開發具蛋白酶活化特性之前驅抗體藥物降低類風濕性關節炎治療的副作用

Development of a protease-activated antibody that reduces the side-effects of Rheumatoid Arthritis (RA) therapy

李文翰(Wen-Han Lee)

指導教授 ： 鄭添祿

高雄醫學大學/生命科學院/生物醫學暨環境生物學系碩士班/碩士(2016年)

摘要

類風溼性關節炎(Rheumatoid arthritis，RA)為一種長期關節腫脹發炎的自體免疫疾病。目前用來治療類風濕性關節炎的治療型抗體藥物Remicade®藉由中和關節炎區過度表現的腫瘤壞死因子(TNFα)來減緩發炎症狀，但由於抗體藥物缺乏對疾病區抗原的選擇性，導致使用後會中和全身性抗原產生副作用，例如：據FDA指出長期使用anti-TNFα的抗體藥物會使病人免疫力下降容易造成細菌及病毒的感染及併發惡性腫瘤的風險，因此開發出能針對選擇性針對疾病區域、降低藥物副作用的標靶藥物便十分重要。目前我們成功開發一個新型的抗體鎖(Hinge)，此抗體鎖可以藉由屏蔽抗體的結合位抑制抗體與抗原的結合能力，並以蛋白酶受質胜肽(Matrix metalloprotease substrate peptide)連結抗體和抗體鎖，建構具有蛋白酶活化專一性的前驅抗體藥物pro-Remicade。前驅抗體唯有在蛋白酶過度表現的疾病區，抗體鎖才能被移除，回復原有抗體功能。此前驅抗體可以增加抗體藥物對於類風濕性關節炎的疾病區域的選擇性，降低副作用。在實驗中我們成功證明抗體鎖(Hinge)可以有效降低pro-Remicade與抗原結合的能力，並且可以透過MMPs作用移除抗體鎖，回復與抗原結合的能力。接著證實pro-Remicade在MMPs活化後可以有效抑制TNFα的下游產物。在活體中，確認pro-Remicade抗體與Remicade有相似的藥物動力學。我們在自發性產生關節炎的hTNFα基因轉殖鼠中確認pro-Remicade會選擇性活化於關節疾病區，並且可以有效治療小鼠關節發炎的情形。在李斯特菌感染小鼠的模式中，pro-Remicade可以有效降低感染後小鼠的副作用。另外，在臨床上長期使用Remicade會產生的anti-Remicade抗體中和體內抗體將低藥物療效，pro-Remicade可以有效避免anti-Remicade抗體的辨認。我們相信透過抗體鎖可建構出更具有選擇性的前驅抗體藥物(pro-antibody)，做為新型的治療性抗體藥物，對未來各種疾病的治療也具極大助益。

關鍵字

類風溼性關節炎 ； 基質金屬蛋白酶腫瘤壞死因子α ； 前驅抗體藥物

並列摘要

Rheumatoid Arthritis (RA) is a long-lasting autoimmune disorder that primarily affects joints. Remicade® (anti-TNFα) is an effective antibody drug for Rheumatoid Arthritis (RA) therapy. Remicade can neutralize TNFα to releive inflammention in RA. However, the U.S. Food and Drug Administration (FDA) had reported that long-term systemic injection of anti-TNFα Ab. (Remicade®) leads to serious side effects such as severe infections and malignancy. To reduce the side effects and selectively target the disease site, we have successfully developed a novel Antibody-Locker” (Hinge) which could efficiently block the antigen binding site of Ab and inhibit its binding activity. We use a protease substrate peptide as a linker to connect the antibody locker and generate a protease-activated Hinge Remicade(pro-Remicade). The protease substrate peptide can only be hydrolyzed by protease MMPs which are over-expressed in the disease region. Then the pro-antibody can specifically target to the disease region to improve the selectivity of the RA therapy and to reduce side-effect. We successfully demonstrated that the antibody locker (Hinge) can decrease the binding capacity of the pro-Remicade. We also confirm that the pro-Remicade can be cleaved by MMPs treatment which revives its binding capacity. The downstream signaling of TNFα was inhibited by pro-Remicade Ab after cleavage with MMPs. The pro-Remicade have a similar pharmacokinetics compared to Remicade alone in normal mice. We also showed that pro-Remicade can be active specifically in the disease area (paw), and can significantly relieve the inflammation symptoms of hTNFα transgenic mice. In Listeria infection mice model, pro-Remicade can raise the survival rate of infected mice. In clinical, long-term inject Remicade will produce anti-Remicade antibody. Anti-Remicade antibody will neutralize Remicade to reduce the therapeutic effect. Pro-Remicade can avoid anti-Remicade recognized and revive it’s function after MMPs treatment. We believe that this novel strategy of using an Antibody-Locker will provide a break-through in the development of antibody drugs and provides a "new generation of Ab drugs" that makes a selectively targeted therapy possible.

並列關鍵字

(RA) ； Matrix metalloproteinase (MMP) ； TNFα ； pro-antibody drug

參考文獻

Bsargent. "Top 10 Best Selling Biologics of 2014." Genetic Engineering News and Drug Prescribing Information, on March 19, 2015

2. Alamanos, Y., Voulgari, P. V., & Drosos, A. A. (2006, December). Incidence and prevalence of rheumatoid arthritis, based on the 1987 American College of Rheumatology criteria: a systematic review. In Seminars in arthritis and rheumatism (Vol. 36, No. 3, pp. 182-188). WB Saunders.

3. Arend, W. P. (2001, April). Cytokine imbalance in the pathogenesis of rheumatoid arthritis: the role of interleukin-1 receptor antagonist. In Seminars in arthritis and rheumatism (Vol. 30, No. 5, pp. 1-6). WB Saunders.

4. Fiehn, C., Neumann, E., Wunder, A., Krienke, S., Gay, S., & Müller-Ladner, U. (2004). Methotrexate (MTX) and albumin coupled with MTX (MTX-HSA) suppress synovial fibroblast invasion and cartilage degradation in vivo. Annals of the rheumatic diseases, 63(7), 884-886.5. Fleischmann, R. M. "Progressive multifocal leukoencephalopathy following rituximab treatment in a patient with rheumatoid arthritis." Arthritis & Rheumatism 60.11 (2009): 3225-3228.

6. Maini, R. N., Elliott, M. J., Brennan, F. M., & Feldmann, M. (1995). Beneficial effects of tumour necrosis factor‐alpha (TNF‐α) blockade in rheumatoid arthritis (RA). Clinical & Experimental Immunology, 101(2), 207-212.