建構巨量合成人類抗體庫應用於抗體藥物開發

Abstract

單株抗體 (mAb) 及其衍生物已成為發展最快速最蓬勃的藥物之一，為了快速篩選出具有治療疾病潛力的人類抗體，本研究規劃建構一個具高度多樣性 (highly diverse) 的合成人類單鏈 (single chain Fv, scFv) 抗體庫。抗體庫中的抗體序列是建構於高穩定性的骨架上，抗體六個 CDR 的設計是模擬天然人類抗體的多樣性，在完成序列優化後進行抗體基因片段合成。此外，為了提高抗體庫的品質，六個 CDR (包括所有不同長度的 CDR-H3) 都先經過 β-內酰胺酶篩選系統篩選，以剔除無法表達抗體的序列，然後重組六個 CDR 成為 scFv 抗體並進行建構抗體庫。在本研究中，完成設計和建構一個具高度多樣性的合成人類抗體庫，命名為 DSyn-1 (DCB Synthetic-1) 抗體庫，此抗體庫之變異區 (variable region) 含有約 2.5E-10 的多樣性。為了評估抗體庫應用於藥物開發的潛力，在研究中規劃針對五種和治療疾病相關的抗原進行抗體篩選的實驗，並進一步以 TIM-3 標的為例，評估 TIM-3 專一性抗體的效力，驗證該抗體庫應用於藥物開發的可能性。噬菌體抗體庫淘選 (panning) 測試的結果顯示，該抗體庫能夠分別針對不同的抗原篩選出數十到上百個專一性抗體。此外，三株 TIM-3 專一性抗體 (DCBT3-4、DCBT3-19 和 DCBT3-22) 在 TIM-3 report assay 中，可顯著抑制 TIM-3 的訊號傳遞，EC50 小於 10 nM，與人類 TIM-3 重組蛋白也有很強的結合力，親和力 (KD) 小於 1 nM。這些結果證實了 DSyn-1 是一個很有潛力的抗體庫，可用於篩選治療用抗體，其篩選出的三株新穎 TIM-3 中和性抗體，也非常有潛力能夠繼續開發為臨床治療用的藥物。

Monoclonal antibodies (mAbs) and their derivatives have become one of the fastest expanding classes of pharmaceuticals. To rapidly isolate potent human antibodies, we have constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library by phage display. The library is designed with high stability scaffolds with six complementarity determining regions (CDRs) tailored to mimic natural antibody compositions. The engineered antibody sequences are optimized for codon usage before synthesis. Furthermore, the six CDRs with variable length CDR-H3s were individually subjected to beta-lactamase selection and then recombined for library construction. In this study, we have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5E-10 transformants. Phage library performance was assessed by panning against five therapeutically relevant antigens including the evaluation of TIM-3-specific antibodies for clinical use. Phage library panning results showed that the library has the potential to generate up to hundreds of specific binders for each target. In addition, three TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity in TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Clone DCBT3-22 was further confirmed to be superior with good physicochemical property and a purity of more than 98% without aggregation. The promising results illustrate the utility of the DSyn-1 library for biomedical research applications and potential of the three novel fully human TIM-3-neutralizing antibodies for further clinical development.