Interactions between complement and cellular mediated mechanisms of monoclonal antibody

150 Pages·2017·5.61 MB·English

University of Iowa
Iowa Research Online
Theses and Dissertations
Spring 2010
Interactions between complement and cellular
mediated mechanisms of monoclonal antibody
therapy
Siao-Yi Wang
University of Iowa
Copyright 2010 Siao-Yi Wang
This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/619
Recommended Citation
Wang, Siao-Yi. "Interactions between complement and cellular mediated mechanisms of monoclonal antibody therapy." PhD (Doctor
of Philosophy) thesis, University of Iowa, 2010.
https://doi.org/10.17077/etd.arl8n9mt
Follow this and additional works at:https://ir.uiowa.edu/etd
Part of theImmunology of Infectious Disease Commons
INTERACTIONS BETWEEN COMPLEMENT AND CELLULAR MEDIATED
MECHANISMS OF MONOCLONAL ANTIBODY THERAPY
by
Siao-Yi Wang
An Abstract
Of a thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Immunology
in the Graduate College of
The University of Iowa
1
May 2010
Thesis Supervisor: Professor George J Weiner
1
ABSTRACT
Monoclonal antibodies (mAbs) have become an important part of therapy for a
number of cancers. The first mAb to be approved for clinical use is rituximab, which is
currently used for the treatment of various B cell malignancies. Despite its clinical value,
the mechanisms in which rituximab induces tumor regression are unclear. Growing
evidence suggests that multiple mechanisms involving complement-dependent
cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are involved.
However, the direct interactions between CDC and ADCC have yet to be investigated.
My studies examine the relationship between complement fixation and the
activation of NK cells by utilizing in vitro assays, a syngeneic murine lymphoma model,
and clinical samples from patients. Using these systems, I demonstrate that the initiation
of the complement cascade inhibits NK cell activation and ADCC induced by rituximab
in vitro. I also show that depletion of complement enhances the activation of NK cells
and improves the efficacy of mAb therapy in a murine model. Lastly, I demonstrate that
NK cell activation correlates with decreased complement activity in patients after
rituximab treatment.
The studies described in this dissertation have furthered the understanding of the
1

mechanisms involved in antibody therapy. These results have described a novel
inhibitory role for complement activity in the anti-tumor responses of mAbs.
Furthermore, these findings suggest that strategies to circumvent the inhibitory effect of
complement may improve how current mAbs are used and the how mAbs are designed in
the future.
2
Abstract Approved: ____________________________________
Thesis Supervisor
____________________________________
Title and Department
____________________________________
Date

2
INTERACTIONS BETWEEN COMPLEMENT AND CELLULAR MEDIATED
MECHANISMS OF MONOCLONAL ANTIBODY THERAPY
by
Siao-Yi Wang
A thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Immunology
in the Graduate College of
The University of Iowa
1
May 2010
Thesis Supervisor: Professor George J Weiner
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
PH.D. THESIS
_______________
This is to certify that the Ph.D. thesis of
Siao-Yi Wang
has been approved by the Examining Committee
for the thesis requirement for the Doctor of Philosophy
degree in Immunology at the May 2010 graduation.
Thesis Committee: ___________________________________
George J Weiner, Thesis Supervisor
___________________________________
Gail Bishop
___________________________________
Michael D Henry
___________________________________
Jonathan Heusel
___________________________________
David Lubaroff
ACKNOWLEDGMENTS
I would like to first thank my adviser, Dr. George J. Weiner, for his mentorship
and guidance. He has shown me that it is possible to translate research into medicine and
be successful at b(cid:82)(cid:87)(cid:75)(cid:17)(cid:3)(cid:41)(cid:85)(cid:82)(cid:80)(cid:3)(cid:75)(cid:76)(cid:80)(cid:15)(cid:3)(cid:44)(cid:182)(cid:89)(cid:72)(cid:3)(cid:68)(cid:79)(cid:86)(cid:82)(cid:3)(cid:79)(cid:72)(cid:68)(cid:85)(cid:81)(cid:72)(cid:71)(cid:3)(cid:87)(cid:75)(cid:68)(cid:87)(cid:3)(cid:86)(cid:82)(cid:70)(cid:76)(cid:68)(cid:79)(cid:3)(cid:68)(cid:81)(cid:71)(cid:3)(cid:70)(cid:82)(cid:80)(cid:80)(cid:88)(cid:81)(cid:76)(cid:70)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:86)(cid:78)(cid:76)(cid:79)(cid:79)(cid:86)(cid:3)
(cid:68)(cid:85)(cid:72)(cid:3)(cid:68)(cid:79)(cid:80)(cid:82)(cid:86)(cid:87)(cid:3)(cid:68)(cid:86)(cid:3)(cid:76)(cid:80)(cid:83)(cid:82)(cid:85)(cid:87)(cid:68)(cid:81)(cid:87)(cid:3)(cid:68)(cid:86)(cid:3)(cid:87)(cid:75)(cid:72)(cid:3)(cid:86)(cid:70)(cid:76)(cid:72)(cid:81)(cid:70)(cid:72)(cid:3)(cid:69)(cid:72)(cid:76)(cid:81)(cid:74)(cid:3)(cid:83)(cid:72)(cid:85)(cid:73)(cid:82)(cid:85)(cid:80)(cid:72)(cid:71)(cid:17)(cid:3)(cid:55)(cid:75)(cid:72)(cid:3)(cid:78)(cid:81)(cid:82)(cid:90)(cid:79)(cid:72)(cid:71)(cid:74)(cid:72)(cid:3)(cid:87)(cid:75)(cid:68)(cid:87)(cid:3)(cid:44)(cid:182)(cid:89)(cid:72)(cid:3)(cid:74)(cid:68)(cid:76)(cid:81)(cid:72)(cid:71)(cid:3)
in the past few years will extend beyond the laboratory and into all aspects of my life.
I must also thank my thesis committee, Dr. Gail Bishop, Dr. Michael Henry, Dr.
Jonathan Heusel, and Dr. David Lubaroff for their insight and understanding. Dr. Bishop
has helped me develop personally, as well as scientifically. Dr. Heusel has provided me
with specific advice regarding my research and has helped me achieve my goals in
graduate school. I thank you all.
My work would not be possible if it were not for the help the Weiner lab
members. I would not have been able to spend the days and occasional nights in lab if it
(cid:90)(cid:68)(cid:86)(cid:81)(cid:182)(cid:87)(cid:3)(cid:86)(cid:82)(cid:3)(cid:72)(cid:81)(cid:77)(cid:82)(cid:92)(cid:68)(cid:69)(cid:79)(cid:72)(cid:17)(cid:3)(cid:44)(cid:3)(cid:87)(cid:75)(cid:68)(cid:81)(cid:78)(cid:3)(cid:38)(cid:75)(cid:85)(cid:76)(cid:86)(cid:15)(cid:3)(cid:90)(cid:75)(cid:82)(cid:3)(cid:75)(cid:68)(cid:86)(cid:3)(cid:75)(cid:72)(cid:79)(cid:83)(cid:72)(cid:71)(cid:3)(cid:80)(cid:72)(cid:3)(cid:87)(cid:85)(cid:72)(cid:80)(cid:72)(cid:81)(cid:71)(cid:82)(cid:88)(cid:86)(cid:79)(cid:92)(cid:3)(cid:90)(cid:76)(cid:87)(cid:75)(cid:3)(cid:75)(cid:76)(cid:86)(cid:3)(cid:78)(cid:81)(cid:82)(cid:90)(cid:79)(cid:72)(cid:71)(cid:74)(cid:72)(cid:3)
and technical skills. I also thank Sue, who shares with me a special sense of humor. Her
experience in science has also been extremely helpful. I must also thank Emil for
(cid:71)(cid:76)(cid:86)(cid:68)(cid:74)(cid:85)(cid:72)(cid:72)(cid:76)(cid:81)(cid:74)(cid:3)(cid:90)(cid:76)(cid:87)(cid:75)(cid:3)(cid:72)(cid:89)(cid:72)(cid:85)(cid:92)(cid:87)(cid:75)(cid:76)(cid:81)(cid:74)(cid:3)(cid:44)(cid:182)(cid:89)(cid:72)(cid:3)(cid:72)(cid:89)(cid:72)(cid:85)(cid:3)(cid:86)(cid:68)(cid:76)(cid:71)(cid:3)(cid:82)(cid:85)(cid:3)(cid:71)(cid:82)(cid:81)(cid:72)(cid:17)(cid:3)(cid:44)(cid:3)(cid:81)(cid:82)(cid:90)(cid:3)(cid:78)(cid:81)(cid:82)(cid:90)(cid:3)(cid:87)(cid:75)(cid:68)(cid:87)(cid:3)(cid:75)(cid:72)(cid:3)(cid:90)(cid:68)(cid:86)(cid:3)(cid:77)(cid:88)(cid:86)(cid:87)(cid:3)(cid:87)(cid:72)(cid:68)(cid:70)(cid:75)(cid:76)(cid:81)(cid:74)(cid:3)
2
me how think scientifically. Thanks guys.
I would also like to thank my parents for teaching me the importance of
education. I would never have strived to a(cid:70)(cid:75)(cid:76)(cid:72)(cid:89)(cid:72)(cid:3)(cid:76)(cid:73)(cid:3)(cid:76)(cid:87)(cid:3)(cid:90)(cid:68)(cid:86)(cid:81)(cid:182)(cid:87)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:87)(cid:75)(cid:72)(cid:80)(cid:17)(cid:3)(cid:44)(cid:3)(cid:68)(cid:79)(cid:86)(cid:82)(cid:3)(cid:87)(cid:75)(cid:68)(cid:81)(cid:78)(cid:3)(cid:80)(cid:92)(cid:3)
sister, Peggy, who has always been a great role model for me. Thanks.
Finally, I must thank my fiancée, Anne, who has sacrificed so much for my
success. I will forever be in her debt.
ii
ABSTRACT
Monoclonal antibodies (mAbs) have become an important part of therapy for a
number of cancers. The first mAb to be approved for clinical use is rituximab, which is
currently used for the treatment of various B cell malignancies. Despite its clinical value,
the mechanisms in which rituximab induces tumor regression are unclear. Growing
evidence suggests that multiple mechanisms involving complement-dependent
cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are involved.
However, the direct interactions between CDC and ADCC have yet to be investigated.
My studies examine the relationship between complement fixation and the
activation of NK cells by utilizing in vitro assays, a syngeneic murine lymphoma model,
and clinical samples from patients. Using these systems, I demonstrate that the initiation
of the complement cascade inhibits NK cell activation and ADCC induced by rituximab
in vitro. I also show that depletion of complement enhances the activation of NK cells
and improves the efficacy of mAb therapy in a murine model. Lastly, I demonstrate that
NK cell activation correlates with decreased complement activity in patients after
rituximab treatment.
The studies described in this dissertation have furthered the understanding of the
3
mechanisms involved in antibody therapy. These results have described a novel
inhibitory role for complement activity in the anti-tumor responses of mAbs.
Furthermore, these findings suggest that strategies to circumvent the inhibitory effect of
complement may improve how current mAbs are used and the how mAbs are designed in
the future.

iii
TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................. vi
LIST OF FIGURES .......................................................................................................... vii
CHAPTER I: GENERAL INTRODUCTION ................................................................1

Development of monoclonal antibodies ...........................................................1
Use of monoclonal antibodies in cancer treatment ...........................................2
Mechanisms of tumor lysis ...............................................................................3
Role of complement-dependent cytotoxicity .............................................4
Role of antibody-dependent cellular cytotoxicity .....................................8
Factors that influence the mechanisms of cytotoxicity ...................................15
Interactions between mechanisms ..................................................................17
Additional Fc(cid:74) receptor mediated mechanisms ..............................................19
Rationale for project .......................................................................................20
CHAPTER II: NK CELL ACTIVATION AND ANTIBODY-DEPENDENT
CELLULAR CYTOTOXICITY IS INHIBITED BY
COMPLEMENT ACTIVITY ................................................................31

Abstract ...........................................................................................................31
Introduction .....................................................................................................31
Materials and Methods ...................................................................................34
Antibodies and serum ..............................................................................34
Samples from human subjects .................................................................34
NK cell activation ....................................................................................34
NK cell phenotypic analysis ....................................................................35
Microtiter plate adhesion assay ...............................................................36
Cytotoxicity assay ...................................................................................36
Results.............................................................................................................37
Normal human serum inhibits NK cell activation ...................................37
Inhibitory effects of normal human serum are due to complement
activity .....................................................................................................37
Inhibitory effects of complement activity are not influenced by
target cell viability ...................................................................................38
4
C1q and C3 are necessary for the inhibitory effects of complement
activity .....................................................................................................39
Inhibition of NK cell activation correlates with C3b deposition .............39
Blocking CR3 does not affect inhibition of NK activation .....................40
Complement activity inhibits the binding of NK cell CD16 to
rituximab ..................................................................................................40
Complement activity inhibits NK cell-mediated cytotoxicity .................41
Discussion .......................................................................................................41
CHAPTER III: DEPLETION OF COMPLEMENT ACTIVITY ENHANCES NK
CELL ACTIVATION AND IMPROVES THE EFFICACY OF A
MONOCLONAL ANTIBODY THERAPY IN VIVO .........................67

Abstract ...........................................................................................................67
Introduction .....................................................................................................68
iv
Materials and methods ....................................................................................70
Antibodies and reagents ..........................................................................70
Samples from human subjects .................................................................70
NK cell activation ....................................................................................71
NK cell phenotypic analysis ....................................................................71
38C13 murine lymphoma model .............................................................71
Results.............................................................................................................72
Complement activity is present in extravascular fluid ............................72
Complement activity in extravascular fluid inhibits NK activation ........73
The inhibitory effects of extravascular fluid are dose-dependent
and abrogated with heat inactivation .......................................................73
Depletion of complement enhances NK cell activation ..........................73
Murine serum blocks mAb-induced activation of murine NK cells ........74
Depletion of complement improves the efficacy of monoclonal
antibody therapy in a murine B cell lymphoma model ...........................75
Discussion .......................................................................................................76
CHAPTER IV: NK CELL ACTIVATION CORRELATES WITH DECREASED
COMPLEMENT ACTIVITY IN PATIENTS FOLLOWING
RITUXIMAB TREATMENT ................................................................93

Abstract ...........................................................................................................93
Introduction .....................................................................................................93
Materials and methods ....................................................................................95
Patients and samples ................................................................................95
Determination of rituximab concentration ..............................................95
NK cell phenotypic analysis ....................................................................96
Serum cytokine analysis ..........................................................................96
CH50 Assay .............................................................................................97
Results.............................................................................................................97
Patient characteristics ..............................................................................97
Serum rituximab levels display high individual variability ....................98
Absolute lymphocyte counts decrease after rituximab infusion ..............98
NK cell activation occurred in two patients after therapy .......................98
Complement activity in serum samples was decreased in two
patients after therapy ...............................................................................99
NK cell activation correlates with decreased complement activity .......100
Discussion .....................................................................................................100
5
CHAPTER V: GENERAL DISCUSSION ..................................................................112

New role for complement in antibody therapy .............................................112
Impact on patient care ...................................................................................113
Careful dosing of rituximab ...................................................................113
Depletion of complement ......................................................................114
Design of future antibodies ...................................................................116
Future directions ...........................................................................................118
Conclusion ....................................................................................................120
REFERENCES ................................................................................................................121

v