建立表達人類第九及第十因子之A型血友病小鼠模型作為新興雙特異性抗體藥物之測試平台

Abstract

A型血友病是由凝血第八因子(FVIII)缺乏或功能障礙引起的先天性出血性遺傳疾病，嚴重型患者會出現自發性出血及關節積血等臨床症狀，必須終身接受治療以維持正常生活。然而，大約20-30%的患者會因注射第八因子治療後產生第八因子中和性抗體(FVIII neutralizing antibody)造成治療失效。近年來，隨著雙特異性單株抗體藥物”emicizumab”上市，大幅改善前述的治療困境。類似於第八因子的功能，emicizumab會透過橋接活化的第九因子(FIXa)和第十因子(FX)，使止血功能恢復正常。目前臨床試驗已證明長期使用emicizumab的安全性及療效，但由於emicizumab無法辨識活化與非活化態因子，故只能達到15-20%的第八因子活性，因此患者遭遇急性出血時仍需補充第八因子。據文獻指出，患者出血時給予第八因子治療所誘發抗體的風險高於預防性投予，目前有一臨床試驗正進行收案，旨在評估新診斷未曾接受治療的患者(PUPs)給予emicizumab的同時也預防性投予第八因子，目的在於讓使用emicizumab之PUP，避免暴露於第八因子的時機是發生在出血或手術時，希望藉此減少誘發抗體之風險，然透過臨床試驗獲得數據恐需數年，本篇研究希望建立一小鼠模型來加速取得參考依據。 由於emicizumab會特異性結合人類第九及第十凝血因子，可以使用的動物模型有限，目前唯一被發表的小鼠模型只能用於短期觀察，因此，我利用腺相關病毒(AAV)載體使小鼠長期內源性的表現人類第九及第十因子，實驗室先前已具備攜帶第九因子的AAV載體(AAV-hFIX)。為了製備AAV-hFX，首先，利用反轉錄-聚合酶鏈反應(RT-PCR)方法，獲得第十因子互補DNA(cDNA)，定序出第十因子基因片段後，進行建構表達第十因子之AAV-hFX質體，最後將AAV-hFIX及AAV-hFX注射於兩周大的血友病小鼠，並確認emicizumab可於此模型發揮效果，初步建立一個可用於emicizumab長期試驗的小鼠模型。此外，建立小鼠膝關節損傷模型，以此模擬患者急性出血狀態。將emicizumab測試實驗分為四組；第八因子預防性治療，FVIII需求性治療、合併emicizumab預防性與第八因子預防性治療，及合併emicizumab與FVIII需求性治療，比較不同治療策略對抗體產生風險的影響。結果觀察到，與單獨使用第八因子相比，用emicizumab合併第八因子，無論是第八因子預防性治療還是需求性治療，皆降低第八因子抗體的效價和發生率。此外，emicizumab合併第八因子預防性治療對第八因子抗體的誘發率低於emicizumab合併第八因子需求性治療。本研究顯示emicizumab合併第八因子治療可減少第八因子抗體產生，為臨床治療策略提供參考。另外，本研究建立的小鼠模型未來可用於初步評估emicizumab相關的臨床問題或測試其他新型雙特異性抗體藥物。

Hemophilia A is a hereditary hemorrhagic disorder result from coagulation factor VIII(FVIII) protein dificiency or disfunction. Severe Hemophilia A patients, often present spontaneous bleeding and hemarthrosis, have to receive lifelong treatment to maintain a normal life. However, approximately 20~30% severe hemophilia A patients develop anti-FVIII neutralizing antibodies, termed FVIII inhibitors, which diminish the effect of a FVIII agent and rander the factor replacement therapy ineffective. Recently, with the launch of the bispecific antibody drug emicizumab, the aforementioned treatment dilemma has been greatly improved. Similar to the function of FVIIIa, emicizumab restores normal hemostasis by bridging factor IXa(FIXa) and factor X(FX). Current clinical trials have proven the safety and efficacy of long-term use of emicizumab, but as emicizumab cannot identify the active or inactive state of the factors, it can only achieve 15-20% of FVIII activity, so patients still need to supplement factor VIII when encountering acute bleeding. According to the literature, the risk of antibodies induced by factor VIII therapy is higher than that of prophylactic administration when patients are bleeding. A clinical trial is currently underway to evaluate the use of emicizumab in previously untreated patients (PUPs), and FVIII is also administered prophylactically. The purpose is to make PUPs that use emicizumab can avoid exposure to FVIII during bleeding or surgery, and reduce the risk of inducing antibodies. However, it often takes several years to obtain answers through clinical trials. This study aims to establish a mouse model to speed up the process of acquiring the reference basis. Emicizumab specifically binds both human FIX and FX. Available animal models for testing its efficacies are very limited. The published mouse model can only be used for short-term observation. Hence, I use AAV gene delivery to generate mice that endogenously express human FIX and FX for long term. Our laboratory already has an AAV vector carrying factor IX(AAV-hFIX). To construct AAV-hFX, human FX cDNA was obtained by reverse transcription-PCR, and after DNA sequencing, subcloned into the AAV vector. Finally, both the AAV-hFIX and AAV-hFX was delivered to two-week-old hemophilia A mice, which have demonstrated efficatious for emicizumab function tests, and therefore can be used for long-term trials of emicizumab. Besides, a mouse knee joint injury model was established to simulate the acute bleeding state of hemophilia A patient. Four experimental groups are designed; FVIII prophylaxis or on-demand treatment, group A or B respectively; Emicizumab prophylaxis combimed with FVIII prophylaxis or with FVIII on-demand treatment, group C or D, respectively; to compare the impact of different treatment strategies on the incidence of FVIII inhibitor. As a result, it was observed that prophylactic treatment with emicizumab combined with FVIII, either prophylactic or on-demand treatment, can reduce the titer and incidence of FVIII inhibitors as compared with using FVIII alone. In addition, inhibitors elicited by emicizumab combined with FVIII prophylactic were reduced as compared with emicizumab plus FVIII on-demand. This study suggests that combination of emicizumab and FVIII treatment could reduce FVIII inhibitor induction, which can provide a reference for clinical therapeutic strategies. Also, the mouse model established in this study can be used in the future to initially evaluate clinical problems related to emicizumab or test other newly bispecific antibody drugs.