單株抗體（mAbs）是治療多種疾病的主要方法，通過針對受影響區域中過度表達
的抗原以實現治療效果。如果抗原在正常細胞中也有表達，抗體也可能結合到正常細胞
的抗原上，可能引起副作用。例如，針對程式性死亡-1（PD-1）的單克隆抗體，如 Nivolumab，
已被用於治療非小細胞肺癌、黑色素瘤和腎細胞癌。然而，如果抗體也結合到正常細胞
中表達的 PD-1 上，這些療法可能會導致免疫介導的反應，如肺炎、結腸炎、肝炎和腎
炎。為了解決這個問題，鄭教授的實驗室開發了通用抗體鎖（Ab locks），可以提高抗體
藥物對疾病的選擇性並減少副作用。他們利用鉸鏈（Hinge）結構作為抗體鎖，通過一
個蛋白酶底物肽連接到抗體上。這種方法增加了抗體對疾病區域的選擇性，減少了副作
用。在這項研究中，使用兩個長度不同的肽，Lu09（26 個氨基酸）和 Lu02（34 個氨基
酸），作為抗體鎖，分別附加到 Nivolumab 的 N 端，形成 pro-Lu02-Ab 和 pro-Lu09-Ab。
使用微量熱泳（MST）定量測定了 pro-Lu09-Ab 和 pro-Lu02-Ab 對 PD-1 的結合親和力。
pro-Lu09-Ab 的 Kd值約為 4.7 μM，而 pro-Lu02-Ab 的 Kd值約為 22 μM，遠高於 Nivolumab
的 Kd 值（通過表面等離子共振（SPR）測定約為 1.5-4 nM）。pro-Lu09-Ab 和 pro-Lu02-
Ab 的晶體結構分別以 2.50 Å 和 2.59 Å 的分辨率確定。我們發現，抗體鎖的覆蓋率越
高，其阻止抗原結合的能力就越強。我們對 pro-Lu09-Fab 和 pro-Lu02-Fab 結構進行的
分析顯示，這兩種抗體鎖都佔據了 PD-1 的結合位點。與此同時，pro-Fabs 中 CDR 的構
象也發生了變化。值得注意的是，pro-Lu09-Fab 和 pro-Lu02-Fab 中 FG-loop 的結合位凹
槽較小，而 Ab lock Lu02 幾乎完全覆蓋了它們。儘管 N-loop 結合位的大小保持不變，
但它們在 pro-Lu09-Fab 和 pro-Lu02-Fab 中卻被抗體鎖完全阻擋。pro-Lu09-Fab 和 pro-
Lu02-Fab 中的抗體鎖覆蓋率分別為 39.4%和 64.9%。然而，pro-Lu09-Fab 的 Kd 值小於
pro-Lu02-Fab，說明其與抗原結合更強，但抗體鎖的阻斷能力較弱。因此，我們的結構
分析證實了抗體鎖覆蓋率與其阻斷效能之間的正相關

Monoclonal antibodies (mAbs) are a leading therapeutic approach for numerous diseases, functioning by targeting overexpressed antigens in affected areas to achieve therapeutic efficacy. If the antigen is expressed in normal cells as well, the antibody may also bind to the antigens in normal cells, potentially causing side effects. As an example, monoclonal antibodies targeting programmed death-1 (PD-1), such as Nivolumab, have been used to treat non-small cell lung cancer, melanoma, and renal cell carcinoma. However, these therapies may result in immune-mediated reactions, such as pneumonitis, colitis, hepatitis, and nephritis, if the antibodies bind to PD-1 expressed in normal cells as well. To address this issue, Professor Cheng’s Lab has developed universal antibody locks (Ab locks) that can improve the disease selectivity of antibody drugs and reduce their side effects. They utilized the hinge domain as an antibody lock, which was connected to the antibody through a protease substrate peptide. This approach increased the selectivity of the antibody for the disease region, reducing side effects. In this study, two different peptides of varying lengths, Lu09 (26 amino acids) and Lu02 (34 amino acids), were utilized as Ab locks and were attached to the N-terminus of Nivolumab to form pro-Lu02-Ab and pro-Lu09-Ab, respectively. Micro-scale thermophoresis (MST) was used to quantify the binding affinity of pro-Lu09-Ab and pro-Lu02-Ab to PD-1. The Kd value for pro-Lu09-Ab was about 4.7 μM, while the Kd value for pro-Lu02-Ab was about 22 μM, which were much higher than that of Nivolumab (about 1.5–4 nM determined by surface plasmon resonance (SPR)). The crystal structures of pro-Lu09-Ab and pro-Lu02-Ab were determined at 2.50 Å and 2.59 Å resolution, respectively. We found that a higher coverage rate of the Ab lock resulted in a stronger ability to block antigen binding. Our analysis of the pro-Lu09-Fab and pro-Lu02-Fab structures revealed that both Ab locks occupied the PD-1 binding site. The conformation of the CDRs in the pro-Fabs also underwent changes. Notably, the grooves for the FG-loop were smaller in both pro-Lu09-Fab and pro-Lu02-Fab, and Ab lock Lu02 almost entirely blocked them. Although the size of the grooves for the N-loop remained unchanged, they were completely obstructed by the Ab locks in both pro-Lu09-Fab and pro-Lu02-Fab. The coverage rates of Ab locks in pro-Lu09-Fab and pro-Lu02-Fab were 39.4% and 64.9%, respectively. However, pro-Lu09-Fab had a smaller Kd value than pro-Lu02-Fab, indicating stronger antigen binding but weaker blocking ability of the Ab lock. In summary, our structural analysis confirmed a direct link between the coverage rates of Ab locks and their blocking efficiency.

Contents
中文摘要.............................................................................................................................i
Abstract............................................................................................................................ iii
Acknowledgements............................................................................................................v
Abbreviations....................................................................................................................vi
Chapter 1. Introduction ......................................................................................................4
1.1. Monoclonal antibodies in therapy.......................................................................4
1.1.1. US FDA approved therapeutic monoclonal antibodies............................4
1.1.2. The structure and function of antibody....................................................4
1.1.3. Immune checkpoint inhibitor for targeted immunotherapy in tumor ......5
1.2. The strategies to prevent severe adverse events from mAb therapy...................6
1.3. How to design an Ab lock with high blocking efficacy......................................9
1.4. Aim 10
Tables and Figures of Chapter 1 ......................................................................................11
Table 1.1. Comparison of different masking strategies of pro-Abs.........................11
Table 1.2. The composition of different IgG hinge-based Ab locks........................12
Table 1.3. The blocking abilities of various pro-Abs with different Ab locks.........13
Table 1.4. The cover rate and blocking abilities of various pro-Abs with different Ab
locks.......................................................................................................14
Figure 1.1. The structure of antibody.......................................................................15
Figure 1.2. The effector mechanisms of antibody....................................................16
Figure 1.3. The applications of therapeutic antibodies. ...........................................17
Figure 1.4. The strategies to increase the selectivity of therapeutic mAbs..............18
Figure 1.5. The cover rate of Ab lock. .....................................................................19
Chapter 2. Materials and Methods...................................................................................20
2.1. Materials ...........................................................................................................20
2.2. Antigen binding assay by Microscale thermophoresis (MST)..........................20
2.3. Determination of the 3D structure of Pro-Lu02-Fab and Pro-Lu09-Fab..........21
2.3.1. Crystallization........................................................................................21
2.3.2. X-ray diffraction Data Collection and Processing .................................21
2.3.3. Structure determination..........................................................................22
2.4. Calculation of cover rate...................................................................................22
2.5. Protein-protein docking of pro-Lu02-Fab and pro-Lu09-Fab ..........................23
Tables and Figures of Chapter 2 ......................................................................................24
Table 2.1. The protein sequences of Fab precursors of Nivolumab.........................24
Chapter 3. Results and Discussion...................................................................................25
3.1. Binding affinity between PD-1 and pro-Lu02-Ab and pro-Lu09-Ab...............25
3.2. Crystal structure Determination of pro-Lu09-Fab and pro-Lu02-Fab..............25
3.3. Structural insights into the shielding effects of Ab locks .................................29
3.4. The PD-1 binding status in pro-Fab predicted by docking ...............................30
Tables and Figures of Chapter 3 ......................................................................................31
Table 3.1. The optimization of crystallization condition of pro-Lu09-Fab. ............31
Table 3.2. Data collection and refinement statistics ................................................32
Figure 3.1. Pro-Ab binding assay by MST. .............................................................34
Figure 3.2. The density fit graphs of pro-Lu09-Fab when MR with different template
structures................................................................................................35
Figure 3.3. Comparison of Nivolumab Fab apo form and PD-1 complex...............36
Figure 3.4. The density fit graphs of pro-Lu02-Fab when MR with different template
structures................................................................................................37
Figure 3.5. The final density fit graphs of pro-Lu09-Fab and pro-Lu02-Fab..........38
Figure 3.6. Side view of pro-Lu09-Fab and pro-Lu02-Fab. ....................................39
Figure 3.7. Top view of pro-Lu09-Fab and pro-Lu02-Fab. .....................................41
Figure 3.8. Comparison of pro-Lu09-Fab and pro-Lu02-Fab..................................42
Figure 3.9. Comparison of pro-Lu09-Fab and Nivolumab Fab...............................43
Figure 3.10. Comparison of pro-Lu02-Fab and Nivolumab Fab.............................44
Figure 3.11. B-factor analysis of pro-Lu09-Fab. .....................................................45
Figure 3.12. B-factor analysis of pro-Lu02-Fab. .....................................................46
Figure 3.13. The AlphaFold similations of pro-Lu09-Fv.........................................47
Figure 3.14. The blocking status of pro-Lu09-Fab and pro-Lu02-Fab....................48
Figure 3.15. The coverage of Ab lock Lu09 and Lu02............................................49
Figure 3.16. The docking results of PD-1 to Nivolumab Fab..................................50
Figure 3.17. The docking results of PD-1 to pro-Lu09-Fab. ...................................51
Figure 3.18. The docking results of PD-1 to pro-Lu02-Fab. ...................................52
Chapter 4. Conclusion......................................................................................................53
References........................................................................................................................56

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