新型人類CD47抗體開發及其癌症免疫治療應用

Abstract

先前的研究發現癌症細胞表面蛋白CD47與巨噬細胞表面上的SIRP-α蛋白結合會使得癌症細胞抑制巨噬細胞的吞噬作用進而逃脫免疫細胞的攻擊，而這個結合作用被視為髓細胞特有的免疫檢查點 (myeloid specific immune checkpoint)，目前有研究發現利用抗體去阻斷CD47與SIRP-α蛋白結合能夠促進巨噬細胞對癌症細胞的吞噬作用，然而此抗體會造成紅血球凝集而造成安全上的危害。因此抗體若能同時具有功效及安全性的話則會是個具有癌症治療潛力的抗體，為此，在本研究中，先開發了癌症病人噬菌體Fab抗體庫 (cancer specific phage-displayed Fab library)，且以此找尋到具有高親和力及專一性的抗體，之後將CwP1A1, CwP2F12及BrP1F11抗體可結晶區域片段(Fragment crystallizable region)改成IgG4的型式，進一步分析此三個IgG4 format抗體具有抑制CD47與SIRP-α的結合的能力，也能夠誘導巨噬細胞利用吞噬作用對白血病細胞株 (Leukemia cell line) Jurkat及 HL-60進行毒殺作用，並且不會造成抗體依賴型細胞介導的細胞毒性作用(antibody-dependent cell-mediated cytotoxicity, ADCC) 及細胞凋亡(apoptosis)。之後，紅血球凝集實驗結果發現我們所開發之抗體CwP1A1及BrP1F11不同於目前其他開發的抗體，並不會造成紅血球不正常的凝集，因此，之後若進行臨床試驗時則可以大大的降低其副作用風險。最後，在異體移植白血病及三陰性乳癌細胞株個別於重症聯合免疫缺陷小鼠 (SCID mice) 及裸鼠模式上，CwP1A1抗體具有非常好的抑制腫瘤效果，也因此，我們期許在之後能以此基礎去研發、改良出具有更好抑制腫瘤且具有更低副作用的免疫療法藥物。

The CD47/signal regulatory protein alpha (SIRP-) axis is a key molecular interaction that inhibits antitumor activation by macrophages and myeloid cells, thereby acting as a myeloid-specific immune checkpoint. Blocking CD47/ SIRP- interaction promotes phagocytosis of tumor cells. Currently, CD47 antibody in clinical trial showed RBC agglutination and this effect may cause safety risk issue. In this thesis new human CD47 blocking antibodies were identified from a cancer patient antibody Fab library. The anti-CD47 antibodies exhibited high affinity and specificity against recombinant and cell surface CD47, which are able to inhibit the CD47-SIRP- interaction comparable to Hu5F9-G4, which is a humanized CD47 blocking antibody currently under clinical investigations. The human IgG4-reformatted CD47 antibodies, CwP1A1, CwP2F12, and BrP1F11, were generated dependent on the result of affinity screening. The IgG4-reformatted CD47 blocking antibody were confirmed to be effective in treatment of a cancer since it is found that the IgG4-reformatted CD47 blocking antibody treated cancer cells, such as Jurkat-1 or HL-60 cells, and induced robust phagocytosis activity explicated by polarized THP-1 macrophages as well as peripheral blood mononuclear cell (PBMC), but not antibody-dependent cell-mediated cytotoxicity (ADCC) and apoptosis of tested cancer cells, in vitro. Furthermore, CwP1A1 and BrP1F11 did not induce hemagglutination of human RBCs compared to Hu5F9-G4. Finally, CwP1A1 showed the good anti-tumor activities in both hematologic and solid tumor cell lines mouse xenograft model. In the future, we will further confirm the safety of these antibodies and further study these antibodies in clinical trials.