novel monoclonal antibodies targeting conformational erbb2 epitopes

253 Pages·2012·9.34 MB·English

NOVEL MONOCLONAL ANTIBODIES TARGETING
CONFORMATIONAL ERBB2 EPITOPES

A THESIS SUBMITTED TO
THE DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS
AND THE GRADUATE SCHOOL OF ENGINEERING AND SCIENCE
OF BILKENT UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

OVER PAGE
1.1COVER PAGE

BY
CEYHAN CERAN
OCTOBER 2012

i
1.2 DEDICATION PAGE

To my loving, understanding, caring, self-sacrificing,
smart, wise, imaginative, helpful and inspiring mother, Reyhan
ii
1.3 SIGNATURE PAGE
I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope and in quality, as a thesis for the degree of Doctor of Philosophy.

____________________
Prof. Dr. Mehmet Öztürk

I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope, and in quality, as a thesis for the degree of Doctor of Philosophy.

____________________
Prof. Dr. Tamer Yağcı

I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope, and in quality, as a thesis for the degree of Doctor of Philosophy.

____________________
Assoc. Prof. Dr. Işık Yuluğ

I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope, and in quality, as a thesis for the degree of Doctor of Philosophy.

____________________
Assoc. Prof. Dr. İhsan Gürsel

I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope, and in quality, as a thesis for the degree of Doctor of Philosophy.

____________________
Assist. Prof. Dr. Elif Erson Bensan

Approved for the Graduate School of Engineering and Science

____________________
Prof. Dr. Levent Onural
Director of the Graduate School of Engineering and Science
iii
ABSTRACT
NOVEL MONOCLONAL ANTIBODIES TARGETING
CONFORMATIONAL ERBB2 EPITOPES
1.4ABSTRACT
Ceyhan Ceran
Ph.D. in Molecular Biology and Genetics
Supervisor: Prof. Dr. Mehmet Öztürk
October 2012, 233 pages

ERBB2 is a tyrosine kinase receptor which can act as homodimers or
heterodimers with other members of the ERBB family. Nearly 30% of breast cancers
overexpress ERBB2, which can be effectively targeted by anti-ERBB2 monoclonal
antibodies. Trastuzumab directed against an epitope on subdomain IV of the
extracellular domain (ECD) of ERBB2 is a clinically used therapeutics but the
response rate is poor and acquired resistance is frequent. Pertuzumab that binds to
subdomain II and inhibits receptor dimerization is another promising therapeutics
under clinical trials. Anti-ERBB2 antibodies directed to novel epitopes are
potentially useful tools for replacement and combinatorial therapies. We produced
five new anti-ERBB2 antibodies, all directed against epitope(s) present only on the
native ECD. They performed selective growth inhibitory effects depending on the
level of ERBB2 expression and cellular background. When used alone, novel anti-
ERBB2 antibodies displayed modest but significant growth inhibition on SK-BR-3,
BT-474 and MDA-MB-361 cells with ERBB2 overexpression; while no detectable
inhibition was observed on MCF-7 and T47D cells lacking ERBB2 amplification.
When the antibodies were tested in combination with TNF-α, they acted
synergistically on SK-BR-3 cells, producing upto 80% growth inhibition; but
performed antagonistically on BT-474 cells. Detailed investigation of a
representative antibody indicated G1-arrest as the main mechanism of the anti-
proliferative effects exerted on SK-BR-3 cells. Antibody treatment induced
permanent inhibition of DNA synthesis, leading to accumulation of cells at G1-
phase; an effect which was accelerated in the presence of TNF-α. In addition, treated
SK-BR-3 cells displayed inhibition of Akt and ERK1/2 phosphorylation leading to
cyclin D1 accumulation and growth arrest, independently from TNF-α. Novel
antibodies against conformational epitopes present on the extracellular domain of
ERBB2 receptor may serve as new analytical and diagnostic tools, in addition to
being potent anti-cancer bioactive molecules. Cell-dependent synergy and
antagonism between anti-ERBB2 antibodies and TNF-α provide evidence for a
complex interplay between ERBB2 and TNF-α signaling pathways. Such complexity
may drastically affect the outcome of ERBB2-directed therapeutic interventions.

iv
ÖZET
KONFORMASYONAL ERBB2 EPİTOPLARINI HEDEF ALAN
ÖZGÜN MONOKLONAL ANTİKORLAR
1.5 ÖZET
Ceyhan Ceran
Moleküler Biyoloji ve Genetik Doktora Tezi
Tez Yöneticisi: Prof. Dr. Mehmet Öztürk
Ekim 2012, 233 sayfa

Bir tirozin kinaz almacı olan ERBB2, gerek homodimer yapıda, gerekse ERBB
ailesinin diğer üyeleriyle heterodimer oluşturarak etki gösterir. Meme kanserlerinin
% 30’unu oluşturan ve ERBB2 almaç ifadesi yüksek olan vakalar, hedefe yönelik
tedavi kapsamında anti-ERBB2 antikorlarıyla başarılı bir şekilde tedavi
edilebilmektedir. ERBB2’nin hücredışı bölgesindeki IV. alt-bölgeye karşı
geliştirilmiş Trastuzumab klinikte kullanılan bir terapötik olmasına karşın, elde
edilen başarılı yanıt oranı zayıftır ve hastalarda sıklıkla tedaviye karşı bağışıklık
gelişir. II. alt-bölgeye bağlanarak almaç dimerizasyonunu engelleyen Pertuzumab,
klinik çalışmaları yürütülmekte olan diğer bir ümit verici terapötiktir. Yeni epitoplara
karşı geliştirilen anti-ERBB2 antikorları, replasman ve kombine tedavilerde
kullanılabilecek etkin araçlardır. ERBB2 almaçının yalnızca doğal haldeki hücredışı
bölgesine bağlanan beş adet özgün anti-ERBB2 antikoru ürettik. Bunlar, hücredeki
ERBB2 ifade düzeyine ve hücrenin yapısal özelliklerine bağlı olmak üzere değişik
oranda çoğalmayı baskılayıcı özellik gösterdiler. Tek başlarına kullanıldıklarında,
SK-BR-3, BT-474 ve MDA-MB-361 gibi ERBB2 ifadesi yüksek hücrelerin
çoğalmasını mütevazi ancak anlamlı ölçüde baskılarken, MCF-7 ve T47D gibi
normal ifade düzeyine sahip hücrelerde belirgin etki göstermediler. Antikorlar TNF-
α ile birlikte kullanıldıklarında, SK-BR-3 hücrelerinde sinerjik bir etki göstererek
hücre çoğalmasını % 80’e varan oranlarda baskılarken, BT-474 hücrelerinde
antagonistic etki gösterdiler. Antikorlardan seçilen bir tanesiyle yapılan detaylı
inceleme, SK-BR-3 hücrelerinde görülen çoğalma baskılayıcı etkinin ana
mekanizmasının hücre döngüsünün G1-evresinde durma ile ilintili olduğunu ortaya
koymuştur. TNF-α ile birlikte kullanıldığında artan antikor etkisi, DNA sentezini
kalıcı olarak engelleyerek hücrelerin G1-evresinde birikmesini sağlamıştır. Buna ek
olarak, antikor tedavisine tâbi tutulan SK-BR-3 hücrelerinde TNF-α’dan bağımsız
olarak Akt ve Erk1/2 proteinlerinin fosforlanmasındaki azalmayı takiben Cyclin D1
birikmesi gözlenmiş ve hücre çoğalması engellenmiştir. ERBB2 almacının hücredışı
bölgesindeki üç boyutlu yapıya bağlı oluşan epitoplara karşı geliştirilen özgün
antikorlar, anti-kanser özellik taşıyan biyoaktif ajan olma potansiyellerinin yanı sıra,
yeni analitik ve tanısal araçlar olarak da kullanılabilir. Anti-ERBB2 antikorları ve
TNF-α arasında hücre yapısına bağlı olarak görülen sinerji ve antagonism, ERBB2
ve TNF-α arasında karmaşık bir etkileşim olduğunu göstermektedir. Böyle karmaşık
etkileşimlerin, ERBB2 hedefli tedavilere gösterilen yanıtı ciddi ölçüde etkilemesi
mümkündür.
v
ACKNOWLEDGEMENTS
1.6ACKNOWLEDGEMEN
I owe my deepest gratitude to Prof. Tamer Yağcı and Prof. Mehmet Öztürk
for helping me learn the scientific way of thinking, enlightening my route with
invaluable advises and giving me the opportunity to become one of the numerous
scientists they have raised. In addition to being great mentors, I would like to thank
them for always believing in me since 2001, helping me find my way when I was
lost, allowing and more importantly encouraging me to stand on my own feet when
they knew I was experienced enough.
I am indepted to the members of the supervisory committee, Assoc. Prof. Işık
Yuluğ, Asist. Prof Elif Erson Bensan and Assoc. Prof. İhsan Gürsel for their priceless
advice on vital aspects that enabled me save precious time and effort.
I would like to express my very great appreciation to all Bilkent MBG faculty
members who were more than mere instructors for me. I especially would like to
thank Assoc. Prof. Dr. Rengül Atalay and Assoc. Prof. Işık Yuluğ for the fruitful
discussions and guidance they have generously provided whenever I knocked their
door in great despair. I also want to thank Asist. Prof. Can Akçalı not only for the
scientific knowledge I have gained in his enjoyable and fruitful lectures, but also for
his friendship throughout which he has thought me to keep patient when need be and
gave hope when I needed most.
I am grateful to all Gürsel group, especially Assoc. Prof. İhsan Gürsel, Tamer
Kahraman, Gizem Tinçer König and Fuat Yağcı in addition to Tülin Erşahin and
Assoc. Prof. Mayda Gürsel, without whose knowledge and assistance the flow
cytometric analysis in this study would not have been successful.
I want to send my special thanks to all past and present members of Yağcı
and Öztürk groups; especially Nuri Öztürk, Mine Mumcuoğlu, Şerif Şentürk, Haluk
Yüzügüllü, Özge Gürsoy Yüzügüllü, Hani Alotaibi, Ayça Arslan Ergül, Sevgi
Bağışlar, Ender Avcı, Emin Öztaş, Fatih Semerci, Eylül Harputlugil, Mustafa
Yılmaz, Pelin Gülay, Çiğdem Özen, Gökhan Yıldız, Dilek Çevik, Ayşegül Örs and
Derya Soner for all material, knowledge and experience exchange that made research
easy, enjoyable and fruitful throughout my PhD life; but above everything, for
absolute friendship.
vi
I would like to thank all graduate friends at Bilkent MBG, especially Ebru
Bilget Güven, Emre Onat and Işıl Nalbant Çevik for sharing invaluable knowledge
and experience.
I am especially grateful to Şükrü Atakan, Nilüfer Sayar, Gurbet Karahan,
Sinem Yılmaz Özcan, Kerem Mert Şenses, İrem Gürbüz, Muammer Üçal, Damla
Gözen and Derya Soner for being wonderful friends who were always there in good
and bad times both academically and non-academically. It is a pleasure for me to
have them all as life-long friends.
I cannot thank Bilge Kılıç enough for she was always helpful, generous,
understanding, sympathetic, kind and thoughtful. I am honoured to have her as one
of my best friends in life.
I would like to express my gratefulness to Sevim Baran and Füsun Elvan who
were always supporting and encouraging even at times of shortest notice, Tülay
Arayıcı, Abdullah Ünnü, Turan Daştandır and Yavuz Ceylan who were always there
to provide the perfect research environment.
And above all, I would like to thank my parents Reyhan Ceran and Abdullah
Ceran for their continuous uphold, patience, care, sympathy, unconditional and
endless love at all times.
This work was supported grants from both TUBITAK and State Planning
Office.
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TABLE OF CONTENTS
1.7 TABLE OF CONTENTS
COVER PAGE .............................................................................................................. i
DEDICATION PAGE .................................................................................................. ii
SIGNATURE PAGE ................................................................................................... iii
ABSTRACT ................................................................................................................ iv
ÖZET............................................................................................................................ v
ACKNOWLEDGEMEN ............................................................................................. vi
TABLE OF CONTENTS .......................................................................................... viii
LIST OF TABLES .................................................................................................... xiii
LIST OF FIGURES .................................................................................................. xiv
ABBREVIATIONS ................................................................................................. xvii

1 INTRODUCTION ....................................................................................... 1
1.1 Breast Cancer ............................................................................................... 1
1.2 ERBB2 ......................................................................................................... 1
1.3 Anti-ERBB2 Antibody Based Targeted Therapy of ERBB2
Overexpressing Breast Cancers.................................................................................. 10
1.3.1 Mechanism of Action of anti-ERBB2 Antibody Treatment .............. 12
1.3.1.1 Increased Autophosphorylation, Internalization, Degradation and
Down-regulation of ERBB2 ....................................................................................... 13
1.3.1.2 Growth Inhibition Modulated Through PI3K and MAPK Pathways14
1.3.1.3 Inhibition of ERBB2 Shedding ..................................................... 14
1.3.1.4 ADCC ........................................................................................... 15
1.3.1.5 Inhibition of DNA Repair (after chemotherapy or radiation) ....... 16
1.3.1.6 Inhibition of Angiogenesis............................................................ 16
1.3.2 Response to Trastuzumab .................................................................. 17
1.3.3 Mechanisms of Trastuzumab Resistance ........................................... 18
1.3.3.1 Variations in ERBB2-ECD ........................................................... 20
1.3.3.2 Activating Mutations on ERBB2 Transmembrane and Kinase
Domain……………. .................................................................................................. 21
1.3.3.3 Inhibition of Receptor Downregulation ........................................ 22
1.3.3.4 Receptor Masking ......................................................................... 23
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1.3.3.5 Crosstalk Among Other ERBB Family Members ........................ 23
1.3.3.6 Constitutive Activation of Effector Proteins in PI3K Pathway .... 24
1.3.3.7 Increasing Signalling From Other Receptors................................ 26
1.3.3.8 Fc Receptor Status ........................................................................ 27
1.3.3.9 DNA Methylations ........................................................................ 27
1.3.3.10 Other Factors................................................................................. 27
1.3.3.11 Complexity Accompanying ERBB2 gene amplifications ............ 28
1.4 Alternative Therapeutics for Treatment of ERBB2 Overexpressing Breast
Cancer..... ................................................................................................................... 28
1.5 TNF-α and ERBB2..................................................................................... 31
2 OBJECTIVES AND RATIONALE ........................................................... 35
2.1 Aim of the Study ........................................................................................ 35
2.2 Rationale .................................................................................................... 35
3 MATERIALS AND METHODS ............................................................... 37
3.1 MATERIALS ............................................................................................. 37
3.1.1 General Chemicals ............................................................................. 37
3.1.2 Instruments ......................................................................................... 39
3.1.3 Cell Lines and Tissue Culture Reagents ............................................ 40
3.1.4 Animal Experiments........................................................................... 44
3.2 SOLUTIONS AND MEDIA ...................................................................... 45
3.2.1 Cell Culture Solutions ........................................................................ 45
3.2.2 General Solutions ............................................................................... 46
3.2.3 Cell Lysis Buffers .............................................................................. 49
3.2.4 SDS-PAGE Solutions ........................................................................ 50
3.2.4.1 Conventional SDS-PAGE Gel Buffers ......................................... 50
3.2.5 Solutions for Staining the Proteins on SDS-Polyacrylamide Gels .... 51
3.2.6 Solutions for Staining Total Proteins on PVDF Membranes ............. 52
3.2.7 Western Blotting Solutions ................................................................ 52
3.2.8 ELISA Solutions ................................................................................ 52
3.2.9 Immunofluorescence (IF) Solutions ................................................... 53
3.2.10 Agarose Gel Electrophoresis Solutions .............................................. 53
3.2.11 Bacterial Culture Media and Solutions .............................................. 54
3.2.12 Sulforhodamine (SRB) Based Growth Assay Solutions .................... 55
ix
3.2.13 Antibody Purification Buffers using Protein G Columns .................. 55
3.2.14 Flow Cytometry and Cell Cycle Analysis Buffers ............................. 56
3.2.15 SAβ-Gal Staining Solutions ............................................................... 57
3.3 METHODS ................................................................................................ 59
3.3.1 Cell Culture ........................................................................................ 59
3.3.1.1 Thawing Cell Lines ....................................................................... 59
3.3.1.2 Expansion of Adherent Cell Lines ................................................ 60
3.3.1.3 Expansion of Suspension Cell Lines ............................................ 60
3.3.1.4 Freezing Cell Lines ....................................................................... 61
3.3.1.5 Transient Transfection of Huh-7 Cells ......................................... 61
3.3.2 Generation of anti-ERBB2 Monoclonal Antibodies .......................... 63
3.3.2.1 Immunization of BALB/c mice against human ERBB2 ............... 63
3.3.2.2 Harvesting splenocytes and preparation of cells........................... 63
3.3.2.3 Preparation of Hybridoma Clones and Subcloning ...................... 64
3.3.3 Indirect ELISA ................................................................................... 67
3.3.4 Antibody Isotyping and Purification of anti-ERBB2 Antibodies ...... 68
3.3.4.1 Collecting Antibodies from Hybridoma Supernatants.................. 68
3.3.4.2 Preparation and Collection of Ascites from Mice ........................ 69
3.3.4.3 Isotyping of Monocolonal Antibodies .......................................... 69
3.3.4.4 Purification of Monoclonal Antibodies by FPLC using ProteinG
Columns………… ..................................................................................................... 70
3.3.5 Cloning ERBB2-deletion Constructs ................................................. 71
3.3.5.1 Preparation of ERBB2-deletion Constructs .................................. 71
3.3.5.2 PCR-Amplification of human ERBB2 regions ............................. 78
3.3.5.3 Restriction Digestion of PCR Products ........................................ 79
3.3.5.4 Ligation of Digested Fragments ................................................... 80
3.3.5.5 Preparation of Competent DH5α E. coli Cells .............................. 80
3.3.5.6 Transformation of Ligated Plasmids to DH5α.............................. 81
3.3.5.7 Plasmid Isolation from transformed DH5α................................... 82
3.3.5.8 Selection of Clones by Diagnostic Digest .................................... 82
3.3.5.9 Sequence Verification of Constructed Plasmids ........................... 83
3.3.6 Cell Lysis and Protein Quantification ................................................ 83
3.3.7 Immunoprecipitation (IP) ................................................................... 84
x