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공학석사 학위논문

Development of Therapeutic Antibody Screening Method

Using GPCR-embedded Nanodisc

GPCR이 포함된 나노디스크를 통한 약물항체 스크리닝

2022년 2월

서울대학교 대학원 바이오엔지니어링전공

김 수

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Abstract

Development of Therapeutic Antibody Screening Method

Using GPCR-embedded Nanodisc

Soo Kim Interdisciplinary Program in Bioengineering The Graduate School Seoul National University

G protein-coupled receptor (GPCR) targeting drug

development has been a major topic in the pharmaceutical industry over decades, while mainly chemical molecules are currently employed in the clinical field due to complexity in development schemes for contemporary biopharmaceutics. In developing a therapeutic antibody against GPCR, antigen

preparation and its mass production are the main bottlenecks of the scheme. Here, we design E.coli-expressed reconstituted GPCR using a nanodisc (ND) platform, assemble a kit to screen antibodies and derivatives, and perform its capability to . For antibody screening, indirect ELISA is the methodological basis in which antibody to identify serves as primary antibody, and an

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enzyme-conjugated secondary antibody serves as an indicator.

We validated this kit by identifying a well-known therapeutic antibody, Mogamulizumab (MOG), to the cc-chemokine receptor 4 (CCR4)-embedded ND from a pool of antibodies against the CCR family. This new approach suggests that any type of fully reconstituted GPCR can be mass-produced and used for screening purposes, shifting to the new paradigm in antibody drug discovery.

Keywords: therapeutic drugs, mass production, antibody screening, GPCR, ND, ELISA

Student number: 2020-21101

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요약 (국문 초록) ... 37

List of Tables and Figures

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List of Tables and Figures

Table 1. Quantitative comparison between CCR4 mono-

expression and CCR4 + rraA co-expression ... 16

Figure 1. Schematic diagram of the research ... 3 Figure 2. Schematic of nanodisc assembly ... 11 Figure 3. Expression and purification of

CCR4 produced in E.coli. ... 14 Figure 4. Comparison between CCR4 mono-expression and CCR4 + rraA co-expression ... 15 Figure 5. Expression, purification, and truncation of MSE1E3D1 produced in E.coli ... 17 Figure 6. FPLC chromatogram of CCR4-ND during size

exclusion chromatography ... 19 Figure 7. DLS size analysis of CCR4-ND ... 20 Figure 8. Schematic of selection test ... 22 Figure 9. Absorbance reading of MOG + Anti-CCR5 antibody against CCR4-ND ... 23 Figure 10. Schematic of testing antibody-to-absorbance

relationship ... 25 Figure 11. Absorbance reading of increasing concentration of MOG against CCR4-ND ... 26 Figure 12. Schematic of reliability test ... 28 Figure 13. Absorbance reading of MOG + anti-CCR antibodies against CCR4-ND ... 29

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Chapter 1 Introduction

G-protein-coupled receptors (GPCR), comprising one of the largest membrane protein family, is easily one of the most important targets for pharmaceuticals. Each of approximately 400 non-olfactory GPCRs plays key role in homeostasis, and its mutation is an indication of various diseases such as immune disorders and cancer. So far, the FDA approved 475 drugs targeting 108 GPCRs, and yet many of them are small molecules [1]. Until now, only 2 anti-GPCR antibodies are approved by the FDA, and 15 are under clinical trials [2].

The main challenge for anti-GPCR antibody discovery is access to pristine antigen. FDA-approved antibodies were derived from peptide or soluble extracellular domains (ECD) [3- 5], and those that have reached phase III clinical trial were derived from receptor overexpressing cell [6]. The former cases are exclusive for GPCR that has large ECD, and the latter cases are resource-intensive. Ongoing effort has been made to tackle this problem, and nanodisc (ND) is one of the alternative solutions.

ND is a soluble lipid bilayer system, originally designed for structural and functional study of membrane proteins [7-9].

It is a way to reconstitute purified membrane proteins in the most stabilized and natural form using E.coli expression system [10].

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Also, further studies revealed practical usage of this format such as immunogen [11] and biosensor [12].

In this study, we investigate the possibility of this format through developing GPCR-embedded ND as an antigen and ascertain its potential in screening therapeutic antibody candidates by adopting ELISA technique (Fig. 1). We used one of the approved anti-GPCR antibody, Mogamulizuamb (MOG) [13], and its target c-c chemokine receptor type 4 (CCR4) [14- 16] to validate our study.

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Fig 1. Schematic diagram of the research

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Chapter 2 Materials and Methods

2.1 Expression and purification of CCR4 CCR4 Expression

Rosetta 2 (DE3) competent cells were transformed with pET-DEST42/CCR4 and pBAD33.1/RraA vectors. The former vector is ampicillin-resistant and engineers CCR4 gene to have 6 histidine tag at C-terminal. Transformed cells were

inoculated into 5 mL of Luria-Bertani (LB) broth (37°C) with one-thousandth LB amount of ampicillin and chloramphenicol (Sigma-Aldrich, USA), and scaled-up to 6 L. In 6 L volume, LB broth was added with 0.2% arabinose to induce the

expression of rraA proteins, followed by the addition of 1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) to induce CCR4 proteins when OD600 value reached to 0.4~0.5. After additional 4 hours of cell growth, the cells were collected and pelleted using centrifuge at 7000 rpm, 4°C for 10 minutes.

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CCR4 Purification

Pelleted cells were prepared in three steps: cell lysis, solubilization, and separation by detergent. 2 L worth of pelleted cells were resuspended in 30 mL of PBS with 2 mM EDTA. Cells were lysed by sonication (35% amplitude, 5 seconds pulse for 5 minute), and centrifugated at 12000 rpm, 4°C for 20 minutes. This step is repeated one extra time.

Pelleted cell lysates were solubilized by 25 mL of solubilization buffer (0.1 M Tris-HCl, 20 mM Sodium dodecyl sulfate (SDS), 1 mM EDTA, 100 mM Dithiothreitol (DTT), pH 8.0) for

overnight at 25°C. Solubilized samples were dialyzed against 4 L of dialysis buffer (0.1 M sodium phosphate (pH 8.0), 10 mM sodium dodecyl sulfate (SDS)) using 10 K MWCO dialysis cassette (Thermo Scientific, USA) for overnight. Dialyzed samples were filtered with 0.22 µm bottle top filter (Jetbiofil, Korea).

Filtered proteins were loaded onto HisTrap HP column (Cytiva) equilibrated by SDS-binding buffer (0.1 M sodium phosphate (pH 8.0), 10 mM SDS). Unintended proteins and wastes were washed with SDS-washing buffer (0.1 M sodium phosphate (pH 7.0), 10 mM SDS), gradually over 20 minutes.

Then, SDS-elution buffer (0.1 M sodium phosphate (pH 6.0), 10 mM SDS) was applied to column for protein elution. Eluted sample’s buffer was changed to HEPES buffer (20 mM HEPES- NaOH, 100 mM NaCl, pH 8.0) by using a desalting column

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NaOH, 100 mM NaCl, pH 8.0) by using a desalting column (Cytiva)[19. 20]. The purified CCR4 proteins were characterized by western blot and gel-staining [17, 18].

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2.2 Expression and purification of MSP1E3D1 MSP1E3D1 Expression

Rosetta 2 (DE3) competent cells were transformed with pET-28a/MSP1E3D1. The vector is kanamycin-resistant and engineers MSP1E3D1 gene to have 6 histidine tag at C-

terminal. Transformed cells were inoculated into 5 mL of LB broth (37°C) with one-thousandth LB amount of kanamycin (Sigma-Aldrich, USA), and scaled-up to 6 L. In 6 L volume, LB broth was added with 1 mM of IPTG to induce MSP1E3D1 proteins when OD600 value reached to 0.4~0.5. After additional 4 hours of cell growth, the cells were collected and pelleted using centrifuge at 7000 rpm, 4°C for 10 minutes.

MSP1E3D1 Purification

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MSP1E3D1 Purification

1 L worth of pelleted cells were resuspended in 20 mL of His- binding buffer (20 mM imidazole (Alfa Aesar), 20 mM Tris, 500 mM NaCl (Junsei, Japan), pH 8.0). Cells were lysed by

sonication (30% amplitude, 5 seconds pulse for 5 minute), and centrifugated at 12000 rpm, 4°C for 20 minutes. Lysed samples were filtered with 0.22 µm bottle top filter.

Filtered proteins were loaded onto HisTrap HP column

equilibrated by His-binding buffer (20 mM imidazole, 20 mM Tris, 500 mM NaCl, pH 8.0). Unintended proteins and wastes were washed with His-washing buffer (50 mM imidazole, 20 mM Tris, 500 mM NaCl), pH 8.0), for 20 minutes. Then, His- elution buffer (350 mM imidazole, 20 mM Tris, 500 mM NaCl, pH 8.0) was applied to column for protein elution. Eluted sample’s buffer was changed to HEPES buffer by using a desalting column.

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MSP1E3D1 his-tag truncation

One hundredth MSP1E3D1’s molar amount of TEV protease was added to MSP1E3D1 proteins in HEPES buffer and

incubated in water bath at 37°C for 4 hours. Heated sample was then loaded onto HisTrap HP column equilibrated by HEPES buffer. Immediately after sample load, any unbound MSP1E3D1 proteins were eluted and bound his-tag and TEV proteases were successively removed from the column. The purified and truncated MSP1E3D1 proteins were characterized by

Coomassie blue staining.

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2.3 Assembly of CCR4-ND

Three components were used to assemble CCR4-ND: purified CCR4 and MSP1E3D1, and synthetic phospholipid as illustrated in Fig 2. In this experiment, 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC) was used as phospholipid. Three components were mixed at molar ratio of 1:20:2300

(CCR4:MSP1E3D1:DMPC). The mixture was initially incubated in shacking incubator for 2 hours at 25°C, 170rpm, then 0.6 g of Bio-Beads (Bio-Rad, USA) were added per 1 ml of the

mixture and incubated for overnight at same condition.

Next day, the mixture was filtered onto 0.45 µm bottle-top filter to remove Bio-Beads and applied to HisTrap HP column equilibrated by HEPES buffer. Unbound empty nanodisc or non-GPCR embedded particles were removed by continuous flow. Bound GPCR-embedded particles were eluted by HEPES elution buffer (20 mM HEPES-NaOH, 100 mM NaCl, 350 mM imidazole, pH 8.0). Then, eluted particles were applied to a size exclusion chromatography (Superdex 200 Increase 10/300 GL, Cytiva, USA) equilibrated with HEPES buffer [10, 21]. The peak representing the size of ND was collected and

characterized by DLS [22]. ND was also quantified through absorbance reading at 280 nm.

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Figure 2. Schematic of nanodisc assembly

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2.4 Antibody screening using CCR4-ND

Screening tests were done by binding various amount of CCR4- ND to nickel-coated 96-well plate (Thermo Fisher, USA). For each test, 100 µL of CCR4-ND solution with given molarity was added onto each well and incubated on rocking table for 2 hours at room temperature. Any unbound CCR4-ND or other wastes were removed by washing twice with excess amount of 0.5%

PBS-T. Then, 100 µL of antibodies for selection were added and incubated on rocking table for 1 hour. After washing twice with excess amount of 0.5% PBS-T, 100 µL of antibodies for detection were added and incubated for another 1 hour. Washed twice again with 0.5% PBS-T, 100 µL of Tetramethylbenzidine (TMB) was added to each well and incubated for 30 minutes at 37°C under darkness. Finally, the absorbance at 450 nm was measured from Microplate reader (TECAN, USA).

Chapter 3

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Chapter 3 Results and Discussion

3.1 Production of CCR4 and His-tag truncated MSP1E3D1

In case of CCR4, we co-tranformed the competent cell with pBAD33.1/rraA vector, which contains rraA protein, an inhibitor of the mRNA-degrading activity of the E.coli RNase E, to

maximize the translational capacity. [23-25]. Production of CCR4 as well as the enhancement of production level was confirmed by western blot and gel staining(Fig. 3,4. Based on 280 nm absorbance reading, co-expression of rraA protein increased the production of CCR4 by almost triple (Table 1).

Production of His-tag truncated MSP1E3D1 was also confirmed by SDS-PAGE gel staining (Fig. 5). His-tag truncated

MSP1E3D1 has a size of 29.6 kDa.

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Figure 3. Expression and purification of CCR4 produced in E.coli On the left is western blot analysis of purified CCR4 On the right is SDS-PAGE analysis of purified CCR4.

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Figure 4. Comparison between CCR4 mono-expression and CCR4 + rraA co-expression

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Table 1. Quantitative comparison between CCR4 mono- expression and CCR4 + rraA co-expression

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Figure 5. SDS-PAGE analysis of purified MSP1E3D1.

3.2 Reconstitution of CCR4-ND

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3.2 Reconstitution of CCR4-ND

CCR4-ND was finally purified by size exclusion chromatography using AKTA FPLC system (Cytiva). FPLC chromatograms in Fig 6 showed single peak of CCR4-ND’s retention volume ranging from 11 mL to 15 mL. This result suggested CCR4-ND assembly mixture’s homogeneity and demonstrated the optimization of the process. We further characterized CCR4-ND by quantifying the size of the samples via dynamic light scattering (DLS) measurement (Fig. 7). The average size of the particle, which is 19.8 nm, indicated that CCR4-ND was successfully assembled during the process.

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Figure 6. FPLC chromatogram of CCR4-ND during size exclusion chromatography

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Figure 7. DLS size analysis of CCR4-ND

3.3 Kit validation

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3.3 Kit validation

We performed three tests aiming different perspectives to validate this kit.

3.3.1 Selection of target antibody

To verify the desired function of the kit, we added two antibodies, MOG from human host (3.35 nM) and anti-CCR5 antibody (LSBio, USA) (3.35 nM) from goat host. Each well in this kit was loaded with 100 µL of 16 nM to 256 nM. The schematics and result were illustrated in Fig 8 and 9. The absorbance of MOG starts to show linear curve at around 50 nM. Meanwhile, the absorbance of MOG was increased as the CCR4-ND’s molarity increased, but the absorbance of anti- CCR5 antibody didn't change.

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Figure 8. Schematic of selection test

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0 0.1 0.2 0.3 0.4 0.5 0.6

10 100

Absorbance (450nm)

CCR4 Nanodisc concentration (nM)

MOG+CCR5 Ab - CCR4-ND ELISA

Human (Mog) Goat (CCR5)

Figure 9. Absorbance reading of MOG + Anti-CCR5 antibody against CCR4-ND

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3.3.2 Antibody-to-absorbance relationship

To understand how absorbance behaves as antibody amounts changes, we tested the kit by adding MOG from human host ranging from 0.1 nM to 3.35 nM. Each well in this kit was loaded with 100 µL of 128 nM. As illustrated in Fig 10 and 11, absorbance value rose in linear manner as antibody added increased.

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Figure 10. Schematic of testing antibody-to-absorbance relationship

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0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2

0.1 1 10

Absorbance (450nm)

MOG Ab concentration (nM)

MOG - CCR4-ND ELISA

Figure 11. Absorbance reading of increasing concentration of MOG against CCR4-ND

3.3.3 Reliability test

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3.3.3 Reliability test

To check whether the kit functions adequately in stressed condition, we tested the kit by adding MOG from human host ranging from 0.1 nM to 3.35 nM and anti-CCR antibodies from goat (CCR1,2,5,6,7) and mouse (CCR3) hosts (each with 3.35 nM, total value of 23,35 nM). Each well in this kit was loaded with 100 µL of 128 nM. As illustrated in Fig 12 and 13, MOG absorbance showed linear trend and was higher than others absorbance.

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Figure 12. Schematic of reliability test

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0 0.05 0.1 0.15 0.2 0.25 0.3

0.1 1 10

Absorbance (450nm)

Ab concentration (nM)

CCR Antibodies - CCR4-ND ELISA

Human (MOG) Goat (CCR2,3,5,6,7) Mouse (CCR1)

Figure 13. Absorbance reading of MOG + anti-CCR antibodies against CCR4-ND

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Chapter 4 Conclusion

GPCR’s inherent structural complexity was hard to mimic in vivo and so industry has been using peptides of extracellular domain to screen anti-GPCR antibody candidates. We offer a new way of screening anti-GPCR antibody via whole GPCR reconstituted into nanodisc platform. By using the entirety of GPCR, antibody derivatives screened from this system would have more specificity to complex structure of GPCR and affinity to the target than those from conventional way.

This research demonstrates the function of the kit in three criteria. The efficiency of the kit was verified by screening antibody of interest from two closely related antibodies. The usefulness of the kit was verified by rising HRP signals as the amount of antibody of interest increases. The reliability of the kit was finally verified by successfully screening antibody of interest from pool of antibodies where the amount of non-binding antibodies was 6-fold that of antibody of interest. Therefore, this kit can be useful in discovering antibody candidates for all unexplored GPCR.

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국 문 초 록

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국 문 초 록

GPCR이 포함된 나노디스크를 통 한 약물항체 스크리닝

김 수 서울대학교 대학원 협동과정 바이오엔지니어링 전공

G 단백질 연결 수용체 (G protein-coupled receptors, GPCR)를 타겟으로 하는 약물의 개발은 제약 산업에서 중요한 주제이며, 현재까지 대부분의 경우 소분자 합성 의약품들이 개발되어 사용되어지고 있다. 그러나 바이오의약품, 특히 항체의 개발은 큰 진전이 없이 2021년 기준 단 2종만이 미국 FDA에 승인받은 상태이다. 이는 항체 약물개발에 항원인 GPCR을 자연적인 형태로 만들어 개발에 사용하기 어려움이 있고 설사 어렵게 만들었다 하여도 그 방식들이 대량생산을 하는데 어려움을 가졌기 때문이다. 본 연구는,

E.coli-발현 시스템을 사용하여 나노디스크 플랫폼으로 재조합한

GPCR을 기반으로 한 키트를 제작하고 항체를 스크리닝 하였다. 이 키트는 간접적 효소면역측정법 (Indirect ELISA) 방식을 기반으로 설계되어, 항원인 GPCR이 포함된 나노디스크가 표면에 붙어있는 상태에서 확인하고자 하는 항체들을 분별한 뒤 특정 파장을 유도하는 표시 항체를 후처리 하는 방식으로 진행하였다. 제작된 키트는 미국 FDA에서 승인받은 항체인 Mogamulizumab (MOG)과 6 종류의 대- 케모카인 수용체 항체들, 그리고 타겟이 되는 GPCR인 cc-

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그리고 타겟이 되는 GPCR인 cc-chemokine receptor type 4를 항원으로 사용하여 그 기능을 확인하였다. 대량생산이 가능한 나노디스크 방식의 재조합 GPCR은 약물개발 방식에 새로운 패러다임을 여는데 기여할 것이다.

주요어: 약물, 대량생산, 항체 스크리닝, G 단백질 연결 수용체, 효소면역측정법, 나노디스크

학번: 2020-21101

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