. The humanized anti-system is described in detail in the previously mentioned patent application EP 0712863 B1 and U.S. Patent 5,891,996, and in some scientific publications, for example, Mateo et al., Immunotechnology 3; 71-81, 1997. Detailed methods for obtaining such antibodies are also described in these documents. The interaction model described by the inventors also explains the results of clinical trials from cancer patients in which the therapeutic effects of the target agents of the present invention are evaluated. It is known that the interaction of EGF with the extracellular domain of EGFR induces a cascade of signal transductions that trigger the cell division of the growth factor. Autophosphorylation of the EGFR intracellular domain is one of the first biochemical events in this signaling cascade. In order for autophosphorylation to occur, the extracellular region of the receptor must first dimerize. It is also known that EGF simultaneously binds to domains I and III of the extracellular region of EGFR to stabilize the activated receptor configuration, wherein domain II is prepared to bind to the second monomer to form a dimer. . All EGFR-targeted monoclonal antibodies that have been clinically tested and have demonstrated therapeutic effects on tumors are molecules that act as EGF antagonists by inhibiting receptor dimerization and subsequent phosphorylation. Most of these antibodies inhibit the dimerization of the receptor by creating a steric hindrance that prevents the formation of the activated receptor. Other antibodies bind directly to domain II of EGFR 201011046, thus directly inhibiting this dimerization. Surprisingly, the authors of the present invention have discovered that blocking EGF binding to its receptor thereby inhibiting the cell division signal produced by such a ligand, while at the same time allowing the receptor to adopt its dimerizable activation configuration is possible, thus Allows basic levels of receptor phosphorylation. It is known in the art that EGFR molecules on the cell membrane are found to exhibit a balance between an activated configuration and an inactive configuration. Studies have shown that between 5% and 10% of membrane receptors exhibit an activated configuration. Thus, the acceptor molecule of this moiety can form a dimer and undergo subsequent phosphorylation. This basal level of phosphorylation of the receptor is not sufficient to trigger cell proliferation, but is necessary for cell survival to maintain quiescence. Thus, the use of molecules having the features described herein will have great utility in the treatment of tumors that exhibit EGFR. The therapeutic use of the target molecule of the present invention inhibits tumor proliferation induced by EGFR while preventing adverse reactions caused by currently used EGF antagonists. In a therapeutic agent having the properties described herein, a monoclonal antibody against the extracellular region of EGFR is a preferred embodiment of the invention, as previously described in US 5,89 1,996 and EP 07 1 2863. The humanized monoclonal antibody hR3 is particularly preferred. Even though there are prior art therapeutic agents that recognize the same target, the therapeutic and therapeutic features of the therapeutic agents of the present invention provide therapeutic advantages over those previously described. The invention also relates to a method of inhibiting EGF-dependent tumor growth characterized by the effect of administering a therapeutic dose of a therapeutic agent which induces a steady state of disease rather than a surprising tumor regression. Other objects of the invention are therapeutic compositions comprising at least one aqueous solution of such cytostatic inhibitors, which compositions are useful for treating tumors or other diseases associated with EGFR dysregulation. In the pharmaceutical composition of the present invention, the concentration of the inhibitor (particularly the antibody) is in the range of 10 to 200 mg/ml, more specifically in the range of 50 to 150 mg/ml. Furthermore, the present invention encompasses such EGFR inhibitors (particularly the humanized monoclonal antibody hR3 (Nimotuzumab) in combination with radiation therapy or other therapeutic agents such as chemotherapeutic agents for the treatment of malignant tumors Use according to the present invention, the administration of the target inhibitor of the present invention may be oral, parenteral (intravenous or intramuscular), topical, transdermal or by inhalation. Another object of the present invention is a A method for selecting an EGFR inhibitor having cytostatic activity. The method is mainly based on the inclusion of propidium iodide in a cell according to the embodiment of the present invention, wherein the cell is previously subjected to permeability treatment. And fixed. φ [Embodiment] Example Example 1: crystal structure of hR3 Fab fragment The crystal structure of the hR3 Fab fragment is determined by 2.5A resolution, and has been refined to crystallographic R factor/free r factor respectively. 21.5 % and 28.8% of good stereochemistry. The overall crystal structure of hR3 Fab is similar to that of other Fab fragments, 14 - 201011046, but two of them are in the variant domain of the heavy chain. Except for the features. An obvious feature relates to the CDR1 of the heavy chain, which does not correspond to any of the basic configurations of the CDR described, but instead presents a short alpha helix at the bottom of the loop. Figure 1 shows the hR3 Fab fragment Representative crystal structure in which the CDR1 characteristics of the heavy chain are emphasized. The crystal structure of the hR3 variant region is compared with the structure of all similar fragments deposited in the protein database as of May 2007 using a computer program. Of these, only two antibodies have similar characteristics at the bottom of CDR H1 (PDB code Ib2w ([lb2w]) and 2 cmr ([2 cmr]). One of the two structures corresponds to the implantation of a humanized antibody with CDRs. 2: Antibody-receptor interaction assay using the Biacore device, the hR3 Fab fragment binds to the extracellular domain of the epidermal growth factor receptor (EGFR) with a dissociation constant (KD) of 2.1 x 1 〇·8 Μ binding. In this assay, the commercial antibody cetuximab © (Cetuximab) was used as a control. The KD値 obtained from the cetuximab Fab fragment was 1.8 χ1〇·9 Μ, similar to the previously reported 尼. The KD of the monoclonal antibody is one higher than the one. The magnitude is mainly due to its & „ too low, and its illusion 7/ is only slightly lower (Table 1). The difference in affinity between these two antibodies may explain their differences in biological effects, especially This is the difference in the adverse effects of the two antibodies used to systematically treat cancer patients. -15- 201011046 Table 1: ____
Fab_K0„(UMs、 Koff(l/s)_KD(M) hR3 5.2xl04 1 .1 x 1 O'3 2.1 x 1 O'8 西妥昔單抗 3·1χ 1 06 5.8xl〇-3 1.8xl〇-9 此外，hR3被發現會與西妥昔單抗競爭與EGFR之結 合。結合抑制試驗係藉由利用西妥昔單抗之ELISA技術進 行，已知西妥昔單抗與EGFR之結構域III專一性及排他 性地結合。在此實驗中，ELISA板係以EGFR之胞外區域 _ 包被，接著與固定濃度之生物素化hR3 Fab片段及不同濃 度之西妥昔單抗Fab片段共同培養。結果觀察到，由該生 物素化hR3 Fab片段所提供之信號（以鹼性磷酸酶共軛之 鏈黴親和素顯色）隨西妥昔單抗Fab之濃度增加而減少。 此外，利用過度表現EGF受體之 A431細胞進行 FACS實驗。該細胞係與不同濃度比例之hR3及西妥昔單 抗Fab片段共同培養，類似 ELISA實驗之情況。該等 FACS實驗證實，hR3和西妥昔單抗競爭與EGFR之結合 〇 〇 由ELISA及FACS所得到之結果顯示，該二種抗體所 辨識之表位重疊或彼此非常接近，且二種抗體與EGFR同 時結合是不可能的。根據這些結果以及從先前技藝得知之 西妥昔單抗係與EGFR之結構域ΠΙ結合，我們可能推論 出hR3係與和西妥昔單抗相同之EGFR結構域也就是結構 域III結合之結論。 -16 - 201011046 實施例3:抗體一受體複合體之模型 hR3-EGFR胞外區域複合體之理論模型係根據hR3 Fab片段之晶體結構以及來自蛋白質資料庫之二個EGFR 結構（代號1YY9及IIVO)加以建構。該模型之建構首先 利用RosettaDock程式對接該二個分子，然後利用NAMD 程式在水箱中進行分子動力學模擬，以最佳化該複合體之 結構。 〇 該得到之模型係顯示於圖1。圖A顯示該模型之全景 ，EGFR呈現其不活化構型，其中結構域I及III之間的距 離遙遠。圖B亦顯示該模型之全景，但是EGFR呈現其活 化構型，其中結構域I及ΠΙ靠近，形成對配位體具高親 和性之結合位置。此圖證明根據我們的模型，hR3能與 EGFR結合而不抑制二聚化及後續信號傳遞所需之活化構 型。圖C放大顯示hR3/EGFR介面之分子交互作用。 圖2A顯示hR3如何與西妥昔單抗競爭與EGFR結構 ® 域III之結合，因爲彼等所辨識之表位重叠’然而圖2B顯 示抗體hR3阻斷EGF與該受體之結合。 實施例4 :與EGFR之結構域I結合之Fv抗體片段之理論 模型 該模型（如圖3所示）係藉由手動對接Fv抗體片段 之結構與結構域1上之選擇區域而獲得’其中結合之抗體 允許該受體採取其活化構型’也就是能讓結構域111接近 結構域I，使結構域II得以進行二聚化。另一方面，該 -17- 201011046 EGF (在圖3中以紅色表示）無法插入其結合位置因爲該 抗體形成立體阻礙（在圖3中可清楚看見二個結構間之重 叠）。EGFR與EGF之複合體之結構係得自蛋白質資料庫 代號1IVO，而Fv片段係由分子模型建構。 實施例5:抗EGFR單株抗體對腫瘤細胞A431及H125之 細胞毒性作用 A431及H125細胞（2xl05 )係生長於含有10%胎牛 φ 血清（FBS ) DMEN : F12培養基之24孔板中。12小時後 ，該細胞係於含有人EGF(500皮克/毫升）之1% FBS DMEN : F12培養基中經單株抗體西妥昔單抗（濃度7至 175奈莫耳）或hR3(濃度70至1750奈莫耳）之處理， 接著培養48小時。重複該相同處理及48小時培養一次。 之後，也就是開始該處理後之96小時，藉由碘化丙啶（ 10微克/毫升）染色並以FACS測定細胞死亡之量。未經 處理之細胞被包括在該實驗中以作爲最低死亡對照組。 在二個細胞系中，所有測試濃度之西妥昔單抗相較於 hR3誘發較高程度之死亡（圖4)。應注意的是，即使使 用高於西妥昔單抗10倍濃度之hR3仍得到該結果，因此 彼等於親和性上之差異可由濃度上之差異補償。 實施例6 :抗EGFR單株抗體對腫瘤細胞A431及H125之 細胞毒性與細胞生長抑制效應 A43 1及H125細胞（2.5X105)係生長於含有10%胎牛 -18- 201011046 血清（FBS) DMEN: F12培養基之6孔板中。12小時後 ，該細胞係於含有人EGF(500皮克/毫升）之1% FBS DMEN : F12培養基中經單株抗體西妥昔單抗或hR3(濃 度7至175奈莫耳）或EGFR酪胺酸激酶抑制劑AG1478 (濃度10微莫耳）之處理，接著培養48小時。重複該相 同處理及48小時培養一次。爲了分析經處理96小時後之 細胞週期及DNA段裂（晚期細胞凋亡之標記），該細胞 ❹ 於4 °C以甲醇：丙酮（4: 1)之混合液固定並以碘化丙啶 (400微克/毫升）及RNAse ( 100微克/毫升）染色。利用 FACS進行分析，收集至少20,000個事件。該資料係利用 WinMDI2.8 及 ModFit3.0 程式處理。 如圖5所示，相較於未處理之細胞，該二種單株抗體 (西妥昔單抗：175奈莫耳，hR3: 1750奈莫耳）在A43 1 (A)及H125(B)細胞中誘發類似之G0-G1期細胞數增 加及G2-M及S期中之細胞相對減少。該效應類似用來作 ©爲細胞週期停止之陽性對照之AG1478抑制劑所獲得者。 表1 ( A431 )及2 ( H125 )顯示在該實驗所使用之廣泛範 圍之hR3及西妥昔單抗濃度中，不同細胞週期之期中之細 胞百分比。 然而，在該等抗體誘發細胞凋亡之能力方面，雖然該 二種抗體對AW1及H125細胞具有類似之抗增生效應， 西妥昔單抗在所有測試濃度中相較於hR3誘發較高百分比 之細胞凋亡細胞（圖5，表2及3)。 201011046 A431 細麵期(％) 無處理 AG1478 西妥昔單抗(奈莫耳） 尼妥珠單抗(奈莫耳） 10微莫耳 7 35 70 140 175 70 350 700 1400 1750 G〇/G, 46.21 79.82 56.57 58.08 62.19 64.87 70.89 56.08 58.98 60.32 64.68 70.01 G2/M 8.89 3.11 8.01 8.04 7.46 6.09 5.03 8.12 7.87 7.98 6.21 5.12 S 44.90 17.06 35.42 33.88 30.35 29.04 24.08 35.08 33.17 31.7 29.11 24.87 凋亡細胞(％) 3.50 42.75 3.96 10.78 19.67 25.45 31.19 3.62 3.94 4.07 4.65 5.50Fab_K0„(UMs, Koff(l/s)_KD(M) hR3 5.2xl04 1 .1 x 1 O'3 2.1 x 1 O'8 Cetuximab 3·1χ 1 06 5.8xl〇-3 1.8xl〇 -9 In addition, hR3 was found to compete with cetuximab for binding to EGFR. Binding inhibition assays were performed by ELISA using cetuximab, known as cetuximab and EGFR domain III Specificity and exclusive binding. In this experiment, the ELISA plate was coated with the extracellular region of EGFR, followed by co-culture with a fixed concentration of biotinylated hR3 Fab fragments and different concentrations of cetuximab Fab fragments. As a result, it was observed that the signal provided by the biotinylated hR3 Fab fragment (developed with alkaline phosphatase-conjugated streptavidin) decreased as the concentration of cetuximab Fab increased. The AGF cells of the EGF receptor were subjected to FACS experiments. The cell lines were co-cultured with different concentrations of hR3 and cetuximab Fab fragments, similar to the ELISA assay. These FACS experiments confirmed that hR3 competes with cetuximab. Binding to EGFR, results obtained by ELISA and FACS showed that the two antibodies were The recognized epitopes overlap or are very close to each other, and simultaneous binding of the two antibodies to EGFR is not possible. Based on these results and the prior art know-how of cetuximab binding to the domain of EGFR, we may infer The hR3 line is identical to the EGFR domain identical to cetuximab, which is the binding of domain III. -16 - 201011046 Example 3: Model of antibody-receptor complex Theoretical model of hR3-EGFR extracellular domain complex Based on the crystal structure of the hR3 Fab fragment and the two EGFR structures (codes 1YY9 and IIVO) from the protein database, the model was constructed by first docking the two molecules with the RosettaDock program and then using the NAMD program to perform the molecule in the water tank. Kinetic simulations to optimize the structure of the complex. The resulting model is shown in Figure 1. Panel A shows a panoramic view of the model, EGFR exhibits its inactive configuration, where domains I and III The distance is far away. Figure B also shows a panoramic view of the model, but EGFR exhibits its activated configuration, in which domain I and ΠΙ are close together, forming a high affinity for the ligand. Binding position. This figure demonstrates that according to our model, hR3 binds to EGFR without inhibiting the activation configuration required for dimerization and subsequent signaling. Figure C shows a magnified molecular interaction of the hR3/EGFR interface. Figure 2A shows hR3 How to compete with cetuximab for binding to EGFR structure® domain III because they recognize epitope overlaps. However, Figure 2B shows that antibody hR3 blocks EGF binding to this receptor. Example 4: Theoretical model of Fv antibody fragment binding to domain I of EGFR. This model (shown in Figure 3) was obtained by manually docking the structure of the Fv antibody fragment with the selected region on domain 1. The antibody allows the receptor to adopt its activated configuration', that is, to allow domain 111 to be close to domain I, allowing domain II to dimerize. On the other hand, the -17-201011046 EGF (indicated in red in Figure 3) could not be inserted into its binding position because the antibody formed a steric hindrance (the overlap between the two structures is clearly seen in Figure 3). The structure of the complex of EGFR and EGF was obtained from the protein database code 1IVO, and the Fv fragment was constructed from a molecular model. Example 5: Cytotoxicity of anti-EGFR monoclonal antibodies against tumor cells A431 and H125 A431 and H125 cells (2xl05) were grown in 24-well plates containing 10% fetal bovine φ serum (FBS) DMEN:F12 medium. After 12 hours, the cell line was subjected to monoclonal antibody cetuximab (concentration 7 to 175 nmer) or hR3 (concentration 70) in 1% FBS DMEN:F12 medium containing human EGF (500 pg/ml). Treatment to 1750 Naim) followed by incubation for 48 hours. This same treatment was repeated and cultured for 48 hours. Thereafter, 96 hours after the start of the treatment, staining with propidium iodide (10 μg/ml) and measuring the amount of cell death by FACS. Untreated cells were included in the experiment as the lowest death control group. In both cell lines, all tested concentrations of cetuximab induced a higher degree of death than hR3 (Figure 4). It should be noted that even if hR3 is used at a concentration 10 times higher than that of cetuximab, the difference in affinity can be compensated for by the difference in concentration. Example 6: Cytotoxicity and cell growth inhibitory effect of anti-EGFR monoclonal antibody against tumor cells A431 and H125 A43 1 and H125 cells (2.5X105) were grown in 10% fetal bovine-18- 201011046 serum (FBS) DMEN: F12 medium in a 6-well plate. After 12 hours, the cell line was subjected to monoclonal antibody cetuximab or hR3 (concentration 7 to 175 nmer) or EGFR cheese in 1% FBS DMEN:F12 medium containing human EGF (500 pg/ml). Treatment with the amino acid kinase inhibitor AG1478 (concentration 10 micromoles) followed by incubation for 48 hours. This same treatment was repeated and cultured for 48 hours. To analyze the cell cycle and DNA segmentation (marker of late apoptosis) after 96 hours of treatment, the cells were fixed at 4 °C with a mixture of methanol:acetone (4:1) and propidium iodide ( 400 μg/ml) and RNAse (100 μg/ml) staining. Use FACS for analysis to collect at least 20,000 events. This data is processed using WinMDI2.8 and ModFit3.0 programs. As shown in Figure 5, the two monoclonal antibodies (cetuximab: 175 nmer, hR3: 1750 nmer) are at A43 1 (A) and H125 (B) compared to untreated cells. The cells induced an increase in the number of G0-G1 phase cells and a relative decrease in cells in the G2-M and S phases. This effect is similar to that obtained by AG1478 inhibitor, which is a positive control for cell cycle arrest. Tables 1 (A431) and 2 (H125) show the percentage of cells in different cell cycle periods in the broad range of hR3 and cetuximab concentrations used in this experiment. However, in terms of the ability of these antibodies to induce apoptosis, although the two antibodies have similar antiproliferative effects on AW1 and H125 cells, cetuximab induces a higher percentage of all tested concentrations compared to hR3. Apoptotic cells (Fig. 5, Tables 2 and 3). 201011046 A431 Fine noodle period (%) No treatment AG1478 Cetuximab (Nymole) Nimotuzumab (Nemo) 10 micromoles 7 35 70 140 175 70 350 700 1400 1750 G〇/G, 46.21 79.82 56.57 58.08 62.19 64.87 70.89 56.08 58.98 60.32 64.68 70.01 G2/M 8.89 3.11 8.01 8.04 7.46 6.09 5.03 8.12 7.87 7.98 6.21 5.12 S 44.90 17.06 35.42 33.88 30.35 29.04 24.08 35.08 33.17 31.7 29.11 24.87 Apoptotic cells (%) 3.50 42.75 3.96 10.78 19.67 25.45 31.19 3.62 3.94 4.07 4.65 5.50
表2。經不同濃度之西妥昔單抗或hR3處理之A431細胞 之細胞週期及凋亡細胞之百分比分佈。 H125 細胞週期(％) 無處理 AG1478 10微莫耳 西妥昔單抗(奈莫：？> 尼妥珠單抗(奈莫耳） 7 35 70 140 175 70 350 700 1400 1750 G〇/G, 50.68 74.55 51.41 55.22 57.69 60.69 69.46 53.89 54.63 55.52 58.34 68.55 G2/M 10.25 8.79 10.02 9.56 9.34 9.22 9.05 11.45 9.89 9.68 9.46 9.28 S 39.07 16.66 38.57 35.22 33.27 30.09 21.54 34.66 35.48 34.80 32.20 22.17 凋亡細胞(％) 7.18 31.67 10.35 16.62 20.15 23.59 25.48 7.24 8.30 10.63 11.40 13.67 表3。經不同濃度之西妥昔單抗或hR3處理之H125細胞 之細胞週期及凋亡細胞之百分比分佈。 ❹ 實施例7:在異種移植無胸腺小鼠中之抗腫瘤活性 無胸腺小鼠（8至10週齡）係得自査理士河（ Charles River )實驗室（德國蘇爾茨費爾德）。該鼠於經 相關機構核准且遵守目前法規標準之場所中以無菌條件飼 養維持，且該鼠之用途係經當地專責機構核准。爲了產生 腫瘤，自生長中之（subconfluent)培養以0.25%胰蛋白酶 及0.05% EDTA處理以收集細胞。添加10%胎牛血清培養 -20- 201011046 基以終止胰蛋白酶消化。只有超過90%存活率之單細胞懸 浮液被用於注射。 每組8隻動物接種來自U87MG細胞系之細胞。107細 胞被經皮下接種至左腹脅，同時2x1 04細胞在立體定位裝 置之協助下被經腦內接種至右大腦半球。在實驗期間監測 小鼠體重。每週測量腫瘤大小三次。腫瘤體積係利用下式 計算：0.5x(大直徑>χ(小直徑）2。相對腫瘤體積（RTV)係 φ 將每天之中位數體積參照第一測量値（設定爲Ο計算。 當對照組之腫瘤重量超過全動物重量之10%時，所有動物 被犧牲。亦測定顱內腫瘤之大小。就此目的而言，收集腦 並急速冷凍於2-甲基-丁烷中。製備連續冷凍切片（10毫 米），並以甲苯酚紫染色。利用顯微鏡（蔡司Axioskop ) 之協助測量腫瘤直徑及周長，接著計算腫瘤體積。 皮下腫瘤被冷凍儲存於-80 °C以供額外分析。所有處 理在腫瘤注射後3天開始。動物每週以單株抗體hR3或西 ® 妥昔單抗（50毫克/公斤/劑量）經腹腔內投予處理三次。 在放射線治療組中，動物暴露至總劑量3.0格雷之全身照 射（TBI )，從腫瘤接種後72小時開始，在3週期間分成 每週1 · 0格雷。其他組別之動物係經抗體加放射線治療之 組合處理。在這些組別中，抗體係於放射線治療前6小時 投予。另一有10隻動物之對照組被包括於實驗中。該些 動物接受PBS以取代抗體。 圖6顯示由抗EGFR單株抗體造成人腫瘤細胞系 U87MG之敏感化，該細胞系經皮下異種移植至無胸腺 201011046 NMRI小鼠。該抗體係於三週期間以50毫克/公斤/劑量每 週三次經腹腔內投予。以放射線治療之動物接受3.0格雷 之總劑量，在3週期間分成每週1.0格雷。抗體之投予係 以黑色箭頭表示，放射線以虛線箭頭表示。在所示時間測 定腫瘤體積。相較於僅接受放射線治療及未經治療之動物 ，放射線與抗體之組合治療導致顯著（p<〇. 05)延緩腫瘤 生長。相較於單獨以抗體治療之組，與hR3之組合亦延緩 腫瘤生長。使用克魯斯卡-瓦立斯檢定；在該圖中，顯示 φ 統計差異性之符號如下：（*)相較於PBS具顯著性，（+ )相較於單獨抗體具顯著性，（〇）相較於放射線具顯著 性。 圖7顯示由抗EGFR單株抗體所產生之人腫瘤細胞系 U8 7MG之敏感化，該細胞系經原位異種移植至NMRI裸鼠 。所有治療（hR3、西妥昔單抗、放射線治療、hR3加放 射線治療、西妥昔單抗加放射線治療及PBS )始於腫瘤接 種後3天。該抗體係於三週期間以50毫克/公斤/劑量每週 @ 三次經腹腔內投予。以放射線治療之動物接受3.0格雷之 總劑量，分成每週1.0格雷。來自接受抗體加放射線治療 之小鼠經分析之腦切片顯示腫瘤大小顯著減少（p<〇.05 ) 。相較於僅接受放射線治療，hR3與放射線治療之組合導 致腫瘤大小顯著降低（p<〇.〇5 )。使用曼-惠特尼檢定； 在此圖中，顯示統計差異性之符號如下：（* )相較於 PBS具顯著性，（+ )相較於放射線具顯著性。 -22- 201011046 血管形成（CD31/PECAM-1) 使腫瘤樣本於室溫中解凍10分鐘，且固定於3.7%三 聚甲醛中15分鐘。之後，內源性過氧化酶係以0_〇3%過 氧化氫（加州卡賓特利亞達可（Dako)公司）阻斷1 5分鐘 ，接著該樣本於室溫中與經1: 100稀釋之抗CD31/ PECAM01 一級抗體培養2小時。經過清洗後，樣本與該 對應之HRP共軛二級抗體培養30分鐘以偵測抗體-抗原 e 交互反應。最後，切片以3,30-二胺基聯苯胺（DAB )( 加州卡賓特利亞達可（Dako)公司）作爲發色體檢視，該樣 本經固定並分析CD3 1染色。代表性腫瘤切片以40倍目鏡 放大之光學顯微鏡（蔡司 Axioskop 40 )識別。在腫瘤中 之內皮微血管面積係以從5個顯微鏡視野所得到之平均値 估計。內皮微血管總面積係利用軟體Axio Vison 4.5 (蔡 司）計算。以hR3處理導致血管面積顯著減少（ρ<〇·〇ΐ) (圖 8 )。 O CD133。爲了測定CD 133之表現，使腫瘤樣本於室溫 中以丙酮固定15分鐘。之後’內源性過氧化酶係以 0 · 0 3 %過氧化氫（加州卡賓特利亞達可公司）阻斷3 0分鐘 ，非專一性結合係以2 0 %胎牛血清於室溫中阻斷2 0分鐘 。之後，該樣本於室溫中與經1:10稀釋之抗CD 133/1 AC133 —級抗體（美天旎生技）培養1小時。接著該樣本 經過清洗，並與過氧化酶共軛鏈黴親和素於室溫中培養i 小時。最後’腫瘤切片以DAB發色體（加州卡賓特利亞 達可公司）檢視，該樣本經固定並分析CD133染色。代表 -23- 201011046 性腫瘤切片以40倍目鏡放大之光學顯微鏡（日本奧林巴 斯）檢視。各分析組別之CD 133陽性細胞百分比係以觀察 到最強染色之5個視野所對應之切片中之陽性細胞平均數 決定。 圖9顯示在經hR3(h-R2 3)、西妥昔單抗（C225) 、該等抗體之一與放射線之組合、或單獨放射線（RT)處 理之 U87MG人腫瘤異種移植之無胸腺NMRI小鼠中， CD133表現之免疫化學分析結果。相較於單獨放射線治療 φ 之處理，以hR3處理導致CD 133陽性細胞百分比顯著降 低（ρ<0·05 )。相較於單獨以放射線治療處理及未經處理 之動物，hR3與放射線治療之組合顯著降低此組中之 CD 133陽性細胞百分比（p<0.05 )。使用曼-惠特尼檢定； 在此圖中，顯示統計差異性之符號如下：（*)相較於 PBS具顯著性，（+)相較於放射線具顯著性。 統計分析。利用G r a p h P A D程式第4 · 0版（美國加州 聖地牙哥GraphPAD )計算平均値及標準差。統計分析係 φ 利用相同程式進行。組別間差異之顯著性利用曼一惠特尼 檢定及部恩（Dunn)多重比較檢定評估。若ρ<〇.則認 爲差異具顯著性。 實施例8 :單株抗體hR3 (尼妥珠單抗）與低劑量卡銷（ carboplatin)之組合之抗腫瘤效應 在該等實驗中’無胸腺NMRI小鼠（8至1〇週齡）經 皮下接種人來源之非小細胞肺癌（NSCLC )腫瘤。第〇天 -24- 201011046 時，動物接種腫瘤片段。當腫瘤可被偵測時（接種後10 至12天），動物於6週期間以50毫克/公斤之單株抗體 hR3每週三次及/或每週劑量50毫克/公斤之卡鉑進行腹腔 內注射。P B S係用來作爲對照。每週測量二次腫瘤之大直 徑及小直徑，且該腫瘤體積係利用公式VT = 0.5x(小直徑）2 X大直徑計算。相對腫瘤體積係根據下式計算： RTV = VT/VT no*。圖10顯示相對腫瘤體積及彼之標準差 〇 【圖式簡單說明】 圖l:hR3/EGFR複合體之模型《Α)卡通式模型全景 ，顯示呈現其不活化構型之EGFR胞外區域（EGFR爲藍 色，hR3之Fv片段爲黃綠色）《Β)類似圖A，但EGFR 之胞外區域係呈現活化構型，其不受hR3結合之阻礙。C )放大hR3與EGFR之結構域III之間介面之交互作用。 ®氫鍵係以虛線表不。 圖2: A) hR3抑制西妥昔單抗之結合。西妥昔單抗之 Fv片段係以紅色表示（卡通圖示）；EGFR爲藍色且hR3 之Fv片段爲黃綠色。B ) hR3抑制EGF之結合，但允許採 取活化受體構型。EGFR及hR3之圖A顏色同；EGF係以 紅色表示。該圖顯示EGF之C端撞到尼妥珠單抗之輕鏈 ，EGFR之結構域I接近抗體但不衝突。 圖3 :與EGFR (藍色）之結構域I結合之Fv抗體片 段（綠色）之理論模型。該抗體阻止EGF (以紅色表示） -25- 201011046 之結合，但允許該活化EGFR構型。 圖4 : hR3相較於西妥昔單抗較不具細胞毒性。A4 3 1 及H125細胞係經西妥昔單抗（濃度7至175奈莫耳）或 hR3 (濃度70至1 750奈莫耳）處理96小時，之後以碘化 丙啶染色，然後由FACS分析。各長條代表計算3孔之死 亡細胞百分比之平均値。該些實驗重複三次並產生類似之 結果。 圖5: hR3及西妥昔單抗對A4 31及H125細胞具有類 參 似之細胞生長抑制功效，然而西妥昔單抗在這些細胞誘發 較高之細胞凋亡效應。A431及H1 25細胞係經西妥昔單抗 (175奈莫耳）、尼妥珠單抗（ 1750奈莫耳）或AG1478 抑制劑（1 〇微莫耳）處理96小時，然後以碘化丙啶染色 並經FACS分析。各圖顯示二個區域。在虛線內之區域對 應活細胞。各細胞週期中之該等細胞之百分比被顯示。在 實線內之區域對應細胞凋亡之細胞，彼等佔細胞總數之百 分比亦被顯示。該些實驗重複三次並產生類似之結果。 β 圖6:由抗EGFR單株抗體造成人腫瘤細胞系U87MG 之敏感化，該細胞系經皮下異種移植至無胸腺NMRI小鼠 。始於腫瘤接種三天後之處理包括：〇hR3 ( h-R3 ) 、△ 西妥昔單抗（C225 ) 、放射線治療（RT) 、#hR3加放 射線治療（h-R3 + RT ) 、▲西妥昔單抗加放射線治療（ C225 + RT)、及□作爲對照之 PBS ( PBS )。 圖7 :由抗EGFR單株抗體所產生之人腫瘤細胞系 U 8 7MG對放射線治療之敏感化，該細胞系經原位異種移 -26- 201011046 植至無胸腺NMRI小鼠。 圖8: CD31於U87MG人腫瘤中之表現之免疫化學分 析，該腫瘤經異種移植至無胸腺NRMI小鼠，並以hR3 ( h-R3 )、西妥昔單抗（C225 )、放射線治療（RT ) 、hR3 加放射線治療（h-R3+RT ) 、C225加放射線治療（ C225 + RT )、或 PBS 處理。 圖9: CD133於U87MG人腫瘤中之表現之免疫化學 φ 分析，該腫瘤經異種移植至無胸腺NRMI小鼠，並以hR3 (h-R3 )、放射線治療（RT ) 、hR3加放射線治療（h- R3+RT )、或 PBS 處理。 圖10:單株抗體hR3與低劑量卡鉑之組合之抗腫瘤 功效。接種人來源之非小細胞肺癌（NSCLC )腫瘤之無胸 腺小鼠係於6週期間以每週三次之5 0毫克/公斤劑量之 hR3抗體及每週50毫克/公斤劑量之卡鉑處理。該圖顯示 相對腫瘤體積與彼之標準差。 -27-Table 2. The cell cycle and percentage distribution of apoptotic cells of A431 cells treated with different concentrations of cetuximab or hR3. H125 Cell cycle (%) No treatment AG1478 10 micromolecoxibumab (Nemo:?> Nimotuzumab (Nemo) 7 35 70 140 175 70 350 700 1400 1750 G〇/G, 50.68 74.55 51.41 55.22 57.69 60.69 69.46 53.89 54.63 55.52 58.34 68.55 G2/M 10.25 8.79 10.02 9.56 9.34 9.22 9.05 11.45 9.89 9.68 9.46 9.28 S 39.07 16.66 38.57 35.22 33.27 30.09 21.54 34.66 35.48 34.80 32.20 22.17 Apoptotic cells (%) 7.18 31.67 10.35 16.62 20.15 23.59 25.48 7.24 8.30 10.63 11.40 13.67 Table 3. Percentage distribution of cell cycle and apoptotic cells of H125 cells treated with different concentrations of cetuximab or hR3. 实施 Example 7: in xenograft athymic mice Antitumor activity Athymic mice (8 to 10 weeks old) were obtained from the Charles River laboratory (Sultsfeld, Germany). The rats were approved by the relevant agencies and complied with current regulatory standards. The site is maintained under aseptic conditions, and the use of the mouse is approved by a local specialized agency. In order to produce tumors, subconfluent culture is 0.25% trypsin. Treatment with 0.05% EDTA to collect cells. Add 10% fetal bovine serum culture-20-201011046 base to stop trypsin digestion. Only single cell suspensions with more than 90% survival were used for injection. Each group of 8 animals was inoculated from U87MG Cells of the cell line. 107 cells were subcutaneously inoculated into the left flank, while 2x1 04 cells were inoculated intracerebrally into the right cerebral hemisphere with the aid of a stereotactic device. Mouse body weight was monitored during the experiment. Tumor size was measured weekly. Three times. The tumor volume was calculated using the following formula: 0.5x (large diameter > χ (small diameter) 2. Relative tumor volume (RTV) system φ The median volume per day was calculated with reference to the first measurement 値 (set to Ο. When the tumor weight of the control group exceeded 10% of the total animal weight, all animals were sacrificed. The size of the intracranial tumor was also determined. For this purpose, the brain was collected and rapidly frozen in 2-methyl-butane. Frozen sections (10 mm) were stained with cresyl violet. Tumor diameter and circumference were measured using a microscope (Zeiss Axioskop), followed by calculation of tumor volume. Subcutaneous tumors were stored frozen. -80 ° C for additional analysis. All treatments started 3 days after tumor injection. Animals were treated intraperitoneally three times a week with monoclonal antibody hR3 or cetuximab (50 mg/kg/dose). In the radiation treatment group, animals were exposed to a total dose of 3.0 Gray's whole body irradiation (TBI), starting at 72 hours after tumor inoculation and dividing into 1 +/- Gray per week for 3 weeks. Animals of other groups were treated with a combination of antibody plus radiation therapy. In these groups, the anti-system was administered 6 hours prior to radiation therapy. Another control group of 10 animals was included in the experiment. These animals received PBS to replace the antibody. Figure 6 shows the sensitization of human tumor cell line U87MG by anti-EGFR monoclonal antibody, which was subcutaneously xenografted into athymic 201011046 NMRI mice. The anti-system was administered intraperitoneally three times a week at 50 mg/kg/dose. Animals treated with radiation received a total dose of 3.0 gres and were divided into 1.0 gres per week for 3 weeks. The administration of antibodies is indicated by black arrows and the radiation is indicated by dashed arrows. Tumor volume was measured at the indicated times. The combination of radiation and antibody resulted in significant (p<0.05) delaying tumor growth compared to animals receiving only radiation therapy and untreated animals. The combination with hR3 also delays tumor growth compared to the group treated with antibodies alone. Using the Kruska-Walris test; in this figure, the sign showing the statistical difference of φ is as follows: (*) is significantly more significant than PBS, (+) is significant compared to the individual antibody, (〇 ) is significant compared to radiation. Figure 7 shows the sensitization of the human tumor cell line U8 7MG produced by anti-EGFR monoclonal antibody, which was xenotransplanted in situ into NMRI nude mice. All treatments (hR3, cetuximab, radiation therapy, hR3 plus radiation therapy, cetuximab plus radiation therapy, and PBS) started 3 days after tumor implantation. The anti-system was administered intraperitoneally at a rate of 50 mg/kg/dose per week for three times. Animals treated with radiation received a total dose of 3.0 gres, divided into 1.0 gres per week. Brain sections analyzed from mice receiving antibody plus radiation therapy showed a significant reduction in tumor size (p<〇.05). The combination of hR3 and radiation therapy resulted in a significant reduction in tumor size compared to radiation therapy alone (p<〇.〇5). Using the Mann-Whitney test; in this figure, the symbols showing statistical differences are as follows: (*) is significant compared to PBS, and (+) is significant compared to radiation. -22- 201011046 Angiogenesis (CD31/PECAM-1) Tumor samples were thawed at room temperature for 10 minutes and fixed in 3.7% paraformaldehyde for 15 minutes. Thereafter, the endogenous peroxidase was blocked with 0_〇3% hydrogen peroxide (Dako, Calif.) for 15 minutes, then the sample was diluted 1:100 at room temperature. The anti-CD31/PECAM01 primary antibody was incubated for 2 hours. After washing, the sample was incubated with the corresponding HRP conjugated secondary antibody for 30 minutes to detect antibody-antigen e interaction. Finally, the sections were examined with 3,30-diaminobenzidine (DAB) (Dako, Calif.) as a color body, and the samples were fixed and analyzed for CD3 1 staining. Representative tumor sections were identified by a 40x eyepiece magnification optical microscope (Zeiss Axioskop 40). The endothelial microvascular area in the tumor was estimated from the mean 値 obtained from 5 microscope fields. The total area of endothelial microvessels was calculated using the software Axio Vison 4.5 (Cai Si). Treatment with hR3 resulted in a significant reduction in vascular area (ρ<〇·〇ΐ) (Fig. 8). O CD133. To determine the performance of CD 133, tumor samples were fixed in acetone for 15 minutes at room temperature. After that, 'endogenous peroxidase was blocked with 0. 03% hydrogen peroxide (Cabbindia Dacco, California) for 30 minutes, and non-specific binding system with 20% fetal bovine serum at room temperature. Block for 20 minutes. Thereafter, the sample was incubated with a 1:10 diluted anti-CD 133/1 AC133-grade antibody (Mitsubishi Biotech) for 1 hour at room temperature. The sample was then washed and incubated with the peroxidase conjugated streptavidin for 1 hour at room temperature. The final 'tumor sections were examined with DAB chromophore (Cabicon, CA) and the samples were fixed and analyzed for CD133 staining. Representative -23- 201011046 Sexual tumor sections were examined with a 40-fold eyepiece magnification optical microscope (Olympus, Japan). The percentage of CD 133 positive cells in each analysis group was determined by the average number of positive cells in the sections corresponding to the 5 fields of view in which the strongest staining was observed. Figure 9 shows the athymic NMRI of U87MG human tumor xenografts treated with hR3 (h-R2 3), cetuximab (C225), one of these antibodies in combination with radiation, or radiation alone (RT). The results of immunochemical analysis of CD133 in mice. Treatment with hR3 resulted in a significant decrease in the percentage of CD 133 positive cells compared to treatment with ray alone (ρ < 0.05). The combination of hR3 and radiation therapy significantly reduced the percentage of CD 133 positive cells in this group compared to radiation treated and untreated animals alone (p<0.05). Using the Mann-Whitney test; in this figure, the signs showing statistical differences are as follows: (*) Significantly compared to PBS, (+) is more significant than radiation. Statistical Analysis. The mean enthalpy and standard deviation were calculated using the G r a p h P A D program version 4.0 (Graph PAD, San Diego, CA). The statistical analysis system φ is performed using the same program. Significant differences between groups were made using the Mann Whitney test and the Dunn multiple comparison test. If ρ < 〇. then the difference is significant. Example 8: Antitumor effect of a combination of monoclonal antibody hR3 (nicotuzumab) and low dose cartridge (carboplatin) in these experiments 'Athymic NMRI mice (8 to 1 week old) subcutaneously Non-small cell lung cancer (NSCLC) tumors from human origin were inoculated. On the day of the second day -24- 201011046, the animals were inoculated with tumor fragments. When the tumor can be detected (10 to 12 days after inoculation), the animal is intraperitoneally administered with 50 mg/kg of monoclonal antibody hR3 three times a week and/or weekly dose of 50 mg/kg carboplatin during 6 weeks. injection. The P B S system was used as a control. The large diameter and small diameter of the secondary tumor were measured weekly, and the tumor volume was calculated using the formula VT = 0.5x (small diameter) 2 X large diameter. The relative tumor volume is calculated according to the following formula: RTV = VT/VT no*. Figure 10 shows the relative tumor volume and the standard deviation of each other [Simplified illustration] Figure 1: Panorama of the cartoon model of the hR3/EGFR complex model, showing the EGFR extracellular domain (EGFR) showing its inactive configuration In blue, the Fv fragment of hR3 is yellow-green. "Β" is similar to Figure A, but the extracellular region of EGFR exhibits an activating configuration that is not hindered by hR3 binding. C) Amplification of the interaction between hR3 and domain III of EGFR. The ® hydrogen bond is indicated by a dotted line. Figure 2: A) hR3 inhibits the binding of cetuximab. The Fv fragment of cetuximab is shown in red (cartoon illustration); EGFR is blue and the Fv fragment of hR3 is yellow-green. B) hR3 inhibits the binding of EGF, but allows the adoption of an activated receptor configuration. Figure A of EGFR and hR3 is the same color; EGF is expressed in red. The figure shows that the C-terminus of EGF hits the light chain of nimotuzumab, and the domain I of EGFR is close to the antibody but does not conflict. Figure 3: Theoretical model of the Fv antibody fragment (green) bound to domain I of EGFR (blue). This antibody blocks the binding of EGF (indicated in red) -25 to 201011046, but allows for the activation of the EGFR configuration. Figure 4: hR3 is less cytotoxic than cetuximab. The A4 3 1 and H125 cell lines were treated with cetuximab (concentration 7 to 175 nmer) or hR3 (concentration 70 to 1 750 nmer) for 96 hours, then stained with propidium iodide and then analyzed by FACS . Each strip represents the average 値 of the percentage of dead cells in the 3-well count. These experiments were repeated three times and produced similar results. Figure 5: hR3 and cetuximab have similar cytostatic effects on A4 31 and H125 cells, whereas cetuximab induces a higher apoptotic effect in these cells. A431 and H1 25 cell lines were treated with cetuximab (175 nmer), nimotuzumab (1750 nmol) or AG1478 inhibitor (1 〇 micromolar) for 96 hours, then with propidium iodide Pyridine staining and analysis by FACS. Each figure shows two areas. The area within the dotted line corresponds to the living cells. The percentage of such cells in each cell cycle is shown. The cells in the solid line correspond to apoptotic cells, and their percentage of the total number of cells is also displayed. These experiments were repeated three times and produced similar results. β Figure 6: Sensitization of human tumor cell line U87MG by anti-EGFR monoclonal antibody, which was subcutaneously xenografted into athymic NMRI mice. Treatments starting three days after tumor inoculation include: 〇hR3 (h-R3), △ cetuximab (C225), radiation therapy (RT), #hR3 plus radiation therapy (h-R3 + RT), ▲West Rituximab plus radiation therapy (C225 + RT), and □ as a control PBS (PBS). Figure 7: Sensitivity to radiation therapy by human tumor cell line U 8 7MG produced by anti-EGFR monoclonal antibody, which was transplanted into athymic NMRI mice by in situ xenograft -26-201011046. Figure 8: Immunochemical analysis of the expression of CD31 in U87MG human tumors xenografted to athymic NRMI mice with hR3 (h-R3), cetuximab (C225), radiation therapy (RT) ), hR3 plus radiation therapy (h-R3+RT), C225 plus radiation therapy (C225 + RT), or PBS. Figure 9: Immunochemical φ analysis of the expression of CD133 in U87MG human tumors, which were xenografted into athymic NRMI mice and treated with hR3 (h-R3), radiation therapy (RT), hR3 plus radiation (h) - R3+RT ), or PBS processing. Figure 10: Antitumor efficacy of a combination of monoclonal antibody hR3 and low dose carboplatin. Athymic mice vaccinated with human-derived non-small cell lung cancer (NSCLC) tumors were treated with a 50 mg/kg dose of hR3 antibody three times a week and a 50 mg/kg dose of carboplatin per week for 6 weeks. The figure shows the relative tumor volume and its standard deviation. -27-