Measuring Binding Kinetics of Therapeutic Antibodies to Membrane Receptors Using Nanohole ...

112 Pages·2016·4.03 MB·English

by  Luke Jordan

Measuring Binding Kinetics of
Therapeutic Antibodies to
Membrane Receptors Using
Nanohole Array SPR Biosensors

A DISSERTATION
SUBMITTED TO THE FACULTY OF
THE UNIVERSITY OF MINNESOTA BY

Luke Jordan

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

Dr. Sang-Hyun Oh, Adviser

April 2016
Copyright Luke Jordan 2016
Front Matter
Acknowledgements
First, I would like to thank my adviser, Sang-Hyun Oh, for his dedicated mentoring and
generous provision to learn a multitude of fabrication, characterization, and analytical
methods to perform scientific research. Second, I would like to thank my mentor, Nathan
Wittenberg, who taught me so many practical things in the lab and for helping plan
experiments and analyze data. I also would like to thank two former lab members,
Hyungsoon Im and Si-Hoon Lee, who strengthened my work ethic and generously shared
their expertise of micro- and nanofabrication. I also would like to thank all my wonderful
current and former lab mates along the way who made this a wonderful endeavor: Shailabh
Kumar, Avijit Barik, Yong-Sang Ryu, Tim Johnson, Stephen Olson, Daehan Yoo, Xiaoshu
Chen, Lauren Otto, Nathan Lindquist, Antoine Lesuffleur, Jonah Shaver, Dmitriy Zhukov,
Sudhir Cherukulappurath, Jincy Jose, Dan Klemme, and Dan Mohr.
In addition, I want to thank my collaborators at the Mayo Clinic who provided valuable
samples, discussion, and a noble cause to spend time on: Moses Rodriguez, Arthur
Warrington, Xiaohua Xu, Jens Watzlawik, Bharath Wootla, Aleksandar Denic, and Brent
Wright.
I would also like to thank the numerous scientists and technicians in the Minnesota
Nanofabrication Center, the Characterization Facility, and University Imaging Center who
taught me to use their sophisticated instruments and machines and helped solve arising
technical problems, in particular Tony Whipple, Greg Haugstad, and John Oja.
Lastly, I would like to thank my funding sources: the 3M Company for a fellowship
and an opportunity to get to know their innovative company, and the Minnesota Partnership
for Biotechnology and Medical Genomics and National Institutes of Health for grants to
conduct this research.
i
Abstract
In the field of drug discovery, two important metrics of candidate drugs are their binding
affinity and kinetics to target receptors. Dr. Moses Rodriguez and his colleagues at the
Mayo Clinic have found monoclonal IgM antibodies exhibiting therapeutic effects for
multiple sclerosis and amyotrophic lateral sclerosis in animal models, and therefore desired
to obtain the kinetic profiles of these antibodies to their targets. Dr. Sang-Hyun Oh’s lab
at the University of Minnesota specializes in designing and fabricating plasmonic devices,
and have developed a nanohole array sensor coated with silicone dioxide which permits
formation of cell mimicking supported lipid bilayers. The focus of this dissertation has
been to build these devices and develop assays to measure the binding between these
antibodies and receptors in cell extracts and supported lipid bilayers. The first antibody to
measure was rHIgM22, which binds to myelin membrane. We did not know the receptor,
so we used myelin extracts which would include the unknown receptors, and attached these
particles to the sensor surface by passive immobilization. To reduce particle size into the
sensor detection window, we extruded the particles through pores of known dimensions.
After immobilization we measured binding with antibodies. Unfortunately, binding with
rHIgM22 was undetectable, but a similar antibody, mouse IgM O4, which also binds to
myelin and has a therapeutic effect, did bind consistently and gave K = 2.6 ± 3.6
D, apparent
nM, k = 2.5 ± 0.0l × 104 M-1s-1, and k = 6.6 ± 0.3 × 10-5 s-1. The second antibody to
a d,slow
measure was rHIgM12, which binds to neuronal membranes. We found rHIgM12 binds to
the gangliosides GT1b and GD1a, but not GM1. These gangliosides were incorporated
into supported lipid bilayers (5 mol %) and binding to the antibodies was measured.
Binding of rHIgM12 to GT1b gave K = 24.8 ± 7.9 nM, k = 2.19 ± 0.196 × 104 M-
D, apparent a
1s-1, and k = 4.72 ± 1.15 × 10-4 s-1. Binding of rHIgM12 to GD1a gave K =
d,slow D, apparent

42.3 ± 20.6 nM, k = 1.79 ± 0.516 × 104 M-1s-1, and k = 4.43 ± 1.38 × 10-4 s-1.
a d,slow
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Table of Contents
Front Matter ......................................................................................................................... i
Acknowledgements .............................................................................................................. i
Abstract ............................................................................................................................... ii
Table of Contents ............................................................................................................... iii
List of Figures ......................................................................................................................v
List of Tables .................................................................................................................... vii
List of Abbreviations and Acronyms ............................................................................... viii
List of Publications ..............................................................................................................x
1 Introduction ...................................................................................................................1
1.1 Brief Overview ....................................................................................................1
1.2 Scope of Dissertation ..........................................................................................1
1.3 Outline of Chapters .............................................................................................2
2 Background ...................................................................................................................3
2.1 Medical Purpose ..................................................................................................3
2.2 Affinity and Kinetics .........................................................................................12
2.3 The Sensor .........................................................................................................21
2.4 Supported Lipid Bilayers ..................................................................................31
3 Kinetics of IgM O4 and rHIgM22 to Myelin Particles ...............................................36
3.1 Contributions .....................................................................................................36
3.2 Introduction .......................................................................................................36
3.3 Materials and Methods ......................................................................................41
3.4 Results ...............................................................................................................47
3.5 Discussion .........................................................................................................60
3.6 Conclusions .......................................................................................................68
4 Kinetics of rHIgM12 to Gangliosides .........................................................................70
4.1 Contributions .....................................................................................................70
4.2 Introduction .......................................................................................................70
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4.3 Materials and Methods ......................................................................................73
4.4 Results ...............................................................................................................80
4.5 Discussion .........................................................................................................86
4.6 Conclusions .......................................................................................................91
5 Overall Impact of Dissertation ....................................................................................92
References ..........................................................................................................................93
Appendix A Shear Force Driven Lipid Bilayer ...............................................................99

iv
List of Figures
Figure 1: Multiple sclerosis (MS) damages neurons and oligodendrocytes. ..................... 4
Figure 2: Amyotrophic lateral sclerosis (ALS) damages motor neurons and the
muscles they innervate. ..................................................................................... 5
Figure 3: rHIgM22 increases myelin in a mouse model of MS. ........................................ 6
Figure 4: rHIgM22 increases body movement in mouse model of MS. ............................ 8
Figure 5: Neurons follow patterns of immobilized rHIgM12. ........................................... 9
Figure 6: rHIgM12 increases movement in mouse model of ALS. ................................. 10
Figure 7: rHIgM22 and rHIgM12 bind to different cell types. ........................................ 11
Figure 8: Schematic of SPR instrument and data obtained during a binding kinetics
experiment....................................................................................................... 15
Figure 9: Drugs with the same dissociation equilibrium constant, K , may have very
D
different kinetic rate constants, k and k . ....................................................... 20
a d
Figure 10: Surface plasmon polariton at a metal surface. ................................................ 22
Figure 11: Binding kinetics experimental setup. ............................................................. 26
Figure 12: Microfluidic SPR nanohole array device. ...................................................... 27
Figure 13: Microfluidic channels and nanohole array surface of the SPR chip. ............. 29
Figure 14: Imaging spectrometer schematic and data for multiplexed experiments. ...... 30
Figure 15: Extrusion process creates defined particle size. ............................................. 32
Figure 16: FRAP experiments confirm supported lipid bilayer formation. ..................... 33
Figure 17: Extracted myelin particles were too large for SPR, so extrusion was used
to reduce their sizes. ........................................................................................ 39
Figure 18: SPR chip coated with extruded myelin particles interacting with IgM O4
or rHIgM22. .................................................................................................... 40
Figure 19: Typical SPR binding curves between IgM O4 or rHIgM22 and extruded
myelin particles. .............................................................................................. 48
Figure 20: Troubleshooting non-binding of rHIgM22 to myelin: testing new
rHIgM22. ........................................................................................................ 51
Figure 21: Troubleshooting non-binding of rHIgM22 to myelin: testing a higher
concentration of rHIgM22. ............................................................................. 52
v
Figure 22: Troubleshooting non-binding of rHIgM22 to myelin: testing extrusion
through larger pores. ....................................................................................... 53
Figure 23: Troubleshooting non-binding of rHIgM22 to myelin: testing new myelin. .. 54
Figure 24: Troubleshooting non-binding of rHIgM22 to myelin: testing unextruded
myelin. ............................................................................................................ 55
Figure 25: Immunohistochemistry experiment to detect binding between rHIgM22
and myelin. ...................................................................................................... 57
Figure 26: Kinetic binding curves of mouse IgM O4 to surfaces incubated with and
without myelin particles. ................................................................................. 59
Figure 27: Structure of major brain gangliosides. ........................................................... 71
Figure 28: SPR chip coated with SLB doped with ganglioside receptors interacting
with rHIgM12. ................................................................................................ 73
Figure 29: FRAP experiment confirms SLBs have formed from vesicles doped with
gangliosides..................................................................................................... 81
Figure 30: Transmission spectra from SPR chip during kinetic experiment between
rHIgM12 and GT1b. ....................................................................................... 83
Figure 31: SPR kinetic curves of binding between rHIgM12 and gangliosides. ............. 85
Figure 32: Shear force from a high-speed solution can move a supported lipid
bilayer. ............................................................................................................ 99

vi
List of Tables
Table 1 Myelin Binding Experiment Conditions Tested and Results.............................. 50
Table 2 Mouse IgM O4 Binding Constants to Myelin and Sulfatide in SLB .................. 61
Table 3 Calculated Kinetic Rate and Thermodynamic Constants ................................... 86
Table 4 Binding Properties of Natural Autoantibodies of Isotype IgM ........................... 87
Table 5 Binding Affinity of Various Drugs ..................................................................... 89

vii
List of Abbreviations and Acronyms
AFM atomic force microscopy
Ag silver
Al O aluminum oxide
2 3
ALD atomic layer deposition
ALS amyotrophic lateral sclerosis
Au gold
CCD charged coupled device
Chol cholesterol
CNP 2',3'-cyclic-nucleotide 3'-phosphodiesterase
Cryo-SEM cryogenic scanning electron microscopy
Cryo-TEM cryogenic transmission electron microscopy
EAE Experimental Autoimmune Encephalomyelitis
EDTA ethylenediaminetetraacetic acid
egg PC L-α-phosphatidylcholine from chicken eggs
ELISA enzyme-linked immunosorbent assays
FIB focused ion beam
FRAP fluorescence recovery after photobleaching
GD1a disialoganglioside
GM1 monosialoganglioside
GPMV giant plasma membrane vesicle
GT1b trisialoganglioside
GUV giant unilamellar vesicle
HSA human serum albumin
IgM Immunoglobulin M
K apparent equilibrium dissociation constant
D, apparent
k association kinetic constant
a
k fast dissociation kinetic constant
d, fast
k slow dissociation kinetic constant
d, slow
MAG myelin associated glycoprotein
MBP myelin basic protein
MOG myelin oligodendrocyte glycoprotein
MP methylprednisolone
MS multiple sclerosis
NSL nanosphere lithography
PDMS polydimethylsiloxane
PLP proteolipid protein
PTFE polytetrafluoroethylene
PVP poly(N-vinylpyrrolidone)
Rho-DMPE 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine
rhodamine B sulfonyl
viii