Antibody-Drug Conjugates and Cellular Metabolic Dynamics 9811956375, 9789811956379

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Shuqing Chen · Jinbiao Zhan Editors

Antibody-Drug Conjugates and Cellular Metabolic Dynamics

Antibody-Drug Conjugates and Cellular Metabolic Dynamics

Shuqing Chen • Jinbiao Zhan Editors

Antibody-Drug Conjugates and Cellular Metabolic Dynamics

Editors Shuqing Chen College of Pharmaceutical Sciences Zhejiang University Hangzhou, Zhejiang, China

Jinbiao Zhan School of Medicine Zhejiang University Hangzhou, Zhejiang, China

This book project is supported by “B&R Book Program”. ISBN 978-981-19-5637-9 ISBN 978-981-19-5638-6 https://doi.org/10.1007/978-981-19-5638-6

(eBook)

Jointly published with Zhejiang University Press © Zhejiang University Press 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publishers, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publishers nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publishers remain neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Cancer is the leading cause of morbidity and death in the world. According to statistics from the World Health Organization (WHO), out of 56.4 million deaths worldwide in 2015, 8.8 million were due to cancer, that is, nearly one-sixth of deaths were caused by cancer. In developed countries, people’s living standards are relatively high, sanitary conditions are better, medical technology is advanced, and the number of people dying from other diseases is decreasing year by year. This is prominently reflected in the increase in cancer incidence and mortality. This shows that among the many diseases that affect people’s lifespan, cancer is the most intractable disease and a challenge to modern medicine. Commonly used clinical tumor treatment methods are mainly surgery, radiotherapy, and chemotherapy, which have serious side effects. Generally, the quality of life of patients declines sharply after starting treatment. The toxicity of molecular targeted drugs is less than that of general chemotherapy drugs, and the immediate effect is also good. However, usually within 3–6 months, tumor cells will develop resistance to the drug and progress quickly. Antibodies for the treatment of tumors have also been on the market for clinicians to choose from, but their overall effect is still too mild. Scientists have long proposed the idea of combining more toxic chemotherapeutics with mildly targeted antibody to form a drug for the treatment of tumors. This type of drugs are called antibody-drug conjugates (ADCs). It has both strong cytotoxicity and precise tumor cell targeting. It is a form of drug with the smallest side effects and the highest efficacy in theory. The concept of ADC began with the idea of “magic bullet” proposed by Paul Ehrlich in 1913, to use of a carrier (antibody) with specific targeting of tumor to bring toxins into the lesions, thereby the toxins can only effect in the target tissue site. Limited by outdated monoclonal antibody preparation technology, early ADC research used polyclonal antibodies, so the development was very slow. In 1975, Kohler and Milstein pioneered the hybridoma technology, using hybridoma cells to prepare murine monoclonal antibodies, allowing the monoclonal antibodies to be prepared in large quantities in a short time, thus making ADC research an epochmaking progress. In the late 1980s, emerging molecular biology developed rapidly. v

vi

Preface

People could recombinantly express immunoglobulin (Ig) molecules, realizing the artificial modification of natural antibody molecules, and accelerating the development of antibodies and ADCs. Throughout the history of ADC development, there have been three generations of technological changes. The first-generation ADC used traditional chemotherapeutics as part of the toxic molecules. Because small molecules were not toxic enough or ADCs were not stable enough, systemic toxins were released (too many side effects), and most of them ended in failure. The second-generation ADC used more toxic small molecules (such as donotoxins, maytansinoids that act on tubulin, and calicheamicins act on DNA) to overcome the weaknesses of the first-generation ADC, and two ADCs have been approved by the Food and Drug Administration (FDA) for marketing. However, the secondgeneration ADC adopts traditional chemical conjugation technology, which is conjugated small molecules through antibody lysine or interchain disulfide bonds, and its drug-antibody ratio (DAR) uniformity is poor (0–8 or even higher), which brought complex problems of drug distribution, metabolism, and excretion in the body. In addition, linker of the second-generation ADC has poor stability and is easily lysed in the blood, causing serious toxic and side effects. Therefore, the thirdgeneration ADC came into being, and the site-specific conjugation technology achieved unprecedented development. First is the ThioMab technology that introduces antibody site-directed mutations into free cysteine conjugation sites, and then the improved ThioBridge bridging technology on this basis (even 100% uniformity of the conjugation product DAR can be achieved). The bio-orthogonal chemical conjugation technology introduces a succinic acid stop codon, inserts an unnatural amino acid in the antibody sequence, and is used in conjunction with click chemistry and other technologies to achieve rapid site-specific conjugation. In addition, enzymatic conjugation methods using sortase A (SrtA), glycosyltransferase, and transglutaminase have also been gradually developed. In order to further broaden the scope of application of ADCs, some small toxic molecules such as pyrrolobenzodiazepine (PBD), 7-ethyl-10-hydroxycamptothecin (SN-38), α-amanitin have also been tried to conjugate antibodies. ADC R&D technology is gradually developing and maturing. In 2000, the US FDA approved the first ADC to go on the market, which was Pfizer’s Mylotarg® (gemtuzumab ozogamicin). The drug targets the CD33 and is used to treat acute myeloid leukemia (AML). It was withdrawn from the market in June 2010 due to clinically poor efficacy and increased patient mortality. Later studies on CD33 found that the single nucleotide mutation rs12459419 (C> T; Ala14Val) in exon 2 caused the truncated splice variant to lack the epitope recognized by the CD33 antibody hP67.6, that is, the IgV domain, making the antibody part of Mylotarg® cannot recognize the CD33 antigen and is ineffective. After reducing the dose and changing the indications, Mylotarg® was again approved for marketing by the FDA in September 2017. In the meantime, Seattle Genetics’ Adcetris® (brentuximab vedotin, targeting CD30), Roche’s Genentech’s Kadcyla® (ado-trastuzumab emtansine, T-DM1; targeting HER2), and Pfizer’s Besponsa® (inotuzumab ozogamicin, targeting CD22) was launched in 2011, 2013, and 2017, respectively. Kadcyla® is also effective for HER2-positive breast cancer patients, who are non-sensitive to trastuzumab, and

Preface

vii

reduces adverse reactions. Since its launch, the sales volume has continued to grow, and its annual sales in 2018 reached $1 billion. The success of Kadcyla® has pushed ADC research to a climax, and the pharmaceutical industry’s enthusiasm for ADC development continues to rise. In September 2018, the US FDA-approved AstraZeneca’s Lumoxiti® (moxetumomab pasudotox-tdfk) that targets CD22 and treats hairy cell leukemia; in June 2019, the US FDA approved Genentech’s targeting CD79b, Polivy® (polatuzumab vedotin-piiq) for the treatment of diffuse large B-cell lymphoma. As of October 2019, more than 200 ADCs have entered clinical trials in the world. ADCs are composed of three parts: antibody, linker, and effector molecule. The choice of each part and the combination of each part can affect the final drug product of ADC. Coupled with the complexity of the human body environment, the drug product of ADCs is facing severe challenges. Whether the rational design of ADCs has rules to follow, what are the key issues that affect the druggability of ADCs, and how to evaluate the impact of these key issues on the druggability of ADCs have all become issues that must be solved urgently in the process of ADC research and industrialization. The laboratory of Precision Medicine and Biopharmaceutics and Biotechnology Drugs, College of Pharmaceutical Sciences, Zhejiang University, is one of the first laboratories to enter the field of ADC research in China. It has not only explored sitespecific conjugation technology and enzymatic conjugation technology, but also conducted research on novel ADC targets and on non-antibody-targeted molecular conjugates. The concept of XDC (X-drug conjugates) was first proposed, and the designed novel ADC has been transferred to pharmaceutical companies for subsequent development. In 2014, the research team was funded by the National Natural Science Foundation of China, “The studies on mechanisms of cellular metabolic dynamics of antibody-drug conjugates (No. 81430081)” to carry out related cellular drugs in the design of novel ADC metabolic kinetics studies to evaluate the influence of various related factors on the druggability of novel ADCs, so as to improve the success rate of novel ADC design. Since undertaking this project, the research team has explored many factors and obtained many research results. Based on the existing project research results and related academic papers published at home and abroad, we have compiled this book to review the research results in this field and provide references for researchers in the field of ADC. Thanks to the National Natural Science Foundation of China for supporting the project (No.81430081; No.81872784)! Thanks to all members of the laboratory’s research team for their hard work over the past few years, as well as the efforts made by Keying Liang, Saleem M., Kalim M., Jun Lai, Xiaoyue Wei, Wenbin Zhao, Wenhui Liu, Jiansheng Fan, Chixiao Qiu, Xuefei Bai, Baoying Shi, Ying Shen, and others in the process of writing this book! Hangzhou, Zhejiang, China October 2019

Shuqing Chen Jinbiao Zhan

Contents

1

Antibody-Drug Conjugates: Basic Concepts and Structures . . . . . . Jinbiao Zhan

2

Relationship Between Target and Specific Action of Antibody-Drug Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jun Lai and Shuqing Chen

13

The Internalization and Therapeutic Activity of Antibody Drug Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jiansheng Fan and Shuqing Chen

25

3

1

4

The Internalization and Intracellular Trafficking of ADCs . . . . . . . Keying Liang, M. Saleem Khan, M. Kalim, and Jinbiao Zhan

35

5

Distribution and Metabolism of Antibody-Drug Conjugates . . . . . . Xuefei Bai and Shuqing Chen

45

6

Application of Antibody Fragments in ADCs . . . . . . . . . . . . . . . . . Wenhui Liu and Shuqing Chen

55

7

Novel Targeting Carriers in Antibody-Drug Conjugates . . . . . . . . . Xiaoyue Wei and Shuqing Chen

69

8

Site-Specified Conjugating Technology and Application . . . . . . . . . Ying Shen, Baoying Shi, and Shuqing Chen

83

9

Determination of Drug-to-Antibody Ratio of ADCs . . . . . . . . . . . . . 101 Chixiao Qiu and Shuqing Chen

10

Pharmacokinetic Study of Antibody-Drug Conjugates . . . . . . . . . . 117 Wenbin Zhao and Shuqing Chen

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Chapter 1

Antibody-Drug Conjugates: Basic Concepts and Structures Jinbiao Zhan

1.1

Basic Concepts

One hundred years ago, Paul Ehrlich, a Germany scientist, proposed “magic bullet” concept in which he used antibody to deliver drugs. With the progress of biotechnology, now his ideal has become reality. Antibody targeted therapy has become a hot spot in the global cancer treatment. Besides the traditional humanized antibodies, in recent years, many novel formats of antibodies have been generated through a perfect combination of genetic engineering and chemical synthesis. Among them, as a highly effective antibody drug, antibody-drug conjugate (ADC) which composed of a monoclonal antibody and cytotoxin attracts much attention. Generally, an ADC consists of three parts: a monoclonal antibody, a molecular linker, and an effector molecule. In terms of different effector molecules, ADCs can be divided into three categories- chemical conjugates (small cytotoxins as payload), immunotoxins (protein toxins used for conjugation), radioimmunoconjugates (radionuclides conjugated to antibodies). If there is no specific instruction in this book, ADCs refer to the chemical conjugates in which monoclonal antibody has been linked with a small molecule cytotoxin. Compared to chemotherapeutic drugs and conventional monoclonal antibodies, ADCs combine the specific targeting of monoclonal antibodies with the high activity of small molecule cytotoxins. The capability of ADCs to kill tumor cells depends in large part on their internalization efficiency into the cell and cytotoxicity of small molecular drugs released from conjugates. After endocytosis, ADC is mainly degraded in lysosome, then released cytotoxic drugs which lead to tubulin filaments disrupting, or double-stranded DNA damaging etc., and eventually cause targeted cancer cell death.

J. Zhan (*) School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chen, J. Zhan (eds.), Antibody-Drug Conjugates and Cellular Metabolic Dynamics, https://doi.org/10.1007/978-981-19-5638-6_1

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Table 1.1 Antibody-drug conjugates approved by US FDA Company Seattle genetics

Drug name Adcetris® (brentuximab vedotin)

Target CD30

Cytotoxin Auristatin MMAE

Genentech/ Roche

Kadcyla® (ado-trastuzumab emtansine, T-DM1) Mylotarg® (gemtuzumab ozogamicin)

HER2 (ErbB2)

Maytansinoid DM1

CD33

Calicheamicin

Acute myelogenous leukemia (AML)

Pfizer

Besponsa® (inotuzumab ozogamicin)

CD22

Calicheamicin

AstraZeneca

Lumoxiti® (moxetumomab pasudotox-td-) Polivy® (polatuzumab vedotin-piiq)

CD22

Truncated PE38

B-cell precursor acute lymphoblastic leukemia (B-ALL) and other B-cell hematologic diseases Hairy cell leukemia (HCL)

CD79b

MMAE

Wyeth/ Pfizer

Genentech/ Roche

Indications Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL) HER2positivebreast cancer

Diffuse large B-cell lymphoma (DLBCL)

Approval date Accelerated approval in Aug, 2011. Full approval in Aug 2015

Approval in Feb, 2013

First Accelerated approval in May, 2000 by FDA, withdrawal in 2010 and re-approval in 2017 Approval in Aug, 2017 by FDA

Approval in Sep, 2018 by FDA Approval in Jun, 2019 by FDA

CD clusters of differentiation, MMAE monomethyl auristatin E; DM1 methyl maytansine 1, HER2 human epidermal growth factor receptor 2

Gemtuzumab ozogamicin (Mylotarg), an anti-CD33 mAb-calicheamicin conjugate was the first ADC approved by the FDA in 2000. From 2000 to 2019, a total of six ADCs have been approved. Over 200 clinical trials are underway (www. ClinicalTrials.gov). Among them, at least 20 are in phase II–III stages and about half of them targeted to haematological malignancies and solid tumors (see Tables 1.1 and 1.2) [1–3].

Indatuximab ravtansine Denintuzumab Mafodotin Coltuximab ravtansine Naratuximab emtansine SAR408701 SAR428926 Anetumab Ravtansine

Glembatumumab Vedotin

Biotest pharmaceuticals

Celldex therapeutics

Sanofi Sanofi Bayer

ImmunoGen

ImmunoGen

Seattle genetics

Lorvotuzumab mertansine

Drug Labetuzumab govitecan Belantamab mafodotin PSMA-ADC

Children’s oncology group/National Cancer Institute (NCI)

GlaxoSmithKline/ Iqvia Pty Progenics pharmaceuticals

Company Immunomedics

gpNMB

CEACAM5 LAMP1 Mesothelin

CD37

CD19

CD19

CD138

MMAE

DM4 DM4 DM4

DM1

DM4

MMAF

DM4

DM1

MMAE

PSMA CD56

MMAF

Cytotoxin SN-38

BCMA

Target CEACAM5

Phase II Phase II Phase II

Phase II

gpNMB over-expressing triple negative breast cancer

Phase II

Phase II

Phase II

Phase II

Phase II

Phase II

Phase II

Phase in clinical trials Phase II

Solid tumors Solid tumors Mesothelioma, pancreatic cancer, non-small cell lung cancer

Non-Hodgkin lymphoma

Diffuse large B-cell lymphoma

DLBCL and FL

Pleuropulmonary blastoma, recurrent malignant peripheral nerve sheath tumor, recurrent neuroblastoma refractory rabdomyosarcoma, synovial sarcoma, Wilms tumor Multiple myeloma

Prostate cancer

Multiple myeloma

Conditions Metastatic colorectal cancer

Table 1.2 Antibody-drug conjugates under investigation in phase II and phase III clinical trials

Antibody-Drug Conjugates: Basic Concepts and Structures (continued)

NCT02575781 NCT02561962 NCT02610140, NCT03023722, NCT03455556 NCT01997333

NCT01638936

NCT02592876, NCT02855359 NCT01534715

NCT01638936

NCT01695044, NCT02020135 NCT02452554

NCT03544281

ClinicalTrials. gov Identifier NCT01605318

1 3

Trastuzumab deruxtecan Trastuzumab duocarmazine Enfortumab vedotin Depatuxizumab Mafodotin Sacituzumab Govitecan Mirvetuximab Soravtansine Rovalpituzumab tesirine (Rova-T)

Daiichi Sankyo/ AstraZeneca UK Synthon biopharmaceuticals Astellas Pharma/ Seattle genetics AbbVie/radiation therapy Oncology group Immunomedics SN-38

Trop-2

PBD dimer

MMAF

EGFR vIII

DLL3

MMAE

Nectin-4

DM4

Duocarmazine

HER2

FolRα

Deruxtecan

Cytotoxin DM4 MMAF

Target CA6 ENPP3 (CD203c) HER2

Triple-Negative breast cancer(TNBC), Urothelial cancer Ovarian cancer, primary peritoneal cancer Fallopian tube cancer SCLC

Ureteral cancer, urothelial cancer, bladder cancer Glioblastoma, Gliosarcoma,

Metastatic HER2 positive breast cancer

Conditions TNBC Metastatic renal cell Carcinoma HER2 positive breast cancer

Phase III

Phase III

Phase III

Phase III

Phase III

Phase III

Phase III

Phase in clinical trials Phase II Phase II

NCT03033511 NCT03061812

NCT02631876

NCT02574455

NCT02573324

NCT03474107

NCT03262935

NCT03734029

ClinicalTrials. gov Identifier NCT02984683 NCT02639182

CEACAM5 carcinoembryonic antigen-related cell adhesion molecule 5, BCMA B-cell maturation antigen, MMAF monomethyl auristatin F, DM4 methyl maytansine 4, LAMP1 lysosome-associated membrane protein 1, CA6 carbonic anhydrase 6, EGFR epidermal growth factor receptor, DLL3 delta-like protein 3

ImmunoGen/Merck Sharp & Dohme Corp AbbVie

Drug SAR566658 AGS-16C3F

Company Sanofi Astellas Pharma

Table 1.2 (continued)

4 J. Zhan

1

Antibody-Drug Conjugates: Basic Concepts and Structures

1.2

5

Monoclonal Antibodies Used for ADC Development

Monoclonal antibody is the “guidance unit” which can carry the toxin payloads to the cancer cell surface. Theoretically, other molecules working as delivery vectors also can replace the antibody action. For example, tumor necrosis factor-related apoptosis-inducing ligand (TRIAL) can carry the toxic payload-monomethyl auristatin E (MMAE) and specifically bind to death receptor 4 (DR4) and death receptor 5 (DR5) on the cell membrane, resulting in anticancer effect (see Chaps. 2 and 6) [4]. Selection of antibodies depends on the target of disease. In principle, any biomolecules present on the surface of cancer cell, including polypeptides, proteins, carbohydrates and lipids, can be targets of ADCs. Ideal targets are the antigens which are expressed only on the surface of tumor cells and not on that of normal cells. Generally speaking, a tumor-specific antigen or tumor-associated antigen that is not or poorly expressed in normal tissues can be a candidate target antigen. For example, differentiation antigens or their receptors expressed on malignant tumor cells may serve as targets for antibody therapy. Human leukocyte differentiation antigens, also called clusters of differentiation (CD antigens), have become important targets for haematological malignancies in the past 5 years [2, 3, 5]. Among them, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD70, CD79, CD203 and CD138 are frequently used for targeting antigens (see Tables 1.1, 1.2, and 1.3). For solid tumors, often-used targets include: (1) Receptors of growth factors and protein antigens, such as epithelial growth factor receptor (EGFR), human epithelial growth factor receptor-2 (HER-2), trophoblast cell-surface antigen 2 (Trop-2), mesothelin, folate receptor 1 (FolR1), prostate specific membrane antigen (PSMA), Delta-like ligand 3 (DLL3), glycoprotein nonmetastatic melanoma protein B (gpNMB), CA6, etc. [1]. ADCs can inhibit cell growth and induce apoptosis of cancer cells through blocking the growth signaling pathways. T-DM1 is the first Table 1.3 Other CD antigens used for the treatment of hematological malignancies Target CD56

ADC drug Lorvotuzumab mertansine

Cytotoxin DM1

CD70

SGN-75

MMAF

MDX-1203

Multiple myeloma(MM) Multiple myeloma(MM)

CD74

HLL1-DOX

DNA alkylating cytotoxin drug A Doxorubicin

CD138

BT-062

DM4

conditions Wilms tumor, rhabdomyosarcoma, neuroblastoma and other CD56-expressing tumors Renal cell carcinoma, non-Hodgkin lymphoma (NHL)

Phase in Clinical trials (Identifier) Phase II (NCT02452554) Phase I (NCT0101591) Phase I (NCT00944905)

Phase I, II (NCT01101594) Phase I, II (NCT01638936)

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J. Zhan

successfully approved ADC for solid tumors in which HER-2 has been used for targeting. The antibody portion of T-DM1 would pre-emptively occupy HER-2 and block further interaction between HER-2 and growth factors, thus inhibit the cell proliferation of breast cancer. Moreover, the free toxin DM1 released from lysosomes accelerates the death of cancer cells [6]. T-DM1 has been successful in the treatment of HER2-positive breast cancer and gastric cancer. A number of targets are being developed for other solid tumors. Some of them including EGFR, Trop-2, DLL3, and FolR1, have entered phase III trials (see Table 1.2). (2) Inhibiting tumor angiogenesis and neovascularization strategy. Angiogenic factors include vascular endothelial growth factors (VEGFs), VEGF receptors (VEGFRs), endoglin, etc. Endoglin (CD105) is a type I membrane glycoprotein of the vascular endothelial cells. It mediates the signal transduction of transforming growth factor-β (TGF-β) through forming a complex with the signaling receptors. Antibody drug conjugates and immunotoxins against endoglin are being evaluated for safety and efficacy in patients with glioblastoma multiform [7]. An intact antibody has large molecular weight (such as IgG, MW 150 kD). Although intact antibodies have the advantage of longer in vivo half-life, smallmolecule antibody fragments may be more likely to penetrate solid tumors. Recently, antibody fragments such as single-chain antibody fragment/single-chain variable fragment (scFv), antigen binding fragment (Fab), F(ab’)2, minibody, nanobody and diabody, have widely used for ADC design. The selection of antibody and target plays a key role in the success of ADC [5]. The ADC activity is influenced by the number of antigen molecules expressed on tumor cell surface, the endocytosis ability of antigens, the binding affinity of antibody to antigen, the internalization efficiency of ADC complex and the circulation of antigen molecules etc. Ideal antibodies or antibody fragments for ADC synthesis can effectively recognize and bind to most antigens on tumor surfaces, and can be efficiently endocytosed by tumor cells. After endocytosis, they can be transported to the lysosomes, and then be degraded and releases cytotoxins that destroy the tumor cells. Furthermore, the released toxin molecules can also cause “bystander effect” to expand the killing range of tumor cells [8].

1.3

Linkers

A Linker is a crucial component for successful ADC construction. Two criteria should be met. (1) The linker must have sufficient stability in plasma so that the toxin payload will not be prematurely liberated and causes undesired damage to healthy tissues. (2) the linker must be able to release drug payload efficiently and causes anticancer effects once the ADC molecules arrive in target tumor tissues [9]. In fact, depending on different antibodies and toxin payloads, optimal linkers need to be designed. Sometimes the same linker endows distinctive properties to different ADCs.

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Antibody-Drug Conjugates: Basic Concepts and Structures

7

After over 20 years of practice and improvement, linker technologies between monoclonal antibodies and cytotoxic drugs have been optimized. According to the difference in stability, linkers can be divided into two categories: cleavable linkers and noncleavable linkers [9, 10]. Cleavable linkers mainly include chemically cleavable linkers and enzymatically cleavable linkers. Chemically cleavable linkers include acid-labile linkers (hydrazone) and disulfide linkers. Enzymatically cleavable linkers are mainly peptide linkers and β-glucuronide linkers, which can be hydrolyzed by enzymes located in cytoplasm. In ADC development, several noncleavable linkers such as thioether linkers have been explored. Hydrazone Linker One of acid-labile linkers is designed to take advantage of differences in metabolism between the normal tissue and tumor tissue (weak acidity), as well as differences in pH in blood circulation and organelles. The hydrazone linker is not stable in the circulation system. It was reported that after 24 h, approximately 5–6% of the cytotoxin would fall off. However, most of them are still concentrated in the tumor site and released in the lysosomes (pH 5.0) of the tumor cells. Disulfide Linker A frequently used linker in ADC construction, which is designed according to the characteristic that disulfide bonds can be selectively cleaved in the reductive environment of the cell and remain stable in the oxygen-rich bloodstream. At present, disulfide linkers are commonly used in the synthesis of site-specific ADCs. Cysteine (Cys) residues can be introduced into different positions of antibody light chain or heavy chain by gene engineering. Then the sulfhydryl group on Cys is used for coupling reaction. To improve its stability in the blood circulation system, one or two methyl groups are often introduced to the end of disulfide linkage in the blood circulation system. In cells, especially in tumor cells that are relatively hypoxic, the concentration of glutathione (GSH) is so high that disulfide bonds in ADCs will break down and cytotoxin payloads can be released successfully. Peptide Linker One of enzymatically cleavable linkers has good stability since there are various inhibitors of proteases in plasma and proteases cannot work in the unfavorable pH environment. In cells, especially in lysosomes of tumor cells, there are highly active proteases, cathepsins, that can cleave specific dipeptide bonds. The dipeptide linker consisting of Val-Cit (VC)is the patented technology developed by Seattle Genetics and now it is one of the most widely used linkers. In addition, some researchers in recent years have adopted LPETG pentapeptide as specific tag for sortase A-mediated synthesis of site-specific ADCs [11]. Some relative studies have also been carried out in our laboratory. Glucuronide Linker It is another type of enzymatically cleavable linkers. The glucuronic acid linkers act like the dipeptide linker. The β-glucuronide glycosidic bond in ADCs is hydrolyzed by β-glucuronidase (GUSB) in the tumor cells to release cytotoxins. This type of linker has strong hydrophilicity, which can reduce the aggregation of ADCs and improve the stability of ADCs in the circulation system.

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J. Zhan

Thioether Linker It is a new type of noncleavable linkers with super stability, which can only be broken by the complete degradation of polypeptide chains of the monoclonal antibody in lysosomes. In tumor cells, the antibody portion of ADCs can be completely degraded in lysosomes, releasing Lys-toxin molecules which possess similar activity to free toxins. This type of linkers has been successfully employed in T-DM1 synthesis, showing greater stability and therapeutic efficacy. Bifunctional Linker The above linkers can be used not only alone but also in combination. Currently the commonly used bifunctional crosslinkers include N-succinimidyl-3-(2-pyridyldithiolpro) pionate (SPDP, disulfide linker), Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, thioester) etc. Bifunctional linkers are usually conjugated to the Lys and Cys residues of the antibody polypeptide chains. In order to enhance therapeutic effects, a spacer molecule would be inserted to improve activity of the antibody and drug-release efficiency [9, 10]. In recent years, it has also been reported that unnatural amino acids were introduced into the antibody polypeptide chain, and then the site-specific conjugating approach was used to synthesize ADCs [12]. In conclusion, it is the key to improve the efficacy of ADCs by selecting the appropriate linkers, proper number of toxin molecules at the appropriate conjugating site, and optimizing the synthetic process.

1.4

Effector Molecules

In theory, toxic substances (such as chemicals, toxins, radionuclides, etc.) that have high cytotoxicity can act as effector molecules for ADCs. However, the number of effector molecules that can reach tumor cells through the circulation system and the tissue barrier of the solid tumor is very limited. Therefore, the selected effector molecules need to be highly active, usually with EC90 less than 1 nmol/L [13]. In addition, the mechanism of action of the effector molecules must be very clear for safety considerations. Based on the mechanism of action, the commonly-used effector molecules of ADCs can be divided into the following two classes.

1.4.1

Tubulin Inhibitors

Tubulin inhibitors including auristatins and maytansines have been widely explored for ADC synthesis. Auristatins are synthetic pentapeptides derived from natural tubulin polymerase inhibitor dolastatin-10. Among them, MMAE (monomethyl auristatin E) and MMAF (monomethyl auristatin F) are commonly used as payloads of ADCs, and their structures are shown in Fig. 1.1a, b. In Vitro analysis shows that MMAE is a highly toxic cytotoxin (EC90 less than 1 nmol/l), and MMAF was a modified product of MMAE. MMAF differs from MMAE since a phenylalanine moiety introduced at its C-terminus, contributing to its membrane impermeability.

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Antibody-Drug Conjugates: Basic Concepts and Structures

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Fig. 1.1 Structures of tubulin inhibitors. (a) Structure of MMAE. (b) Structure of MMAF. (c) Structure of DM1. (d) structure of DM4

For this reason, the cytotoxicity of MMAF itself is very low, which increases ADC safety in the blood circulation. However, when MMAF is coupled with the monoclonal antibody and internalized into tumor cells, it can produce high cytotoxicity like MMAE [13]. Maytansine analogs used for ADC construction mainly include maytansinoid DM1 and DM4, their structures are shown in Fig. 1.1c, d. Maytansine was first isolated in 1972 from the East African shrubs Maytenus ovatus (now known as Maytenus serrata). This compound is able to inhibit tubulin assembly and induces cell apoptosis. Currently, DM1 and DM4 are usually semi-synthesized from ansamitocin which can be obtained by microbial fermentation. The cytotoxicity of these analogs is similar to that of maytansine, and they have extremely potent cytotoxic effects to many cells.

1.4.2

DNA-Damaging Antibiotics

Antibiotics such as calicheamicin, duocarmycins and pyrrobenzodiazepine (PBD) derivatives that can damage DNA structures are often used to constructure ADCs [13, 14]. Calicheamicins are a family of enediyne antitumor antibiotics

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Fig. 1.2 Structures of DNA-damaging antibiotics. (a) Structure of calicheamicin γ. (b) Structure of duocarmycin A. (c) Structure of anthramycin

originally extracted from Micromonospora echinospora, whose structures contain carbohydrate chain, enediyne moiety, and aglycon consisting of bicycle tridec-9ene-2, 6-diyne system with a labile methyl trisulfide. Calicheamicins bind the minor groove of the specific sequence of double-stranded DNA and generate 1,4-benzenoid diradical that damages the phosphodiester backbone of DNA, causing cytotoxicity. Of them, calicheamicin γ has the highest activity and is commonly used in ADC synthesis (see Fig. 1.2a for its structure). The natural duocarmycins are isolated from Streptomyces refuineus, including duocarmycin A, adozelesin, CC-1065, etc. They also bind to the minor groove of the DNA double helix and specifically alkylate adenine at the N3 position, exhibiting strong cytotoxicity. The structure of duocarmycin A is shown in Fig. 1.2b.

1

Antibody-Drug Conjugates: Basic Concepts and Structures

11

Pyrrolobenzodiazepines (PBDs) are another class of anti-tumor antibiotics isolated from streptomyces and bind to minor groove of DNA. Doxorubincin is an early PBD derivative, but not toxic enough. The most commonly used PBD derivative is anthramycin, whose structure is shown in Fig. 1.2c.

1.5

Challenges and Prospective

Antibody-drug conjugate is a new format of antibody drugs. Since its highly specific and high potency, it changes the conventional cancer therapy and significantly improves the survival, duration and quality of life of cancer patients. The successful application of ADCs in solid tumor therapy has accelerated the development of ADCs to a climax, which has attracted wide attention of pharmaceutical enterprises and investment circles. At present, the main challenges in ADC R&D come from technical and patent challenges. From the technical views, the difficulty of ADC development has reached the maximum of biological products. It’s a multi-step and multi-phase processes, that involves knowledge of both biopharmaceutics and chemical pharmaceutics. The processes include the design of ADCs, the production and purification of monoclonal antibodies, the linker selection and conjugation of ADCs, the production and purification of ADCs, the preclinical drug metabolism and safety evaluation, clinical trials and analysis. The most key technical platforms which have been patented include linker technology and selection of effector molecules. In the respects, many high-tech companies have already taken the lead. Samples include that Seattle Genetics owns vc-MMAE platform, Immunogen has tumor-activated prodrug (TAP) conjugating technology, and Syntarga/Synthon Inc. possesses Duocarmycin prodrug design platform etc. How to find novel conjugating approaches and new effector molecules, and to avoid patent restrictions is a big challenge. The new generation of linker technology must be able to efficiently control the number and coupling sites of effector molecules, increase the stability of ADCs, or decrease the aggregation of ADCs. Discovery of natural sources of effector molecules and the derivatives by chemical modification provide a way to break through patent barriers. It is possible to find potent cytotoxins from natural plants or marine microbes. In addition, the understanding of action mechanism of ADCs is still superficial and needs to be further studied. It includes the metabolism of ADCs in blood circulation, ADC penetrating into tumor tissues, endocytosis and intracellular trafficking of ADCs, and the degradation of ADCs in cells. The detailed studies of the ADC dynamics, can help us identify critical regulatory points for ADC potency, and thus benefit us to find the key for designing an effective ADC.

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References 1. Thomas A, Teicher BA, Hassan R. Antibody-drug conjugates for cancer therapy. Lancet Oncol. 2016;17(6):e254–62. https://doi.org/10.1016/S1470-2045(16)30030-4. 2. Lin L, Ding Q, Tang Q, et al. Antibody-drug conjugates and their application in the treatment of hematological malignancies. Acta Pharm Sin. 2012;47(10):1287–96. 3. Jen EY, Ko CW, Lee JE, et al. FDA approval: Gemtuzumab ozogamicin for the treatment of adults with newly-diagnosed CD33-positive acute myeloid leukemia. Clin Cancer Res. 2018;24:3242. https://doi.org/10.1158/1078-0432.CCR-17-3179. 4. Pan LQ, Wang HB, Xie ZM, et al. Novel conjugation of tumor necrosis factor-related apoptosisinducing ligand (TRAIL) with monomethyl auristatin E for efficient antitumor drug delivery. Adv Mater. 2013;25(34):4718–22. 5. Polson AG, Calemine-Fenaux J, Chan P, et al. Antibody-drug conjugates for the treatment of non-Hodgkin’s lymphoma: target and linker-drug selection. Cancer Res. 2009;69:2358–64. 6. Lewis-Phillips GD, Li G, Dugger DL. Targeting HER2-postive breast cancer with trastuzumabDM1, an antibody-cytotoxic drug conjugate. Cancer Res. 2008;6:9280–90. 7. Barriuso B, Antolín P, Arias FJ, et al. Anti-human endoglin (hCD105) immunotoxin— containing recombinant single chain ribosome-inactivating protein musarmin 1. Toxins. 2016;8(6):184. 8. Kalim M, Chen J, Wang S, et al. Intracellular trafficking of new anticancer therapeutics: antibody-drug conjugates. Drug Des Devel Ther. 2017;11:2265–76. 9. Tsuchikama K, An ZQ. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell. 2018;9(1):33–46. 10. Jain N, Smith SW, Ghone S, et al. Current ADC linker chemistry. Pharm Res. 2015;32:3526– 40. 11. Lai J, Wang Y, Wu S, et al. Elimination of melanoma by sortase A generated TCR like antibody-drug conjugates (TL-ADCs) targeting intracellular melanoma antigen MART-1. Biomaterials. 2018;178:158–69. 12. Axup JY, Bajjuri KM, Ritland M, et al. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci USA. 2012;109:16101–6. 13. Zhu GD, Fu YX. Design of next generation antibody drug conjugates. Acta Pharm Sin. 2013;48 (7):1053–70. 14. Chudasama V, Maruani A, Caddick S. Recent advances in the construction of antibody-drug conjugates. Nat Chem. 2016;8:114–9.

Chapter 2

Relationship Between Target and Specific Action of Antibody-Drug Conjugates Jun Lai and Shuqing Chen

2.1

Introduction

Antibody drug conjugates (ADCs), which combine monoclonal antibody specificity with highly potent chemical, cytotoxic drugs, has recently emerged as a fast-growing and promising biopharmaceutical class. Upon binding with target antigens, ADCs deliver cytotoxic cargo into tumor cells via antigen-mediated internalization, followed by enzyme-catalyzed (e.g., cathepsin B) cytotoxic drug release in late lysosomes. The selection of ADCs target has always been an important part of researchers’ attention. According to statistics, the majority of ADCs targets are tumor associated antigens (TAAs), such as HER2, CD30, etc. Such antigens are not only highly expressed on the surface of tumor cells, but also expressed to a certain extent on the surface of other somatic cells. Therefore, ADCs can kill some normal cells as well as tumor cells. For example, THE HER2-targeted ADC may cause cardiotoxicity [1]. Therefore, this kind of ADCs targets have to face the problem of off-target. However, tumor specific antigens (TSAs) do not have the above problems, such as mutated membrane proteins in the extracellular region and peptides-major histocompatibility complex (pMHC).They can specifically distinguish tumor cells from normal cells, allowing ADCs to specifically target and kill tumor cells. Meanwhile, the specificity and affinity of monoclonal antibodies are highly required. Therefore, this chapter will introduce tumor-associated antigen and

J. Lai Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China Department of Precision Medicine on Tumor Therapeutics, ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou, China S. Chen (*) College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chen, J. Zhan (eds.), Antibody-Drug Conjugates and Cellular Metabolic Dynamics, https://doi.org/10.1007/978-981-19-5638-6_2

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tumor-specific antigen respectively according to the degree of tumor-specificity, and focus on tumor-specific antigen in detail.

2.2

Tumor Associated Antigens

Tumor-associated antigens (TAAs) are proteins that are highly expressed in tumor cells and low or normal expressed in normal somatic cells, among which the more common ones are HER2, CD30, CA125, etc., and are often used in clinical diagnosis and typing of tumors.

2.2.1

Membrane Proteins Associated with Cell Proliferation

Human epidermal growth factor receptor 2 (HER2), a member of the epidermal growth factor receptor family, is encoded by the ERBB2 gene and located on the cell membrane. According to reports, HER2 protein is highly expressed in breast cancer, stomach cancer and other tumor cells. For breast cancer, the expression of HER2 protein is an important factor affecting its prognosis. 25% to 30% of breast cancer patients are affected by the overexpression of HER2 gene in tumor cells in vivo. These tumor cells not only have strong reproduction ability, but also easily develop resistance to some chemotherapy drugs. Currently marketed monoclonal drugs targeting HER2 include Trastuzumab (Herceptin®) and Pertuzumab (Perjeta®), which are based primarily on blocking HER2 dimerization. Trastuzumab’s ado-Trastuzumab emtansine (Kadcyla®) (Fig. 2.1) was approved in 2013 for the treatment of advanced HER2-positive metastatic breast cancer. However, it can also cause pulmonary toxicity (interstitial lung disease, including local acute pneumonia, acute respiratory distress syndrome), infusion-related reactions, allergic reactions, thrombocytopenia, neurotoxicity, and spillage (redness, pain, etc.) at the injection site.

Fig. 2.1 Structure of Trastuzumab’s ado-Trastuzumab emtansine (Kadcyla®)

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2.2.2

15

Carcinoembryonic Antigen

Trophoblast glycoprotein (TPBG), also known as 5T4, is a typical carcinoembryonic antigen, which is expressed on the surface of tumor cells and embryonic cells, and rarely expressed on the surface of other normal somatic cells. Among them, 5T4 antigen was found to be highly expressed in colorectal cancer, gastric cancer, ovarian cancer and other tumors. Studies have shown that the expression of 5T4 in colorectal cancer and gastric cancer reaches 85% and 81% respectively. Therefore, 5T4 as a potential antigen of antibody drugs or ADCs has been widely concerned by researchers [2].

2.2.3

Leukocyte Differentiation Antigen

Leukocyte differentiation antigen is a cell surface marker of lineage (including platelets, vascular endothelial cells, etc.) that appeared or disappeared during normal differentiation and maturation, different stages and activation process. Among them, the cluster of differentiation (CD) is more common in the selection of antibody drug targets. One of these is CD30, which is widely expressed on Hodgkin’s lymphoma cells and is the target of the marketed ADCs Brentuximab Vedotin (Adcetris®). In addition, CD33 and CD22 are targets of two other marketed ADCs (inotuzumab Ozogamicin and Gemtuzumab Ozogamicin).

2.3

Tumor Specific Antigen

The membrane proteins or membrane protein complexes which only exist on the surface of tumor cells are defined as tumor specific antigens, which can be potential markers to specifically distinguish tumor cells from normal somatic cells. Therefore, we suggest that mutated membrane protein complexes on the surface of tumor cells and mutated membrane proteins in the extracellular region on the surface of tumor cells can serve as tumor-specific antigens. As shown in Fig. 2.2, mutated intracellular proteins presented polypeptides containing mutated amino acids to the cell surface through proteasome degradation and presentation by major histocompatibility complex (MHC) class I molecules, forming mutated pMHC; while the extracellular mutated membrane proteins are located directly in the cell membrane. Both of them form tumor specific antigens based on mutated amino acids and become potential targets for antibodies or ADCs. Next, we will focus on the above two types of tumor-specific antigens in detail.

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Fig. 2.2 The formation of mutated membrane proteins and mutated pMHC from mutated genes

2.3.1

Tumor Specific pMHC

pMHC is composed of three parts: MHC class I molecules, β2-microglobulin (β2M), and an antigen peptide composed of 8 to 11 amino acids [3], as shown in Fig. 2.3. Among them, MHC class I molecules are composed of three domains, α1, α2, and α3. The α3 domain is located on the cell membrane, and α1 and α2 domains are located outside the cell membrane. The antigen peptide is located in two large α helices in the α1 and α2 domains. In the major groove; β2-microglobulin interacts with the α1 and α3 domains of MHC class I molecules. In cells, some new proteins that are folded and modified incorrectly after translation, and some proteins that are linked to ubiquitin, are degraded by proteasomes to produce a series of polypeptides ranging in length from 3 to 21 amino acids. Those polypeptides with a length of 8–11 amino acids are transported to endoplasmic reticulum by a polypeptide transporter. After further N-terminal modification, they combine with MHC class I molecules and β2microglobulin to form pMHC, which is transported by Golgi to cell membrane.

2.3.1.1

Tumor-Specific pMHC Under Investigation as Targets of Antibody Drugs

Tumor mutation antigens are a type of mutation antigens that are only expressed in tumor cells. Therefore, drugs targeting tumor mutation antigens can fundamentally

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Fig. 2.3 Structure of pMHC

distinguish tumor cells from normal somatic cells and minimize the toxic side effects caused by drug off-targets. The pMHC can provide such tumor-specific mutation targets for antibody drugs. Using phage display technology, Skora screened out the scFv which targets the human leukocyte antigen (HLA)-A*02/ kirsten rat sarcoma viral oncogene (KRAS) G12V mutant pMHC and HLA-A *03/EGFR L858R mutant pMHC, and the specificity and affinity of the scFv were analyzed and evaluated at the cell level [4]. The results showed that the scFv specifically bind to the mutant pMHC which only contain just one mutant amino acid with the affinity 48.65 nmol/ L, while the wild-type pMHC was unaffinity. This result fully demonstrated that monoclonal antibodies were capable of distinguishing the pMHC with only one amino acid difference, the pMHC composed of tumor-specific mutant peptides could become a very promising kind of targets of antibodies and ADCs.

2.3.1.2

Tumor-Specific pMHC as Potential Targets of Antibody Drugs

The Philadelphia translocation is a specific chromosomal abnormality in chronic myelogenous leukemia (CML) cells, which led to formation of fusion gene BCR-ABL [5].In Philadelphia chromosome positive (ph +) cells, three different fusion proteins, b2a2, e1a2 and b3a2, are created respectively due to the splicing

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variants. Several neo-peptide derived from these fusion proteins junctions have high affinity with MHC I subtypes. Peptide KQSSKALQR which binds to HLA-A03 has been proved immunogenic in vitro, peptides eluted from HLA-A03 transfected K562 cells (ph+) were detected by tandem nanospray mass spectrometry [6]. Cytotoxic T lymphocytes(CTLs) specific for HLA-B08 binding peptide GFKQSSKAL from CML patients and healthy donors have been separated, but the positive frequency was lower than HLA-A03 binding peptide either from healthy donors or patients, which means tumor burden may affect the ability to generate the tumor specific CTLs. For HLA-A02 binding peptide SSKALQRPV, Yotnda raised the specific CTLs successfully lyse the target HLA-A02 transfected CML cells [7]. What’s more, Kessler synthesized the peptides from three fusion proteins and systematically detected degraded peptides after in vitro proteasome digestions by mass spectrometrical analysis, finally identified a peptide AEALQRPVA as an HLA-B61 presented CTL epitope [8]. The B-raf V600E mutation is derived from an amino acid substitution at position 600 from a valine to a glutamic acid. It was reported that 37–50% melanomas harbor V600 mutations [9, 10], and among them, approximately 80–90% mutations are V600E [11, 12]. The V600E mutation in B-raf increases kinase activity and invasiveness, also contributing to melanoma cells reduced apoptosis. Because of the high frequency intracellular expression of V600E mutation in melanomas, it is widely regarded as a potential immunotherapeutic target for T cell. CD4 positive (CD4+) T cell and CD8 positive (CD8+) T cell were generated derived from melanoma patients by mutant peptides stimulation in vivo [13], and specific activity to target cells was observed. Especially for CD8+ HLA-B2705 stricted mutant specific T cell, V600E harbored melanoma cells were selectively recognized and killed. Coincidentally, similar results have been verified in melanoma patients with V600E mutations and HLA-A02 positive. These studies demonstrated that V600E mutant peptides could bind to HLA-B2705 and HLA-A2, and induce CD8+ CTL responses, providing a promising epitope for immunotherapy [14]. G12 mutations are dominant oncogenic mutations in K-ras gene, particularly G12D and G12V mutations were reported in 60–70% pancreatic cancer [15], 20–30% colorectal cancer and 20–30% non-small-cell lung cancer (NSCLC) [16]. Currently, immunotherapeutic approaches on mutant K-ras have been developed. Peptides containing G12V mutation were vaccinated on pancreatic cancer patients, autologous G12V mutation harboring tumor cells were killed by CD8+ specific T cell in vivo, also HLA-B35 restricted peptide VVVGAVGVG was verified as the target for effector T cell. Dendritic cells (DCs), a powerful antigen presenting cells, were used to present G12V K-ras mutant peptide to activate mutant specific T cell which functional against pancreatic cancer cells in vitro and mouse pancreatic tumor model. G12D K-ras mutant DNA vaccine was investigated as a novel immunotherapeutic agent in mouse NSCLC tumor model, the enhanced T-helper 1 immune response and the increasing number of tumor-infiltrating CD8+ T cells were observed after vaccination [17]. Coincidentally, HLA-A1101 restricted G12D mutant epitope specific T-cell receptors (TCR) were generated by immunizing HLA-A1101 transgenic mice and genetic-engineered to human peripheral blood lymphocytes. In mouse xenograft model, the transduced

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TCR-T cell significantly inhibit growth of HLA-A1101 positive tumor carrying the G12D mutation [18]. EGFR T790M was firstly studied as an acquired drug resistance mutation for EGFR tyrosine kinase inhibitors (TKIs) treatment, about 50%–60% NSCLC patients detected this mutation after experiencing disease progression on EGFR TKIs [19, 20]. Recently study revealed that T790M mutation existed in 79.9% EGFR TKI pretreatment NSCLC patients with the frequency ranged from 0.009% to 26.9% [21]. In 2015, osimertinib (Tagrisso) was approved by FDA as the third-generation EGFR TKI, which binds to certain mutant forms of EGFR (T790M, L858R, 19 exon deletion) at about 9-folds lower concentrations than wild-type. However, osimertinib also inhibited the activity of HER2, HER3, HER4, ACK1 and BLK at clinically relevant concentration in in vitro experiments [22]. It can be said that EGFR T790M has been a popular target for anti-tumor drug research in recent years. Researchers hope to find a drug that specifically targets the mutation, so as to specifically kill tumor cells, but not other tissues. Also, two T790M mutation peptides were verified as tumor specific epitope by HLA-A2 restriction [23]. What’s more, mutant specific CTLs were induced by mutant peptides stimulation from PBMCs of HLA-A2+ healthy donors and selectively showed reactivity against the HLA-A2+, T790M harboring NSCLC cells, which demonstrating EGFR T790M mutation could be potential immunotherapy target for EGFR-TKI-resistant patients. Recently, antibodies targeting pMHC derived from P53, preferentially expressed antigen of melanoma (PRAME), alpha fetoprotein (AFP) and other tumor-associated antigens have also been screened out one by one, and are used in preclinical antitumor research [24–27]. This shows that tumor-related and tumor-specific pMHC are getting more and more attentions. Researchers hope that through the discovery of such targets, the selection of anti-tumor drug targets will be broadened, and a solid foundation will be found for finding a new class of anti-tumor drugs and therapies.

2.3.2

Tumor Specific Mutant Extracellular Domain of Membrane Protein

The extracellular domain of transmembrane proteins locate on the cell surface, which could bind to antibodies or antibody derivatives. Once mutation occurred on the extracellular domain of transmembrane protein, a new mutation specific B cell epitope may be generated. The International Cancer Genome Consortium (ICGC) database and the Catalogue Of Somatic Mutations In Cancer (COSMIC) database are two important tumor sequencing database, based on the ICGC database, we analysis 9155 tumor samples, we find 3328 mutations in membrane protein extracellular domain, among them 17 mutations occurs more than 5 and frequency more than 1% (Table 2.1). At present, among ADCs undergoing clinical and pre-clinical research, one ADC targets the tumor-specific antigen—EGFRv variantIII mutation. EGFR variantIII

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Table 2.1 Overview of 17 mutations in membrane protein extracellular domain Gene NOTCH2

Mutation 61G > A

AA_change A21T

Count 23

Project 10

ERBB2 EGFR FGFR2 NOTCH2 CACNA1D ACSL3 FAT4 NOTCH2 GRIN2A FGFR3 KIT KDR KDR LIFR EGFR PDCD1LG2

929C > T 1793G > T 755C > G 112G > A 6319G > A 2041C > T 8054G > A 57C > G 3199C > T 742C > T 2447A > T 3299C > T 3095G > A 955G > A 865G > A 490A > C

S310F* G598V* S252W* E38K E2107K R681C R2685Q* C19W R1067W* R248C* D816V S1100F R1032Q* E319K* A289T T164P

15 15 9 8 8 7 6 5 5 5 5 5 5 5 5 5

6 2 1 4 5 2 3 5 3 2 3 2 2 4 1 1

Remarks 8.74% bladder cancer, 6.67% thyroid cancer 3.88% bladder cancer 4.48% glioblastoma 3.66% endometrial cancer

0.9% cutaneous melanoma 3.88% bladder cancer

1.19% cutaneous melanoma 1.25% rectal cancer

FGFR2 fibroblast growth factor receptor 2, CACNA1D L-type voltage-dependent calcium channel subunit alpha-1 D, ACSL3 long-chain acyl-CoA synthetase 3, GRIN2A glutamate receptor, ionotropic, N-methyl D-aspartate 2A, LIFR leukemia inhibitory factor receptor PDCD1LG2 programmed cell death 1 ligand 2

(EGFRvIII) mutation results from exons 2–7 deletion of EGFR, which has been identified in glioblastoma and NSCLC [28].Recent 20 years, EGFRvIII was regarded as a tumor specific epitope and lots of efforts were made to develop a effective drug, especially in monoclonal antibody industry. AMG595, a highly selective EGFRvIII-specific ADC which was developed by Amgen showing a potent activity on tumor growth inhibition [29]. It is the first ADC targeting a tumor specific epitope being tested in phase I clinical trial. In addition, the EGFR S492R mutation is also a potential tumor specific ADCs target, which is a point mutation in membrane protein extracellular domain which dramatically influence the binding between cetuximab and EGFR [30]. This mutation was detected in metastatic colorectal cancer (mCRC) biopsies and mCRC cell line after cetuximab treatment while all pre-treatment biopsies and cells were wild type for EGFR and K-ras, and verified in vitro for the cetuximab resistance [31]. Further more, a sensitive droplet digital PCR-based method was established to detect EGFR S492R mutation in circulating free DNA (cfDNA) in plasma sample of mCRC patients treated with panitumumab and cetuximab, the S492R mutation was detected in 16% patients (46/285) treated with cetuximab and 1% patients (3/261) treated with panitumumab [32]. The above researches indicated the EGFR

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S492R mutation may be a unique acquired drug resistance mutation after cetuximab treatment which is not related to the primary resistance, and the occurrence frequency of post-cetuximab treatment make it a potential tumor-specific antigen for mutant specific antibody-based therapy.

2.4

Conclusion

In summary, we find that most of the targets targeted by ADCs currently undergoing clinical trials are still tumor-associated antigens, but many tumor-specific antigens are already undergoing clinical or preclinical research. With the development of sequencing technology and detection technology, more primary tumor-specific antigens and acquired tumor-specific antigens will be discovered and identified. Therefore, in the selection of antibodies and ADCs targets, we are more inclined to choose tumor-specific antigens to reduce the side effects caused by target selection.

References 1. Poon KA, Flagella K, Beyer J, Tibbitts J, Kaur S, Saad O, et al. Preclinical safety profile of trastuzumab emtansine (T-DM1): mechanism of action of its cytotoxic component retained with improved tolerability. Toxicol Appl Pharmacol. 2013;273:298–313. 2. Sapra P, Damelin M, DiJoseph J, Marquette K, Geles KG, Golas J, et al. Long-term tumor regression induced by an antibody-drug conjugate that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells. Mol Cancer Ther. 2013;12:38. 3. York IA, Rock KL. Antigen processing and presentation by the class I major histocompatibility complex. Annu Rev Immunol. 1996;14:369–96. 4. Skora AD, Douglass J, Hwang MS, Tam AJ, Blosser RL, Gabelli SB, et al. Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes. Proc Natl Acad Sci. 2015;112:9967–72. 5. Rowley JD. A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and giemsa staining. Nature. 1973;243:290. 6. Clark RE, Dodi IA, Hill SC, Lill JR, Aubert G, Macintyre AR, et al. Direct evidence that leukemic cells present HLA-associated immunogenic peptides derived from the BCR-ABL b3a2 fusion protein. Blood. 2001;98:2887. 7. Yotnda P, Firat H, Garcia-Pons F, Garcia Z, Gourru G, Vernant JP, et al. Cytotoxic T cell response against the chimeric p210 BCR-ABL protein in patients with chronic myelogenous leukemia. J Clin Invest. 1998;101:2290–6. 8. Kessler JH, Bres-Vloemans SA, van Veelen PA, de Ru A, Huijbers IJG, Camps M, et al. BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes. Leukemia. 2006;20:1738. 9. Hodis E, Watson Ian R, Kryukov Gregory V, Arold Stefan T, Imielinski M, Theurillat J-P, et al. A landscape of driver mutations in melanoma. Cell. 2012;150:251–63. 10. Krauthammer M, Kong Y, Ha BH, Evans P, Bacchiocchi A, McCusker JP, et al. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat Genet. 2012;44: 1006.

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11. Rubinstein JC, Sznol M, Pavlick AC, Ariyan S, Cheng E, Bacchiocchi A, et al. Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032. J Transl Med. 2010;8:67. 12. Lovly CM, Dahlman KB, Fohn LE, Su Z, Dias-Santagata D, Hicks DJ, et al. Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials. PLoS One. 2012;7:e35309. 13. Andersen MH, Fensterle J, Ugurel S, Reker S, Houben R, Guldberg P, et al. Immunogenicity of constitutively active V599E BRaf. Cancer Res. 2004;64:5456–60. 14. Somasundaram R, Swoboda R, Caputo L, Otvos L, Weber B, Volpe P, et al. Human leukocyte antigen-A2–restricted CTL responses to mutated BRAF peptides in melanoma patients. Cancer Res. 2006;66:3287–93. 15. Laghi L, Orbetegli O, Bianchi P, Zerbi A, Di Carlo V, Boland CR, et al. Common occurrence of multiple K-RAS mutations in pancreatic cancers with associated precursor lesions and in biliary cancers. Oncogene. 2002;21:4301. 16. Roberts PJ, Stinchcombe TE. KRAS mutation: should we test for it, and does it matter? J Clin Oncol. 2013;31:1112–21. 17. Weng TY, Yen MC, Huang CT, Hung JJ, Chen YL, Chen WC, et al. DNA vaccine elicits an efficient antitumor response by targeting the mutant Kras in a transgenic mouse lung cancer model. Gene Ther. 2014;21:888. 18. Wang QJ, Yu Z, Griffith K, Hanada K-i, Restifo NP, Yang JC. Identification of T-cell receptors targeting KRAS-mutated human tumors. Cancer Immunol Res. 2016;4:204. 19. Yu HA, Arcila ME, Rekhtman N, Sima CS, Zakowski MF, Pao W, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFRmutant lung cancers mechanisms of acquired resistance to EGFR-TKI therapy. Clin Cancer Res. 2013;19:2240. 20. Ji W, Choi C-M, Rho JK, Jang SJ, Park YS, Chun S-M, et al. Mechanisms of acquired resistance to EGFR-tyrosine kinase inhibitor in Korean patients with lung cancer. BMC Cancer. 2013;13:606. 21. Watanabe M, Kawaguchi T, Isa S-i, Ando M, Tamiya A, Kubo A, et al. Ultra-sensitive detection of the pretreatment EGFR T790M mutation in non–small cell lung cancer patients with an EGFR-activating mutation using droplet digital PCR. Clin Cancer Res. 2015;21:3552. 22. Gao X, Le X, Costa DB. The safety and efficacy of osimertinib for the treatment of EGFR T790M mutation positive non-small-cell lung cancer. Expert Rev Anticancer Ther. 2016;16: 383–90. 23. Yamada T, Azuma K, Muta E, Kim J, Sugawara S, Zhang GL, et al. EGFR T790M mutation as a possible target for immunotherapy; identification of HLA-A*0201-restricted T cell epitopes derived from the EGFR T790M mutation. PLoS One. 2013;8:e78389. 24. Li D, Bentley C, Anderson A, Wiblin S, Cleary KLS, Koustoulidou S, et al. Development of a T-cell receptor mimic antibody against wild-type p53 for cancer immunotherapy. Cancer Res. 2017;77:2699. 25. Chang AY, Dao T, Gejman RS, Jarvis CA, Scott A, Dubrovsky L, et al. A therapeutic T cell receptor mimic antibody targets tumor-associated PRAME peptide/HLA-I antigens. J Clin Invest. 2017;127:2705–18. 26. Mathias MD, Sockolosky JT, Chang AY, Tan KS, Liu C, Garcia KC, et al. CD47 blockade enhances therapeutic activity of TCR mimic antibodies to ultra-low density cancer epitopes. Leukemia. 2017;31:2254. 27. Liu H, Xu Y, Xiang J, Long L, Green S, Yang Z, et al. Targeting alpha-fetoprotein (AFP)–MHC complex with CAR T-cell therapy for liver cancer. Clin Cancer Res. 2017;23:478. 28. Wikstrand CJ, Hale LP, Batra SK, Hill ML, Humphrey PA, Kurpad SN, et al. Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res. 1995;55:3140.

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29. Hamblett KJ, Kozlosky CJ, Siu S, Chang WS, Liu H, Foltz IN, et al. AMG 595, an AntiEGFRvIII Antibody–Drug Conjugate, Induces Potent Antitumor Activity against EGFRvIIIExpressing Glioblastoma. Mol Cancer Ther. 2015;14:1614. 30. Montagut C, Dalmases A, Bellosillo B, Crespo M, Pairet S, Iglesias M, et al. Identification of a mutation in the extracellular domain of the epidermal growth factor receptor conferring cetuximab resistance in colorectal cancer. Nat Med. 2012;18:221–3. 31. Esposito C, Rachiglio AM, La Porta ML, Sacco A, Roma C, Iannaccone A, et al. The S492R EGFR ectodomain mutation is never detected in KRAS wild-type colorectal carcinoma before exposure to EGFR monoclonal antibodies. Cancer Biol Ther. 2013;14:1143–6. 32. Newhall K, Price T, Peeters M, Kim TW, Li J, Cascinu S, et al. Frequency of S492R mutations in the epidermal growth factor receptor analysis of plasma dna from metastatic colorectal cancer patients treated with panitumumab or cetuximab monotherapy. Ann Oncol. 2014;25:ii109.

Chapter 3

The Internalization and Therapeutic Activity of Antibody Drug Conjugates Jiansheng Fan and Shuqing Chen

3.1

Introduction

Unlike small molecules such as amino acids and ions, antibody conjugate drugs (ADCs), a class of protein macromolecules, usually be transported into the cell in a different way. ADCs need to be wrapped on the surface of cell membrane before enter the cytoplasm as a vesicle structure. a process called endocytosis. Apart from some cells (such as macrophages, monocytes, etc.), endocytosis is used by cells to engulf over 100 um diameter particles [1]. In terms of function mechanism, it can be divided into macropinocytosis, clathrin-mediated endocytosis (CME), caveolinmediated endocytosis, clathrin- and caveolin-independent endocytosis [2]. Endocytosis of ADCs and antibodies is mainly completed through receptor-mediated endocytosis, that is, through CME [3, 4]. CME widely exists in mammalian cells and plays a role in the process of ingesting some essential nutrients, such as low density lipoprotein (LDL) receptor binding LDL, transferrin receptor binding transferrin [5]. CME could affect the signal pathway by regulating cell surface receptors. It also provides support for intercellular signal transmission in the growth of tissues and organs [6]. The endocytosis process is first mediated by receptors, which recruit adaptor proteins and combine receptor ligands complex with clathrin before transport of them across the cell membrane. After that, the surface of cell membrane invaginates to form a den.

J. Fan Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China Department of Precision Medicine on Tumor Therapeutics, ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou, China S. Chen (*) College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chen, J. Zhan (eds.), Antibody-Drug Conjugates and Cellular Metabolic Dynamics, https://doi.org/10.1007/978-981-19-5638-6_3

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Vacuoles coated with clathrin would separate from cell membrane and interact with endosomes by GTPase-activating protein [7]. After entering the endosome, the receptor protein complex has two pathways: recycled back to cell surface [8]; retained in late endosomes and shuttled into lysosomes. During this trafficking process, once ADCs enter the lysosome, the antibody would be degraded and cytotoxic agents would be released in the cells. Although some ADCs in the early research choosing extracellular cleavable linkers to release cytotoxic drugs in the tumor microenvironment, which could exert its activity without internalization [9, 10], FDA approved ADCs and most ADCs in clinical trials must bind to the target cell surface antigen before efficiently internalize into the cytoplasmic to release cytotoxic drugs to kill the target cells. Therefore, the internalization and internalization efficiency of ADCs impact the drug trafficking in tumor cells and the subsequent cytotoxic activity, which closely related to the activity of ADCs. How to evaluate the internalization of ADCs and improve the internalization efficiency of ADCs has important guiding significance for the development of new ADCs in the future.

3.2

Experimental Steps for Evaluating Internalization

Determine rate of internalization and intracellular localization after internalization are two aspects for evaluating internalization. Determine rate of internalization: Antibodies could not be internalized into cell after binding to cell surface at 4 °C. While being transferred to 37 °C, the internalization of antibodies on the cells could be observed. The fluorescence intensity difference of cell surface antibodies labeled with fluorophores are measured by flow cytometry under two temperatures. The rate of internalization could be calculated. Intracellular localization of internalized antibodies: ADC is internalized into cells and degraded in lysosomes to release the cytotoxic agents. Lysosomes and antibodies were labeled with different fluorescences and localized by confocal microscope.

3.2.1

Materials

FITC (fluorescein isothiocyanate)-labeled goat anti-human IgG (H + L) second antibodies; Alexa Fluor 555 labeled goat anti-mouse IgG (H + L); Anti-LAMP-1 (lysosome-associated membrane protein) mouse monoclonal antibody; 4′,6diamidino-2-phenylindole, DAPI; Fixation solution for cells: 4% paraformaldehyde in PBS (phosphate buffer saline); Permeabilization solution ((0.1% TritonX-100 and 0.2% bovine serum albumin (BSA) in PBS); Freezing centrifuge; Flow cytometry and high-resolution confocal microscopy, etc.

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3.2.2

27

Internalization Assay by Flow Cytometry

The cells were centrifuged to remove the medium and incubated with certain concentrations of antibody or ADC in 1% BSA-PBS at 4 °C for 30 mins. PBS was used as negative control. Wash twice with ice cold PBS to remove antibody or ADC that do not bind to the cell surface. The cells were incubated at 4 or 37 °C for 2 h. After washing twice with PBS, cells were stained with FITC-labeled goats anti-human IgG (H + L) secondary antibody diluted 1:250 and incubated on ice for 30 mins. Wash twice and resuspend in PBS. The mean fluorescence intensity (MFI) on cell surface was detected by flow cytometry. The internalization rate of ADC is calculated by formula below: Internalization% =

3.2.3

ð1 - ðMFI of sample incubated at 37 ° CÞ= ðMFI of control sample incubatedat 4 ° CÞÞ × 100%

Intracellular Localization Assay by Confocal Microscope

The cells were incubated with certain concentration of ADC or antibody at 37 °C for some time (typically 1 h, 4 h and 24 h), centrifugation at 1000 R / min for 5 mins. To fix the cells on the slide (make sure the cells do not adhere to each other), add 4% paraformaldehyde after rinse the cells with PBS 3 times. Allow cells to fix for 15 mins and then wash twice with PBS. Add 0.1% TritonX-100, 0.2% BSA in PBS at room temperature and incubate for 15 mins to permeabilize cells. After wash cells 3 times with PBS, anti LAMP-1 mouse monoclonal antibody diluted with 1% BSA-PBS was added to the cells and incubated for 45 mins. Again, wash cells 3 times with PBS, cells were stained with FITC -labeled goat anti-human IgG (H + L) and Alexa Fluor 555-labeled goat anti-mouse IgG (H + L). Allow to incubate in dark for 30 mins and wash slides 3 times in PBS. The nuclei were stained with DAPI in dark for 3 mins before washing with PBS 3 times. Anti-quencher solution was dropped on the slide, covered the slide and visualized the subcellular localization of antibody or ADC by confocal laser scanning microscope (Fig. 3.1).

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Fig. 3.1 Trafficking and subcellular localization of anti-CD20 ADC. Daudi cells were treated with ADC at 37 °C for 6 h. Cells were fixed as above, ADC and lysosome-associated membrane protein1 (LAMP-1) were detected with different fluorochrome-conjugated secondary antibodies. Nuclei were stained with DAPI (blue). Arrow indicated co-localization of ADC (green) and lysosomes (red)

3.3

Strategies to Improve Internalization Efficiency

At present, most ADCs target antigens on the cell surface, which means the ADCs needed to be internalized into cells and transported into lysosomes to release cytotoxic drugs and further kill target cells to exert their antitumor activity. Therefore, improving the internalization efficiency of ADCs is a key to the efficacy of them. ADC is composed of targeted antibody, small molecule toxins and linkers. Considering the mechanism of action and structure of ADCs, the key to improve the internalization efficiency depends mainly on the characteristics of specific antibody molecules. There are actually many solutions mainly focus on the construction of antibodies. First of all, choosing the antibodies meets the requirements in antibody screening. The antibody internalization process is related to the characteristics of antigen and the dissociation rate between antibody and antigen. Antibodies with high affinity do not means high internalization efficiency [11]. In addition, modifying the structure of screened antibody to further improve its internalization efficiency is also a hot topic in current ADC research.

3.3.1

Identifying Antibody Binding Epitopes

For specific determined antibody, different binding epitopes of target protein sometimes have a great impact on internalization of antibody. CD56 is over expressed in neuroblastoma, multiple myeloma and small cell lung cancer. Anti-CD56 humanized ADC—IMGN901 from Immunogen is mainly focus on small cell lung cancer and has been in clinical trials. CD56 was proved to be one of optional targets for ADCs [12]. Feng et al. [13] identified two full human antibodies targeting CD56— m900 and m906. The affinities of these two antibodies were similar, however, their

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binding epitopes were different. While m900 binds to membrane-distal domain of the extracellular region of CD56, m906 binds to membrane-proximal domain. Compared with m900, m906 showed higher internalization efficiency and induced significant down-regulation of cell surface CD56 more efficiently after binding to CD56. Two ADCs were constructed by conjugating both antibodies with pyrrolobenzodiazepine (PBD) respectively. m906 ADC also displayed more potent killing activity. However, the difference of receptor-mediated internalization efficiency caused by different binding epitopes on same antigen is correlated with the characteristic of target protein. It has been found a similar phenomenon in HER2, a fully studied ADC target. For example, extracellular binding epitope of anti-HER2 pertuzumab is far away from the membrane and does not overlap with that of trastuzumab. Similarly, pertuzumab showed better HER2 internalization efficiency as m906 [14]. This phenomenon is more obvious in a study on anti-HER3 antibodies. Antibody A2 binds to membrane-proximal domain, while A3 binds to the membrane-distal domain. The experiment showed that A2 could induce internalization of receptors, but not A3 [15]. This suggests that for some targets, such as CD56, HER2 and HER3, antibodies binding to membrane-distal domain of the extracellular region have higher internalization efficiency. One potential explanation is that the dimerization of these receptors is influenced by the binding epitopes of antibodies.

3.3.2

Antibody Drug Conjugate Toxins

Depending on the characteristic of antigen, whether the antibody is coupled with small molecule cytotoxic agents will affect the internalization of antibody. For some antigens, such as CD30, the internalization efficiency of anti- CD30 ADC brentuximab vedotin is same as antibody [16]. While for other antigens, such as CD20, the internalization efficiency of antibody has been significantly improved [17], and difference of linkers will also affect the internalization of antibody [18]. This shows that even if the binding characteristics of antigen and antibody are clarified in the early stage, the conjugation process of ADC would also affect its internalization efficiency.

3.3.3

Application of Bispecific Antibody

Application of bispecific antibody to ADC also improves the internalization and lysosome transportation of ADC. This bispecific ADC usually in the form of full antibody could retain its longe half-life in the blood. When targeting two antigens as a bispecific ADC, one target with lower internalization could be dragged into the cell by the high internalization of another target. This drag-type ADC can not only improve the internalization efficiency of low internalization antigens, but also reduce the possibility of antigens recycling to cell surface. Devay et al. [19] combined

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amyloid precursor like protein 2 (APLP2) and HER2 to form a bispecific antibody. APLP2 could effectively interact with HER2 to generate a complex which are internalized and transported into lysosomes [20]. In another anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) antibody, APLP2 could bind antibody and promot its degradation after entering lysosome [21]. In order to evaluate the internalization efficiency of APLP2 antibody conjugate drug alone, it was compared with anti-Trop 2 ADC. In vitro cytotoxicity in SKOV3 cells showed that, although the expression level of Trop-2 on cell surface was twice that of APLP2, anti-APLP2 ADC showed lower EC50 value and higher cell killing efficiency. This suggests that the improvement of the lysosome travel efficiency of ADC could indeed improve the efficacy of ADC. Combination of APLP2 and HER2, a target with low internalization efficiency, a bispecific ADC was formed. The lysosomal degradation of HER2 has also been significantly improved due to the high lysosomal targeting efficiency of APLP2 in the cell. This phenomenon has been observed in a variety of cell lines with different APLP2: HER2 ratio. De Goeij et al. [22] constructed a bispecific ADC targeting CD63 and HER2 in a same way. While the anti-HER2 binding domain provides tumor specificity, antiCD63 binding domain can promote internalization. CD63, also known as lysosome associated membrane protein 3 (LAMP3), belongs to four transmembrane protein superfamily. It exists on some cell surfaces and is mainly expressed on intracellular vesicles, such as the membrane of endosomes and lysosomes. CD63 regulates the transportation of other proteins in the endocytic pathway [23]. Targeting CD63 can promote internalization and lysosomal transport efficiency. Compared with monovalent control antibodies targeting HER2 or CD63, this bispecific structure could be transported into lysosomes of HER2 positive tumor cells more effectively. This bispecific ADC also exhibited excellent tumor killing in in vitro and in vivo experiments. To avoid this bispecific ADC targeting cells expressing only CD63, such as platelets and granulocytes in peripheral blood, the affinity of anti-CD63 antibody was attenuated, which could maintain the specificity and improve the safety of this bispecific ADC [23, 24]. Choosing a target widely expressed and could be internalized into lysosome targets efficiently to drag other tumor associated antigen are solutions above. In order to reduce the non-specific killing of this ADC, it necessary to modify the affinity of high internalization binding domains or allow the ADC binding antigens in a pH-dependent manner and exhibit its activity in a tumor microenvironment expressed two antigens simultaneously. Construction of a bispecific ADC with two tumor associated antigens is another idea. Although simultaneously expressing two tumor associated antigens shrink the applicable tumor targets, it also reduced the non-specific killing effect resulted by widely expressed target. Similarly, Regeneron Pharmaceuticals constructed a bispecific ADC targeting HER2 and prolactin receptor (PRLR) [25]. PRLR is expressed in some kind of breast cancer and is related to the pathogenesis of breast cancer. Therefore, as same as HER2, PRLR is also a tumor associated antigen [26, 27]. The expression level of PRLR on the surface of breast tumor cells is lower than that of HER2. However, unlike HER2, which is often recycling to the cell surface [28], PRLR could be

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continuously transported into lysosomes and degraded [29]. This characteristic of PRLR make this bispecific ADC could drag the anti-HER2 part with low internalization efficiency into lysosomes and then release small molecule cytotoxic agents more effectively. Compared with anti-HER2 or anti-PRLR ADC, the bispecific ADC targeting PRLR and HER2 also kill breast tumor cells more effectively. The ligand of mesenchymal epithelial transition factor (MET), also known as c-Met, is hepatocyte growth factor (MET) secreted by stromal cells. MET is associated with drug resistance in various cancers. Lee et al. [30] combining the MET with HER2 or EGFR. Although it was not conjugated to obtain ADC, this bispecific antibody could also exhibit drag function in presence of MET, and promote the degradation of the whole bispecific antibody. MET and EGFR are tumor associated antigens in this bispecific format. Overexpression of both proteins plays an important role in tumor growth and metastasis. Since the signal pathways of MET and EGFR are interrelated, inhibiting one target alone would activate another pathway in tumor cell, targeting both antigens could achieve a better therapeutic effect [31]. In a study of EGFR- MET bispecific antibody, it has been found that this bispecific antibody could promote the interaction between EGFR and MET and dissociate heat shock protein 90 (Hsp90) from MET-EGFR complex. As a chaperone, HSP90 plays a protective role in the process of lysosomal degradation. It is reasonable for the degradation of EGFR and MET simultaneously after this bispecific antibody binding to both targets and the good antitumor activity of this antibody.

3.3.4

Application of Biparatopic Antibody

Biparatopic ADC is also a kind of ADC with bispecific structure. it does not target two antigens at the same time, but different epitopes on the same antigen. Until now, targeting different epitopes of same target simultaneously mainly focus on EGFR and HER2, which all belong to ErbB family. One successful example is an anti-HER2 biparatopic ADC in clinical research. This biparatopic antibody format was not used in ADC at first. Robert et al. [32] found that a antibody targeting two nonoverlapping epitopes of carcinoembryonic antigen (CEA) exhibited higher binding activity and in vivo antitumor activity compared with the original antibody. Another research shows that combining several non-competitive anti EGFR antibodies could synergistically reduce the EGFR on tumor cell surface and show better antitumor activity and longer survival time in tumor-bearing mouse model [33, 34]. Further research have shown that the combination of multi-epitope antibodies makes the receptors on the cell surface interact cross linked and form a larger antibody-antigen complex [35]. Spangler et al. [36] found that multi-epitope antibodies could inhibit the antigen in endosomes recycling back to cell membrane and promote the down-regulation of EGFR on the cell surface after receptor-antigen is internalized. As same as EGFR, the combination of anti-HER2 multi-epitope antibodies would also down-regulate cell surface receptors and promote internalization [37]. Based on

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this principle, MedImmune identified a full human anti-HER2 antibody—39S whose binding epitope is different from trastuzumab or pertuzumab. Fusing the scFv form of Trastuzumab at the N- terminal of heavy chain of 39S obtained a new antibody. Since the HER2 binding epitope of 39S is nonoverlap with trastuzumab, this new antibody is a tetravalent anti-HER2 biparatopic antibody. The relative molecule mass of antibody is larger than conventional antibody, about 240 kDa [38]. Although the relative molecular weight of this biparatopic antibody is large, its internalization efficiency on BT-474 cells is higher than that of trastuzumab or pertuzumab, even higher than that of mixture from both trastuzumab and pertuzumab. Therefore, this biparatopic format is not equivalent to the mixing of antibodies targeting different epitopes. Further mechanism studies showed that once the biparatopic antibody binding with the different epitopes on HER2, the N-terminal tetravalent structure of biparatopic ADC (MEDI4276) would induce HER2 receptor clustering on the cell surface, which form a larger HER2- ADC complex cluster. The clustering of HER2 receptor at the cell surface could promote the internalization of receptor and inhibit its recycling from lysosome to the cell surface, which improves ADC enter lysosomal trafficking of ADC significantly [39]. It has been found that down-regulation of HER2 in some T-DM1 resistant cell lines. In addition, heterodimerization of HER2 and HER3 is another reason for T-DM1 resistance. Benefit from high internalization efficiency of MEDI4276 and the hindrance of HER2-HER3 dimerization by binding epitope of 39S, MEDI4276 could kill T-DM1-resistant HER2 low expression tumor cells, which could expand the application scope of anti-HER2 ADC in future clinical trials.

3.4

Conclusion

The internalization efficiency of ADC is a key factor for its activity. Due to the low internalization efficiency of some target proteins, they are not considered as a suitable target for ADCs. The internalization efficiency of screened antibodies specific for different epitopes of some target proteins might be quite different, and the activities of ADCs derived from them could be quite different. Therefore, choosing an appropriate antibody as the target part of ADC could effectively improve its internalization efficiency and enhance its antitumor activity. In addition, multi-specific or bispecific antibodies are currently hot research topics in antibody drugs. ADCs can also have multi-target characteristics. The combination of high and low internalization efficiency targets not only strengthens the specificity of ADC, but also strengthens its antitumor activity, which could widen the pharmaceutical application of ADCs.

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Chapter 4

The Internalization and Intracellular Trafficking of ADCs Keying Liang, M. Saleem Khan, M. Kalim, and Jinbiao Zhan

4.1

Introduction

ADC binds to the target antigen expressing on the tumor cell surface and triggers a series of cell responses, including internalization and intracellular trafficking. The extracellular and intracellular trafficking of ADC include the following processes, (1) ADC traffics to the tumor microenvironment through the intravenous blood circulation; (2) monoclonal antibody (mAb) component of the ADC binds to the tumor-specific or tumor-associated antigens and forms the ADC-antigen complex (AAC); (3) AAC internalizes and transports to lysosomes to be degraded through the endosome-lysosome pathway; (4) free payloads diffuse towards specific targets like DNA and microtubules, resulting in cell death. Sometimes the small molecule drug is effective for the neighboring non-target antigen-expressing tumor cells, which is called bystander killing effect. However, if each of the above steps is associated with an efficiency of 50%, only 1%–2% of the administered does of small molecule drug will reach the intracellular target [1]. Internalization is thought to be a critical process of the antibody-based therapeutics. Upon internalization, the toxic payload can be released to exert the tumor cell killing effect. Thus, the internalization rate and extent of ADC is important to its therapeutic efficacy and need to be explored in-depth. Meanwhile, giving insights

K. Liang · M. S. Khan · M. Kalim Department of Biochemistry, Cancer Institute of the Second Affiliated Hospital (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China e-mail: [email protected] J. Zhan (*) School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chen, J. Zhan (eds.), Antibody-Drug Conjugates and Cellular Metabolic Dynamics, https://doi.org/10.1007/978-981-19-5638-6_4

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into the trafficking mechanism could help us to provide guidelines to optimize ADCs for favorable characteristics and improve ADCs efficacy and safety. In this chapter, we will discuss the intracellular trafficking mechanism of ADC from three parts: the endocytosis pathway, the intracellular transport and the release of drug. We will also give a brief introduction of the methods in ADC internalization study.

4.2

The Endocytosis Pathways of ADC

According to the size, polarity or hydrophobicity, extracellular molecules can be internalized through different pathways. Small molecules, like amino acids and ions, can transport through diffusion and ion channels, however, macromolecules such as mAbs and ADCs, need to transport through energy dependent pathway, including receptor-mediated endocytosis and receptor-independent endocytosis (Fig. 4.1).

Fig. 4.1 Three endocytosis pathways of ADC. (a) Clathrin mediated endocytosis; (b) Caveolae mediated endocytosis; (c) Macropinocytosis. The first two endocytosis pathways (a, b) are regarded as receptor-mediated endocytosis and (c) is receptor-independent endocytosis. CCP clathrin coated pits; EE early endosome, LE late endosome, MVB multi-vascular body, RE recycling endosome, TGN trans-Golgi network

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4.2.1

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Clathrin Mediated Endocytosis (CME)

Clathrin mediated endocytosis (CME) is the most common and best studied pathway in eukaryotic cells. CME mediates the vesicular trafficking of a wide range of cargo molecules. Internalization of extracellular molecules is mediated by the formation of clathrin-coated pits (CCPs) and clathrin-coated vesicles (CCVs). Specifically, adaptor protein 2 (AP2) and clathrin will be recruited to the plasma membrane where ADC-antigen complexes aggregate. The ADC-antigen complexes are wrapped and recessed into the cell to form CCPs /CCVs. Dynamin, a GTPase, forms the helix around the neck of the vesicle and therefore drives the release of CCPs/CCVs from the plasma membrane [2]. Actually, CME is divided into multistep process. Some other proteins are also involved in the formation and release of the vesicles, such as phosphatidylinositol-4,5-bisphosphate (PIP2), RhoA, Epsins, EGFR pathway substrate 15 (EPS15) and clathrin-coatomer protein I, II (COPI, II) [3]. The roles of different proteins regulated in CME are showed in Table 4.1. After the formation and maturation of the CCPs/CCVs, the vesicles lose their clathrin coat and enable the ADC-antigen complex to fuse with the early endosome (pH 5.9–6.0). The early endosome subsequently converts into the late endosome with the lower pH. The late endosome then fused with lysosome (pH 4.0–5.0), where the ADC is degraded to release the cytotoxic drug.

4.2.2

Caveolae Mediated Endocytosis (CavME)

Caveolae mediated endocytosis (CavME) and CME are both receptor-mediated endocytosis. The two endocytosis pathways may have different signaling pathways and endocytic fortune, but they share the same receptors, for example, EGFR and TGF-β [5, 6]. Table 4.1 Proteins regulated in CME [4] Protein AP-2 and clathrin ARF6 GTPase Epsins and endophilin Dynamin Amphyphisin Epsins and epsin-homologydomain proteins Lipid kinases and phosphatases SNARE and Rab proteins

Functions The component of CCPs/CCVs Regulates the recruitment of AP-2 and clathrin Induce the bending of the membrane Promotes vesicle budding from a donor membrane Binds endophilin, AP-2 and clathrin Link certain receptors to coat complexes Help the formation of vesicles Facilitate newly formed endosome fuses with pre-existing sorting endosomes

ARF ADP-ribosylation factor 6, SNARE soluble N-ethylmaleimide-sensitive factor-attachment protein receptors, Rab Ras-like proteins from rat brain

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Caveolae, as the specific subtype of lipid raft, is enriched of cholesterol and sphingolipid with the diameter of 55–60 nm [7]. Caveolin (CAV) is the main scaffolding protein of caveolae. CAV is a 21-kDa integral membrane protein that was first identified by E.Yamada in 1955 [8]. There are three types of mammalian CAV isoforms: CAV-1, CAV-2 and CAV-3. Although CAVs have been considered to be the major proteins in CavME, work in the recent years has identified other proteins as functional components of caveolae. For example, the cavin protein family, dynamin, protein kinase C, Src family kinases [2]. Actin and dynamin play crucial roles in membrane bedding and fission of vesicles. Formation of these caveolae driven by CAVs leads to recruitment of some functional proteins and attachment with cholesterol in the plasma membrane. Thereby, ADC-antigen complexes are internalized through CavME and bud off from the plasma membrane to form the special organelle, which is called caveosome [9–11]. Caveosome may attach with early endosome to initiate their action.

4.2.3

Macropinocytosis

Macropinocytosis is a type of receptor-independent endocytosis pathway, which is different from the above two receptor-mediated pathways. Without the recruitment of some foremost molecules like clathrin, caveolin, and dynamin, this process is mainly responsible for the internalization of larger particles with a diameter greater than 1 μm. Macropinocytosis as well as phagocytosis are actin-dependent process. Actin in plasma membrane plays a fundamental role in protrusions formation, encapsulation, internalization and recycling. Actin polymerization and ruffling at the cell membrane margins lead to the protrusions formation, and then actin-rich ruffles close to form the intracellular vesicle, called macropinosome [12]. Like caveosome, macropinosome has the ability to fuse with lysosome or recycle to the plasma membrane. In addition, some other proteins, such as tyrosine kinase, Ras-related C3 botulinum toxin substrate 1 (RAC1), Rab53, cell division control protein 42 (Cdc42), p21-activated kinase (Pak1), WAVE complex, ARF6, and nexin play the important roles in macropinocytosis [2, 13–15].

4.3

Intracellular Trafficking of ADC

ADC trafficking through the receptor-mediated and receptor-independent endocytosis pathways leads to the formation of different vesicles (CCPs/CCVs, caveosomes and macropinosomes). These intracellular vesicles carrying ADC-antigen complexes will process for sorting. The majority of them will be transport from endosome to lysosome (Fig. 4.2).

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Fig. 4.2 Intracellular trafficking of ADC

4.3.1

Early Endosome (EE)

It has been reported that early endosome (EE) is categorized into sorting endosome and endocytic recycling compartment (ERC) [4, 16]. The pH is maintained at 5.9–6.0 in EE lumen. The newly formed vesicles lose the clathrin or caveolae coat, and fuse to form the more acidic EE. Meanwhile, the proteins in Rab GTPase family, such as Rab5 and Rab 11 will release form the vesicles. The EE can also recycle back to the cell surface directly [2]. Some recycling molecules transport back to the plasma membrane directly, and others may transport through the ERC organelle. This recycling mechanism help to regulate the endocytosis and keep the protein balance on the plasma membrane.

4.3.2

Late Endosome (LE)

Late endosome (LE) is also called multi-vesicular bodies (MVBs). The loss of Rab5 and the acquisition of Rab7 represents the transition from EE to LE [17]. Ultimately, EE will fuse with lysosome in a rapid manner.

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Lysosome

Lysosome is responsible for the degradation of endocytosed content. When late endosome carrying ADC cargo fuses with lysosome, linker degradation and drug release will be triggered in lysosome. Lysosome is characterized as an acidic environment at pH around 4.0–5.0 and contains high content of proteolytic enzymes, such as cathepsins and collagenases. The acidification of lysosomes is maintained because of protonation, which means that protons are pumped from the cytosol to the lysosome lumen through the ATPase [18]. Therefore, a specific vacuolar H+ ATPase inhibitor, bafilomycin A1 can significantly inhibit the degradation of ADC. Engineering the ADCs is enable the payload to entry into the lysosome for degradation, so a possible strategy towards achieving this is to design reliable linkers according to the lysosomal property. For example, hydrazones and proteolytic cleavable linkers respectively respond to low pH and proteolytic enzymes, to release the cytotoxic drugs in lysosome.

4.4

Drug Release

Once ADCs are degraded in lysosome, small molecule drugs are released to the cytoplasm to accomplish the cytotoxic effect. Often these cytotoxic drugs conjugated to mAbs are designed based on specific targets like DNA and microtubules. Traditionally, chemotherapy drugs like doxorubicin and methotrexate have only 1–2 effectiveness because of the off-target toxicity, but using conjugated antibodies their potency increases up to 100–1000 fold [19]. Auristatins and Maytansinoids are the major types of microtubule-targeting drugs which cause the cell cycle arrest in dividing cells. Auristatins inhibit the tubulin formation by binding to the β-subunit or interfering with α-β tubulin dimer in the cytoplasm, and as a result microtubules are not able to separate during anaphase [20]. Compare to Auristatins, the mechanism of Maytansines is slightly different. They usually bind to the plus end of tubulin, inhibit polymerization in tubulin dimmer, and result in cell arrest at metaphase [21]. DNA damaging drugs, such as calicheamicins, pyrrolobenzodiazepines (PBD), duocarmycins are often used in ADC conjugation. They can insert into the minor groove of the double-stranded DNAs and further induce apoptosis and cell death. The main used drugs in ADC development are monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), DM1 and DM2. Among them MMAE is the most commonly used payload in ADC approved by FDA. Additionally, some ADCs can be effective for not only the target antigenexpressing tumor cells, but also the neighboring non-target antigen-expressing cells. The diffusion of the small molecule drugs to the bystander cells is called the bystander killing effect [22]. Transport of free drugs to neighboring cells is mediated by the p-glycoprotein-mediated efflux mechanism [21]. For example, MMAE

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penetrates the neighboring cell membranes through passive diffusion, and results in bystander killing effect. However, MMAF has the limitation to enter the surrounding cells because it contains a charged carboxylic acid terminus [23]. Similarly, maytansine derivate DM1, cannot elicit bystander killing effect because their positive charge prevents them from penetrating the negatively charged cell membrane [24]. In tumor microenvironment, heterogeneous tumor cells and tumor-infiltrating immune cells are surrounding. Inevitably, the extracellular release of free drugs may have effects on immune cells like macrophages and lymphocytes. Therefore, the bystander killing effect is thought to be a double-edged sword.

4.5

Methods in Tracing the Intracellular Transport of ADC

Confocal microscopy and flow cytometry are suitable methods to detect the internalization of ADC. Here, we will briefly introduce these two methods.

4.5.1

Flow Cytometry Analysis to Analyze Endocytosis

A quantitative flow cytometry assay is commonly used to determine the endocytosis efficiency. Mean fluorescence intensity (MFI) of the ADC binding to the cell surfaces is determined for different time intervals, so the endocytosis rate of ADC can be calculated by removing the contribution of surface fluorescence (4 °C control) and normalizing the MFI for the number of fluorophores per antibody. The main protocol can be found at 3.2.2.

4.5.2

Confocal Microscopy to Monitor Internalization and Intracellular Trafficking of ADC

Internalization and intracellular trafficking of ADC are commonly visualized by the use of confocal microscopy. During the digital recording of labeled fluorescent specimens, two or more of the emission signals can overlap in the final image due to their close proximity within the microscopic structure, this is known as colocalization. Using this immunofluorescence technique, colocalization between internalized ADC and intracellular organelles can be clearly observed. Different organelles can be marked by specific antibodies. For example, lysosomal associated membrane protein 1 (LAMP1) is usually used as a lysosome marker and early endosome antigen 1 (EEA1) is a marker of EE [25]. The main protocol can be found at 3.2.3.

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The Colocalization of Internalized ADC and Lysosome

To give an example, an anti-HER2 monoclonal antibody (Herceptin®, trastuzumab) and the corresponding ADC (Kadcyla®, T-DM1) were used to study the internalization process. The confocal images showed that neither Herceptin® nor Kadcyla® can be transported to lysosome as short as 1–2 h. Both of them were colocalized with lysosome when HER2-positve cells were treated for 8 h (Fig. 4.3). Besides, the internalization rate of Kadcyla® was more effective than Herceptin®.

4.6

Conclusions

In this chapter, we summarize the studies on endocytosis pathways and intracellular trafficking of ADC, and provide a perspective on its importance to internalization. Although ADCs have been developed rapidly in the recent years, the trafficking mechanism of ADC remains poorly understood. This trafficking mechanism could help us to understand the target antigen selection, intracellular mechanism of action and physiochemical characteristics of ADC, through which we can design and develop the highly safe and efficient therapeutics for cancer treatment. To sum up,

Fig. 4.3 The colocalization of lysosome and internalized Herceptin®/Kadcyla®

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new ADC bullets can be developed, by better understanding the intracellular trafficking and endocytosis pathways.

References 1. Teicher BA, Chari RV. Antibody conjugate therapeutics: challenges and potential. Clin Cancer Res. 2011;17(20):6389–97. 2. Mosesson Y, Mills GB, Yarden Y. Derailed endocytosis: an emerging feature of cancer. Nat Rev Cancer. 2008;8(11):835–50. 3. Singh HP, Kaur I, Gullaiya S. Antibody drug conjugates: a leap ahead in cancer treatment. J Drug Deliv Therap. 2014;4:52–9. 4. Maxfield FR, Mcgraw TE. Endocytic recycling. Nat Rev Mol Cell Biol. 2004;5(2):121–32. 5. Di GG, Le CR, Goodfellow AF, et al. Distinct endocytic pathways regulate TGF-β receptor signalling and turnover. Nat Cell Biol. 2003;5(5):410–21. 6. Sigismund S, Woelk T, Puri C, et al. Clathrin-independent endocytosis of ubiquitinated cargos. Proc Natl Acad Sci U S A. 2005;102(8):2760–5. 7. Williams TM, Lisanti MP. The caveolin proteins. Genome Biol. 2004;5(3):214. 8. Yamada E. The five structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol. 1955;1(5):445–58. 9. Kalim M, Chen J, Wang S, et al. Intracellular trafficking of new anticancer therapeutics: antibody-drug conjugates. Drug Design Develop Therap. 2017;11:2265–76. 10. Lajoie P, Nabi IR. Lipid rafts, caveolae, and their endocytosis. Int Rev Cell Molecul Biol. 2010;282:135–63. 11. Parton RG, Kai S. The multiple faces of caveolae. Nat Rev Mol Cell Biol. 2007;8(3):185–94. 12. Araki N, Johnson MT, Swanson JA. A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J Cell Biol. 1996;135(5):1249–60. 13. Innocenti M, Gerboth S, Rottner K, et al. Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes. Nat Cell Biol. 2005;7(10):969–76. 14. Schafer DA, D’Souza-Schorey C, Cooper JA. Actin assembly at membranes controlled by ARF6. Traffic. 2010;1(11):896–907. 15. Nobes C, Marsh M. Dendritic cells: new roles for Cdc42 and Rac in antigen uptake? Curr Biol. 2000;10(20):R739–41. 16. Hoffmann RM, Coumbe BG, Josephs DH, et al. Antibody structure and engineering considerations for the design and function of antibody drug conjugates (ADCs). Onco Targets Ther. 2018;7(3):712–9. 17. Poteryaev D, Datta S, Ackema K, et al. Identification of the switch in early-to-late endosome transition. Cell. 2010;141(3):497–508. 18. Luzio JP, Pryor PR, Bright NA. Lysosomes: fusion and function. Nat Rev Mol Cell Biol. 2007;8(8):622–32. 19. Andrew L, Lauren P, Allison S, et al. Factors affecting the pharmacology of antibody-drug conjugates. Antibodies. 2018;7(1):10. 20. Francisco JA, Cerveny CG, Meyer DL, et al. cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood. 2003;102(4): 1458–65. 21. Peters C, Brown S. Antibody-drug conjugates as novel anti-cancer chemother-apeutics. Biosci Rep. 2015;35(4):e00225.

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22. Xu S. Internalization, trafficking, intracellular processing and actions of antibody-drug conjugates. Pharm Res. 2015;32(11):3577–83. 23. Doronina SO, Mendelsohn BA, Bovee TD, et al. Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. Bioconjug Chem. 2006;17(1):114–24. 24. Erickson HK, Park PU, Widdison WC, et al. Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing. Cancer Res. 2006;66(8):4426–33. 25. Tang Y, Wang X, Ma X, et al. Progress on internalization study of antibody-drug conjugates. Chin J New Drugs. 2017;26(10):1130–6.

Chapter 5

Distribution and Metabolism of Antibody-Drug Conjugates Xuefei Bai and Shuqing Chen

5.1

Preface

To avoid early degradation through oral administration, antibody-drug conjugate (ADC) is generally administrated intravenously. Travelling with blood stream, ADC is distributed to the whole body. The antibody in ADC is an essential part of the whole entity, which also lead the in vivo distribution of ADC [1]. Once given intravenously, ADC will firstly be carried to tissues with aplenty blood flow, like liver, and its distribution volume is about 50 mL/kg around this time. During continuous in vivo circulating, ADC is gradually distributed into other tissues and makes ADC concentration decreased in blood, and at this time the distribution volume is about 150–200 mL/kg [2–4]. However, the whole body distribution leads to some extend of non-specific degradation and therefore the early releasing payload might cause toxic effect. For example, liver cells are able to utilize enzymes to process small molecule part through phase I and phase II metabolism [5], and excrete the final product. According to clinical tests, only 0.003–0.008% of dosed ADC can reach tumor site [6]. In all, ADC should be considered as an entity that consist of antibody, linker, and cytotoxic drug when it comes to absorption, distribution, metabolism and excretion [7]. Depending on endocytosis, ADC in human blood can be taken in by cells possessing metabolic enzymes and distributed to such tissue will cause plenty of

X. Bai Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China Department of Precision Medicine on Tumor Therapeutics, ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou, China S. Chen (*) College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chen, J. Zhan (eds.), Antibody-Drug Conjugates and Cellular Metabolic Dynamics, https://doi.org/10.1007/978-981-19-5638-6_5

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ADC being degraded. For example, the enzyme in liver cells is responsible for elimination of the antibody part; the glutathione concentrated in endothelium cells are responsible for degradation of disulfide linkers which cause small molecular drug releasing early [7]. In contrast, through binding to neonatal Fc receptor (FcRn), antibody part can be recycled in blood system and being protected by this process which also prolongs the half time of ADC in human body [8]. Although tumor blood vessels are thought to be more permeable than normal ones, it’s not easy for ADC to get access to tumor site. The possible reason might be as follow. The extracellular lymphatic fluid in tumor tissue barely returns to vascular system compared to normal tissue, which lead to increased osmotic pressure and interstitial fluid pressure, and therefore block the blood capillary and prevent ADC entering solid tumor. Besides, the hydrophobic medicine losses the way deep inside tissue for lymphatic vessels blocked in tumor [9]. As for fragment-based antibody—single chain variable fragment (scFv), which is more permeable to tissue than full-length antibody, its distribution at tumor tissue is still less than 1%, or even 1‰. The number of target antigens on cell surface is one of the factors to affect distribution and systemic clearance of antibody-drug conjugates. Although there are some targeted antigens that are highly expressed on tumor cells, they are not private to tumors, and still can be sequestered by those antigens on normal cells. Boswell et al. [10] had studied a chondroitin sulphate proteoglycan named TENB2 that is overexpressed on prostate cancer cells, and is also expressed in intestines with relative low level. Researchers found that although being lower expressed, TENB2 in intestines is responsible for rapid target-mediated clearance of the anti-TENB2 antibody because of the large surface area of intestines, a clearance pathway that is independent of renal and hepatic clearance. The penetration of therapeutic antibody to solid tumor depends on updating and internalizing rate of antigens. When updating rate is fast, targets that is distributed outside tumor might not saturated by drugs, which makes it hard for drugs to penetrate into deep tumor part. By contrast, the updating rate can reach a balance with internalizing rate when antigens on cell surface is slowly presented, which is benefits for drugs to penetrate into deep part of solid tumors.

5.2 5.2.1

The Mechanism of Action of ADC in Cells The Release of Cytotoxic Payloads

Mediated by antibody part of ADCs, whole ADC could be internalized into tumor cells. Early endosomes would firstly encapsulate the ADCs, and then become late endosomes after maturation process, subsequently, the endosomal vesicle would fuse with the lysosomal compartment and finally undergo lysosome degradation of ADCs [11]. The whole process from internalization to drug release inside cells would last for hours, and within the course, a concentration balance of cytotoxic drugs can be reached on both side of cell membranes.

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The release of cytotoxic payloads is associated with the type of linkers. Design of linkers need to meet the requirement as follow: high stability in circulation and efficient release of cytotoxic payload metabolite. Linkers with wide application contain cleavable linkers and uncleavable linkers [12]. Cleavable linker technology enables the selective release of an unmodified payload in the target cell, rather than in circulation, such as peptidic linkers and β-glucuronic acid linkers. Uncleavable linkers have no inbuilt trigger to be cleaved, and the payload part can only be released by proteolysis of antibody part. The released payload contains the drug, linker and an amino acid appendage [13]. This kind of linker is exemplified by thioether linker. A successful case is the ado-trastuzumab emtansine (T-DM1), the first approved ADC for treatment of solid tumors, which is conjugated by the noncleavable SMCC thioether linker. Despite T-DM1 cannot exert by-stander effect, the ADC itself has potent anti-tumor activity with high safety and stability in circulation, which broaden its therapeutic window [11, 12, 14]. brentuximab vedotin is another example of ADC that is approved by FDA for clinical use. The ADC consists of monomethyl auristatin E (MMAE) linked to CD30 antibody by a valinecitrulline dipeptide linker. The dipeptide linker is designed to be cleaved in lysosomes and release the MMAE payload to exert highly efficient by-stander effect [15]. Payload and antibody can therefore be conjugated with different linkers and more investigations should be given to find out the best drug candidate [16]. The traditional mechanism of action of ADC is internalization into cytosolic of tumor cells. However, a non-internalizing mechanism of action is also possible in case that target antigen has a nature of poor internalization rate. In this situation, linker cleavage and payload release are designed to occur in the extracellular tumor environment [17].

5.2.2

Mechanism of Action and Metabolism of Cytotoxic Payloads

The payloads are the ultimate effector components in ADCs, and therefore they have to be ultra-toxic in order to keep sensitive to kill tumor cells when drug concentration is very low in tumor tissues. In early days, researchers used anti-tumor drugs that had been approved for marketing, for instance. Doxorubicin [18], Vinca alkaloid [19], Methotrexate [20]. These drugs are insufficient in potency with human subjects which contributes to the lack of clinical utility, ADC payloads targeting either DNA or tubulin of cells are in universe use. These agents are highly potent that are too toxic to be clinically useful on their own. For example, Maytansinoids has a median effective dose (EC50) of 10-5–10-4 μg/mL [21], which is considered as the best choice of ADC payload. Auristatin- and maytansine- antibody conjugates obtain wide application in pre-clinical and clinical use, and their mechanism of action has been discoveried. Maytansinoid is released from tumor cells and metabolized to payload-antibody

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component with further methylation on thiol group to generate ultimate hydrophobic metabolites. As for uncleavable linker, there is no further modification on payload but exert cytotoxic effect in its cognate form. When the released payloads diffuse into blood circulation, the liver cells are responsible for de-toxic of the payload molecules through phase I and II reaction, which prevent severe hepatic toxicity. The target-independent metabolism of ADC is also observed in normal cells, which lead to drug release and off-target toxicity. Besides, driven by instability of dipeptide linker, this kind of ADC is hard to obtain precise in vivo preclinical data. Dorywalska et al. had found high concentration of carboxylesterase 1 (Ces 1) in mice serum, which led to early disassociation of antibody-payload component (antibody is named C16, and linked with Aur0101 by dipeptide linker VC-PABC) before ADC reaching tumor tissue. VC-PABC linker cleavage happened in 12 hours with 40%, which greatly decrease the quantity of ADC that entered tumor sites. Fortunately, there is no homologous enzyme in human serum.

5.2.3

Metabolism of Antibody Parts in ADCs

Upon accumulation in tumor tissue, ADCs entry tumor cells through endocytosis. The mAb part is metabolized by proteolytic machinery to its constituent amino acid. There has the chance that antibody part in ADC is susceptible to systemic clearance and payload is therefore released in non-target cells or tissues. But antibody usually has a long circulation time with FcRn recycle mechanism, for example, Maylotarg® has a half life about 14 days [22].

5.3

Methods and Procedures in Studying Distribution and Metabolism of ADC

The focus of studying pharmacokinetics of ADCs is two agents: (1) payload-derived analytes, containing unconjugated small molecular drugs, derivatives, conjugated small molecular drugs and premature release of payload; (2) antibody-derived analytes, containing free antibody and conjugates with different numbers of payloads. Each analytes mentioned above could provide reliable data that demonstrating the distribution and metabolism of ADCs, which helps in realizing the process of distribution and metabolism in order to optimize the design and therapeutic strategies of ADCs.

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Ligand Binding Assay

Enzyme-linked immunosorbent assay (ELISA) is a typical assay that suitable for testing affinity of large molecules with tertiary structures [23, 24], and it is also a common method to quantitively and qualifiedly analyze conjugated antibodies in ADC [22, 25]. Concentration of antibody-derived analytes could be readily and precisely measured. This method provides information on total antibody, even if payload drop off, which means the quantity of antibodies and ADCs. The following procedure of ELISA is exemplified by Boswell’s [4] research work. 1. Total antibody ELISA Coating plates with anti-idiotypic antibody 5093 or donkey antihuman Fc and incubating plates overnight at 4 °C. The plates were washed 3 times with 0.05% Tween-20 in PBS buffer (pH 7.4). The diluted plasma sample was added to each well and incubating the plate at room temperature for 2 hours. Then the plate was washed six times. A detection antibody, either goat antihuman IgG antibody conjugated to horseradish peroxidase or goat antihuman Fc conjugated to horseradish peroxidase, was added to the wells and incubated on a shaker for 1 h at room temperature. The plates were washed 6 times and developed using TMB peroxidase substrate. Incubating the plate with detection antibody at room temperature for 10–20 min until being terminated with 50–100 μL 1 mol/L H2SO4 and the reaction fluid is detected for absorbance at 450 nm. 2. Conjugated antibody ELISA Coating plates with anti-MMAE antibody and incubating the plate overnight at 4 °C. the plate was washed 3 times with 0.05% Tween-20 in PBS buffer (pH 7.4). Diluted standards and the ADC or TDC plasma samples were added to the wells and incubated for 2 h at room temperature. The plates were washed 6 times and a detection antibody, either goat antihuman IgG antibody conjugated to horseradish peroxidase or goat antihuman Fc conjugated to horseradish peroxidase, was added on a shaker for 1 h at room temperature. All plates were developed using TMB peroxidase substrate. Incubating the plate with detection antibody at room temperature for 10–20 min until being terminated with 50–100 μL 1 mol/L H2SO4 and the reaction fluid is detected for absorbance at 450 nm. The sensitivity of ELISA method could be improved when utilizing high affinity pair of biotin-aviditin [26]. Besides, there is a method to detect metabolites by ELISA. The plate was coated with protein-DM4 conjugates and using maytansinoid metabolites in blood sample to compete with biotinylated anti-maytansinoid antibody, and the binding substances were finally detected with streptavidin-HRP conjugate. The signal is from remaining biotinylated anti-Maytansinoid antibody after saturating the maytansinoid catabolites in blood sample.

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Liquid Chromatography-Tandem Mass Spectrometry Assay

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method used in identify and detect chemical compounds. As the technique developed, LC-MS/ MS can also be used in investigation of protein molecules, for example, qualitative analysis of peptide presented by MHC molecule. LC-MS/MS has lower throughput and lower sensitivity than ELISA analysis, but it can resolve unknown metabolites while ELISA analysis is more suitable for known materials [27]. When detected using LC-MS/MS, chemical compounds in samples are concentrated with streptavidin-coated magnetic beads and dehydrated with the stream of nitrogen. The LC/MS/MS platform can also deliver a superior specificity of the analyte, which is particularly valuable for the quantitation of payload metabolites which may also be required during the development process. Conjugated payload was measured using a hybrid immunocapture (IC)-LC–MS/ MS approach. The procedure can be described as following: First, the ADC is immunocaptured on biological matrix. The captured antibody is biotinylated at a molar ratio of 12:1. The biotinylated capture reagent was immobilized on cartridges coated with streptavidin at a flow rate of 5 μL/ min. The ADC calibration standards, QC and study samples in matrix were diluted 1:1 using DPBS and then slowly loaded on the cartridges at a flow rate of 2 μL/min. The captured analyte was eluted from the cartridges using 12 mmol/L hydrochloric acid at a flow rate of 5 μL/min then immediately neutralized using 250 mmol/L ammonium bicarbonate. The eluted samples were incubated with the specific enzyme for 3 h at RT to release the payload molecules that were conjugated to the antibody. The incubation was terminated by adding cold acetonitrile containing the stable isotope labeled internal standard of payload. Next, a reverse phase HPLC column (for example, Acquity UPLC BEH C18, Waters) was used for chromatographic separation of unconjugated chemical compounds. The mobile phases consisted of mobile phase A (5 mmol/L ammonium bicarbonate containing 0.005% ammonium hydroxide in 95:5 water: acetonitrile) and mobile phase B (5: 95 water: acetonitrile) at a flow rate of 0.8 ml/min. The gradient started with 30% B and increased to 60% B in 1.00 min, then changed to 95% B in 0.05 min and held at 95% B for 0.45 min, then switched back to 30% B in 0.05 min and held at 30% B until the run was stopped at 2.00 min. Payload and the internal standard were detected using multiple reaction monitoring in positive ion electrospray mode with the transitions of m/z 771 → 98 and m/z 781 → 98, respectively.

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Radiolabeled Antibody Assay

To study the distribution of ADC in human body, radiolabeled antibody part or chemical molecule part is used to show their trace. Erickson et al. used 3H-labeled maytansinoid, which was moderated at the C-20 methoxy group, to generate huC242-SPDB-[3H] DM4. COLO 205 cells were exposed to 3H-labeled conjugates for different time periods and samples were treated with acetone. The radioactivity was measured from acetone extracts and therefore metabolites of maytasinoid were traced, which turned out to be lysine-N-SMCC-[3H]DM1. Another example to study metabolites of ADC using radiolabeling assay is present. Cohen et al. [28] labeled two tubulysin analogues with 131I, which were low cytotoxic TUB-OH and high cytotoxic TUB-OMOM. The radiolabeled compounds were next conjugated to 89Zrtrastuzumab, and the resulting dual-labeled ADCs were detected for in vivo distribution at different time points. In this study, the researchers demonstrated that the coupling method produced stable radiolabeled ADCs, which is suitable for studying different pharmacokinetics behaviors with different DARs of ADC. The procedures of radiolabeling is as follows: Tubulysin analogues TUB-OH or TUB-OMOM were dissolved in H2O/MeCN (1/1) at a concentration of 3.1 μg/μL. Hundred microliters of TUB-OH (450 nmol) or TUB-OMOM (415 nmol) was incubated with 30 μL MeCN, 100 μL 0.5 mol/L phosphate buffer (pH 7.2), 3 to 100 MBq131I (volume: 4–8 μL), and 25 μL (50 μg; 2 mg/mL) chloramine-T, by shaking in a Thermomixer at 550 rpm at room temperature. After 2 minutes, the reaction was stopped with 10 μL Na2SO3 (20 μg; 20 mg/mL) and a 1-μL sample was taken and diluted to 20 μL with H2O/MeCN (7/3) for HPLC analysis of the radioiodinated tubulysin and assessment of radiolabeling efficiency. Unreacted 131I and reduced chloramine-T were removed by use of a Seppak tC2 Light column (Waters), via loading of the mixture on the column and washing with 10 mL H2O. Subsequently, 131I-TUB-OH or 131I-TUBOMOM (the radiolabeled final product) was eluted from the Seppak with 2 mL MeCN.

5.4

Summary

After entering human body, antibody part is more likely to be degraded by proteases universally existed in cells or blood stream. For chemical compounds, there have the drug metabolizing enzymes in liver to process phase I and phase II metabolism. The metabolites are more hydrophilic than cognate compounds by reducing active groups, which have reduced cytotoxicity and can be more easily excreted from body. The metabolism of ADC and resulting metabolites are important for drug discovery and drug design. Currently, ligand binding technique and liquid chromatographymass chromatography technique provides us methods to study metabolism pathway and metabolites of ADCs. More efforts are still needed in developing

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biotechnologies to provide more strategies for studying ADC metabolism, which will promote universe and deep level applications.

References 1. Tabrizi M, Bornstein GG, Suria H. Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease. AAPS J. 2010;12(1):33–43. 2. Mould DR, Green B. Pharmacokinetics and pharmacodynamics of monoclonal antibodies. BioDrugs. 2010;24(1):23–39. 3. Tabrizi MA, Tseng CML, Roskos LK. Elimination mechanisms of therapeutic monoclonal antibodies. Drug Discov Today. 2006;11(1–2):81–8. 4. Boswell CA, Mundo EE, Zhang C, et al. Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats. Bioconjug Chem. 2011;22 (10):1994–2004. 5. Kraynov E, Kamath AV, Walles M, et al. Current approaches for absorption, distribution, metabolism, and excretion characterization of antibody-drug conjugates: Anindustry white paper. Drug Metabol Disposit. 2016;44(5):617–23. 6. Sedlacek HH, Seemann G, Hoffmann D, et al. Antibodies as carriers of cytotoxicity. Basel: Karger; 1992. 7. Ducry L, Stump B. Antibody-drug conjugates: linking cytotoxic payloads to monoclonal antibodies. Bioconjug Chem. 2010;21(1):5–13. 8. Stapleton NM, Andersen JT, Stemerding AM, et al. Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential. Nat Commun. 2011;2(1):599. 9. Thurber GM, Schmidt MM, Wittrup KD. Antibody tumor penetration: transport opposed by systemic and antigen-mediated clearance. Adv Drug Deliv Rev. 2008;60(12):1421–34. 10. Boswell CA, Mundo EE, Firestein R, et al. An integrated approach to identify normal tissue expression of targets for antibody-drug conjugates: case study of TENB2. Br J Pharmacol. 2013;168(2):445–57. 11. Erickson HK, Park PU, Widdison WC, et al. Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing. Cancer Res. 2006;66(8):4426–33. 12. Casi G, Neri D. Antibody-drug conjugates: basic concepts, examples and future perspectives. J Control Release. 2012;161(2):422–8. 13. Tsuchikama K, An ZQ. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell. 2018;9(1):33–46. 14. Lewis Phillips GD, Li G, Dugger DL, et al. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res. 2008;68(22):9280–90. 15. Okeley NM, Miyamoto JB, Zhang X, et al. Intracellular activation of SGN-35, a potent antiCD30 antibody-drug conjugate. Clin Cancer Res. 2010;16(3):888–97. 16. Polson AG, Caleminefenaux J, Chan P, et al. Antibody-drug conjugates for the treatment of non-Hodgkin’s lymphoma: target and linker-drug selection. Cancer Res. 2009;69(6):2358–64. 17. Gébleux R, Wulhfard S, Casi G, et al. Antibody format and drug release rate determine the therapeutic activity of noninternalizing antibody-drug conjugates. Mol Cancer Ther. 2015;14 (11):2606–12. 18. Saleh MN, Sugarman S, Murray J, et al. Phase I trial of the anti-Lewis Y drug immunoconjugate BR96-doxorubicin in patients with Lewis Y-expressing epithelial tumors. J Clin Oncol. 2000;18(11):2282–92.

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19. Petersen BH, Deherdt SV, Schneck DW, et al. The human immune response to KS1/4desacetylvinblastine (LY256787) and KS1/4-desacetylvinblastine hydrazide (LY203728) in single and multiple dose clinical studies. Cancer Res. 1991;51(9):2286–90. 20. Elias DJ, Kline LE, Robbins BA, et al. Monoclonal antibody KS1/4-methotrexate immunoconjugate studies in non-small cell lung carcinoma. Am J Respir Crit Care Med. 1994;150(4):1114–22. 21. Cassady JM, Chan KK, Floss HG, et al. Recent developments in the maytansinoid antitumor agents. Chem Pharm Bull. 2004;52(1):1–26. 22. Sanderson RJ, Hering MA, James SF, et al. In vivo drug-linker stability of an anti- CD30 dipeptide-linked auristatin immunoconjugate. Clin Cancer Res. 2005;11(2 Pt 1):843–52. 23. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry. 1971;8(9):871–4. 24. Lequin RM. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem. 2005;51(12):2415–8. 25. Francisco JA, Cerveny CG, Meyer DL, et al. cAC10-vcMMAE, an anti-CD30- monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood. 2003;102(4): 1458–65. 26. Salomon PL, Singh R. Sensitive ELISA method for the measurement of catabolites of antibodydrug conjugates (ADCs) in target cancer cells. Mol Pharm. 2015;12(6):1752–61. 27. Zhang S, Jian W. Recent advances in absolute quantification, of peptides and proteins using LC-MS. Rev Anal Chem. 2014;33(1):31–47. 28. Cohen R, Vugts DJ, Visser GW, et al. Development of novel ADCs: conjugation of tubulysin analogues to trastuzumab monitored by dual radiolabeling. Cancer Res. 2014;74(20):5700–10.

Chapter 6

Application of Antibody Fragments in ADCs Wenhui Liu and Shuqing Chen

6.1

Introduction

Cancer is one of the greatest threats to human health in the twenty-first century, approximately 8.8 million people die of cancer annually. Over the last several decades, several new technologies have revolutionized cancer treatments. As a milestone among therapeutic methods, antibody-based drugs have achieved remarkable success in clinical cancer treatment. In 2018, there were more than 60 monoclonal antibody drugs approved by the FDA for marketing [1]. Traditional monoclonal antibody drugs are generally of the IgG type, with a relative molecular mass of about 150 kDa and a long half-life. For blood tumor, the monoclonal antibody can fully contact with tumor cells, and its good pharmacokinetic properties further ensure its full play of efficacy in the treatment of blood tumor. However, for solid tumors, due to the relatively large molecular weight of traditional IgG drugs, antibodies need to pass through many obstacles to get from the intravenous injection site to the tumor site. Therefore, the amount of antibody drug reaching the tumor site is very small, only about 0.01% of the injected dose [2, 3]. About 2/3 of the marketed monoclonal antibodies are used for hematoma treatment, while only 1/3 are used for solid tumors. In addition, among the five ADCs on the market, only Kadcyla® (ado-trastuzumab emtansine) is used for the treatment of solid tumors. Among human cancer types, 85% of cancer types are solid tumors. Therefore, there is an urgent need for clinically effective antibody drugs for the treatment of solid tumors.

W. Liu Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China Department of Precision Medicine on Tumor Therapeutics, ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou, China S. Chen (*) College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chen, J. Zhan (eds.), Antibody-Drug Conjugates and Cellular Metabolic Dynamics, https://doi.org/10.1007/978-981-19-5638-6_6

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For solid tumors, the amount of antibody exposure at the tumor site is a key factor determining its anti-tumor activity, and the amount of antibody exposure at the tumor site is mainly determined by the following factors: the penetration and the retention characteristics of the antibody at the tumor site, as well as the appropriate pharmacokinetic properties of the antibody. The relative molecular weight, valence, charge and affinity of the antibody are the key factors that determine these factors [3, 4]. In recent years, more and more researchers have focused on the development of antibody fragments, such as single-chain variable fragments (scFv), antigenbinding fragments (Fab), multivalent scFv (diabody), nanobodies, and minibodies (scFv-CH3) etc. The antibody fragments retain the affinity and specificity of the antibody, and also have lower immunogenicity and faster penetration of tumors. It is an ideal choice to break through the limitations of traditional antibody treatment of solid tumors. In addition, the marketed antibody drugs (including monoclonal antibodies and ADCs) mainly target tumor-associated antigens (TAA). This type of antigen is highly expressed in tumor tissues, but also low expressed in some normal tissues [5], causing on-target off-tumor effects. Traditional antibody drugs have a longer half-life, which also increases the probability of on-target off-tumor effects. Compared with traditional antibodies, antibody fragments have a shorter half-life, which can reduce the side effects caused by on-target off-tumor effects. This chapter mainly focuses on the application of antibody fragments in ADCs.

6.2

Antibody Fragment Drug Conjugates (AFDCs)

This chapter mainly selects a monoclonal antibody (Ofatumumab (OFA)) approved by the FDA for the treatment of chronic leukemia [6] as the research object. On the basis of OFA, the miniaturization was carried out, and two miniaturized antibodies Fab and Fab-CH3mut were constructed using genetic engineering technology. Among them, CH3mut was modified by Ying [7] on the basis of CH3 with mutating 4 amino acids and introducing a pair of disulfide bonds at the N-terminus and C-terminus. CH3mut retains most of the affinity of the Fc fragment to FcRn, is a novel fragment used to modify the pharmacokinetic properties of proteins. The small toxin molecule selected in this chapter is MMAE, and the selected conjugation method is Sortase A enzyme-mediated conjugation [8–10] .

6.2.1

Materials

Triple glycine-modified toxin Gly3-val-cit-PABC-MMAE; Expression vector PET28a(+)-sortase A plasmid; Cell lines Ramos, Daudi, K562, HEK293F; E. coli Rosetta (DE3), DH5α; Ni-NTA purification column, Protein L column; CCK-8 (Cell Counting Kit-8) detection kit, Annexin V-FITC apoptosis kit; Anti-Fab mouse monoclonal antibody, FITC-labeled goat anti-mouse IgG (H + L); Immune-deficient BALB/c Nude nude mice (6 weeks old).

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6.2.2

Methods

6.2.2.1

Expression and Purification of Fab and Fab-CH3mut Miniaturized Antibodies

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The light and heavy chain variable regions of OFA, CH1, and the CH3mut DNA sequences reported in the literature, were selected as templates. Genetic engineering technologies were used to obtain recombinant fragments Fab-L, Fab-VH-CH1-LPETG-His6 and Fab-VH-CH1-CH3mut-LPETG-His6 (corresponding Fab, Fab-CH3mut antibody fragments are shown in Figure 6.1a). The fragment was digested with two restriction enzyme EcoR I and Not I, and inserted into the expression vector pFUSE-CHIg-hG1. After the verification of the inserted fragments by sequencing, the corresponding plasmid with the correct gene sequence was extracted and transfected into HEK293F cells. The cell supernatant was collected 4 days later and purified with a Ni column.

Fig. 6.1 Structure of antibody fragments and the synthesis of antibody conjugates. (a) A schematic showing the domain compositions of the engineered antibody fragments, Fab and Fab-CH3mut; (b) The fundamental depiction of the Sortase A enzyme-mediated coupling of antibody fragments with small molecule toxins

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Preparation of Fab-vcMMAE and Fab-CH3mut-vcMMAE

Sortase A is a protein expressed in Staphylococcus aureus. It can specifically recognize the LPXTG polypeptide sequence, and add molecules and peptides with glycine at the N-terminus to the C-terminus of the protein through nucleophilic addition. Using the property of sortase A, toxic small molecules containing glycine at the N-terminus can be conjugated to the carboxyl end of the antibody with LPETG modified specifically, as shown in Fig. 6.1b. In a 10 mL reaction system, 6 μmol/L Fab or Fab-CH3mut, 300 μmol/L GGG-vcMMAE [In GGG-vcMMAE, GGG represents 3 glycine residues, and vc represents a dipeptide (Valine-citrulline linker) that can be cleaved by the lysosomal enzyme cathepsin B, MMAE is a highly toxic small molecule drug], 50 mmol/L Sortase A in 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 5 mmol/L CaCl2 (pH 7.4) solution, were reacted at 37 °C for 12 h. The reaction product was purified by a Protein L column, eluted with the elution buffer, adjusted to pH 7.0 with 1 mol/L Tris, concentrated by ultrafiltration with a 10 kDa ultrafiltration tube and replaced with PBS, finally stored at -20 °C for later use. SDS-PAGE (SDS polyacrylamide gel electrophoresis) analysis of Fab, Fab-CH3mut and conjugation products is shown in Fig. 6.2.

6.2.2.3

Affinity of Fab, Fab-CH3mut, OFA, Fab-vcMMAE, Fab-CH3mut-vcMMAE and OFA-vcMAME

1. Collect 1 × 107 Daudi cells (CD20 positive). Dilute Fab, Fab-CH3mut, OFA, Fab-vcMMAE, Fab-CH3mut-vcMMAE and OFA-vcMMAE to a certain concentration (200 nmol/L, 160 nmol/L, 80 nmol/L, 40 nmol/L and 20 nmol/L) and then incubated with Daudi cells for 30 min at 4 °C. 2. After washing 3 times with PBS, add 200 μL of anti-Fab mouse monoclonal antibody to each tube, and incubate at 4 °C for 30 min.

Fig. 6.2 SDS-PAGE analysis of Fab, Fab-CH3mut, OFA and their conjugates (Fab-vcMMAE, Fab-CH3mut-vcMMAE, OFA-vcMMAE)

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Fig. 6.3 Flow cytometry determination of the affinity of Fab, Fab-CH3mut, OFA and their conjugates (Fab-vcMMAE, Fab-CH3mut-vcMMAE, OFA-vcMMAE)

3. After washing with PBS for 3 times, add 200 μL of FITC-labeled goat anti-mouse IgG (H + L) to each tube. The cell were incubate at 4 °C for 30 min, and measured with flow cytometry after washing with PBS for 3 times. 4. The affinity of the antibody conjugates detected by flow cytometry is represented by the average fluorescence intensity of FITC after the second antibody labeling. 5. As shown in Fig. 6.3, the affinity of Fab, Fab-CH3 and OFA and their conjugates were almost the same; Compared with the OFA-vcMMAE, the affinity of Fab-vcMMAE and Fab-CH3mut-vcMMAE is slightly decreased, but basically retains the affinity of the IgG based antibody.

6.2.2.4

In Vitro Anti-Tumor Activity of Fab-vcMMAE, Fab-CH3mut-vcMMAE, OFA-vcMMAE

1. Collect CD20-positive cells Ramos and Daudi, then cells were cultured at 3000 cells/well with 100 μL/well RPMI-1640 medium [containing 10% FBS (fetal bovine serum)] respectively. 2. Add different concentrations of Fab, Fab-CH3mut, Fab-vcMMAE and Fab-CH3mut-vcMMAE diluents to the 96-well cell culture plate, 100 μL/well, 3 copies of each concentration in parallel, the cells were cultured for 4 days in a CO2 incubator (CO2 concentration Level is 5%) at 37 °C. 3. Add 50 μL of 25% CCK-8 (blank RPMI-1640 medium dilution) to each well, incubate in a CO2 incubator at 37 °C for 2 to 4 h, and detect OD450 with a microplate reader. Use Graphpadprism software to calculate the 50% inhibiting concentration (IC50) of the conjugates.

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Fig. 6.4 Anti-tumor activity of Fab, Fab-CH3mut, OFA and their conjugates (Fab-vcMMAE, Fab-CH3mut- vcMMAE, OFA-vcMMAE) on Ramos, Daudi and K562 cell lines

4. The results in Fig. 6.4 show that compared with Fab and Fab-CH3mut, Fab-vcMMAE and Fab-CH3mut-vcMMAE have significantly improved lethality on CD20 positive cells. Among them, the IC50 of Fab-vcMMAE and OFA-vcMMAE is not much different; the IC50 value of Fab-CH3mut-vcMMAE is 5 to 10 times of that of Fab-vcMMAE and OFA-vcMMAE.

6.2.2.5

Apoptosis-Inducing Activity of Fab-vcMMAE, Fab-CH3mut-vcMMAE and OFA-vcMMAE on Tumor Cell

1 × 105 cells/mL Ramos were incubated with 20 nmol/L Fab, Fab-CH3mut, OFA, Fab-vcMMAE, Fab-CH3mut-vcMMAE or OFA-vcMMAE for 48 h. After centrifugation and discarding the medium, the cells were washed with PBS, resuspended in 100 μL of binding solution containing Annexin V-FITC and propidium iodide (PI). Then, the cells were incubated at room temperature for 15 min, and supplemented with 400 μL of binding buffer. Finally, the flow cytometry was used to detect the percentage of apoptosis. It can be seen from the results in Fig. 6.5 that Fab, Fab-CH3mut, and OFA have very weak killing effects on tumor cells, and the percentages values of apoptotic and dead cells of them are basically the same. Fab-vcMMAE and Fab-CH3mutvcMMAE induced cell apoptosis obviously. The percentages values of apoptotic cells/dead cells are 24.2%/23.7% and 15.4%/20.9%, respectively, which is little different from the percentage of OFA-vcMMAE 21.8%/30.4%.

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Fig. 6.5 Apoptosis-inducing activity of Fab, Fab-CH3mut, OFA and their conjugates (Fab-vcMMAE, Fab-CH3mut-vcMMAE, OFA-vcMMAE) on Daudi cells

6.2.2.6

In Vivo Biodistribution of Fab, Fab-CH3mut and OFA

1. Fab, Fab-CH3mut, OFA were conjugated to the small molecule GGG-(PEG)3-N3 (GPN) according to the reaction system in 6.2.2.2; the conjugates Fab-GPN, Fab-CH3mut-GPN, OFA-GPN were purified by Protein L column. 2. The equivalent amount of Fab-GPN, Fab-CH3mut-GPN, OFA-GPN and dibenzocyclooctyne-Cy5 (DBCO-Cy5) were reacted overnight in PBS at room temperature. The products were centrifuged with a 10 kDa ultrafiltration tube and replaced with PBS, and finally obtain Fab-Cy5, Fab-CH3mut-Cy5, OFA-Cy5. 3. The CD20-positive Ramos cells were collected by centrifugation, then washed twice with PBS and resuspended the cells to 7 × 107 cells/mL with PBS. 4. 7 × 106 CD20-positive Ramos cells were injected subcutaneously into the right flanks of the nude mice to establish imaging models. When the volume of tumors grew up to approximately 1000mm3, an equivalent molar quantity (7 nmol/L) of Fab-GPN-Cy5, Fab-CH3mut-GPN-Cy5 or OFA-GPN-Cy5 were injected into the tumor-bearing mice via the tail vein. The time-dependent in vivo distribution, antibody-targeted specificity and the rate of the antibody fragments penetration into the tumors were observed by using a Maestro In vivo Imaging System

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Fig. 6.6 In vivo distribution of Cy5 labeled Fab, Fab-CH3mut and OFA in tumor-bearing mice

(Cambridge Research Instrumentation Inc., USA). All the images were calculated and analyzed using the CRi Maestro Image software. 5. As shown in Fig. 6.6, at the tumor site, Fab-Cy5 has obvious aggregation at 4 h, Fab-CH3mut-Cy5 has obvious aggregation at about 8 h, and OFA-Cy5 has obvious aggregation at 24 h. Obviously, the rate of antibody entry into the tumor site is Fab>Fab-CH3mut>OFA. From the perspective of tumor aggregation, Fab-CH3-Cy5 has obvious aggregation at 8 h, but the aggregation decreases slightly with time; Fab-Cy5 and OFA-Cy5 have their maximum aggregation at 12 h and 24 h, respectively, but the aggregation of the two did not decrease over time. Therefore, Fab has the advantages of both OFA and Fab-CH3mut, not only has a faster rate of penetration of the tumor, but also can maintain a higher amount of aggregation for a longer period of time.

6.2.2.7

Antitumor Activity and Toxicity of Fab-vcMMAE, Fab-CH3mut-vcMMAE and OFA-vcMMAE in Vivo

Eight-week-old female BALB/c Nude mice were inoculated subcutaneously with 7 × 106 Ramos cells into the right flank of the nude mice. When the volume of the tumor grew to 80–150 mm3, the mice were randomly divided into nine groups with six mice per group: the control groups included a saline group and a Fab-HER2vcMMAE group, and the treatment groups included Fab, Fab-CH3mut, OFA, FabvcMMAE (55 nmol-vcMMAE/kg, 5 mg/kg), Fab-vcMMAE (110 nmolvcMMAE/kg, 10 mg/kg), Fab-vcMMAE (220 nmol-vcMMAE/kg, 20 mg/kg), Fab-CH3mut-vcMMAE (110 nmol-vcMMAE/kg, 13 mg/kg) and OFA-vcMMAE (110 nmol-vcMMAE/kg, 30 mg/kg) groups. Then mice were treated intravenously with the abovementioned drugs once every 3 days for four times (q3d × 4). The volume of the tumors and the body weight of the mice were

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Fig. 6.7 In vivo efficiency and toxicity of Fab-vcMMAE, Fab-CH3mut-vcMMAE, OFA-vcMMAE. (a) In vivo antitumor activity of Fab-vcMMAE, Fab-CH3mut-vcMMAE, OFA-vcMMAE; (b) Body weight monitoring of mice after drug administration; (c) Toxicity evaluation via H&E stained histological sections of primary organs

measured using calipers and an electronic balance, respectively, every 3 days. The volume of the tumors was calculated using the formula: tumor length×tumor width×tumor width/2. The results (shown in Fig. 6.7) indicated that the Fab and Fab-CH3mut did not have a significant antitumor efficiency compared with the PBS-control at the dosage of 220 nmol/kg (20 mg/kg-Fab and 25 mg/kg-Fab-CH3mut), a finding that was in line with the in vitro results. Regarding the Fab-vcMMAE treatment, there was a dose-dependent effect among the doses of 55 nmol-vcMMAE/kg (5 mg/kg), 110 nmol-vcMMAE/kg (10 mg/kg) and 220 nmol-vcMMAE/kg (20 mg/ kg). The tumors were eliminated in four of the six mice treated with the highest dose of Fab-vcMMAE group (220 nmol-vcMMAE//kg) at 12 days, and no recurrence was observed in these mice during the observation period (Fig. 6.7a). The tumor growth inhibition was more obvious in the mid-level Fab-vcMMAE dose group than in the mid-level Fab-CH3mut-vcMMAE dose group. These two groups manifested a

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similarly slower tumor growth rate in the first 12 days, while after the finish of the administration, the Fab-CH3mut-vcMMAE group showed a faster tumor growth rate than the Fab-vcMMAE group (110 nmol-vcMMAE/kg), which might be due to the lower retention time and accumulation of Fab-CH3mut-vcMMAE in tumor tissue. The tumors of the OFA-vcMMAE group (110 nmol-vcMMAE/kg, 30 mg/kg) were eliminated in all six mice at the 12 days after treatment and did not relapse during the observation period. In contrast, tumors in the nonbinding Her2-Fab-vcMMAE group (110 nmol-vcMMAE/kg, 10 mg/kg) grew rapidly, similar to the tumors in the PBS group. Body weight, as an important index of the in vivo toxicity was monitored in the nine treated groups. The results showed that the body weights of all treated groups did not change significantly compared with the body weights of the control groups (Fig. 6.7b). In addition, the toxicities to the main organs in the nine groups were evaluated. The results indicated that no obvious histomorphologic alterations were found in any sections of organs (Fig. 6.7c), except in the 110 nmolvcMMAE/kg OFA-vcMMAE group, which showed an evident hepatic steatosis.

6.3

Overview of Antibody Fragments

Although antibody drugs (monoclonal antibodies and ADCs) have achieved significant results in clinical treatment of malignancies, there are still shortcomings. For example, the uneven distribution or poor penetration of antibody drugs in the tumor site leads to poor efficiency or prone to recurrence [4, 11]. In addition, the long halflife of antibody drugs (>10 days), can cause off-target effects, resulting in a narrow treatment window [12, 13] or higher background when used for contrast imaging due to a slower clearance rate in the body. Therefore, there is an urgent need for safer and more effective antibody drugs in clinical practice. In addition, the traditional single form of IgG based antibody drugs cannot meet clinical needs, and the diversification of antibody forms is also a product of clinical needs. Early antibody fragments (such as scFv, Fab) retain the specificity and affinity of the antibody, but due to their short half-life and poor anti-tumor activity, they are mainly used for imaging diagnosis of tumors. Up to 2019, the FDA has approved six Fab forms of antibody fragments for the market [14]. Among them, Arcitumomab is used for angiography of colorectal cancer, Digibind and DigiFab are used for the treatment of digoxin poisoning, CroFab is used for the treatment of rattlesnake poisoning, Abciximab and Ranibizumab are used for the treatment of cardiovascular diseases and eye diseases, respectively [15, 16]. It can be seen from this that there are few applications of antibody fragments in tumor therapy, and development is still needed. In order to improve the anti-tumor activity of the antibody fragments, researchers tried to modify them: (1) By fusing the antibody fragment with human serum albumin (HSA), Fc fragment or polyethylene glycol (PEG) to improve its pharmacokinetic properties [17, 18]. (2) Genetic engineering is carried out on the basis of Nanobodies (including VHH, V-NAR, VH), monovalent Fab and scFv to

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Application of Antibody Fragments in ADCs

VHH

Diabody

V-NAR

VH

Fab-CH2/CH3

scFv

Fab

VHH-Fc

65 2F(ab)

scFv-Fc

scFv-HSA/PEG

3F (ab)

Fig. 6.8 The structure of different antibody fragments

prepare multivalent antibody fragments, such as diabody, trivalent antibody (tribody), minibody, etc. (Fig. 6.8). (3) Prepare antibody fragment-drug conjugate (AFDC) by conjugating antibody fragments with different small molecules. For example, antibody fragments conjugated with radioactive elements can be used for tumor diagnosis and targeted therapy of tumors; conjugating with enzymes can prepare prodrugs; coupling with small toxin molecules can be used for targeted therapy of tumors. The expression of antibody fragments is different from that of IgG based antibodies. IgG based antibodies are generally limited to mammalian expression systems. In addition to mammalian expression systems, prokaryotic E. coli expression systems [19] and eukaryotic yeast expression systems [10] can also be selected for antibody fragments expression. In addition, for Fab or F (ab’)2 forms of antibody fragments, they can be obtained by pepsin or papain digestion [20]. In terms of antibody preparation, miniaturized bispecific antibodies have obvious advantages over IgG based antibodies. At present, the only bispecific antibody on the market, Blinatumomab, is the scFv form. The versatility of the expression system and the low cost of optimization process are also the driving force for the development of antibody fragments. Antibody fragments are very different from the IgG based antibodies in structure and composition, and their conjugation methods are also different from those of IgG based antibodies. The conjugation methods of traditional antibodies mainly include random coupling of cysteine and arginine, glycosyl side chain conjugation, sitespecific conjugation of unnatural amino acids, mutant cysteine, and enzymatic conjugation. Among these conjugation methods, the conjugate sites of the glycosylated side chain, cysteine and arginine are mostly located in the constant region of the antibody, while antibody fragment (such as scFv, diabody etc.) without a constant region. In addition, for the site-specific conjugation of cysteine mutations,

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it may affect the activity or affinity of the antibody, if the mutation occurs in the variable region of the antibody. Therefore, in recent years, some site-specific conjugation methods (such as sortase A enzymatic conjugation, SNAP-tag® technology [21], etc.) have been widely used in the conjugation of antibody fragments. At present, there are more and more research on AFDCs. Antikor Pharmaceuticals is committed to the research of AFDCs and has developed anti-HER2-scFv-aurisatin. The DAR of anti-HER2-scFv-aurisatin can reach 10 after optimization, and a higher DAR has no effect on its pharmacokinetic properties. However, further clinical research data has not been reported [25]. At present, the extensively studied antibody fragments mainly target CD20, CD19, HER2, CEA and other targets, and the expression forms are also diverse. Different forms of antibody fragments have different properties, which also determine their most suitable use. ScFv (25 kDa) and Fab (50 kDa), as monovalent binding fragments, have a faster tumor penetration and metabolism rate, but their retention time in the tumor site is very short, so they are mainly used in radiography of the tumor. Ideal antibodies for tumor therapy (especially antibodies against solid tumors) need to have the following characteristics: appropriate molecular size to ensure appropriate pharmacokinetic properties, faster tumor penetration rate, and longer retention time in tumor sites. Bivalent or multivalent antibody fragments (such as diabody, tribody, etc.) can be used for tumor diagnosis and treatment. And F (ab’)2 and minibody (scFv-CH3dimer, scFv2-Fc, etc.) is an ideal fragment for tumor targeted therapy. Of course, the application of antibody fragment does not stop there. Using gene therapy methods, the antibody fragments are expressed in situ— intrabody, which can be used for anti-viral or anti-tumor therapy by regulating transcription factors [22]. For non-endocytic ADCs [23], the tumor environment is mainly used to separate the antibody from the small molecule drug, so as to exert the anti-tumor activity of the small molecule drug. The choice of antibody fragment will greatly reduce the potential toxicity. As a natural ligand [24], antibody fragments can be used to modify nano-formulations to improve targeting and reduce toxicity. There are only dozens of antibody fragments currently in preclinical research, and we believe that more antibody fragments will enter clinical applications in the near future.

6.4

Summary

Antibody fragments retain the affinity and specificity of the IgG based antibodies, and have the advantages of high tumor penetration rate. They have great prospects in the development of AFDCs, especially in the treatment of solid tumors. However, antibody fragments also have shortcomings such as short half-life and poor stability. At present, research on antibody fragments is mainly focused on tumor diagnosis and imaging, and the research on therapeutic antibody fragment drugs is in infancy. With the deepening of research, the factors and mechanisms that affect the druggability of antibody fragments will be clearly elucidated. We can use the advantages and avoid

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weaknesses of miniaturized antibodies in the development of AFDCs and other antibody drugs, thereby increasing the possibility of success in tumor treatment.

References 1. Carter PJ, Lazar GA. Next generation antibody drugs: pursuit of the ‘high-hanging fruit’. Nat Rev Drug Discov. 2018;17:197–223. 2. Sedlacek HH, Seemann G, Hoffmann D, et al. Antibodies as carriers of cytotoxicity. Basel: Karger; 1992. 3. Deonarain MP, Yahioglu G, Stamati I, et al. Emerging formats for next-generation antibody drug conjugates. Expert Opin Drug Discovery. 2015;10:463–81. 4. Yan L, Hsu K, Beckman RA. Antibody-based therapy for solid tumors. Cancer J. 2008;14:178– 83. 5. Poon KA, Flagella K, Beyer J, et al. Preclinical safety profile of trastuzumab emtansine (T-DM1): mechanism of action of its cytotoxic component retained with improved tolerability. Toxicol Appl Pharm. 2013;273:298–313. 6. Gupta IV, Jewell RC. Ofatumumab, the first human anti-CD20 monoclonal antibody for the treatment of B cell hematologic malignancies. Ann N Y Acad Sci. 2012;1263:43–56. 7. Ying T, Chen W, Feng Y, et al. Engineered soluble monomeric IgG1 CH3 domain: generation, mechanisms of function, and implications for design of biological therapeutics. J Biol Chem. 2013;288:25154–64. 8. Hagemeyer CE, Alt K, Johnston AP, et al. Particle generation, functionalization and sortase A-mediated modification with targeting of single-chain antibodies for diagnostic and therapeutic use. Nat Protoc. 2015;10:90–105. 9. Massa S, Vikani N, Betti C, et al. Sortase A-mediated site-specific labeling of camelid singledomain antibody-fragments: a versatile strategy for multiple molecular imaging modalities. Contrast Media Mol Imaging. 2016;11:328–39. 10. Kornberger P, Skerra A. Sortase-catalyzed in vitro functionalization of a HER2- specific recombinant fab for tumor targeting of the plant cytotoxin gelonin. MAbs. 2014;6:354–66. 11. Jain RK. Delivery of molecular medicine to solid tumors: lessons from in vivo imaging of gene expression and function. J Control Release. 2001;74:7–25. 12. Gorovits B, Krinos-Fiorotti C. Proposed mechanism of off-target toxicity for antibody-drug conjugates driven by mannose receptor uptake. Cancer Immunol Immunother. 2013;62:217–23. 13. Litvak-Greenfeld D, Benhar I. Risks and untoward toxicities of antibody-based immunoconjugates. Adv Drug Deliv Rev. 2012;64:1782–99. 14. Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat Biotechnol. 2005;23:1126–36. 15. Leung K. Abciximab microbubbles. Molecular Imaging and Contrast Agent Database (MICAD). Bethesda (MD): National Center for Biotechnology Information (US); 2004. 16. Hernandez L, Lanitis T, Cele C, et al. Intravitreal aflibercept versus ranibizumab for wet age-related macular degeneration: A cost-effectiveness analysis. J Manag Care Specialty Pharm. 2018:1–9. https://doi.org/10.18553/jmcp.2018.24.7.608. 17. Kontermann RE. Strategies to extend plasma half-lives of recombinant antibodies. BioDrugs. 2009;23(2):93–109. 18. Zhang H, Wang Y, Wu Y, et al. Therapeutic potential of an anti-HER2 single chain antibodyDM1 conjugates for the treatment of HER2-positive cancer. Signal Transduct Target Ther. 2017;2:17015. 19. Akbari V, Sadeghi HM, Jafarian-Dehkordi A, et al. Optimization of a single- chain antibody fragment overexpression in Escherichia coli using response surface methodology. Res Pharm Sci. 2015;10:75–83.

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20. Zhou Z, Zhang J, Zhang Y, et al. Specific conjugation of the hinge region for homogeneous preparation of antibody fragment-drug conjugate: a case study for doxorubicin-PEG-anti-CD20 fab’ synthesis. Bioconjug Chem. 2016;27:238–46. 21. Woitok M, Klose D, Niesen J, et al. The efficient elimination of solid tumor cells by EGFRspecific and HER2-specific scFv-SNAP fusion proteins conjugated to benzylguanine-modified auristatin F. Cancer Lett. 2016;381:323–30. 22. Sanz L, Blanco B, Alvarez-Vallina L. Antibodies and gene therapy: teaching old ‘magic bullets’ new tricks. Trends Immunol. 2004;25:85–91. 23. Gebleux R, Wulhfard S, Casi G, et al. Antibody format and drug release rate determine the therapeutic activity of noninternalizing antibody-drug conjugates. Mol Cancer Ther. 2015;14: 2606–12. 24. Alibakhshi A, Abarghooi Kahaki F, Ahangarzadeh S, et al. Targeted cancer therapy through antibody fragments-decorated nanomedicines. J Control Release. 2017;268:323–34. 25. Deonarain MP. Antikor biopharma presentation. World ADC Summit: San Diego; 2014.

Chapter 7

Novel Targeting Carriers in Antibody-Drug Conjugates Xiaoyue Wei and Shuqing Chen

7.1

Introduction

Paul Ehrlich first proposed the concept of “magic bullet” by stating that “the optimal agent would combine high parasitotropism with low organotropism”, when he was screening antibacterial compounds in 1908. Selective drugs are needed which specifically bind to pathological targets, without affecting the host cells in other tissues and organs, to ensure drug safety [1]. The concept of “magic bullet” was initially proofed in infection treatment, where Salvarsan (also known as 606) was used to treat syphilis successfully. Since then, “magic bullet” was applied to cancer treatment, but it never came true until people got to know more about carcinogenesis, and found out the exact cancer targets [2]. Nowadays, many kinds of targeting therapeutic, like small molecule chemotherapeutic drugs, antibodies and antibodydrug conjugates (ADCs), had come into clinical applications, with increasing defined cancer specific targets. ADC consists of cancer-targeting antibody, linker and payload. Payloads are usually cytotoxins that interfere with RNA, DNA or microtubulin to inhibit cell proliferation, but had no cancer cell selectivity. After conjugated to antibody, cytotoxins can be brought into tumor and the kill cancer cells within the local lesion. The encouraging clinical outcome of ADC has proved it to be a successful “magic bullet” that combine “targeting carrier” and “effective warhead” together as a targeting therapy. Whether other biomolecules with cancer targeting properties can be used as carrier for bringing warhead into caner location?

X. Wei Institute of Drug Metabolism and Pharmaceutical Analysis, Hangzhou, China Department of Precision Medicine on Tumor Therapeutics, ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou, China S. Chen (*) College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chen, J. Zhan (eds.), Antibody-Drug Conjugates and Cellular Metabolic Dynamics, https://doi.org/10.1007/978-981-19-5638-6_7

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In this chapter, our group from Laboratory of Precision Medicine and Biotechnology Drug, School of Pharmacy, Zhejiang University will explore the feasibility based on a case study: tumor-necrosis-factor (TNF)-related apoptosis-inducing ligand (TRAIL) as a novel tumor targeting carrier for cytotoxin MMAE conjugation in cancer therapy.

7.2 7.2.1

The Feasibility of TRAIL as a Conjugate Carrier TRAIL and its Receptor

TRAIL, like TNF, CD40 ligand (CD40L) and Fas ligand (FasL), belong to TNF super family members, induces cell apoptosis upon binding to its receptor. TRAIL is a type II transmembrane proteins, consists of 281 amino acids, the N-terminal 1–17 amino acids locate inside the cell, and the C-terminal 39–281 amino acids extend outside to the cell, where the 114–281 amino acids are the dominant receptor binding domain. Studies showed that TRAIL widely express on many tissues in human body, such as spleen, lung, prostate and peripheral lymphocytes, but not in brain and testis. Certain proteinase can hydrolysis membrane-bound TRAIL, which lead to soluble TRAIL shedding from cell surface without transmembrane, intracellular domain and signal peptide. Both membrane-bound and soluble TRAILs induce cell apoptosis in vitro and in vivo in a trimer form, by binding TRAIL receptor and activating cellapoptosis pathway [3]. To date, 5 TRAIL receptors had been identified, including TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and OPG (osteoprotein). And they are classified into 3 groups according to different function: death receptor, decoy receptor and soluble receptor (Fig. 7.1). 1. Death receptor (DR) DR4 (TRAIL-R1) and DR5 (TRAIL-R2), they are transmembrane receptors with intact ectodomain and intracellular death domain (DD), can be activated by TRAIL, induce cell apoptosis. Besides, overexpression of DR4 or DR5 can induce cell apoptosis directly in a TRAIL-independent way [4, 5]. 2. Decoy receptor (DcR) DcR1 (TRAIL-R3) and DcR2 (TRAIL-R4), also transmembrane receptors, while DcR1 has no cytoplasmic death domain and DcR2 has only 1/3 death domain, resulting in cell-apoptosis uninducible effect even binding to TRAIL [5, 6]. 3. Soluble receptor OPG is a secreted protein, binds to TRAIL with weak affinity, doesn’t involve in apoptosis pathway. OPG binds to other TNF family members and osteoclast differentiation factor, thus regulate bone balance by inhibiting osteoclasts differentiation and formation, increasing bone density [6, 7].

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Fig. 7.1 TRAIL and its receptor

7.2.2

Cell Apoptosis Inducing and Anti-Tumor Activity of TRAIL

Cell apoptosis is a highly active control mechanism of cell death for the body’s homeostasis, to better adapt to the living environment, it is important to the regulation of cell proliferation and differentiation. DR4 or DR5 homo-trimerizes upon binding to the ligand TRAIL, recruits adaptor Fas-associated death domain protein (FADD) and caspase 8 to death domain motifs in the C-terminus of the receptors, forming the death inducing signalling complex (DISC). Caspase 8 is activated by FADD, then stimulate downstream apoptotic signal. There are two ways of apoptosis defined by different mechanism: extrinsic apoptotic pathway and intrinsic apoptotic pathway. Activation of caspases 8 lead to caspase 3, caspase 6, and caspase 7 signal activation downstream, trigger the extrinsic apoptotic pathway; or activate the Bcl2 homology domain 3 interacting domain death agonist (BID) to trigger the intrinsic mitochondrial apoptotic pathway [8, 9]. DcR1, DcR2 and OPG cannot stimulate apoptosis, while they can regulate TRAIL functioning though competitive binding to TRAIL. Data has showed that TRAIL can induce apoptosis in many tumor cells while normal cells are well tolerant, this is benefit from the fact that DR4 and DR5 express highly on many tumor cells, while express limited or no on normal cells. Thus, TRAIL has received widely attention in antitumor therapy, for example,

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recombinant human TRAIL used as a antitumor agent. Untag soluble TRAIL114–281 (Dulanermin) binds to death receptors with high affinity, but has very fast renal clearance due to too small relative molecular mass, it has only 30 min-half live in non-human primates [10]. Fusion tags such as Flag, leucine/isoleucine zipper, and tenascin C (TNC) derived TRAIL to become multimer, this would enhance serum stability and antitumor activity, but increasing toxicity of recombinant TRAIL in other normal tissues. While untag TRAIL showed much weaker adverse effect in vivo [11]. N-terminal HSA fusion or PEGylation improved PK properties of TRAIL and decrease toxicity [12]. Most cancer cells were sensitive to TRAIL when screening in vitro, there still 20% cancer cells were resistant to TRAIL treatment [13]. Furthermore, some cells obtained resistant after TRAIL treatment [14], this might be caused by many reasons, for example, expression level changes in TRAIL signaling proteins or receptors, or mutation [15, 16]. The clinical application of TRAIL has been limited due to primary and acquired resistance. As the therapeutic effect of TRAIL monotherapy is not satisfactory, combination with chemotherapeutic agents is a common treatment option. Chemotherapeutic drugs kill cancer cells and sensitize TRAIL-resistant cells to TRAIL. For example, TRAIL combination with gemcitabine, oxaliplatin or irinotecan showed significantly more effective than TRAIL monotherapy [17]. Dulanermin, developed by Shanghai GeBaiDe Biotechnology Co., Ltd., has completed its phase III study after more than 10 year clinical trials, and exerted excellent safety and therapeutic effect combined with cisplatin for treatment of advanced NSCLC, was expected to be approved [18]. Others reported that TRAIL can be fused with small antibody fragment such as scFv, nanobody to form a bispecific protein. For example, the antiCD19 scFv-sTRAIL bispecific fusion protein selectively induces apoptosis of B lymphocytic leukemia cells [19]; the anti-EGFR nanobody fusion named ENb2TRAIL suppressed cell proliferation while simultaneously inducing apoptosis [20]. Hutt et al. [21] designed a hinge-crossing Fc fusion dimer containing 6 TRAIL and 2 scFv, which improved antitumor activity in vitro and in vivo. Current research results have demonstrated the advantage of combination and bispecific fusion protein on overcome TRAIL resistance, but their clinical application prospects need to be evaluated due to very limit toxicity study in vivo.

7.2.3

Study on TRAIL as a Cytotoxin Carrier

DR4 and DR5 are over express on many malignancies, including colon, breast, lung, pancreas, liver, kidney, and ovary cancers, while express at very low level or not in normal tissue [22, 23]. Besides, TRAIL receptor internalizes quickly when interact with TRAIL, right after apoptotic signal stimulating. These fulfill the common conditions of ADC, where antibody target to cancer cells specifically, internalize upon antigen recognition, release toxin in lysosome and kill cell. TRAIL binds to

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DR4/DR5 in the same way as antibody recognize cell surface antigen, which makes it a suitable carrier molecule for toxin conjugation. Site-specific conjugation is the most common used technique nowadays, of which using site-direct mutagenesis to generate free cysteine for chemical coupling has been well established. So, when designing TRAIL conjugate, we chose site-specific conjugation. Amino acids fragment 114–281 or 95–281 of TRAIL are usually used in cancer treatment, the former is the critical function domain, and the latter is more favorable for chemical coupling owing to the extend N-terminus. The only free cysteine at 230 is occupied for trimerizing through Zn2+ chelating, another cysteine was generated by site-direct mutation in order to maintain TRAIL’s function. The Asn 109 is a potential glycosylation site, and it’s outside the activity domain, thus we mutated Asn 109 to Cys 109, called N109C [24]. Theoretical molecular weights of TRAIL N109C is 21.6 kDa, it can be express in soluble form in the E. coli prokaryotic expression system. Our group was the first to conjugate cytotoxin MMAE with TRAIL N109C via a cleavable valine-citrulline (VC) linker, implementing the idea of using TRAIL as carrier to bring cytotoxin into tumor [25]. After that, PEGylation by PEG-5000 was performed to increase TRAIL half life in vivo, for the purpose of enhancing antitumor activity and improving safety [26, 27].

7.2.3.1

Materials

Expression vector of pET28a-N109C and pET28a-TRAIL95–281, E. coli prokaryotic expression system, reductant TCEP-HCl (tris(2-carboxyethyl)phosphine hydrochloride), MC-Val-Cit-PABC-MMAE(vcMMAE), methoxy poly(ethylene glycol) maleimide-5 K, PEG-5 K, reaction terminating agents N-acetyl-L-cysteine, Balb/c athymic nude mice (6 weeks).

7.2.3.2

Experiments [28]

1. Preparation of recombinant TRAIL: cDNA coding recombinant human TRAIL 95–281 and variant TRAIL N109C were cloned and inserted into expression vector pET28a(+) to get expression vector of pET28a-N109C and pET28aTRAIL95–281, plasmid were transfected into E.coli Rosetta (DE3), isopropyl-β-D-thiogalactopyranoside (IPTG) was applied to induce TRAIL expression. As poly histidine cluster on the surface of TRAIL can bind to Ni2+, HisTrap™ HP affinity column (GE Healthcare) was used in the first purification step, followed by ion exchange or hydrophobic column purification. In order to stabilize TRAIL structure during process, Zn2+ was added to increase TRAIL trimer formation.

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Fig. 7.2 Preparation and Characterization of TRAIL conjugates. (a) Strategy of the comodification of N109C with PEG-5 K and vcMMAE. (b) conjugates analyzed by SDS-PAGE. Reaction efficiency was around 90%, PEG/MMAE ratio on TRAIL trimer was computed using grayscale values of each brand

2. Preparation of TRAIL conjugates: TRAIL N109C was diluted in PBS at 1 mg/ mL, ten-fold molar equivalents to N109C of TCEP-HCl was added and incubated at 37 °C for 1.5 h to reduce N109C for releasing free cysteine at Cys 109. After reduction, certain molar equivalents of PEG-5 K was added and incubated for 30 min, followed by vcMMAE, reaction continued for 1 h longer, 20-fold molar excess N-acetyl-L-cysteine was added to quench the reaction. Reaction mixture was slowly stirring throughout the conjugation process. All PEG-TRAIL-MMAE conjugates were centrifuged at 12,000 g for 2 min and concentration measured by UV spectrophotometer. 5 μg of each was analyzed by 12% SDS-PAGE under reducing and non-reducing condition. As showed in Fig. 7.2, TRAIL trimer conjugated with different ratios of PEG and MMAE was obtained by modified the feeding ratios of PEG-5 K and vcMMAE. 3. In vitro antitumor activities of TRAIL and PEG-TRAIL-MMAE conjugates: DR4 or DR5 high expression cancer cell line such as NSCLC cell line NCI-H460 was

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chosen as model. Cells in logarithmic growth phase was seeded in 96-well cell culture plates at 103–104 cells/100 μL/well, after cells adhesion, TRAIL and conjugates were sterilized by 0.22 μm membrane filter and diluted into serial concentrations in medium, added into 96-well plate at 100 μL/well. Experiment was performed at three parallels, cells treated with medium only was set as negative control. After 72 h, medium was removed and cell viability was determined by a Cell Counting Kit-8 or 3-(4,5-dimethylthiazo-2-yl)-2,5diphenyletrazolium bromide (MTT). Relative cell viability was calculated using the following equation: Relative cell viabilityð%Þ OD450 of cells exposure to TRAIL or TRAIL conjugates - OD450 of blank medium = OD450 of negative control - OD450 of blank medium × 100% Sample concentration was in accordance with the abscissa, and the ordinate represented relative cell viability for cell-killing activity for result analysis using GraphPad Prism 6. As showed in Fig. 7.3, the IC50 values of TRAIL N109C and PEG: MMAE = 0:3, 1:1, 1:2,and 2:1 conjugates were calculated to be 0.63 nM, 0.14 nM, 0.23 nM, 0.31 nM, and 0.80 nM respectively, indicating that the in vitro antitumor activities of TRAIL conjugates increased along with MMAE payload incresing. 4. In vivo antitumor activities of TRAIL and PEG-TRAIL-MMAE conjugates: Xenograft tumors were developed in BALB/c athymic nude mice using NCI-H460 cells. When average tumor volume reached 190 mm3, mice were grouped randomly, and treated with different conjugates, using PBS as vehicle. Before dosing, samples were diluted in PBS and sterilized by 0.22 μm Fig. 7.3 In vitro antitumor activities of TRAIL and it’s conjugates

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Fig. 7.4 Evaluation of antitumor efficacy of TRAIL and its conjugates

membrane filter after inoculation. Mice were treated with 20 mg/kg of the above samples once every 3 days for four times (q3d × 4) intravenously. Tumor volumes were continuously monitored until the end of the experiment and calculated by the formula V = (L × W2)/2; L and W refer to longitudinal and transverse tumor diameters, respectively. The body weight of mice was also monitored simultaneously. Antitumor efficacy was usually presented by tumor volumes (Fig. 7.4), tumor recurrence and mice survival. From Fig. 7.4, we can see that TRAIL conjugated with PEG: MMAE = 0:3 exhibited dramatic tumor growth inhibition activity, while exceeding the MTD (maximum-tolerated dose) of mice, resulting in some mice death over the duration of treatment. As depicted in other groups, antitumor activity of TRAIL conjugates enhanced along with the ratio of payload MMAE increasing. 5. Safety evaluation of TRAIL and TRAIL Derivatives (Fig. 7.5): body weight, serum biochemical index and histological examination are common parameters to assess toxicity of drugs. Mice were fasted for 12 h before blood sampling and then sacrificed. Serological markers indicating hepatic function include total bilirubin (TBIL), albumin (ALB), alanine transaminase (ALT), and aspartate transaminase (AST), indicating renal function include blood urea nitrogen (BUN), and creatinine (CREA). Tissues isolating from mice should be

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Fig. 7.5 Safety evaluation of TRAIL and its conjugates

immediately fixed by 10% neutral-buffered formalin to avoid non-pathological damage for the following morphological examination. The fixed tissues were paraffin-embedded and stained by H&E (Hematoxylin and Eosin), the sections were examined by light microscope. As showed in Fig. 7.5, there was no significant different of all parameters between treatment group and control, suggesting that TRAIL conjugates were well tolerant in mice.

7.3

Review Study on Other Novel Carriers

Besides recombinant human TRAIL mentioned above, there are plenty of studies about variety kinds of “targeting carrier” for toxin conjugation. Such as peptide, designed ankyrin repeat protein (DARPin), aptamer, nanobody, scFv, probody, and bispecific antibody, several novel carriers are depicted in Fig. 7.6 bellow. 1. Peptide: Traditional chemotherapeutics like doxorubicin (DOX), paclitaxel (PTX) and camptothecin (CPT) have very low solubility, coupling to peptide will improve their LogP, and bring drugs to tumor location if the peptide is capable of specific targeting. Studies have demonstrated that DOX conjugated to the RGD motif of integrin αvβ3 improved antitumor activity and decreased adverse toxicity to liver and heart. Others suggested that DOX or PTX modified with guanidine-rich targeting peptide attributed to avoid drug efflux by

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Fig. 7.6 Illustration of some common new carriers used for ADC design

P-glycoprotein, thus increased drug uptakes in tumor. Coupling to cytotoxin that can be reduced by cellular reductant glutathione and fluorescence that linked by caspase recognized sequence, could combine therapy and intracellular tracking together. Peptide is easy to permeate into tumor tissue owing to small molecule weight, so it is usually applied to imaging in clinical [29]. 2. DARPin: DARPin is derived from natural anchor protein, it has 4–5 repeat motifs, and relative molecular weight at 14–18 kDa, which can be produced by prokaryotic bacteria. DARPin has excellent target specificity and affinity, structural stability and flexibility, and very large libraries, which is suitable to screen binders to various targets, and applied to clinical diagnosis and therapy. In disease diagnosis, it was reported that a DARPin targeting HER2 with high specificity and affinity, was used to detect HER2 expression level in paraffin-embedded tissue sections, and it showed comparable sensitivity to the method approved by FDA, even more excellent specificity. As for clinical therapy, two DARPins fused with rigid linker as an anti-HER2 biparatopic binder recognized two different epitopes on HER2, and fixed monomer HER2 proteins in a structural distance to prevent their dimerization and stimulation, so as to inhibit cancer cell proliferation [30]. In other study, Simon and his colleagues [31] conjugated cytotoxin MMAF to the C-terminus of an anti-EpCAM (epithelial cell adhesion molecule) DARPin Ec1, then added mouse serum albumin (MSA) to the N-terminus of Ec1 to increase its serum area under curve(AUC) and tumorspecific delivery of drugs. 3. Aptamer: aptamer is single strand oligo that fold into particular steric conformation following the base pairing principle, its screening is based on systematic evolution of ligands by exponential enrichment technique (SELEX), and has wide binding ranges from small molecule, like metallic ion or organic molecule,

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to large molecule, like peptide and protein. As heated and chemical denaturation of nucleotide is reversible, it is more convenient for preservation and transportation than protein, thus is very attractive in clinical diagnosis and therapy. There are many aptamers recognizing some hot tumor targets under development, including PSMA, MUC1, integrin αvβ3、HER2、EGFR、protein tyrosine kinase 7 (PTK7), et al. Because of small molecular weight, short half life, lack of functional activity or prone to be cleavage by nuclease, aptamer is rarely used in clinical application. The only PEGylation anti-VEGF aptamer approved by FDA is used in age-related macular degeneration. In terms of drug conjugation, aptamer is capable of coupling with chemotherapeutic drug, RNA, peptide/ protein, photosensitizer and thermal sensitizer, for delivering these materials into tumor [32]. 4. Nanobody and scFv: Both of them are designed for miniaturized antibody fragment. In peripheral blood of some camels, antibody that has only two heavy chains, called heavy chain antibody was found to have the ability to recognize antigen. It is consist of single variable domain located on a heavy chain (VHH), CH2 and CH3 domain, of which VHH domain alone in stable and high affinity enough for antigen binding. VHH antibody, also known as nanobody, has very small molecular weith at 15 kDa, high solubility and stability, and good permeation over full-length antibody, and can be humanized to human heavy chain antibody. Nanobody is usually used to design bispecific antibody, or conjugated with peptide, cytotoxin and nanoparticles [33]. One classical application of cytotoxin conjugation is that anti-mouse MHC II nanobody VHH7 conjugated with DM1, the conjugation showed higher internalization rate, shorter half life, safer, and stronger antitumor efficacy than full-length antibody conjugate [34]. ScFv is derived from variable region of antibody heavy chain and light chain, and fused together via flexible linker, it is about 30 kDa, double to nanobody. ScFv is eliminated quickly in vivo and are not as stable as nanobody. When conjugated with toxin DOX, scFv transferred and permeated into tumor, but showed unsatisfying antitumor efficacy [35]. 5. Probody: The idea of probody came from pro-drug, its variable region of antibody is masked by small peptide to block antigen recognition, the flexible linker and protease cutting motif between the peptide and antibody ensure probody stability in circulation and activated in tumor. To improved tumor-specific release of antibody, motif sequence of protease expressed or activated specifically in tumor is preferred, such as urokinase-type plasminogen activator(uPA), membrane type-serine proteinase 1(MT-SP1) and endopeptidase. Cellular libraries of peptide substrates (CLiPS) was a efficient screening approach to determine protease specific peptide. In an early study of probody PB1 based on antiEGFR antibody Cetuximab, PB1 performed similar binding affinity and cellgrowth inhibition activity to parent Cetuximab in vitro. On animal model, PB1 could not bind to EGFR, and had no function in circulation, thus showed no skin toxicity as Cetuximab did at the same dose. While after permeating into tumor, masking peptide was removed by protease, the antigen recognition site of PB1 was exposure to bind EFGR presented on cancer cells, making it an efficient

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therapeutic in tumor xenograft model. Furthermore, PB1 demonstated longer half life and higher AUC than Cetuximab when testing using cynomolgus monkey [36]. To date, probodys targeting some hot antigens like cytotoxic T-lymphocyte antigen-4(CTLA-4), programmed death 1(PD-1)/programmed death ligand-1 (PD-L1), and integrin subunit alpha 3(ITGA3) are under development by CytomX, who owns the proprietary technique of probody. Also, CytomX cooperated with ImmunoGen or Pfizer, on antibody conjugation technique for probody-drug conjugate development (PDC). Anti-CD166 PDC licensed by ImmunoGen is the first PDC that came into clinical trial. In China, the first PDC is a fully human IgG1anti-EGFR probody derived from Panitumumab conjugated to SMCC-DM1. It was developmed by Zhangjiang Biotechnology, called PanP-DM1, showed significant anti-tumor efficacy in preclinical study [37]. 6. Bispecific antibody: bispecific antibody contains two different antigens reorganization domains (variable region), that contributes to increase tumor targeting or improve ADC internalization and payload releasing inside cancer cell. Researchers in Regeneron conjugated DM1 to bispecific antibody recognizing HER2 and prolactin receptor (PRLR), to enhance ADC internalization and intercellular degradation through PRLR-mediating fast endocytosis, thus increased ADC’s cell killing [38]. To address the situation that T-DM1 works only on 20%–25% patients with HER2 high expression (HIC 3+), MedImmune launched an anti-HER2 biparatopic antibody derived from Trastuzumab and Pertuzumab, to make a tubulin inhibitor tubulysin coupling biparatopic ADC named MEDI4276. Datas suggested that MEDI4276 bound to HER2 stronger than monoclonal antibody, it was effective even in HER2 low expression, T-MD1 treated relapsed or refractory breast cancer model, and now it is in phase I/II [39, 40]. In conclusion, there are several purposes of developing new ADC carriers, including (1) utilizing the excellent physical and chemical properties of new carrier to improve conjugate stability, (2) taking advantages of smaller molecular weight or higher affinity of new carrier to increase tumor conjugate permeation, (3) to improve safety capitalized on prodrug property of probody, (4) to enhance internalization and payload release rate in the help of higher affinity or biparatopic bispecific antibody.

7.4

Summary

The major role of carrier in ADC is to bring high potency cytotoxin into tumor, carrier is very important to ADC efficacy and safety, and that it determines the clinical application of ADC. TRAIL can be an optimal ADC carrier based on its high affinity, specific and stability. Theoretically, any molecule with specific targeting ability is potential ADC carrier. With the arising of natural ligand, aptamer or peptide applied to ADC conjugation, their unique properties enable ADC more vibrant and plentiful, and closer to clinical application. And also, new carrier expression system

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and conjugation method applicable to novel carrier are essential to develop new ADC.

References 1. Witkop B. Paul Ehrlich and his magic bullets, revisited. Proc Am Philos Soc. 1999;143(4): 540–57. 2. Valent P, Groner B, Schumacher U, et al. Paul Ehrlich (1854-1915) and his contributions to the foundation and birth of translational medicine. J Innate Immun. 2016;8(2):111–20. 3. Almasan A, Ashkenazi A. Apo2L/TRAIL: apoptosis signaling, biology, and potential for cancer therapy. Cytokine Growth Factor Rev. 2003;14(3–4):337–48. 4. Pan G, O’rourke K, Chinnaiyan AM, et al. The receptor for the cytotoxic ligand TRAIL. Science. 1997;276(5309):111–3. 5. Pan G, Ni J, Wei YF, et al. An antagonist decoy receptor and a death domain- containing receptor for TRAIL. Science. 1997;277(5327):815–8. 6. LeBlanc HN, Ashkenazi A. Apo2L/TRAIL and its death and decoy receptors. Cell Death Differ. 2003;10(1):66–75. 7. Zauli G, Rimondi E, Nicolin V, et al. TNF-related apoptosis-inducing ligand (TRAIL) blocks osteoclastic differentiation induced by RANKL plus M-CSF. Blood. 2004;104(7):2044–50. 8. Holoch PA, Griffith TS. TNF-related apoptosis-inducing ligand (TRAIL): a new path to anticancer therapies. Eur J Pharmacol. 2009;625(1–3):63–72. 9. Thorburn A. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway signaling. J Thorac Oncol. 2007;2(6):461–5. 10. Kelley SK, Harris L, Xie D, et al. Preclinical studies to predict the disposition of Apo2L/ tumor necrosis factor-related apoptosis-inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety. J Pharmacol Exp Ther. 2001;299(1):31–8. 11. Lawrence DA, Shahrokh Z, Marsters SA, et al. Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions. Nat Med. 2001;7(4):383–5. 12. Muller N, Schneider B, Pfizenmaier K, et al. Superior serum half life of albumin tagged TNF ligands. Biochem Biophys Res Commun. 2010;396(4):793–9. 13. Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest. 1999;104(2):155–62. 14. Maksimovic-Ivanic D, Stosic-Grujicic S, Nicoletti F, et al. Resistance to TRAIL and how to surmount it. Immunol Res. 2012;52(1–2):157–68. 15. Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer genetics and chemotherapy. Cell. 2002;108(2):153–64. 16. Dimberg LY, Anderson CK, Camidge R, et al. On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based therapeutics. Oncogene. 2013;32 (11):1341–50. 17. Ganten TM, Koschny R, Sykora J, et al. Preclinical differentiation between apparently safe and potentially hepatotoxic applications of TRAIL either alone or in combination with chemotherapeutic drugs. Clin Cancer Res. 2006;12(8):2640–6. 18. Stuckey DW, Shah K. TRAIL on trial: preclinical advances in cancer therapy. Trends Mol Med. 2013;19(11):685–94. 19. Stieglmaier J, Bremer E, Kellner C, et al. Selective induction of apoptosis in leukemic B-lymphoid cells by a CD19-specific TRAIL fusion protein. Cancer Immunol Immunother. 2008;57(2):233–46. 20. Van de Water J, Bagci-Onder T, Agarwal AS, et al. Therapeutic stem cells expressing variants of EGFR-specific nanobodies have antitumor effects. Proc Natl Acad Sci U S A. 2012;109(41): 16642–7.

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21. Hutt M, Fellermeier-Kopf S, Seifert O, et al. Targeting scFv-fc-scTRAIL fusion proteins to tumor cells. Oncotarget. 2018;9(13):11322–35. 22. Koornstra JJ, Kleibeuker JH, van Geelen CM, et al. Expression of TRAIL (TNF-related apoptosis-inducing ligand) and its receptors in normal colonic mucosa, adenomas, and carcinomas. J Pathol. 2003;200(3):327–35. 23. Ichikawa K, Liu W, Zhao L, et al. Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity. Nat Med. 2001;7(8):954–60. 24. Pan L. The design of TNF-related apoptosis-inducing ligand (TRAIL) conjugates and studies on their specific antitumor effect. Hangzhou: Zhejiang University; 2014. 25. Pan LQ, Wang HB, Xie ZM, et al. Novel conjugation of tumor-necrosis-factor- related apoptosis-inducing ligand (TRAIL) with monomethyl auristatin E for efficient antitumor drug delivery. Adv Mater. 2013;25(34):4718–22. 26. Pan LQ, Zhao WB, Lai J, et al. Hetero-modification of TRAIL trimer for improved drug delivery and in vivo antitumor activities. Sci Rep. 2015;5:14872. https://doi.org/10.1038/ srep14872. 27. Wei X, Yang X, Zhao W, et al. Optimizing multistep delivery of PEGylated tumor- necrosisfactor-related apoptosis-inducing ligand-toxin conjugates for improved antitumor activities. Bioconjug Chem. 2017;28(8):2180–9. 28. Wei X. Studies on the design of novel-carrier and novel-target based antibody-drug conjugates. Hangzhou: Zhejiang University; 2018. 29. Wang Y, Cheetham AG, Angacian G, et al. Peptide-drug conjugates as effective prodrug strategies for targeted delivery. Adv Drug Deliv Rev. 2017;110–111:112–26. 30. Pluckthun A. Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy. Annu Rev Pharmacol Toxicol. 2015;55:489–511. 31. Simon M, Frey R, Zangemeister-Wittke U, et al. Orthogonal assembly of a designed ankyrin repeat protein-cytotoxin conjugate with a clickable serum albumin module for half-life extension. Bioconjug Chem. 2013;24(11):1955–66. 32. Zhu G, Niu G, Chen X. Aptamer-drug conjugates. Bioconjug Chem. 2015;26(11):2186–97. 33. Bannas P, Hambach J, Koch-Nolte F. Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics. Front Immunol. 2017;8:1603. https://doi.org/10.3389/ fimmu.2017.01603. eCollection2017 34. Fang T, Duarte JN, Ling J, et al. Structurally defined αMHC-II nanobody-drug conjugates: a therapeutic and imaging system for B-cell lymphoma. Angew Chem Int Ed Engl. 2016;55(7): 2416–20. 35. Zhao S, Zhao G, Xie H, et al. A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth. Braz J Med Biol Res. 2012;45(3):230–7. 36. Desnoyers LR, Vasiljeva O, Richardson JH, et al. Tumor-specific activation of an EGFRtargeting probody enhances therapeutic index. Science Translational Medicine. 2013;5(207): 207–144. https://doi.org/10.1126/scitranslmed.3006682. 37. Yang Y, Guo Q, Chen X, et al. Preclinical studies of a pro-antibody-drug conjugate designed to selectively target EGFR-overexpressing tumors with improved therapeutic efficacy. MAbs. 2016;8(2):405–13. 38. Andreev J, Thambi N, Perez Bay AE, et al. Bispecific antibodies and antibody-drug conjugates (ADCs) bridging HER2 and prolactin receptor improve efficacy of HER2 ADCs. Mol Cancer Ther. 2017;16(4):681–93. 39. Steve Coats. Development of a HER2 bispecific antibody-drug conjugate in breast and gastric cancers. MedImmune, 2017. [2018-05-17]. http://worldadc-europe.com/wp-content/uploads/ sites/104/2017/02/11.30-S-Coats.pdf. 40. NIH U.S. National Library of Medicine. A phase 1/2 study of MEDI4276 in adults subjects with select HER2-expressing advanced solid tumors. (MEDI4276). 2018. [2018-05-17]. https:// clinicaltrials.gov/ct2/show/NCT02576548.

Chapter 8

Site-Specified Conjugating Technology and Application Ying Shen, Baoying Shi, and Shuqing Chen

8.1

Introduction

The conjugation site of toxic small molecules is a very important consideration when designing ADCs. According to the different conjugation sites and DARs of toxic small molecules of ADCs, ADCs can be divided into two categories: homogeneous and heterogeneous. Heterogeneous ADCs usually link toxic small molecules directly to lysine or reduced cysteine residues of the antibody. There are more than 80 lysines on a humanized antibody, and cytotoxic drugs are randomly conjugated by lysine, and the resulting ADC is connected with 0 to 8 drugs on each antibody, and the conjugation site can occur on about 40 different lysine residues on light and heavy chains, which may produce more than one million ADCs [1]. In addition, by controlling the reaction conditions, the interchain disulfide bonds of monoclonal antibodies can be reduced while the intra-chain disulfide bonds can still maintain their structure, and then the toxic small molecules can be connected to the reduced thiols through different chemical reactions, such as Michael addition, a-halo carbonyl alkylations and disulfide formation [2–4]. Compared with the lysine linkage mode, reduction of the four interchain disulfide bonds of the antibody produces eight potential conjugation sites, reducing the heterogeneity caused by conjugation, but the number of drugs linked on each antibody still varies from 0 to 8 [2, 5]. Studies have shown that the heterogeneous ADCs produced by non-specified conjugation technology are quite different from the

Y. Shen · B. Shi Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China Department of Precision Medicine on Tumor Therapeutics, ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou, China S. Chen (*) College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chen, J. Zhan (eds.), Antibody-Drug Conjugates and Cellular Metabolic Dynamics, https://doi.org/10.1007/978-981-19-5638-6_8

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homogeneous ADCs produced by site-specified conjugation in terms of in vivo activity and stability, so the development of site-specified conjugation technology is of great significance in the study of ADCs [6]. Site-specified conjugation is the conjugation of a determined number of drugs to a determined site of the antibody, ensuring consistency between batches, minimizing the heterogeneity of the ADC and making its properties more predictable. Homogeneous ADCs prepared by site-specified conjugation techniques have higher efficacy and less toxicity in vivo compared with heterogeneous ADCs. Currently, the DAR of site-directed conjugation techniques is generally controlled at 2 or 4 [7]. At present, the commonly used site-directed conjugation strategies mainly include three types: engineered cysteine, inserted non-natural amino acids, and enzyme-chemical conjugation.

8.1.1

Engineered Cysteine

Reactive thiols of cysteine play an important role in the structure and function of many proteins and can also be used for site-specified conjugation of ADCs. Traditional conjugation methods require partial reduction of the interchain disulfide bonds of monoclonal antibodies, and due to the different number of drugs conjugated and conjugation sites per antibody, heterogeneous ADC mixtures connecting 0 to 8 small molecules are produced, and the interchain disulfide bonds of antibodies are also destroyed. To obtain a homogeneous ADC while maintaining the integrity of the inter-chain disulfide bonds, a cysteine can be inserted at a specific position of the antibody by genetic engineering techniques. For successful site-specified introduction of cysteine, appropriate sites need to be selected that cannot affect the structure of the antibody and its binding to the antigen. Junutula et al. [8] first inserted cysteines at various sites of the Fab fragment of trastuzumab (trastuzumab-Fab 4D5) and selected mutant Fab that maintained the affinity of the original antibody by phage display technology. Light chain V110C and heavy chain A114C were selected as the optimal cysteine mutation sites, respectively. Recombinant expression of engineered heavy chain A114C anti-MUC16 antibody (THIOMAB), conjugated to toxic small molecule MMAE under mild reduction-reoxidation conditions, can synthesize site-specified conjugated ADCs, of which 99.7% are conjugated to THIOMAB, while 92.1% are conjugated to THIOMAB (TDC) with DAR of 2. Compared with ADCs synthesized by traditional conjugation methods, TDCs synthesized using antibody THIOMAB greatly improve homogeneity and preserve antitumor activity in vivo while improving tolerance and reducing systemic toxicity. In addition, maleimidocapronic (MC)-based disulfide bridging reagents, such as dibromomaleimide (DBM), can reconnect the reduced disulfide bonds and maintain the original structure and function of the antibody (Fig. 8.1). When the bridging reagent carries a drug, the number of conjugated drugs can be reduced from the original maximum of 8 to 4, which can reduce the aggregation caused by excessive hydrophobic drugs. Behrens et al. [9] conjugated MMAF to trastuzumab in a bridging manner to synthesize a conjugated product with DAR of 4, which has

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Fig. 8.1 Maleimide conjugation and bridging conjugation [9]

better in vivo activity and pharmacokinetic properties than traditional ADCs linked through maleamide.

8.1.2

Insertion of Unnatural Amino Acids

Inserting unnatural amino acids with bioorthogonal reactive groups in the antibody sequence, such as selenocysteine (Sec) and p-acetylphenylalanine (pAcPhe), is another effective method to mediate site-specified conjugation of drugs. The structure of Sec is very similar to that of cysteine (Cys), except that in Sec, the sulfur atom in Cys is replaced by selenium atom. Selenohydride (-SeH) is more nucleophilic than thiol (-SH), and can be covalently coupled to electrophilic compounds selectively even with the interference of other natural amino acid side chains. Normally, UGA is a transcription stop codon, but when there is a selenocysteine insertion sequence (SECIS) at the 3 ′ UTR (untranslated region) of the gene, the secondary structure of the mRNA will be formed to prevent the termination of transcription and Sec can be inserted at the UGA. By inserting UGA and SECIS at the 3 ′ end of the gene, Sec can be inserted at the C-terminus of the antibody, and its selenohydrogen group can be site-specified conjugated with iodoacetamide or maleimide derivatives [10]. Li et al. [11] introduced one or two Sec at the C-terminus of the antibody to conjugate a fluorescently labeled iodoacetamide derivative, resulting in site- specified conjugates with a DAR of 1 or 2 (Fig. 8.2). Using this method, the interchain disulfide bonds of the antibody were not disturbed, the intact structure was maintained, and 80% of the expressed antibody had Sec inserted. However, when three Sec were inserted in the sequence, the expression of the antibody dramatically decreased. Site-directed conjugation of the drug can also be achieved by introducing the unnatural amino acid pAcPhe. pAcPhe contains a keto group that can be linked to the alkoxyamine of the drug via an oxime. The method of introducing pAcPhe into the antibody: first, an amber stop codon (UAG) was introduced at the appropriate site of the antibody, and then this cDNA was co-expressed with aminoacyl tRNA synthetase (aaRS). Properly paired mutant aaRS can load pAcPhe to tRNA, after which pAcPhe is introduced into the amber codon site of the antibody. Tian et al. [12] used

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Fig. 8.2 Site- specified conjugation by insertion of selenocysteine [11]

Fig. 8.3 Site-specified conjugation by insertion of p-azidomethyl-L-phenylalanine [13]

this method to synthesize a conjugate of pAcPhe-containing trastuzumab with MMAF. The conjugate has similar pharmacokinetic properties to unconjugated trastuzumab; it has higher in vitro antitumor activity and better pharmacokinetic properties than ADCs conjugated through cysteine. In addition to pAcPhe, p-azidomethyl-L-phenylalanine (pAMF) has also been introduced into antibodies (Fig. 8.3) to achieve site-directed conjugation of ADCs [13].

8.1.3

Enzyme-Chemical Conjugation

Due to the wide applicability of orthogonal reactions, combining the obvious advantages of both enzymatic and chemical methods, the use of enzyme-chemical conjugation is a very potential strategy for site-directed conjugation of antibodies. Among them, the commonly used enzymes include glycosyltransferase, glutamine transferase, sortase A, and formylglycine synthetase. Glycosyltransferases (glycotransferases) are a large group of proteins that catalyze the transfer of glycosyl groups from activated donors to sugar receptors (glycoproteins or glycolipids). The position 297 of the Fc fragment of the humanized antibody is a conserved asparagine (Asn) N-glycosylation site, which can be deglycosylated to the G0 glycoform. Subsequently, the mutated glycosyltransferase can efficiently transfer ketogalactan here. The ketone group of ketogalactose can be linked to other molecules by orthogonal reactions [14]. This method can be applied

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to site-directed conjugation of ADCs. For example, Zhu et al. [15] introduced reactive ketogalactose through a mutated glycosyltransferase on the N-sugar chain of the Fc fragment of monoclonal antibody m860 against HER2 (different from the HER2 epitope targeted by trastuzumab), which was then oxime-linked with alkoxyamine-modified MMAF to obtain m860 ADC with a DAR of 4 (2 drugs attached to each heavy chain). The effect of m860ADC on HER2-positive cells was powerful and superior to that of T-DM1. Microbial transglutaminase (mTG) is an enzyme from Streptoverticillium mobaraense that catalyzes the acyl transfer reaction between the side chain amide group of glutamine and the lysine amino group to form a stable isomeric peptide bond [16]. Strop et al. [17] introduced glutamine tags (LLQG) into different sites of the light and heavy chains of anti-EGFR, anti-HER2, and anti-M1S1 antibodies and conjugated fluorescein and monomethyl auristatin D (MMAD) to obtain conjugates with DAR of 1.2 to 2. These conjugations had a wider therapeutic window on rats compared to ADCs that were non-site-specified conjugations via cysteine (mean DAR of 3.6). Sortase A (SrtA) is an enzyme from Staphylococcus aureus that catalyzes the transpeptidation reaction and can specifically recognize the LPXTG sequence at the end of the target protein (X is any of the natural amino acids). SrtA uses the thiol group of its cysteine at position 184 to attack the peptide bond between threonine (T) and glycine (G) to form a thiointermediate. Toxic small molecules modified by three to five glycines in the reaction system can affinity attack the thiointermediate and form new peptide bonds to obtain site-specified conjugation products (see Sect. 8.3 of this chapter for details). Pan et al. [18] used SrtA to produce an anti-CD20MMAE conjugate with a DAR of 1.4, which could have an IC50 as low as 5 pg/mL on CD20+ Romas cells and also have a good antitumor effect in vivo. Formylglycine synthase (FGE) can effectively recognize CXPXR sequence (C is cysteine, P is proline, R is arginine, X is usually any of serine, threonine, alanine and glycine), oxygenate cysteine (C) into formylglycine (fGly), and bring aldehyde tag. This aldehyde group can produce a stable carbon carbon bond through the Pictet-Spengler reaction, which can be conjugated with cytotoxic drugs (Fig. 8.4). Drake et al. introduced the CXPXR sequence into 8 sites (1 site on the light chain and 7 sites on the heavy chain) in the constant regions of trastuzumab light and heavy chains, respectively, and co-expressed with FGE intracellularly, resulting in 8 antibody molecules carrying 2 aldehyde tags. Subsequently, three of these sites were selected to couple with the toxic small molecule maytansinoid, confirming that the conjugation site is closely related to plasma stability, in vivo half-life, and in vivo anti-tumor efficacy, while the heavy chain C-terminus is the optimal site. Comparison of this with an ADC conjugated by lysine residues confirmed the better safety of this ADC.

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Fig. 8.4 Site-specified conjugation by Formylglycine Synthetase [19]

8.2

Site-Specified Conjugation of Mutated Cysteine

Cysteine in monoclonal antibodies is widely used in the ligation of toxic small molecules during the preparation of ADCs. Cysteines are involved in the formation of intra-chain disulfide bonds and inter-chain disulfide bonds in antibodies, but they do not have much effect on the basic structure of antibodies. Inter-chain disulfide bonds have been found to be more sensitive to reducing agents than intra-chain disulfide bonds. It is therefore possible to reduce the inter-chain disulfide bonds while the intra-chain disulfide bonds remain stable by controlling the reaction conditions using reducing agents such as DTT or TCEP. Each monoclonal antibody has four inter-chain disulfide bonds, which results in eight thiols, resulting in each antibody being able to connect between 0 and 8 small toxic molecules, resulting in heterogeneous antibody-drug conjugates. However, site-specified conjugation by mutating cysteine can be achieved to obtain a homogeneous ADC.

8.2.1

Principle of Site-Specified Conjugation of Mutated Cysteine

Mutant cysteine ligation technology was first developed by Genentech. Junutula et al. [8] tried to mutate a series of amino acids with little effect on antibody structure and its affinity to antigen to cysteine to form a new reactive antibody (named Thiomab), and then selected the mutant Fab with unchanged affinity to antigen by phage display technology (PHESELECTOR). In the presence of reducing agents DTT or TCEP, the introduced cysteine residues, inter-chain disulfide bonds in the synthesized Thiomab were reduced while the intra-chain disulfide bonds remained stable. Subsequently, under the oxidation of copper sulfate or dehydroascorbic acid,

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the opened interchain disulfide bonds are regenerated, but the cysteine introduced by genetic engineering is still in a reduced state. Thus, reduction of only the mutated cysteine produces free thiols that are able to be linked to small molecules, enabling site-specified conjugation. At present, multiple mutation sites have been reported to be able to be used for site-directed conjugation of ADCs, such as light chain V110C, light chain V205C and heavy chain A114C. These sites can still form a stable antibody structure after mutation and do not affect the affinity of antibody to antigen while successfully expressed.

8.2.2

Main Experimental Materials

Light and heavy chain vectors pFUSE2-CLIg-hk (phk) and pFUSE-CHIg-hG1 (phIgG1) with constant region of human antibody; oxidant DHAA (dehydroascorbic acid); reducing agent tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCL); toxic small molecule with MC-VC-PABC-MMAE (vcMMAE) with linker.

8.2.3

Main Experimental Procedures [20] (DAR = 2 or DAR = 4)

8.2.3.1

Acquisition of Cysteine Mutant Antibodies

According to the antibody light and heavy chain constant panel plasmids of pFUSE2-CLIg-hk (phk, κ light chain), pFUSE-CHIg-hG1 (phIgG1, IgG1 heavy chain), the heavy chain A114C and light chain V205C point mutations were performed by overlapping PCR. The light chain and heavy chain were ligated into the pMH3 vector, respectively, and stably transfected into CHO-K1 cells to obtain the mutant antibody (Fig. 8.5) [20].

8.2.3.2

Synthesis and Analysis of Ab-vcMMAE

The mutant antibody (1 mg/mL) was reduced with 50 eq of TCEP (final concentration is 0.1 mg/mL) for 3 h. The ultrafiltration tube was concentrated and eluted with PBS to remove TCEP. Oxidize with 20 eq of DHAA (final concentration is 0.023 mg/mL) for 3 to 5 h. The ultrafiltration tube was concentrated and eluted with PBS to remove DHAA. After reaction with 10 eq of MC-VC-PABC-MMAE for 1 h, the ultrafiltration tube was concentrated and eluted with PBS to remove small molecule conjugates to obtain the ending product. The ligation of Ab-vcMMAE can be analyzed by SDS-PAGE, UV spectrophotometry, DTNB (5,5 ′ -dithiobis (2-nitrobenzoic acid)) method or mass spectrometry.

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Fig. 8.5 Obtain antibodies containing two and four cysteine mutations [20]

8.3

Site-Specific Conjugation Through Sortase A

SrtA was first discovered in Staphylococcus aureus and consists of 206 amino acids. SrtA is a type II cell membrane topology. The N-terminus located in the cytoplasm anchors SrtA on the cell membrane. The C-terminus located in the cytoplasm is the key core region for catalyzing the transpeptide reaction. Due to the high specificity of the transpeptidation mediated by SrtA (Fig. 8.6) [21], SrtA is widely used in nanoparticle labeling, protein markers and protein fusion [22, 23]. Although SrtA can be conjugated to a series of peptide substrates containing oligoglycine while maintaining a certain specificity, its use is limited due to its poor wild-type kinetic properties. For this reason, our laboratory compared the catalytic activity of a variety of wild-type and mutant SrtA (Fig. 8.7), and selected the wild-type SrtA (ΔN59) with the highest catalytic efficiency [18].

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Fig. 8.6 Transpeptidation mediated by Sortase A [21]. Note: C (Cysteine) at position 184 of SrtA nucleophilically attacks the peptide bond between T (threonine) and G (glycine) in the peptide sequence of LPXTG (X is any of the natural amino acids). When G is released, the carboxy terminal peptide is exposed to form a covalent thio intermediate. The intermediate can capture the polyglycine substrate in solution and form a new peptide bond with threonine

Fig. 8.7 Various mutants of SrtA [18]. SrtA (ΔN25), SrtA (ΔN59) and SrtA (ΔN109) are wildtype SrtA with 25, 59 and 109 amino acid residues truncated at the N-terminal; SrtA (3M) is SrtA (ΔN59) with three site mutations: P94S, D160N, D165A; SrtA (4M) is SrtA (ΔN59) with four site mutations: P94S, D160N, D165A, and K196T

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8.3.1

Direct Enzymatic Approach

8.3.1.1

Materials

pET28a-SrtA (ΔN59) expression plasmid; Escherichia coli Rosetta (DE3) expression host; Recombinant OFA antibody (light and heavy chain C-terminal with LPETG tag); GGG- vc-PAB-MMAE (referred to as GGG-vcMMAE).

8.3.1.2

Methods

1. Expression and purification of SrtA (ΔN59): Transform the cDNA of SrtA (ΔN59) (containing His tag) into the prokaryotic expression vector pET28a(+), transform it into E. coli Rosetta (DE3), induce expression by IPTG, and use Ni-NTA column for affinity purification. The purified protein product is analyzed for purity by SDS-PAGE. If the purity reaches more than 95%, it can meet the requirements of subsequent experiments. 2. SrtA-mediated conjugation: Add recombinant OFA antibody 2 μmol/L, SrtA 50 μmol/L, GGG-vcMMAE 200 μmol/L and CaCl2 5 mmol/L into the 50 nmol/L Tris and 150 nmol/L NaCl (pH 7.4) reaction systems, react at 37 °C for 48 h. The reaction product is affinity purified with a Protein A column to remove excess small molecules and enzymes in the reaction system.

8.3.2

Chemo-Enzymatic (Srt A) Strategy

Generally, toxic small molecules (such as GGG-vcMMAE, etc.) are soluble in organic solvents and directly conjugated with antibodies, which may affect the structure and activity of antibodies, and these small molecules have relatively large steric hindrance, which is not conducive to the direct catalysis of SrtA (Fig. 8.8). In contrast, small molecules with higher hydrophilicity and smaller relative molecular mass can achieve efficient conjugation. Click chemistry,

Fig. 8.8 Strain-promoted azide-alkyne cycloaddition (SPAAC)

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Fig. 8.9 Chemo-enzymatic (Srt A) strategy to generate ADCs

especially the strain-promoted azide-alkyne cycloaddition (SPAAC), is an emerging tool for protein modification. The reaction of azide with strained cyclooctyne is highly specific, efficient, and proceeds under mild conditions. Therefore, our laboratory uses the chemo-enzymatic (Srt A) strategy to prepare ADCs (Fig. 8.9). Under the catalysis of SrtA, GGG-PEG-N3, a small molecule linker containing bifunctional groups (the C-terminal is an azide group, the N-terminal is an oligoglycine, connected with PEG, referred to as GPN), is introduced into the LPETG position of the antibody. Then, the drug containing cyclooctyne (DBCO-PEG-vc-PABMMAE) and the antibody containing azide were chemically conjugated through SPAAC to prepare a site-specific ADC (OFA-GPN-vcMMAE) [21]. Under mild conditions, 1,4-substituted triazoles are formed in the cycloaddition reaction between azide and DBCO without Cu(I)[2 + 3]. In the first step, the bifunctional small molecule linker is connected to the LPETG-labeled antibodies through SrtA. In the second step, small toxin molecules (paylod) modified with functional groups are chemically conjugated to antibodies through SPAAC.

8.3.2.1

Materials

Recombinant antibody (with LPETG label at the C end of the light and heavy chains); SrtA; GGG-PEG-N3 (referred to as GPN); DBCO-PEG-vc-PAB-MMAE.

8.3.2.2

Methods[24]

After the bifunctional small molecule GPN and the antibody are catalyzed and conjuagetd by SrtA, they are purified with a Protein A column to obtain an antibody containing an azide group. Antibody containing an azide group: DBCO-PEG-vc-PAB-MMAE = 1: 8, react overnight at room temperature in PBS, and ultrafiltration with PBS until the concentration of the small toxin molecule is less than 0.1 nmol/L.

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Identification of Conjugation Sites by Mass Spectrometry Materials

Guanidine hydrochloride; Dithiothreitol (DTT); Iodoacetamide; Trypsin.

8.4.2

Methods[25]

Dilute the sample with guanidine hydrochloride-Tris solution (pH 8.0) to 2 mg/mL, add DTT (20 mmol/L) and react at room temperature for 1.5 h. Then add iodoacetamide (60 mmol/L) and react at room temperature in the dark for 40 min to block free cysteine residues. After the reaction, the reaction solution was ultrafiltered at 4 °C and replaced with a 100 mmol/L Na3PO4 solution (pH 7.4). Then add trypsin at 37 °C for 24 h, and finally stop the reaction with formic acid (1%) for testing. Ultra-high performance liquid separation uses the chromatographic column Acquity UPLC BEH300 C18, the column temperature is 40 °C, and the flow rate is 0.2 mL/min. Mobile phase: Phase A is 0.1% formic acid/water solution, phase B is 0.1% formic acid/acetonitrile solution. The elution gradient is shown in Table 8.1. Xevo G2-S Q-TOF mass spectrometer parameter settings are shown in Table 8.2. Use Biopharmalynx 1.3.3 (Waters) software to analyze the data obtained by liquid chromatograph/mass spectrometer (LC-MS) (Fig. 8.10). Table 8.1 Elution gradient

Time(min) 0 5 45 50 55 60 65

Phase A(%) 95 95 50 5 5 95 95

Phase B(%) 5 5 50 95 95 5 5

Table 8.2 Xevo G2-S Q-TOF mass spectrometer parameter settings Parameter Capillary voltage Quality analysis range Ion source temperature temperature Atomization flow rate

Value 2.5 kV 50 ~ 2000 Da 90 °C 700 L/h

Parameter Cone voltage Collision energy Atomization temperature Internal standard

Value 80 V 20 ~ 45 eV 400 °C Leucine Enkephalin

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Fig. 8.10 UPLC/Q-TOF MS analysis [25]

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Figure 8.10a shows the theoretical trypsin digestion sequence of the C-terminus of the light and heavy chains of the sample. The analysis of GGG-vcMMAE is shown in Fig. 8.10b, and three daughter ions (m/z is 506.41, 686.56 and 718.59 respectively) can be selected to further characterize the linker peptide. The separation and characterization of the sample after trypsin digestion is shown in Fig. 8.10c. The fragment containing GGG-vcMMAE is confirmed by the signal of three daughter ions. As shown in Fig. 8.10d, the C-terminal fragments of the light chain and the heavy chain of the trypsinized sample were found at 38.31 min and 39.73 min, respectively. In the liquid chromatography, the two main fragments of the trypsindigested sample were eluted at 38.31 min and 39.73 min, including the three characteristic daughter ion signals of vcMMAE in the LC/MS data (Fig. 8.10b), indicating that they are GGG-vcMMAE conjugated fragment from the sample. The 38.31 min and 39.73 min fragments of ultra performance liquid chromatographyquadrupole time-of-flight mass spectrometry (UPLC/Q-TOFMS) showed that the relative molecular masses were 2895.476 Da and 1733.998 Da, which matches the expected relative molecular mass of the light and heavy chain C-terminal trypsinized fragments (2895.477 Da and 1735.00 Da). In addition, a series of y ions connected to the complete GGG-vcMMAE molecule were also found in the last two corresponding secondary mass spectra of the frame selection in Fig. 8.10d. And confirm that the GGG-vcMMAE small molecule is indeed attached to the LPET sequence at the carboxyl end of the antibody heavy chain (Fig. 8.10e) and light chain (Fig. 8.10f).

8.5

Conclusion

ADCs synthesized by traditional conjugations are highly heterogeneous, and due to the strong hydrophobicity of cytotoxic drugs, it’s easy to cause aggregation and reduce stability when the number of toxic drug couplings is large (DAR greater than 4). Compared with traditional ADCs, site-specific conjugated ADCs can reduce heterogeneity, improve pharmacokinetic properties, and reduce toxic and side effects, thus broadening the therapeutic window. This chapter mainly introduces some mainstream site-specific conjugation methods and applications, such as mutant cysteine site-specific conjugation and SrtA mediated site-specific conjugation. These site-specific conjugation methods have their own characteristics and are significantly better than traditional non-site-specific conjugation methods. Site-specific conjugation is bound to become the future development trend of ADC.

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References 1. Panowski S, Bhakta S, Raab H, et al. Site-specific antibody drug conjugates for cancer therapy. MAbs. 2014;6(1):1–2. 2. Sun MM, Beam KS, Cerveny CG, et al. Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides. Bioconjug Chem. 2005;16(5):1282–90. 3. Nielsen ML, Vermeulen M, Bonaldi T, et al. Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat Methods. 2008;5(6):459–60. 4. McDonagh CF, Turcott E, Westendorf L, et al. Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment. Protein Eng Des Sel. 2006;19(7):299–307. 5. Doronina SO, Toki BE, Torgov MY, et al. Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat Biotechnol. 2003;21(7):778–84. 6. Boylan NJ, Zhou W, Proos RJ, et al. Conjugation site heterogeneity causes variable electrostatic properties in fc conjugates. Bioconjug Chem. 2013;24(6):1008–16. 7. Hamblett KJ, Senter PD, Chace DF, et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res. 2004;10(20):7063–70. 8. Junutula JR, Raab H, Clark S, et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol. 2008;26(8):925–32. 9. Behrens CR, Ha EH, Chinn LL, et al. Antibody-drug conjugates (ADCs) derived from interchain cysteine cross-linking demonstrate improved homogeneity and other pharmacological properties over conventional heterogeneous ADCs. Mol Pharm. 2015;12(11):3986–98. 10. Caban K, Copeland PR. Size matters: a view of selenocysteine incorporation from the ribosome. Cell Mol Life Sci. 2006;63(1):73–81. 11. Li X, Yang J, Rader C. Antibody conjugation via one and two C-terminal selenocysteines. Methods. 2014;65(1):133–8. 12. Tian F, Lu Y, Manibusan A, et al. A general approach to site-specific antibody drug conjugates. Proc Natl Acad Sci U S A. 2014;111(5):1766–71. 13. Zimmerman ES, Heibeck TH, Gill A, et al. Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjug Chem. 2014;25(2):351–61. 14. Boeggeman E, Ramakrishnan B, Pasek M, et al. Site specific conjugation of fluoroprobes to the remodeled fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection. Bioconjug Chem. 2009;20(6):1228–36. 15. Zhu Z, Ramakrishnan B, Li J, et al. Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar. MAbs. 2014;6(5):1190–200. 16. Yokoyama K, Nio N, Kikuchi Y. Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol. 2004;64(4):447–54. 17. Strop P, Liu SH, Dorywalska M, et al. Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem Biol. 2013;20(2):161–7. 18. Pan L, Zhao W, Lai J, et al. Sortase A-generated highly potent anti-CD20-MMAE conjugates for efficient elimination of B-lineage lymphomas. Small. 2017;13(6):201602267. 19. Carrico IS, Carlson BL, Bertozzi CR. Introducing genetically encoded aldehydes into proteins. Nat Chem Biol. 2007;3(6):321–2. 20. Wei XY. Studies on the design of novel-carrier and novel-target based antibody-drug conjugates. Hangzhou: Zhejiang University; 2018. 21. Xu Y, Jin S, Zhao W, et al. A versatile chemo-enzymatic conjugation approach yields homogeneous and highly potent antibody-drug conjugates. Int J Mol Sci. 2017;18(11).

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22. Matsumoto T, Tanaka T, Kondo A. Sortase A-catalyzed site-specific coimmobilization on microparticles via streptavidin. Langmuir. 2012;28(7):3553–7. 23. Levary DA, Parthasarathy R, Boder ET, et al. Protein-protein fusion catalyzed by sortase a. PLoS One. 2011;69(4):e18342. 24. Xu Y. The design of antibody-drug conjugates and studies on their specific antitumor activities. Hangzhou: Zhejiang University; 2018. 25. Lai J. Exploration of the feasibility of peptide/major histocompatibility complex (pMHC) as antibody-drug conjugates (ADCs) target. Hangzhou: Zhejiang University; 2018.

Chapter 9

Determination of Drug-to-Antibody Ratio of ADCs Chixiao Qiu and Shuqing Chen

9.1

Introduction

ADCs are mainly composed of three parts: antibody, linker and effector molecule. Among them, monoclonal antibody molecules are susceptible to modifications such as glycosylation and oxidation during expression, purification, and storage due to their relative large molecular weight. Therefore, they are prone to produce isomers which will affect drug conjugation. Different conjugation processes will also affect the efficiency and quantity of drug conjugation. For example, gemtuzumab ozogamicin (Mylotarg®), the first ADC that was approved by the FDA in 2000, uses the stable amide bond formed by the side chain amino group of lysine and butyric acid to link calicheamicin and gemtuzumab [1]. Due to the large amount of lysine on the antibody, the ADC obtained by this conjugation method is heterogeneous. According to research reported by Acchione [2], the average drug-to-antibody ratio(DAR) was 6–14. The DAR refers to the number of small toxic molecules contained in each antibody in the ADC. DAR is an important quality attribute of ADC, because it determines the amount of “payload” that can be delivered to tumor cells, which directly affects safety and efficacy [3]. It will affect the efficacy of the drug when the DAR is low; while the high DAR will make its safety and stability potentially risky, and there are also cases where the efficacy of the ADC decreases with the increase of

C. Qiu Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China Department of Precision Medicine on Tumor Therapeutics, ZJU-Hangzhou Global Scientific and Techaters) software to anological Innovation Center, Hangzhou, China e-mail: [email protected] S. Chen (*) College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chen, J. Zhan (eds.), Antibody-Drug Conjugates and Cellular Metabolic Dynamics, https://doi.org/10.1007/978-981-19-5638-6_9

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DAR. By detecting and controlling DAR, the consistency of ADC production between batches can be ensured, which is very important for the stability of its quality. The measurement of DAR can also be fed back to the process control, which is also crucial for optimizing process parameters. In addition to the heterogeneity of ADC itself, toxic small molecules can be gradually dissociated from ADC through enzymatic hydrolysis or chemical reaction after ADC entering the body, further increasing its diversity in the body. Although the stability of the linker is taken into consideration when designing the ADC, the ADC should theoretically maintain a stable structure in the plasma. But in some special cases the linker will still be disconnected, freeing toxic small molecules. For example, ADCs conjugated by disulfide bonds are stable in the circulatory system and are disconnected under conditions where the intracellular is rich in glutathione and other reducing substances, such as SAR3419 (anti-CD19 antibody conjugation with maytansine) [4, 5], but this type of ADC is more sensitive to endogenous peptides or proteins containing free sulfhydryl groups (such as glutathione and albumin), and linker-toxic small molecules will be transferred to such structures [6, 7]. This ever-changing diversity has brought great difficulties to the study of the drug metabolism kinetics of ADCs. At present, many studies have been working hard to reduce the diversity of ADCs [8–11]. The heterogeneity of ADCs and the dissociation of small molecules in the body make the DAR of ADCs to be different. ADCs from different DAR are metabolized differently in vivo. In the study of cantuzumab mertansine (huC242-DM1), it can be observed that the clearance rate of the ADC in the blood circulation was faster than that of antibodies that were not bound to toxic small molecules [12]. There are also literatures showing that the clearance rate of ADC increases with the increase of DAR [13]. In vivo studies of T-DM1 have shown that the average value of T-DM1 DAR will gradually decrease over time [14]. Common methods for determining DAR include spectrophotometry [13], radiometry [15], hydrophobic liquid chromatography [13], and mass spectrometry [16, 17].

9.2

Experimental Procedures for RP-HPLC and HIC to Determine DAR

Reversed phase high performance liquid chromatography (RP-HPLC) and hydrophobic interaction chromatography (HIC) can be used to determine the DAR of ADCs. Among them, RP-HPLC provides an orthogonal method to determine DAR. At first, this method separates the heavy and light chains of the ADC completely through a reduction reaction, then separates the light and heavy chains and their corresponding drug-carrying forms on RP-HPLC. The weighted average DAR is calculated by using the peak area percentage obtained by integrating the light chain and heavy chain peaks and combining the drug load of each peak. HIC measures the DAR of ADCs by the difference in hydrophobicity of ADCs of different DARs. Usually, the naked antibody is the least hydrophobic and will be

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eluted first, and the more toxic small molecules connected, the higher of the hydrophobicity of the antibody and the longer of the elution time. The area percentages of different peaks are used to represent the relative distribution of ADCs with different DARs. The weighted average DAR is then calculated using the peak area percentage and drug load. Materials and instruments: All solutions and reagents should be prepared with high-purity salt, buffers, high-performance liquid chromatography-grade solvents and ultrapure water (high-performance liquid chromatography-grade or dual deionization). Generally speaking, the mobile phase solution is stored at room temperature and can be used for 1 month. HPLC system: Agilent 1100 or 1200 HPLC system, equipped with binary pump, thermostatic automatic sampler, column chamber with temperature control and diode array detector or other HPLC system with equivalent modules. The temperature of the water bath is controlled at 37 °C ± 2 °C.

9.2.1

Determination of DAR by RP-HPLC

RP-HPLC is a liquid chromatography system composed of a non-polar stationary phase and a polar mobile phase. Since RP-HPLC uses organic solvents and a small amount of organic acids, which will cause great damage to ADCs conjugated via Cys. This type of ADC cannot withstand high denaturation conditions during analysis and will split into antibody fragments. This is because some of the interchain disulfide bonds in the antibody are reduced during the conjugation process and attached to toxic small molecules, and ADCs are bound together through non-covalent hydrophobic interactions. When the Cys-linked ADC is treated with a reducing agent such as dithiothreitol (DTT), the remaining interchain disulfide bonds can be completely reduced, and six forms are produced: the light chain, the light chain with single toxic small molecules attached, heavy chain, the heavy chain with single, two or three toxic small molecules attached respectively. These forms are stable in denatured organic environments and can be analyzed by reversed-phase chromatography columns. According to the integrated area of the light and heavy chain peaks, and taking into account the number of toxic small molecules corresponding to each peak, calculate the peak area percentage to obtain the weighted average DAR.

9.2.1.1

Experimental Conditions and Methods

Column: Varian PLRP-S 100 Å column (150 mm × 25 mm, 8 μm). Mobile phase A is 0.1% trifluoroacetic acid/water. Mobile phase B is acetonitrile. Gradient conditions: 25% mobile phase B (3 min); 25% to 50% mobile phase B (25 min); 50% to 95% mobile phase B (2 min); 95% to 25% mobile phase B (1 min)); 25% mobile phase B(2 min).

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Flow rate: 0.6 mL/min. Column temperature: 80 °C. Detection wavelength: 280 nm, 248 nm and 252 nm. The sample was reduced with DTT at 37 °C for 30 min.

9.2.1.2

Data Processing

Peaks are integrated by using manual or automatic integration tools. According to the ratio of the peak areas of the light and heavy chains, we can calculate the ratio of connected toxic small molecules, and then the ADC’s DAR can be calculated.

9.2.2

Determination of DAR by HIC

HIC takes advantage of the difference of the hydrophobic force between the sample molecules and the stationary phase. When eluting with the mobile phase, the migration speed of each component is different, which can achieve the purpose of separation. Due to the relatively high hydrophobicity of generally toxic small molecules, the residence time on the hydrophobic stationary phase (such as TOSOH Butyl-NPR) increases after the antibody is conjugated to the hydrophobic toxic small molecule. With the decrease of the salt concentration gradient in the mobile phase and the increase of the organic phase, the naked antibody is eluted first, and the antibody with more toxic small molecules will be eluted later. For ADCs linked to toxic small molecules via inter-disulfide cysteine (Cys), HIC will not cause the separation of these forms of antibodies due to the non-denaturing treatment and relatively mild conditions. The integrated area of each peak represents the relative proportion of the number of small molecules with specific toxicity. From the peak area percentage and the corresponding number of toxic small molecules, the weighted average DAR of the ADC can be calculated.

9.2.2.1

Experimental Conditions and Methods

Column: TOSOH Butyl-NPR (4.6 mm × 3.5 cm, 2.5 μm). Mobile phase A is 1.5 mol/L (NH4) 2SO4, 25 mmol/L NaH2PO4 (pH 7.0). Mobile phase B is 75% 25 mmol/L Na3PO4 (pH 7.0) with 25% isopropanol. Gradient conditions: 15 min linear gradient from mobile phase A to mobile phase B. Flow rate: 0.8 mL/min. Column temperature: room temperature. Detection wavelength: 280 nm, 248 nm and 252 nm.

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9.2.2.2

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Data Processing

Peaks are integrated by using manual or automatic integration tools. The ADC’s DAR is calculated based on the percentage of the area occupied by the shifted peak and the number of corresponding toxic small molecules.

9.2.3

Case Analysis [18]

Utilizing the property that SrtA can specifically recognize the short peptide sequence LPXTG and catalyze its transpeptidation reaction with oligoglycine substrates, the OFA antibody (anti-CD20) with the LPETG label can conjugate to the cytotoxic drug GGG-vc-PAB-MMAE modified with oligoglycine (Fig. 9.1). RP-HPLC and HIC analysis are performed on the prepared ADC. According to the above experimental conditions, the following experimental results can be obtained (Figs. 9.2 and 9.3). RP-HPLC was used to detect the conjugation of OFA-vcMMAE under reducing conditions. The result is shown in Fig. 9.2. The heavy chain can be connected to MMAE, but the light chain is not connected to MMAE (this result is consistent with the Western Blot result, which is not shown here). According to the peak area ratio H0/(H0 + H1) = 30%, the calculated DAR of OFA-vcMMAE is about 1.4. Using the gradient elution from mobile phase A to mobile phase B, HIC can separate conjugates of different DARs. The analysis result is shown in Fig. 9.3. The conjugate contains two ADC components, DAR = 1 and DAR = 2. According to its peak area, its DAR is estimated to be around 1.4, which is consistent with the results of RP-HPLC analysis.

Fig. 9.1 Preparation of antibody-conjugated drugs using SrtA

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mAU 160 140

L0

H0

120 100

OFA

80 60

L0

40 20

H0

OFA-vcMMAE

H1

0 0

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Fig. 9.2 RP-HPLC analysis of OFA-vcMMAE light and heavy chain conjugation. Note: L0 is an uncoupled light chain; L1 is a light chain coupled to an MMAE; H0 is an uncoupled heavy chain; H1 is a heavy chain coupled to an MMAE mAU 120

E0

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OFA E1 E2 E0

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OFA-vcMMAE

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Fig. 9.3 HIC analysis of OFA-vcMMAE conjugation. Note: E0, E1 and E2 mean DAR is 0, 1 and 2 respectively

9.3

The Experimental Procedure for the Determination of DAR by LC-MS

Liquid chromatography-mass spectrometry (LC-MS) uses liquid chromatography as the separation system and mass spectrometry as the detection system. The sample is separated from the mobile phase in the mass spectrometer, then it is separated by mass by the mass analyzer of the mass spectrometer after being ionized, and the mass spectrum is obtained by the detector. LC-MS can be used to determine ADC’s DAR and drug load distribution. First, the ADC samples were desalted on a reversedphase liquid chromatography column with an acetonitrile gradient, and then subjected to online mass spectrometry. The mass spectrum is processed and

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converted into a series of zero-charge state masses to correspond to the increase in the number of toxins in ADCs. According to the integral of the mass peak area, the ADC’s DAR and toxin load distribution can be calculated. Compared with RP-HPLC and HIC methods, mass spectrometry-based methods have more advantages in analyzing ADCs conjugated via lysine (Lys), while RP-HPLC and HIC methods are mostly used to analyze ADCs with limited conjugation sites (such as ADCs conjugated via interchain cysteine or engineered cysteine) [3, 19]. LC-MS is essential for the identification of various drug-loaded forms of stable ADCs. Unlike cysteine-linked ADCs, lysine-linked ADCs are stable under the denaturing conditions of typical LC-MS. Therefore, the different drug-loaded forms of lysine-linked stable ADCs can be identified by their precise mass. Compared to ADCs with limited site-specific conjugation, lysine-linked ADCs are more heterogeneous. Thus, the analysis of mass spectrometry data of lysine-linked ADC is challenging. Usually, sample preparation methods such as deglycosylation [20] and removal of C-terminal lysine heterogeneity [21] are used to reduce spectral complexity. The mass spectrum of a lysine-linked ADC usually contains a series of ion peaks with charge states between +45 and +60. The mass spectra are transformed into a series of zero-charge state masses through spectral processing or deconvolution, showing a pattern in which the number of conjugated drugs is increasing. The spectral peak area can be integrated to provide an intuitive method for DAR and drug load distribution calculations. However, this method assumes that the MS ionization efficiency and MS response between different intact drugs are similar. The hydrophobicity of the attached drug and any change in the total net charge of the protein due to the modification of the lysine residues will affect the mass spectrometry response, thereby affecting the determination of DAR and drug load distribution. Therefore, the researchers compared mass spectrometry with ultraviolet-visible light absorption spectroscopy (UV/vis) methods. Studies have shown that there is an acceptable correlation between UV/vis and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) when the full charge envelope of each drug loading situation is taken into account by mass spectrometry [17].

9.3.1

Instruments and Reagents

9.3.1.1

Instruments

Liquid chromatograph: Agilent 1100 binary gradient pump is equipped with a thermal automatic sampler and a column chamber or equivalent. Mass spectrometer: Quadrupole time-of-flight (Q-TOF) hybrid LC-MS/MS system or equivalent standard. High performance liquid chromatography column: PLRP-S (polystyrenedivinylbenzene reverse phase chromatography column), with a specification of 2.1 mm × 150 mm, a particle size of 8 μm, and a pore size of 1000 Å.

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Reagents

Formic acid and trifluoroacetic acid (TFA) with purity greater than 99% or redistilled. It is recommended to use mass spectrometry grade water and acetonitrile. Mobile phase A: 0.1% formic acid and 0.025% TFA in water. Mobile phase B: 0.1% formic acid and 0.025% TFA in acetonitrile. PNGase F: 50000 U/mL (without glycerin). carboxypeptidase B(CpB): 5 mg/mL. Digestion buffer: 1 mol/L 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid (HEPES) or 1 mol/L Tris buffer (pH 8). Sample ADC: Dilute with water to 1 mg/mL.

9.3.2

Method

9.3.2.1

Sample Preparation

Before LC-MS analysis, the ADC needs to be deglycosylated and the heterogeneity of the C-terminal lysine in the antibody heavy chain is removed. Among them, N-terminal deglycosylation is achieved by treatment of ADC with PNGase F; and the removal of C-terminal lysine heterogeneity in the antibody heavy chain can be achieved by treatment of the antibody with CpB. N-terminal deglycosylation: Take 100 μg of ADC and add 2 μL of digestion buffer; add 1 μL of PNGase F and incubate at 45 °C for 1 h; then add TFA to stop the deglycosylation reaction (the final concentration of TFA is 0.2%). If the sample is to be treated with CpB, the reaction is not terminated. CpB treatment of ADC: Take 100 μg of ADC and add 2 μL of digestion buffer; add 1.3 μL of CpB and incubate at 37 °C for 20 min; then add TFA to the mixture (TFA final concentration is 0.2%) to stop the reaction.

9.3.2.2

LC-MS Analysis

The PLRP-S column is pre-equilibrated at 75 °C with a flow rate of 250 μL/min. Make sure that the column is in good condition and verify that a consistent backpressure graph or a stable baseline is achieved with at least two blank runs. In order to perform a complete antibody analysis, the Q-TOF parameters of the MS source must be optimized. The declustering potential (DP) of the mass spectrometer QSTAR is set to 120–140 V, the spray voltage is 4500–5000 V, the source temperature is 350 °C, and the curtain and nebulizer gas parameters are set to 40 each. The mass spectrum should include the high mass range m/z (1000–4000). The expected spectrum envelope of a typical antibody is 2000–3500 m/z (+45 to +60 charge states).

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Table 9.1 Typical liquid phase elution gradient of PLRP-S column

Time(min) 0 10 30 35 36 45

Mobile phase A(%) 10 10 90 90 10 10

109 Mobile phase B(%) 90 90 10 10 90 90

In order to improve accuracy, the mass spectrometer should be calibrated in advance, and the mass spectrometer should be kept in scan mode until the sample analysis is completed. The Q-TOF mass accuracy of intact antibodies is usually less than one part per million. Inject 15–20 μg of ADC solution into the PLRP-S column. A typical liquid phase elution gradient is shown in Table 9.1. In the total ion chromatogram (TIC) curve analyzed by LC-MS, the charge states from +44 to +62 can often be observed, and in most cases, these charge states are included in the deconvolution. For the decompressed (zero charge state) mass spectrum of a specific ADC, the ADC protein sequence and conjugation information can be used to allocate an appropriate amount of drug load (n) to each mass peak in the decompressed mass spectrum [17].

9.3.2.3

Data Processing

In order to calculate the weighted average DAR, the drug load distribution percentage of each antibody is multiplied by the corresponding drug load (n), and the product obtained represents the weighted contribution of each antibody with drug load (n) to the ADC characteristics. To divide the sum of all these products by 100, and the result is the weighted average DAR of the ADC. The result can be correlated and verified with the DAR obtained by the UV/vis method.

9.3.3

Case Analysis [18]

The ADC was prepared through the combination of SrtA catalysis and click chemistry, (For the preparation method of OFA-GPN-vcMMAE, see Sect. 8.3.2). The obtained ADC was performed PR-HPLC and HIC analysis firstly (the conditions are the same as in this Sects. 9.2.1.1 and 9.2.2.1). In addition, it is further analyzed by the following LC-MS experiment. Combine an ultra-high performance liquid chromatograph (Waters ACQUITY UPLC) with a quadrupole-time-of-flight mass spectrometer (Waters XevoG2-S Q-TOF) to determine the relative molecular mass and peptide map coverage, and to determine the conjugation site.

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Relative Molecular Mass of Light and Heavy Chains

Sample processing: Dilute the sample to 2 mg/mL, add 12 mmol/L DTT for reduction (37 °C, 40 min), and then measure the relative molecular mass by LC/MS. For liquid phase separation, a Waters UPLC MassPREPTM desalting column was used, and the column temperature was 80 °C. Mobile phase: Phase A is 0.1% formic acid/water solution, Phase B is 0.1% formic acid/acetonitrile solution. The elution gradient conditions of the liquid phase are shown in Table 9.2. The mass spectrometer parameters are shown in Table 9.3.

9.3.3.2

Peptide Map Detection

Sample preparation: Dilute the sample with guanidine hydrochloride-Tris solution (pH 8.0) to 2 mg/mL, add 12 mmol/L DTT and react for 2.5 h, then add iodoacetamide (final concentration 0.06 mol/L) and avoid light at room temperature react for 40 min to block free cysteine residues, centrifuge at 12000 r/min for 2 h at 4 °C, replace the buffer with 0.01 mol/L Na3PO4 solution (pH 7.4), add trypsin at 37 °C for 24 h, the digestion reaction was terminated with formic acid (final concentration is 1%), and it is to be tested. For liquid phase separation, a Waters UPLC BEH300 C18 column (1.7 Å, 2.1 mm × 150 mm) was used, and the column temperature was 80 °C. The flow rate is 0.2 mL/min. Mobile phase: Phase A is 0.1% formic acid/water solution, Phase B is 0.1% formic acid/acetonitrile solution. The gradient conditions of liquid phase elution are shown in Table 9.4. The mass spectrum parameters are shown in Table 9.5.

Table 9.2 Liquid phase elution gradient

Time(min) 0 1 13 14.5 16 20

Mobile phase A(%) 0 0 10 10 90 90

Mobile phase B(%) 10 10 90 90 10 10

Parameter Ion source temperature Atomization temperature Atomization flow rate Internal standard

Value 120 °C 500 °C 800 L/h Leucine Enkephalin

Table 9.3 mass spectrometer parameter settings Parameter Capillary voltage Cone voltage Quality analysis range Collision energy

Value 2.5 kV 60 V 2000 ~ 8000 Da –

Data analysis: Use Biopharmalynx 1.3.3 (Waters) software to analyze the data obtained by LC/MS

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Table 9.4 Liquid phase elution gradient

Time(min) 0 5 45 50 55 60 65

111

Mobile phase A(%) 95 95 50 5 5 95 95

Mobile phase B(%) 5 5 50 95 95 5 5

Table 9.5 Mass spectrometry parameter settings Parameter Capillary voltage Cone voltage Quality analysis range Collision energy

Value 2.0 kV 150 V 50 ~ 8000 Da 20 ~ 45 eV

Parameter Ion source temperature Atomization temperature Atomization flow rate Internal standard

Value 90 °C 400 °C 700 L/h Leucine Enkephalin

Data analysis: Use Biopharmalynx 1.3.3 (Waters) software to analyze the data obtained by LC/MS. mAU

L0

H0

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OFA-GPN-vcMMAE 0 0

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Fig. 9.4 RP-HPLC analysis of OFA-GPN-vcMMAE light and heavy chain conjugation. Note: L0 is an unconjugated light chain; L1 is a light chain conjuagated to an MMAE; H0 is an unconjugate heavy chain; H1 is a heavy chain conjuagated to an MMAE

9.3.3.3

Results

The prepared ADC was analyzed by RP-HPLC and HIC. According to the above experimental conditions, the following results can be obtained (Figs. 9.4 and 9.5). It can be seen from the results of RP-HPLC that due to the change of the antibody form, the hydrophilicity of the antibody has a certain change, which may affect the retention time, making the light and heavy chain peak times similar, resulting in a merged peak (L1 + H1) appears; at the same time, DTT may not be able to be fully restored. Therefore, it is difficult to estimate the corresponding DAR based on the RP-HPLC results. However, combined with the analysis results of HIC, according to

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mAU 50 E0

40 30 OFA-2G4S-His

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E3 E4 E2 E0

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Fig. 9.5 HIC analysis of OFA-GPN-vcMMAE conjugation. Note: E0, E1, E2, E3, and E4 mean DAR is 0, 1, 2, 3, and 4, respectively

Fig. 9.6 Determination of the relative molecular mass of antibodies and their conjugates. Note: A. OFA-2G4S-His; B. OFA-GPN-vcMMAE

the peak area of each component of OFA-GPN-vcMMAE, the DAR can be calculated to be about 3.3. The relative molecular mass was determined by LC-MS method. Figure 9.6 shows that the relative molecular mass of the light chain increased by 1048.6875 Da, and the relative molecular mass of the heavy chain increased by 1049.461 Da, both of which are consistent with the calculated theoretical molecular mass, indicating that the light chain and heavy chain of antibody are connected to the MMAE through a two-step process. From Figs. 9.7 and 9.8 , it can be seen that the coverage rates of the light and heavy chain peptides of OFA-GPN-vcMMAE are both 100%, indicating that they are consistent with the theoretical amino acid sequence. To determine whether MMAE was conjugated to the light and heavy chain LPETG fragments of OFA antibody as designed, we analyzed the conjugation sites by mass spectrometry (Fig. 9.9). By arranging the results of the secondary mass spectrometry of the peptide fragment, it was confirmed that the fragment was

9

Determination of Drug-to-Antibody Ratio of ADCs

Fig. 9.7 OFA-GPN-vcMMAE light chain peptide coverage

Fig. 9.8 OFA-GPN-vcMMAE heavy chain peptide coverage

Fig. 9.9 Identification of OFA-GPN-vcMMAE conjugation site

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LPET-GPN-vcMMAE, which further showed that the MMAE was conjugated to the target position. Enzymatic-chemical method is used to catalyze and connection of bifunctional small molecule GPN with SrtA, and then click chemistry to connect the antibody with azide group to the cyclooctyne-modified MMAE to obtain OFA-GPNvcMMAE with a DAR of 3.3. From this example, it can be seen that for ADCs with complex structures, RP-HPLC and HIC analysis may not be able to obtain a precise DAR value. Through the LC-MS experiment, we can determine the relative molecular mass and the coverage of the peptide map, and determine the conjugation site to further prove the structure of the ADC.

9.4

Conclusion

DAR is an important quality attribute of ADCs, because it characterizes the number of toxic small molecules carried by each antibody, which has a great impact on the safety and effectiveness of ADCs. There are many methods for the determination of DAR, and this chapter mainly introduces the methods of RP-HPLC, HIC and MS. Due to the different nature of different ADCs, sometimes it is necessary to select the method of determining DAR according to the specific situation, or to further determine the DAR through several different methods.

References 1. Sorokin P. Mylotarg approved for patients with CD33+ acute myeloid leukemia. Clin J Oncol Nurs. 2000;4(6):279–80. 2. Acchione M, Kwon H, Jochheim CM, et al. Impact of linker and conjugation chemistry on antigen binding, fc receptor binding and thermal stability of model antibody-drug conjugates. MAbs. 2012;4(3):362–72. 3. Wakankar A, Chen Y, Gokarn Y, et al. Analytical methods for physicochemical characterization of antibody drug conjugates. MAbs. 2011;3(2):161–72. 4. Carol H, Szymanska B, Evans K, et al. The anti-CD19 antibody-drug conjugate SAR3419 prevents hematolymphoid relapse post induction therapy in preclinical models of pediatric acute lymphoblastic leukemia. Clin Cancer Res. 2013;19:1795–805. 5. Ribrag V, Dupuis J, Tilly H, et al. A dose-escalation study of SAR3419, an anti-CD19 antibody maytansinoid conjugate, administered by intravenous infusion once weekly in patients with relapsed/refractory B-cell non-Hodgkin lymphoma. Clin Cancer Res. 2014;20(1):213–20. 6. Shen BQ, Xu K, Liu L, et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol. 2012;30(2):184–9. 7. Baldwin AD, Kiick KL. Tunable degradation of maleimide-thiol adducts in reducing environments. Bioconjug Chem. 2011;22(10):1946–53. 8. McDonagh CF, Kim KM, Turcott E, et al. Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index. Mol Cancer Ther. 2008;7(9):2913–23. 9. Junutula JR, Bhakta S, Raab H, et al. Rapid identification of reactive cysteine residues for sitespecific labeling of antibody-Fabs. J Immunol Methods. 2008;332(1–2):41–52.

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10. Dornan D, Bennett F, Chen Y, et al. Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma. Blood. 2009;114(13):2721–9. 11. Kudirka R, Barfield RM, McFarland J, et al. Generating site-specifically modified proteins via a versatile and stable nucleophilic carbon ligation. Chem Biol. 2015;22(2):293–8. 12. Xie H, Audette C, Hoffee M, et al. Pharmacokinetics and biodistribution of the antitumor immunoconjugate, cantuzumab mertansine (huC242-DM1), and its two components in mice. J Pharmacol Exp Ther. 2004;308(3):1073–82. 13. Hamblett KJ, Senter PD, Chace DF, et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res. 2004;10(20):7063–70. 14. Kaur S, Xu K, Saad OM, et al. Bioanalytical assay strategies for the development of antibodydrug conjugate biotherapeutics. Bioanalysis. 2013;5(2):201–26. 15. Hinman LM, Hamann PR, Wallace R, et al. Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics. Cancer Res. 1993;53(14):3336–42. 16. Siegel MM, Hollander IJ, Hamann PR, et al. Matrix-assisted UV-laser desorption/ ionization mass spectrometric analysis of monoclonal antibodies for the determination of carbohydrate, conjugated chelator, and conjugated drug content. Anal Chem. 1991;63(21):2470–81. 17. Lazar AC, Wang L, Blättler WA, et al. Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry. Rapid Commun Mass Spectrom. 2005;19(13):1806–14. 18. Yin X. The design of antibody-drug conjugates and studies on their specific antitumor activities. Hangzhou: Zhejiang University; 2018. 19. Valliere-Douglass JF, McFee WA, Salas-Solano O. Native intact mass determination of antibodies conjugated with monomethyl auristatin E and F at interchain cysteine residues. Anal Chem. 2012;84(6):2843–9. 20. Jiang XR, Song A, Bergelson S, et al. Advances in the assessment and control of the effector functions of therapeutic antibodies. Nat Rev Drug Discov. 2011;10(2):101–11. 21. Harris RJ. Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture. J Chromatogr A. 1995;705(1):129–34.

Chapter 10

Pharmacokinetic Study of Antibody-Drug Conjugates Wenbin Zhao and Shuqing Chen

10.1

Introduction

Antibody-drug conjugates are a class of specific drug delivery therapeutics that comprise a monoclonal antibody (mAb) which is connected to small cytotoxin agents via a covalent linker. The ADC pharmacokinetics is determined not only by antibody part, but also by the types of small molecule agents and linkers. Antibody is natural biomacromolecule with large molecular weight that has a longest half-life with IgG isoform in human blood system that is up to 21 days. Fc region of IgG can be specifically bound to Fc neonatal receptor (FcRn) which belongs to MHC I family. The antibody and FcRn complex is taken up by endothelial cells or monocytes through endocytosis. Once entered into the cells, the complex is encapsulated into acidic endosomes, in which IgG binds to FcRn tightly and escape from lysosomes and then circulates to cell surface. The complex is exposed to pH 7.4 extracellular environment that leads to IgG releasing from cell surface and back to circulation. FcRnmediated recycling prolongs half-life of IgG [1]. Besides, when ADC enters human body, there is the chance that ADC develops humoral immunity and anti-antibody defense appears, especially for antibody derived from mouse or human-mouse chimeric antibody, which lead to inactivity or accelerated clearance of ADC [2, 3]. To some extent, the metabolism of ADC molecules is affected by small cytotoxin agents that conjugated to antibodies. Stephen et al. [4] conjugated DM1 with

W. Zhao Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China Department of Precision Medicine on Tumor Therapeutics, ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou, China S. Chen (*) College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chen, J. Zhan (eds.), Antibody-Drug Conjugates and Cellular Metabolic Dynamics, https://doi.org/10.1007/978-981-19-5638-6_10

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Fig. 10.1 Schematic representation and prevalence of the different subpopulations as percentage of the total anti-CD22-MCC-DM1 (a and b, respectively) and anti-CD22-MC-MMAF (c and d, respectively) ADCs [4]

α-CD22 antibody via MCC linker and obtained α-CD22-MCC-DM1, and conjugated MMAF with α-CD22 antibody via MC linker to obtain α-CD22-MC-MMAF. The DAR of two ADCs is 3.4 (Fig. 10.1). The pharmacokinetic study of the two ADCs was conducted in non-tumor-bearing CB-17 SCID mice, which were dosed with anti-CD22 naked antibody at 5 mg/kg, anti-CD22-MCC-DM1 or anti-CD22MC-MMAF at 0.5 and 5 mg/kg as a single intravenous dose. The pharmacokinetic parameters calculated from the different total antibody profiles were equivalent. However, at the same dosing point, the half-life of anti-CD22-MC-MMAF is significantly shorter than that of anti-CD22-MCC-DM1 (Fig. 10.2). The result of this article demonstrated that different small cytotoxin agents and conjugating formats affect pharmacokinetic profiles of ADC. Currently used ADCs, in clinical or in preclinical research, are produced with randomly coupling small cytotoxin to lysine or cysteine and the final product has some certain level of naked antibodies [5]. The FDA approved ADC Adcetris®

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ADC drug: DM1

ADC drug: MMAF

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Fig. 10.2 Mouse plasma pharmacokinetic analysis. Mice were injected with a single IV dose of anti-CD22-MCC-DM1 (a, b) or anti-CD22- MC-MMAF (c, d) at 0.5 (a, c) and 5 mg/kg (b, d) and plasma samples collected over time up to 44 days. Sample analysis was conducted using two different total assay formats: CD22/GxhFc-HRP or RxhFc/GxhFc-HRP and two different conjugated antibody assay formats: CD22/anti-drug-Biotin or anti-drug/CD22-Biotin. For each time point the average concentrations in ng/mL (STDEV for four animals is shown. Total antibody concentrations measured using CD22/GxhFc-HRP format (A1) centrations in ng/mL (STDEV for four animals is shown). Total antibody concentrations measured using CD22/GxhFc-HRP format (A1) were compared to concentrations measured using RxhFc/GxhFc-HRP format (B1). Conjugated antibody concentrations measured using CD22/anti-drug-Biotin format (C1 & C2) were compared to concentrations measured using anti-drug/CD22-Biotin format (D1 & D2) [4]

(rituximab vedotin), which conjugated α-CD30 (named cAC10) to MMAE via SMCC linker, was a mixture that contains different ADC components with DAR 0 ~ 8 [6]. Hamblett et al. [6] produced cAC10-MMAE and separated different components in mixture by HIC-HPLC. It contains naked antibody (DAR = 0), ADC E2 (DAR = 2), ADC E4 (DAR = 4) and ADC E8 (DAR = 8) (Fig. 10.3). when ADCs were respectively applied on SCID mice with the same concentration (10 mg/kg), the pharmacokinetic analysis revealed a shorter half-life for ADC with

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Fig. 10.3 Hydrophobic interaction chromatograph-high performance liquid chromatography chromatograms of partially loaded E4-mixture [6]

Fig. 10.4 Pharmacokinetics of cCA10 and cAC10-antibody-drug conjugates. SCID mice were injected via the vein with 10 mg/kg cAC10, E2, E4, and E8 [6]

larger DAR and with E8 had a significantly shorter half-life (Fig. 10.4). These results demonstrated that drug loading inversely related to clearance of ADCs. The pharmacokinetic property of ADC is also determined by stability of linker. Theoretically, ADC is taken up inside cells and releases drug to exert cytotoxic

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Fig. 10.5 The maleimide exchange from the antibody conjugate and hydrolysis of succinimide ring in the linker are key steps that influence conjugate stability and therapeutic activity [7]

function, however, the linker is liable in blood stream and might cause early release. Shen et al. [7] used anti-HER2/neu antibody trastuzumab for example to investigate the influence of different conjugation sites on ADC’s stability. Researchers introduced cysteine mutation at light chain V205 (LC-V205C), heavy chain A114 (HC-A114C) and Fc396 (Fc-S396C), which was linked with MMAE via MC linker to generate ADC with similar DAR, affinity and internalization rate. Fc-S396C is a highly solvent accessible site that is rapid lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In the contrast, LC-V205C and HC-A114C were predicted to be in partially buried regions that preventing the exchange reaction. Besides, LC-V205 conjugation site appears to be located in a positively charged environment which promoted hydrolysis of the succinimide ring in the linker, thereby promoted antitumor activity (Fig. 10.5). Dorywalska et al. [8] found that a commonly used linker Valine-citrulline (VC) was instable in mouse blood circulation, although human and cynomolgus monkey species appeared to have negligible levels of linker-payload degradation in plasma. They identified Carboxylesterase 1C (Ces 1C) as the enzyme responsible for the extracellular hydrolysis of valine-citrulline-p-aminocarbamate (VC-PABC)-based linkers in mouse plasma. The enzyme could hydrolyze VC linker which was the major factor that caused premature payload releasing in mouse plasma. What’s more, Dorywalska et al. [8] and Anami et al. [9] further modified VC linker that are resistant to Ces 1C cleavage (Table 10.1) (Fig. 10.6).

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Table 10.1 In vitro stability of linker-payload variants in plasma from different species [8] Site A A A A A A A A A F F F F F F F F F

Position LC 200–202 LC 200–202 LC 200–202 LC 200–202 LC 200–202 LC 200–202 LC 200–202 LC 200–202 LC 200–202 LC C-terminus LC C-terminus LC C-terminus LC C-terminus LC C-terminus LC C-terminus LC C-terminus LC C-terminus LC C-terminus

Linker-payload Linker 1-VC-PABCAur0101 Linker 2-VC-PABCAur0101 Linker 4-VC-PABCAur0101 Linker 5-VC-PABCAur0101 (C6) Linker 6-VC-PABCAur0101 Linker 7-VC-PABCAur0101 Linker 8-VC-PABCAur0101 Linker 9-VC-PABCAur0101 Linker 10-VCPABC-Aur0101 Linker 1-VC-PABCAur0101 Linker 2-VC-PABCAur0101 Linker 3-VC-PABCAur0101 Linker 4-VC-PABCAur0101 Linker 5-VC-PABCAur0101 (C6) Linker 6-VC-PABCAur0101 Linker 7-VC-PABCAur0101 Linker 8-VC-PABCAur0101 Linker 9-VC-PABCAur0101

Mouse plasma stability (%) 0

Rat plasma stability (%) 48

Cyno plasma stability (%) 95

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Due to the structure complexity and heterogeneity of antibody-drug conjugates, it is challenging to obtain accurate data for their pharmacokinetic analysis. LC-MS technique which is widely used in PK analysis can detect payload, free cytotoxin and its metabolites, which has a high sensitivity and meanwhile high requirement of detection instruments [10]. The free cytotoxin in blood sample can be separated by reverse phase HPLC followed by quantification process through mass spectrometry analysis in presence of unconjugated small molecules added as internal standards

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Fig. 10.6 Modification of VC-PABC cleavable linker and effect on its stability [8]

Antigen immobilized magnetic beads

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Fig. 10.7 Bioanalytical workflow for quantifying plasma albumin payload adducts by ADC depletion followed by anti-albumin magnetic bead enrichment [12]

[11]. A partial of cytotoxin can be captured by proteins in blood, for example, MC-VC-MMAE released from ADC can be absorbed by albumin in blood, which prevents detection in LC-MS and leads to inaccurate analysis. To resolve the problem, Dong et al. [12] and Shi et al. [13] described a novel immunocapture LC/MS/MS assay to allow quantification of migrated payloads forming adducts with albumin in the plasma samples. Antigen-coupled beads are firstly used to extract ADC and antibody-associated species, while anti-albumin beads to recover the albumin-associated adducts. All protein samples and beads are digested to obtain free payload (Fig. 10.7), which is respectively applied to LC-MS for quantification analysis of payload in each component of blood sample (Table 10.2). Bioanalysis of

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Table 10.2 Summary of measured payload concentrations in each fraction of mouse and human plasma stability samples [12]

ID T1 A B C D E T2 T3

Mouse plasma (n = 2) payload conc. ± SD (ng/mL) T0 Day 6 fraction Initial total 97.1a – Free + deconjugated 1.30 ± 0.04 63 ± 1 Conjugated (first immunocapture) 103 ± 1 16.5 ± 0.5 Conjugated (second immunocapture) 2.2 ± 0.9 0.40 ± 0.04 Albumin adducts 1.1 ± 0.0 13.6 ± 0.1 0.4 ± 0.3 Unknown protein adducts BQLc Remaining conjugated payload 105d 16.9d Total recovered payload 108e 93.7e

Human plasma (n = 2) payload conc. ± SD (ng/mL) T0 Day 6 97.1a – 0.19 ± 0.01 BQLb 105 ± 5 71.8 ± 0.2 2.5 ± 0.7 2.1 ± 0.3 2.1 ± 0.1 16.9 ± 0.1 BQLc 1.4 ± 0.2 108d 74.0d 110e 92.5e

Initial total payload concentration was converted from spiked ADC concentration (10 μg/mL) Below quantitation limit (0.05 ng/mL) c Below quantitation limit (0.2 ng/mL) d Sum of conjugated payload (depleted ADC) fractions = B + C e Sum of all recovered payload fractions = A + B + C + D + E

a

b

antibody–drug conjugates (ADCs) via LC-MS is challenging due to the complex, heterogeneous nature of their structures and their complicated catabolism. Currently LC-MS analysis is mostly used to quantify antibody, signature peptide or small molecule payload [11]. The major limitation in peptide level quantitation is that information about the whole protein molecule is lost upon digestion and any given peptide may not truly represent the whole protein. To overcome the challenges in quantification of protein sample at intact level, researchers utilized a novel strategy called high resolution mass spectrometry (HRMS), which was low sensitivity but there had been growing interests in this method as an important supplementary to traditional LC-MS methods [14, 15]. In purpose of improving the sensitivity of large molecule analysis of HRMS, researchers now use enzymatic digestion of intact antibody or ADC to become Fab, Fc or Lc part with subsequent deglycosylation and remain the structure of protein [16, 17]. ELISA-based methods are often employed for monitoring the total antibody, conjugated antibody and antidrug antibody analytes, which moderately demands instruments and experimental materials [10]. The basic ELISA methods for bioanalysis contains ELISA-sandwich technique and ELISA-based LBA technique (Fig. 10.8a, b) [18]. Stephen et al. [4] collected ADC in mouse plasma by coating soluble CD22 antigen, anti-payload antibody or rabbit anti-human Fc antibody. The captured ADCs were analyzed for their metabolites in vivo by ELISA-sandwich technique. With different coating protein, the detection results were significantly deviated. When coating rabbit anti-human Fc antibody and detected by goat antihuman antibody, the detected half-life appeared to be 2–3 times longer that coating with anti-CD22 antigen and detecting with anti-payload antibody (Table 10.3).

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Fig. 10.8 Illustrations of different assay formats. (a) conventional ELISA, (b) Generic ELISA format, (c) Specific semihomogeneous assay format, (d) Generic semihomogeneous assay format [18]

Choosing proper ELISA method for bioanalysis of ADC is important since the method affect test results significantly. Besides, payload, conjugational position and DAR of ADC can also cause different testing results of ELISA. To decrease the influence of DAR, Kozak et al. [18] developed new ELISA assay named semihomogeneous assay (SHA) with pre-incubated samples, antibody for detection and antibody for capturing, which could be detected afterwards (Fig. 10.8c, d). The structure of ADC during pharmacodynamic process in our body could be altered which will affect the precise of detection [18–20]. Therefore, choosing proper standards to make reference curves is crucial for quantitative measurement of ADC in blood samples. Ligand binding assay is based on soluble antigen while target for ADC is always present on cell surface, which is more difficult in production and therefore limits its application. To break the limitation of traditional LBA, researchers present an in situ detection method based on flow cytometry to detect antibody or ADC. The following section demonstrated detail of the application of ELISA-based and flow cytometry-based method in measuring ADC pharmacokinetics.

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Table 10.3 Different total and conjugated antibody assay formats [4]

Antibody Total antibody

Capture method CD22/ GxhuFcHRP

ADC Anti-CD22MCC-M1 Anti-CD22MC-MMAF

RxhuFc/ Anti-CD22GxhuFc- MCC-M1 HRP Anti-CD22MC-MMF Conjugated antibody

CD22/ antidrug ab

Anti-CD22MCC-M1 Anti-CD22MC-MMF

dose (mg/kg) 0.5 5 0.5 5 0.5 5 0.5 5 0.5 5 0.5 5 0.5 5 0.5 5

AntiAnti-CD22drug MCC-D1 ab/ Anti-CD22CD22MC-MMF biotin CL clearance, Vd apparent volume of distribution

10.2 10.2.1

PK parameter CL t1/2 (mL/(kg
d)) (d) 7.5 11 5.9 9.1 7.5 11 7.4 12 6.2 14 5.3 14 5.6 15 6.1 15 17 6.6 14 6.2 23 4.8 23 5 8.3 9.3 7.2 8.8 15 5.9 16 6.2

Vd (mL/kg) 50 44 50 50 56 47 46 51 60 55 49 52 49 47 48 48

AUC ((d
μg)/ mL) 67 850 67 680 81 950 89 820 30 370 22 210 61 700 33 310

The Application of ELISA-Based Methods in Studying Pharmacokinetic of ADC ELISA-Sandwich Technique

The method uses an anti-idiotype monoclonal antibody to specifically capture the total ADC. Procedures in Stephan et al. [4] is used as example to show how ELISAsandwich technique works.

10.2.1.1

Total Antibody Detection

1. Microtiter plates (96 wells ELI/RIA plate) were coated with rabbit anti-human Fc antibody in F(ab’)2 format at 1 μg/mL in coat buffer (0.05 M carbonate/bicarbonate buffer pH 9.6) for 2 h at 37 °C or overnight at 4 °C. 2. Plates were washed and treated with block buffer (PBS/0.05% Tween 20/0.05% Proclin 300 pH 7.4) for 1–2 h at 37 °C. 3. Standards and samples are diluted in sample diluent (PBS/0.5% BSA/10 ppm Proclin/0.05% Tween 20/0.2%BGG/0.25% CHAPS/5 mmol/L EDTA/0.35 mol/L NaCl, pH 7.4). Assay plates are incubated with 100 μl diluted samples and standards for 1 h at 37 °C.

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4. Assay plates are first washed six times with wash buffer (PBS/0.05% Tween 20) and then incubated for 1 h at 37 °C with the F(ab′)2 fragment goat anti-human IgG, Fcγ fragment specific HRP conjugated diluted to 20 ng/mL. 5. After washing six times with PBST, detection step is done using tetramethylbenzidine (TMB) substrate. 6. Absorbance is measured at 450 nm against a reference wavelength of 620 nm. 7. With the concentration of standard substance as X-axis and OD450/OD620 value as Y-axis, regression analysis was conducted to determine the regression equation. The concentration of antibody or ADC in the sample was calculated according to regression equation, OD450/OD620 and dilution ratio of the sample. 10.2.1.2

Total ADC Detection

Steps (1)–(3) are the same to total antibody detection. 4. Washing plates six times. Biotinylated payload standards and samples are diluted in sample diluent. Incubating diluted anti-payload antibody to each wells for 1 h at 37 °C. 5. Assay plates are first washed six times with wash buffer (PBS/0.05% Tween 20) and then incubated streptavidin-HRP labeled antibody for 1 h at 37 °C. 6. After washing six times with wash buffer, detection step is done using TMB substrate. 7. Absorbance is measured at 450 nm against a reference wavelength of 620 nm. 8. With the concentration of standard substance as X-axis and OD450/OD620 value as Y-axis, regression analysis was conducted to determine the regression equation. The concentration of antibody or ADC in the sample was calculated according to regression equation, OD450/OD620 and dilution ratio of the sample. 10.2.1.3

Active ADC Detection

1. Plates are coated with anti-payload antibody (1 μg/ml) with coating buffer for 2 h at 37 °C or overnight at 4 °C. 2. Pates are washed three times with PBST after which block buffer is added and incubated for 2 h at 37 °C. 3. Standards and samples are diluted in sample diluent. Plates are incubated with 100 μL diluted samples and standards for 1 h at 37 °C. 4. Washing plates six times and then incubated for 1 h with soluble ADC target antigen at 37 °C. Steps (5)–(8) are the same with that in total ADC detection procedures.

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Ligand Binding Assay Based on ELISA

The ELISA-LBA is used in capturing antibody or ADC with soluble antigen, which could be used in detection of antibody and ADC that in active form, while not for inactive ADC or antibody. The following procedures are referred to Stephan et al. [4].

10.2.2.1

Detection of Active Antibody

1. Microtiter plates (96 wells ELI/RIA plate) were coated with extracellular region of antigen protein at 1 μg/mL in coat buffer (0.05 M carbonate/bicarbonate buffer pH 9.6) for 2 h at 37 °C or overnight at 4 °C. 2. Plates were washed and treated with block buffer (PBS/0.05% Tween 20/0.05% Proclin 300 pH 7.4) for 1–2 h at 37 °C. 3. Samples are diluted in sample diluent. Assay plates are incubated with 100 μL diluted samples and standards for 1 h at 37 °C. 4. Assay plates are first washed six times with wash buffer (PBS/0.05% Tween 20) and then incubated for 1 h at 37 °C with the F(ab′)2 fragment goat anti-human IgG, Fc fragment specific HRP conjugated diluted to 20 ng/mL. 5. After washing six times wash buffer, detection step is done using TMB substrate. 6. Absorbance is measured at 450 nm against a reference wavelength of 620 nm. 7. With the concentration of standard substance as X-axis and OD450/OD620 value as Y-axis, regression analysis was conducted to determine the regression equation. The concentration of antibody or ADC in the sample was calculated according to regression equation, OD450/OD620 and dilution ratio of the sample. 10.2.2.2

Detection of Active ADC

Steps (1)–(3) are the same with that in 10.2.2.1. 4. Assay plates are first washed six times with wash buffer (PBS/0.05% Tween 20) and then incubated for 1 h at 37 °C with the biotinylated anti-payload antibody. 5. Assay plates are first washed six times with wash buffer and incubated with HRP-labeled anti-streptavidin antibody. 6. After washing six times wash buffer, detection step is done using TMB substrate. 7. After enzymatic reaction, it is stopped by the addition of 2 mol/L H2SO4. Absorbance was measured at 450 nm against a reference wavelength of 620 nm using a microplate reader. 8. With the concentration of standard substance as X-axis and OD450/OD620 value as Y-axis, regression analysis was conducted to determine the regression equation. The concentration of antibody or ADC in the sample was calculated according to regression equation, OD450/OD620 and dilution ratio of the sample.

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Semihomogeneous Assay

The semihomogeneous assays (SHAs) are developed to accurately measure ADC total antibody concentrations with different DARs, which can ensure 80%–120% recovery. SHAs improve DAR fraction recovery compared to specific ELISA. 1. Sheep anti-human IgG antibody, target ECDs and anti-idiotypic antibody (S) were biotinylated. 2. Plates are coated with streptavidin or anti-biotin antibody for 2 h at 37 °C or overnight at 4 °C. 3. After coating, plates are blocked with 200 ul SuperBlock for 1 h at 37 °C.

10.2.3.1

Specific SHA

4. Approximately equal molar concentrations of HRP-conjugated antihuman IgG antibody and biotin-labeled ECD were mixed and diluted in sample buffer and premixed with shaking at room temperature for 2 h. 5. Aliquots from the above mixture were transferred to plates coated overnight with 2 μg/mL avidin. The complex is captured onto the plates during a 30 min to 2 h incubation at room temperature with shaking. 6. Anti-human IgG-HRP were used to bridge the ADCs. 7. After enzymatic reaction, it is stopped by the addition of 2 M H2SO4. Absorbance was measured at 450 nm against a reference wavelength of 620 nm using a microplate reader. 8. With the concentration of standard substance as X-axis and OD450/OD620 value as Y-axis, regression analysis was conducted to determine the regression equation. The concentration of antibody or ADC in the sample was calculated according to regression equation, OD450/OD620 and dilution ratio of the sample. 10.2.3.2

Non-specific SHA

4. Approximately equal molar concentrations of HRP-conjugated antihuman IgG antibody and biotinylated goat anti-human Fc antibody were diluted in sample buffer and premixed with shaking at room temperature for 2 h. 5. Aliquots from the above mixture were transferred to plates coated overnight with 2 μg/mL avidin. The complex is captured onto the plates during a 30 min to 2 h incubation at room temperature with shaking. 6. Anti-human IgG-HRP were used to bridge the ADCs. 7. After enzymatic reaction, it is stopped by the addition of 2 mol/L H2SO4. Absorbance was measured at 450 nm against a reference wavelength of 620 nm using a microplate reader.

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8. With the concentration of standard substance as X-axis and OD450/OD620 value as Y-axis, regression analysis was conducted to determine the regression equation. The concentration of antibody or ADC in the sample was calculated according to regression equation, OD450/OD620 and dilution ratio of the sample.

10.3

The Application of Flow Cytometry in Studying Pharmacokinetics of Antibody-Drug Conjugates

Soluble antigen is required in conventional LBA, the expression of which makes hard to realize because of the complex protein structure of the target antigen. Compared to whole length antigen, extracellular of antigen can be expressed easier and some antigen is successfully expressed in extracellular part [21]. But most membrane protein (for example, CD20) is still hard to obtain which limits the application of LBA. In the process of developing ADC, the target-positive tumor cell line is always needed. The cell lines express certain amount of antigen on their surface, which represents a natural library of antigen. Utilizing cell surface antigens to replace soluble antigens to establish a LBA method based on flow cytometry and then establishing standard curve could effectively quantify antibody or ADC in samples. The LBA method based on flow cytometry is established by Chen’s Lab [22] (College of pharmaceutical sciences, Zhejiang university) with OFA-HL (antiCD20 antibody) and OFA-MMAE [22, 23] (ADC that is based on OFA-HL) as an example (Fig. 10.9). although this method require higher level of instrument and higher detection limitations compared to traditional LBA method, the new method is certainly affected by factors like fluorescence intensity and antibody affinity. By optimization these factors, the method could meet detection requirement for many sample with lower detection limit. Besides, this method solves the problem of soluble antigen expression and extends the scope of application of the conventional LBA methods. The utilization of antigen on cell surface also ensures the natural structure of antigen which lead to more precise results of quantity of antibody or ADC in samples.

10.3.1

Making Biotinylated Anti-MMAE Antibody

Biotin-LC-hydrazides is used in this chapter to biotinylate anti-MMAE antibody. The antibody could be obtained by hybridoma, phage display, which is not the discussion topic in this book [24, 25]. The procedure is described as following: 1. The anti-MMAE antibody was dissolved in 0.1 mol/L (2-N-morpholino) ethanesulfonic acid (MES) (pH = 5.0) buffer at 5–10 mg/mL.

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Fig. 10.9 (a) Quantitative OFA-HLMMAE flow cytometry method: OFA-HL-MMAE is captured by Daudi cells, recognized by biotinylated anti-MMAE antibody and then detected using PE labelled streptavidin. (b) Quantitative OFA-HL flow cytometry method: OFA-HL is captured by Daudi cells, and then detected using FITC labelled anti-human Fc antibody [22]

2. 25 μL of biotin hydrazide solution (50 mmol/L in dimethyl sulfoxide) and 12.5 μL of the EDC solution (100 mg/mL in 0.1 mol/L MES) per 1 mL of antibody solution were added. 3. The sample was mixed, followed by incubation overnight at room temperature with rotation. 4. The biotinylated antibody was separated from non-reacted material by a Protein A column. 5. The solvent was replaced with phosphate-buffered saline (PBS) using a centrifugal filter. The concentrated protein sample can be stored at -80 °C.

10.3.2

Quantitative OFA-HL and OFA-MMAE with Flow Cytometry Method

The linearity and scope of standard curve are related to antigen express level on tumor cell lines and quantity of tumor cells. When the tumor cell line with more antigen expression or more cells is used, the limit of quantitation (LOQ) and upper limit of quantitation (ULOQ) are consequently improved. While there is a requirement for flow cytometry detection, the number of tumor cells is suitable for 5 × 105–1 × 106.

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Fig. 10.10 (a)The linearity of quantitative OFA-HL flow cytometry method. (b) The linearity of quantitative OFA-HL-MMAE flow cytometry method [22]

1. 8 × 105 Daudi cells were collected (500 g/5 min) in the logarithmic growth phase and washed with PBS three times. 2. Cells were incubated with 200 μL of OFA-HL (diluted in PBS to 4.00, 2.00, 1.00, 0.50, 0.25, 0.20 μg/mL) at 4 °C for 30 min, followed by washing with PBS three times; or, cells were incubated with diluted OFA-HL-MMAE, which concentration is adjusted to 3.00, 2.00, 1.00, 0.50, 0.25, 0.20 μg/mL. 3. Incubating cells with the above antibody or ADC for 30 min at 4 °C. Each sample has 3 repeats. 4. After incubation, cells are washed three times followed by incubation of FITClabeled goat anti human IgG (H + L) for 30 min to OFA-HL group. OFA-HLMMAE group is incubated for biotin-labeled anti-MMAE antibody for 30 min. 5. For OFA-HL group, cells were resuspended in 400 μL of PBS, and the mean fluorescence intensity (MFI) was determined by a Cytomic FC 500MCL. For OFA-HL-MMAE group, cells are incubated for PE-labeled streptavidin for 30 min, and then cells are washed three times followed by detecting for MFI. 6. Linear regression was performed by plotting the average MFI on the Y-axis and the concentration of OFA-HL on the X-axis (Fig. 10.10) [22].

10.3.3

The Lower Limit of Quantification Is Affected by Selection of Fluorescein

The lower LOQ is affected by fluorescein, which can change the sensitivity of detection. The experimental procedure is the same to 10.3.2. When using PE-labeled goat anti-human IgG (H + L) to replace FITC-labeled goat anti-human IgG (H + L), the lower LOQ is down to 25 ng/mL from 200 ng/mL. The sensitivity of this system is improved by eight times (Fig. 10.11).

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Fig. 10.11 The linearity of quantitative OFA-HL flow cytometry method used PE [22]

10.3.4

Quantification of OFA-HL and OFA-HL-MMAE in Blood Samples

For pharmacokinetic detection of ADC or antibody, the drug concentration at different time points of blood samples is the essential content for detection, which is suitable for blood of mouse and human. The flow cytometry-based quantification method of OFA-HL can also be used in detection of active antibody, and flow cytometry-based quantification method of OFA-HL-MMAE can be used in detection of active ADC. The method using tumor cells to capture active antibody or ADCs, which can exclude antibody or ADC that is in inactive form, can help obtain more precise results that represent the drug concentration. Besides, this method is also used in different batch of various samples like urine samples. The detection method is as follow: 1. Blood samples at different time point are diluted to proper concentration (within linear scope of the standard curves) followed by incubation of cells, the procedure of which is described in 10.3.2. The MFI is obtained. 2. Linear regression was performed by plotting the average MFI and concentration. The real concentration in samples can be calculated by multiplying dilution factor with the results of equation, and each sample calculates for three times in repeat. 3. The untreated mouse plasma is used as negative sample and this detection concentration is used as LOD. The detected concentration need to minus the value of negative sample to gain a real concentration. 4. With the calculation results of active antibody or ADC in every time point, the pharmacokinetic parameter can be calculated by software.

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Summary

Pharmacokinetic study has great effect on drugs’ performance of pharmacodynamics. Antibody-drug conjugate is comprised of antibody, payload and linker, the pharmacodynamic properties of which remains that of large molecule proteins and also affected by the character of payload. The traditional analytical methods, like mass spectrometry, high performance liquid chromatography-mass spectrometry and ligand-binding assay, are barely met the requirement of pharmacodynamic study. In recent years, researchers have made innovations upon old technique, and made progression in the sensitivity and precision of those powerful new tools for studying pharmacodymics.

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