chemically self-assembled antibody nanorings (csans): design and characterization of an anti-cd3 ...

171 Pages·2010·10.23 MB·English

CHEMICALLY SELF-ASSEMBLED ANTIBODY NANORINGS (CSANS):
DESIGN AND CHARACTERIZATION OF AN ANTI-CD3 IGM BIOMIMETIC

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

QING LI

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

DR. CARSTON R. WAGNER, ADVISER

OCTOBER 2010
© Qing Li, 2010
ACKNOWLEDGEMENTS

Deepest gratitude to my family, my advisor, and my friends for your enthusiastic support
of my life and my research career, for your love, and for your patience!

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ABSTRACT
Based on our development of a highly efficient protocol for the chemically
controlled self-assembling of protein nanorings, we have sought to exploit our
methodology for engineering multivalent chemically self-assembled antibody-nanorings
(CSANs) for tissue imaging and drug delivery. Two novelDHFR-DHFR-anti-CD3 scFv

fusion proteins were constructed (13DDantiCD3 and 1DDantiCD3). In addition, the two
DHFR cysteines were mutated to either alanine or serine to enhance correct folding. The
protein was expressed in BL21 (DE3) cells, renatured with the SLS-based refolding
protocol and purified by methotrexate affinity chromatography. Incubation of
13DDantiCD3 with the chemical dimerizer, bisMTX, resulted in almost exclusive
formation of the bivalent CSANs while incubation with 1DDantiCD3 resulted in
,
formation of octavalent CSANs. Both antibody nanorings selectively blocked the killing
of the CD3+ human T-leukemia HPB-MLT by a diphtheria-anti-CD3 immunotoxin.
FACS analysis revealed nearly identical dissociation constants for both the self-
assembled and parental monoclonal antibody and a 3-fold lower K for the octavalent
d
species. The chemically dimerized scFv’s were shown to be stable in cell culture at 37oC
and the dimerization was shown to be reversible by the addition of excess amounts of the
non-toxic FDA approved DHFR antagonist trimethoprim. We also demonstrate that,
similar to the parental bivalent anti-CD3 monoclonal antibody (MAB), anti-CD3 CSANs
selectively bind to CD3+ leukemia cells, and undergo rapid internalization through a
caveolin-independent pathway that requires cholesterol, actin polymerization and protein
tyrosine kinase activation. While treatment with the monoclonal antibody leads to T-cell
activation and nearly complete loss (i.e. 90%) of surface displayed T-cell receptor (TCR),
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only 25-30% of the TCR down regulate and no significant T-cell proliferation is observed
after treatment of peripheral blood mononuclear cells (PBMCs) with anti-CD3 CSANs.
Consistent with the proliferation findings, 15-25 % less CD25 (IL-2 receptor) was found
on the surface of PBMCs treated with either the polyvalent or bivalent anti-CD3 CSANs,
respectively, than on PBMCs treated with the parental MAB. Comparative experiments
with F(ab') derived from the MAB confirm that the activation of the T-cells by the MAB
2
is dependent on the Fc domain, and thus interactions of the PBMC T-cells with accessory
cells, such as macrophages. Taken together, our results demonstrate that anti-CD3
CSANs with valencies ranging from 2 to 8 could be employed for radionuclide, drug or
potentially oligonucleotide delivery to T-cells without, as has been observed for other
antibody conjugated nanoparticles, the deleterious affects of activation observed for
MAB. Further the CSAN construct may be adapted for the preparation of other
multivalent scFvs.

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TABLE OF CONTENTS
Acknowledgements………………………………………………………………. i
Abstract………………………………………………………………………....... ii
Table of Contents………………………………………………………………… iv
List of Tables and Schemes……………………………………………………… viii
List of Figures……………………………………………………………………. ix
List of Abbreviations…………………………………………………………….. xii
Chapter One – Introduction: Engineering of Multivalent Antibody………… 1
I. Significance of multivalent Antibody………………………………………… 2
II. Strategies of generating multivalent antibody………………………………… 12
A. Domain-swapping phenomenon…………………………………………. 12
B. Genetically linear fusion…………………………………………………. 14
C. Chemically linking………………………………………………………... 15
D. Self-associating peptides and protein domains…………………………… 17
1. Leucine zipper…………………………………………………….. 17
2. Biotin-streptavidin complex………………………………………. 17
3. Barnase-barstar module…………………………………………… 20
4. Dock and Lock……………………………………………………. 20
5. Shiga-like toxin fusion……………………………………………. 22
6. Knob-into-holes…………………………………………………… 22
7. Antibody Nanoparticle Conjugates……………………………….. 26
III. Method of chemically induced dimerization (CID)…………………………... 27
A. CID system……………………………………………………………….. 27
B. Introduction of DHFR-MTX……………………………………………... 30
C. Hypothesis of DHFR-scFv-MTX………………………………………… 34
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Chapter Two - Plasmid Construction, Prokaryotic Expression and Purification of
DHFR-DHFR-scFv Fusion Protein……………………………………………. 36
I. Introduction……………………………………………………………………... 37
A. Genetically engineering of recombinant fusion protein………………... 37
B. Protein expression systems of recombinant antibody………………….. 39
1. Protein expressed as inclusion body in cytoplasm…………………... 39
2. Protein expressed in periplasm………………………………………. 40
3. Protein expressed in oxidative cytoplasm…………………………… 41
II. Results and Discussion………………………………………………………… 42
A. Plasmid construction and multi-mutant cysteine free reaction…………. 42
B. Fusion protein expression and purification from inclusion bodies……... 46
C. Fusion protein expression and purification from Origami-B cells……... 51
D. DHFR activity assay and anti-CD3 scFv binding assays………………. 52
III. Conclusions…………………………………………………………………… 56
IV. Material and Methods………………………………………………………… 59
A. Construction of p13DD13CD3…………………………………………… 59
B. Multimutagenesis of four cysteines in the two DHFRs …………………. 60
C. Construction of p1DD13CD3 Plasmid…………………………………… 61
D. Protein expression, refolding and purification…………………………… 62
E. DHFR activity assay……………………………………………………… 63
F. In vitro cytotoxicity blocking assay………………………………………. 64
G. In vitro competitive binding assay……………………………………….. 64

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Chapter Three - Self-Assembling Antibodies by Chemical Induction……….. 66
I. Introduction…………………………………………………………………….. 67
A. Methodology for self-assembling antibodies…………………………… 67
B. Endocytosis mechanism study of bivalent anti-CD3-DHFR2................... 70
II. Results and Discussion………………………………………………………… 73
A. Characterization of antibody dimerization by SEC……………………. 73
B. Characterization of binding affinities of self-assembled antibodies by
blocking assay………………………………………………………….. 75
C. Characterization of binding affinities of self-assembled antibodies by
FACS competitive binding assay………………………………………. 78
D. Characterization of disassembly of self-assembled antibodies by SEC... 78
E. Fluorescence labeling of the self-assembled antibodies……………….. 81
F. Cell internalization studies of self-assembled antibodies………………. 83
III. Conclusions…………………………………………………………………… 91
IV. Material and Methods………………………………………………………… 92
A. Size Exclusion Chromatography (SEC)…………………………………. 92
B. In vitro cytotoxicity blocking assay……………………………………… 92
C. In vitro competitive binding assay……………………………………... 92
D. Competitive disassembly of bivalent anti-CD3-DHFR2………………. 93
E. Confocal microscopy…………………………………………………... 94

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Chapter Four - Chemically Self-Assembled Antibody Nanorings (CSANs): Design
and Characterization of an Anti-CD3 IgM Biomimetic……………………… 95
I. Introduction…………………………………………………………………….. 96
II. Results and Discussion………………………………………………………… 99
A. Assemby of multivalent CSANS………………………………………. 99
B. Characterization of CSANs by SLS and AFM…………………………. 101
C. Disassembly of octavalent CSAN……………………………………… 103
D. Binding of anti-CD3 CSANs to cells…………………………………... 103
E. Mechanism of anti-CD3 CSANs cellular uptake……………………….. 108
F. Activation of T-cells by anti-CD3 CSANs……………………………... 113
III. Conclusions…………………………………………………………………… 120
IV. Material and Methods………………………………………………………… 121
A. Construction of p1DD13CD3 plasmid…………………………………. 121
B. Protein expression, refolding and purification………………………….. 121
C. Size exclusion chromatography (SEC)…………………………………. 122
D. Static light scattering analysis………………………………………….. 122
E. Atomic force microscopy analysis……………………………………… 123
F. Competitive disassembly of anti-CD3 CSANs…………………………. 124
G. In vitro competitive binding assay……………………………………... 124
H. Specific binding of octavalent anti-CD3 CSANs to CD3+ cells………. 125
I. Preparation of human PBMC……………………………………………. 126
J. Confocal microscopy……………………………………………………. 126
K. PBMC Proliferation Assay…………………………………………….. 127
L. Analysis of expression of IL-2R (CD25)………………………………. 127
M. T cell receptor modulation…………………………………………….. 128
BIBLIOGRAPHY……………………………………………………………….. 129
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LIST OF TABLES AND SCHEMES

Chapter One – Introduction
Table 1. Examples of therapeutic monoclonal antibodies approved for clinical use. 8

Chapter Three
Scheme 1: Scheme for chemically induced dimerization………………………… 68

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