1 Development of anti-PEG bispecific antibodies for targeting PEGylated nanoparticles to tumour

70 Pages·2017·2.81 MB·English

Development of anti-PEG bispecific antibodies for targeting PEGylated nanoparticles to tumour cells
Tim Ruder
BBiomed. Sc.; Hons.

A thesis submitted for the degree of Master of Philosophy at
The University of Queensland in 2017
Australian Instititute for Bioengineering and Nanotechnology
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Thesis Abstract
Cancer treatment is significantly impaired by the low therapeutic index traditionally exhibited by most
current chemotherapeutics. This is principally a result of a ubiquitous systemic distribution of these
drugs. Recent advances in chemotherapy have seen the development of both antibody conjugated
chemotherapeutics and drug conjugated nanoparticles each with their own unique advantages and
drawbacks. However, to date, there are no approved targeted nanoparticles. Of the seven FDA approved
nanoparticles two are entirely composed of polyethylene glycol (PEG) or are surface PEGylated. Other
FDA approved nanoparticles would likely benefit from PEGylation to diminish aggregation and increase
circulation time. In this study, we designed bispecific antibodies targeting both a PEG chain on a
nanoparticle and cancer-associated cell surface targets. Our initial bispecific antibody designs yielded
one bispecific antibody that successfully binds both the PEG nanoparticle and the epidermal growth
factor receptor (EGFR) antigen on cancer cells. Capture ELISA showed the affinity of the anti-PEG
component to be comparable to the parent monoclonal antibody. We further show that these bispecific
antibodies bind to EGFR overexpressing MDA MB 468 cells both in vitro and in vivo.
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Declaration by author

This thesis is composed of my original work and contains no material previously published or written by
another person except where due reference has been made in the text. I have clearly stated the
contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance,
survey design, data analysis, significant technical procedures, professional editorial advice, and any other
original research work used or reported in my thesis. The content of my thesis is the result of work I have
carried out since the commencement of my research higher degree candidature and does not include a
substantial part of work that has been submitted to qualify for the award of any other degree or diploma
in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have
been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and,
subject to the policy and procedures of The University of Queensland, the thesis be made available for
research and study in accordance with the Copyright Act 1968 unless a period of embargo has been
approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s)
of that material. Where appropriate I have obtained copyright permission from the copyright holder to
reproduce material in this thesis.

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Publications during candidature

Howard CB, Fletcher N, Houston ZH, Fuchs AV, Boase NR, Simpson JD, Raftery LJ, Ruder T, Jones
ML, de Bakker CJ, Mahler SM. Overcoming Instability of Antibody‐Nanomaterial Conjugates: Next
Generation Targeted Nanomedicines Using Bispecific Antibodies. Adv Healthc Mater. 2016
Aug;5(16):2055-68. doi: 10.1002/adhm.201600263. Epub 2016 Jun 10.

Publications included in this thesis

No publications included

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Contributions by others to the thesis

In Vivo Fluorescent Imaging conducted by Nicholas Fletcher; Thurecht Group.
Statement of parts of the thesis submitted to qualify for the award of
another degree

None

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Acknowledgements

"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium,
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incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis
nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur?
Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur,
vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?
At vero eos et accusamus et iusto odio dignissimos ducimus qui blanditiis praesentium voluptatum
deleniti atque corrupti quos dolores et quas molestias excepturi sint occaecati cupiditate non provident,
similique sunt in culpa qui officia deserunt mollitia animi, id est laborum et dolorum fuga. Et harum
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cumque nihil impedit quo minus id quod maxime placeat facere possimus, omnis voluptas assumenda
est, omnis dolor repellendus. Temporibus autem quibusdam et aut officiis debitis aut rerum necessitatibus
saepe eveniet ut et voluptates repudiandae sint et molestiae non recusandae. Itaque earum rerum hic
tenetur a sapiente delectus, ut aut reiciendis voluptatibus maiores alias consequatur aut perferendis
doloribus asperiores repellat."
- Cicero

Gratias vobis ago omnibus habentibus contulit thesi.

Per aspera ad astra.
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Keywords
cd171, cd200, egfr, nanoparticle, bsab mediated targeting

Australian and New Zealand Standard Research Classifications
(ANZSRC)
ANZSRC code: 090399, Biomedical Engineering, 100%

Fields of Research (FoR) Classification
0903 Biomedical Engineering, 100%

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Table of Contents
Thesis Abstract .......................................................................................................................................... 2
Declaration by author ................................................................................................................................ 3
Publications during candidature ................................................................................................................ 4
Publications included in this thesis ........................................................................................................... 4
Contributions by others to the thesis ......................................................................................................... 5
Statement of parts of the thesis submitted to qualify for the award of another degree ............................. 5
Acknowledgements ................................................................................................................................... 6
Keywords .................................................................................................................................................. 7
Australian and New Zealand Standard Research Classifications (ANZSRC) .......................................... 7
Fields of Research (FoR) Classification ................................................................................................... 7
Chapter 1: Introduction and Background ................................................................................................ 12
1.1 Nanoparticles in Cancer Diagnosis and Therapy .......................................................................... 12
1.2 Conjugation of Antibodies and Nanoparticles .............................................................................. 13
1.3 Bispecific Antibodies .................................................................................................................... 14
1.4 Targeting of PEG nanoparticles to Cell surface Targets utilizing BsAbs ..................................... 14
Chapter 2: Methods ................................................................................................................................. 15
2.1.1 BsAb Design: ............................................................................................................................. 15
2.1.2 Cloning: ...................................................................................................................................... 16
2.1.2.1 Restriction digest: ................................................................................................................ 16
2.1.2.2 Ligation: ............................................................................................................................... 16
2.1.2.3 Transformation and Subcloning: ......................................................................................... 16
2.1.3 Transfection and Expression: ..................................................................................................... 17
2.1.4 Purification ................................................................................................................................. 17
2.1.5 SDS-PAGE Electrophoresis: ...................................................................................................... 18
2.1.6 Purity analysis using High-Performance Liquid Chromatography: ........................................... 18
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2.1.7 Spherical molecular size and aggregation analysis using Dynamic Light Scatter: .................... 18
2.1.8 Initial Affinity Screening and EC50 approximation by Enzyme-Linked Immunosorbent Assay:
............................................................................................................................................................. 19
Affinity Screening: .......................................................................................................................... 19
EC50: ............................................................................................................................................... 19
2.1.9 Kinetics Assay and Kd calculation by Biacore Assay: .............................................................. 20
Scouting of optimum receptor and antibody concentration ratios: .................................................. 20
Biacore Kinetics Assay: ................................................................................................................... 20
2.1.10 Flow Cytometry Assays: .......................................................................................................... 20
Binding of BsAb to tumour cell surface targets: ............................................................................. 20
BsAb mediated colocalization of Cy5 labelled PEG nanoparticle and tumour cells ...................... 21
2.1.11 In Vivo Imaging: ...................................................................................................................... 21
2.1.12 Ex Vivo Imaging ...................................................................................................................... 22
Chapter 3: Results and Discussion .......................................................................................................... 22
3.2 Development and design of anti-PEG anti-EGFR Bispecific Antibodies:.................................... 22
3.2.1 Construct 1; anti-EGFR- PEG (Panitumumab-15-2):............................................................. 22
3.2.2 Construct 2; anti-EGFR-PEG (Panitumumab-AGP3): ........................................................... 23
3.2.3 Construct 3; anti-EGFR-PEG (Panitumumab-3.3): ................................................................ 24
3.2.4 Construct 4; anti-EGFR-PEG (Panitumumab-6.3): ................................................................ 24
3.2.5 Construct 5; anti-EGFR-PEG (Panitumumab-E11): .............................................................. 24
3.3 Development of BsAbs targeting novel cancer cell surface targets .............................................. 24
3.3.1 Construct 6; anti-CD171-PEG (L1_9.3Hu – 15-2): ............................................................... 25
3.3.2 Construct 7; anti-CD200-PEG (Samalizumab – 15-2): .......................................................... 26
3.3.3 Construct 8; anti-CD227-PEG (clivatuzumab tetraxetan – 15-2): ......................................... 28
4 Conclusion ........................................................................................................................................... 29
5 Research Environment ......................................................................................................................... 29
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6 Role of Personnel ................................................................................................................................. 29
7 References ............................................................................................................................................ 31
8 Appendix A – Figures .......................................................................................................................... 34
Figure 1: Illustration of the relationship between nanoparticle size and targeting ligand density. . 34
Figure 2: FASTA format sequences of designed BsAbs. ................................................................ 36
Figure 3: Annotated vector map of plasmid NBF 347. ................................................................... 37
Figure 4: SDS-PAGE gel of anti-EGFR-PEG BsAb purification from CHO cell culture supernatant.
......................................................................................................................................................... 38
Figure 5: SDS-PAGE gel of anti-CD171-PEG BsAb purification from CHO cell culture supernatant.
......................................................................................................................................................... 39
Figure 6: SDS-PAGE gel of anti-CD200-PEG BsAb purification from CHO cell culture supernatant.
......................................................................................................................................................... 40
Figure 7: SDS-PAGE gel of anti-CD223-PEG BsAb purification from CHO cell culture supernatant.
......................................................................................................................................................... 41
Figure 8: Isocratic size exclusion HPLC of BsAbs. ........................................................................ 43
Figure 9: Biospecific Antibody Molecular size distribution determined by Dynamic Light Scatter
analysis. ........................................................................................................................................... 46
Figure 10: ELISA of anti-EGFR-PEG(15-2) BsAb binding to the immobilized nanoparticle. ...... 47
Figure 11: ELISA of anti-EGFR-PEG(AGP3) BsAb binding to the immobilized nanoparticle. .... 48
Figure 12: ELISA of anti-EGFR-PEG(3.3) BsAb binding to the immobilized nanoparticle. ......... 49
Figure 13: ELISA of anti-EGFR-PEG(6.3) BsAb binding to the immobilized nanoparticle. ......... 50
Figure 14: ELISA of anti-EGFR-PEG(E11) BsAb binding to the immobilized nanoparticle. ....... 51
Figure 15: ELISA of anti-CD171-PEG BsAb binding to the immobilized nanoparticle and
recombinant receptors. ..................................................................................................................... 52
Figure 16: ELISA of anti-CD200-PEG BsAb binding to the immobilized nanoparticle and
recombinant receptors. ..................................................................................................................... 53
Figure 17: ELISA of anti-CD223-PEG BsAb binding to the immobilized nanoparticle and
recombinant receptors. ..................................................................................................................... 54
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