armed vascular targeting antibodies for the - ETH E-Collection

147 Pages·2014·23.27 MB·English

by  Katrin Gutbrodt

ETH Library
Armed vascular targeting
antibodies for the treatment of
acute myeloid leukemia
Doctoral Thesis
Author(s):
Gutbrodt, Katrin Lisa
Publication date:
2014
Permanent link:
https://doi.org/10.3929/ethz-a-010182294
Rights / license:
In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection.
For more information, please consult the Terms of use.
DISS. ETH NO. 21857

ARMED VASCULAR TARGETING ANTIBODIES
FOR THE TREATMENT OF
ACUTE MYELOID LEUKEMIA

A thesis submitted to attain the degree of

DOCTOR OF SCIENCES of ETH ZURICH
(Dr. sc. ETH Zurich)

presented by

KATRIN LISA GUTBRODT
Master of Science ETH in Biologie
ETH Zurich

born on 03.02.1985
citizen of Bülach ZH

accepted on the recommendation of

Prof. Dr. Dario Neri (examiner)
Prof. Dr. Cornelia Halin Winter (co-examiner)

2014

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“Success is a science;
if you have the conditions, you get the result.”
Oscar Wilde

To those who shape the conditions

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Table of Contents

1. Summary ..................................................................................................... 1
2. Zusammenfassung .................................................................................... 3!
3. Introduction ................................................................................................ 5!
3.1. The selectivity of anticancer drugs .................................................................... 5
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3.2. Antibodies ......................................................................................................... 6
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3.2.1. Antibody structure and function .................................................................. 6
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Antibody-dependent cellular cytotoxicity (ADCC) ......................................................... 8
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Complement-dependent cytotoxicity (CDC) ................................................................. 9
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3.2.2. Engineering antibodies for cancer therapy ............................................... 11
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Engineering the Fc domain of IgGs to modulate effector functions ............................ 12
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Antibody formats and their targeting properties .......................................................... 13
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3.2.3. Therapeutic antibodies and their mechanisms of action .......................... 16
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3.2.4. Antibody targets ....................................................................................... 18
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Vascular tumor targeting ............................................................................................ 20
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3.3. Armed antibodies ............................................................................................ 23
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3.3.1. Immunocytokines ..................................................................................... 24
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Cytokine moieties of immunocytokines ...................................................................... 27
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IL2-based immunocytokines ....................................................................................... 29
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3.3.2 Antibody-Drug Conjugates ........................................................................ 31
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ADC targets, formats and function ............................................................................. 32
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Conjugation strategies and ADC linker technologies ................................................. 34
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Cytotoxic payloads ...................................................................................................... 38
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Clinical stage ADCs .................................................................................................... 41
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3.4. Acute Myeloid Leukemia ................................................................................. 50
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3.4.1. Disease and classification ........................................................................ 51
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3.4.2 Treatment of AML ..................................................................................... 53
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Antibody-based therapies ........................................................................................... 54
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3.5 Aim of thesis .................................................................................................... 56
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4. Results ...................................................................................................... 58!
4.1. Immunochemical analysis of the expression of vascular targeting antigens in
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AML patient specimens. ......................................................................................... 59
4.2. Generation of mouse models of leukemia: analysis of antigen expression and
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targeting by biodistribution studies. ........................................................................ 64
4.2.1 systemic AML models ............................................................................... 64
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4.3. F8-IL2 immunocytokine in combination with cytarabine ................................. 68
4.3.1 Therapeutic effect of F8-IL2 and cytarabine in NB4 xenograft model ....... 68
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4.3.2 Therapeutic effect of F8-IL2 and cytarabine in syngeneic C1498 model .. 70
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4.3.3. Clinical evaluation of F16-IL2 in combination with low-dose cytarabine .. 73
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4.4 Combination of immunocytokines and ADCs ................................................... 75
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4.4.1 Generation and characterization of armed antibody products .................. 76
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4.4.2 Therapeutic effect of F8-IL2 in combination with F8-SS-CH Cem ............ 78
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5. Discussion ................................................................................................ 81!
5.1. Vascular targeting IL2-immunocytokines in combination with cytarabine for the
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treatment of AML ................................................................................................... 81
5.2. Combining IL2-based immunocytokines with ADCs ....................................... 86
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6. Conclusions and outlook ........................................................................ 89!
7. Materials and methods ............................................................................ 94!
7.1 Immunochemical analysis of leukemia specimens .......................................... 94
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7.1.1 Study design and tissues .......................................................................... 94
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7.1.2 Antibodies ................................................................................................. 95
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7.1.3 Immunofluorescence ................................................................................. 95
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Semi-quantitative analysis of immunofluorescence .................................................... 96
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7.1.4 Immunohistochemistry .............................................................................. 96
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7.2 Mouse models of AML ..................................................................................... 97
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7.2.1 Cell lines and animals ............................................................................... 97
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7.2.2 Immunofluorescence of murine tumor and bone marrow specimens ....... 97
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7.2.3 Orthotopic xenograft model ....................................................................... 98
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Verification of disease progression in HL60 model .................................................... 98
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7.2.4 Localized xenograft (chloroma) model ...................................................... 99
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Quantitative biodistribution studies ............................................................................. 99
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7.3 F8-IL2 in combination with cytarabine ........................................................... 100
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7.3.1 Therapeutic agents ................................................................................. 100
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7.3.2 Therapy studies in localized xenograft (chloroma) models ..................... 100
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7.3.3 In vivo depletion of NK cells, CD4+ and CD8+ T cells ............................. 101
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7.3.4 Treatment of a chloroma patient ............................................................. 101
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7.4 F8-IL2 in combination with ADCs ................................................................... 104
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7.4.1 Cell lines, animals, and xenograft models ............................................... 104
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7.4.2 Antibodies ............................................................................................... 104
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7.4.3 Preparation of ADCs ............................................................................... 104
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7.4.4 Characterization of the armed antibody products ................................... 104
7.4.5 Combination therapy and tumor re-challenge in the localized C1498 model
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......................................................................................................................... 105
7.4.6 In vivo depletion of NK cells, CD4+ T cells, and CD8+ T cells ................. 106
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8. Copyright ................................................................................................ 107!
9. List of abbreviations .............................................................................. 108!
10. References ............................................................................................ 110!
11. Appendix ............................................................................................... 124!
11.1 Complete panel of stainings for immunochemical analysis of the expression
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of vascular targeting antigens in AML and ALL patient specimens ..................... 124
11.2 Characterization of the analyzed AML and ALL patients ............................. 128
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11.3 Cytarabine dose finding in subcutaneous NB4 mouse model ..................... 130
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11.4 Generation of traceless site-selective disulfide linked ADCs in the SIP format
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............................................................................................................................. 131
11.5 Expression of an anti-hCD33 antibody in SIP format and analysis of AML
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targeting properties. ............................................................................................. 132
11.6 Sequence of anti!hCD33 antibody in SIP format ........................................ 134
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12. Acknowledgments ............................................................................... 136!
13. Curriculum Vitae .................................................................................. 138!
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1. Summary
“Arming” antibodies with potent payloads (e.g cytotoxic drugs and cytokines),
in order to increase activity at the site of disease and spare normal tissues,
represents an attractive strategy of emerging importance in the field of cancer
therapeutics. The growth and progression of solid cancers depends on the
formation of new blood vessels, which provide accessible and selective
targets for antibody-mediated pharmacodelivery. Indeed, antibodies directed
against neo-vascular antigens have shown selective tumor localization and
potent antitumor activity as antibody-cytokine fusion proteins
(immunocytokines), allowing these products to progress to phase II clinical
trials for the treatment of solid cancers. Recent findings suggest, that these
antibody products may also be applicable for the treatment of hematological
cancers, such as Acute Myeloid Leukemia (AML).
In this thesis, we analyze biopsies of AML and Acute Lymphoid Leukemia
(ALL) patients with the clinical-stage human monoclonal antibodies F8, L19
and F16, directed against markers of tumor angiogenesis. The analysis
revealed that the F8 and F16 antibodies strongly stained 70% of AML and
75% of ALL bone marrow specimens, while chloroma biopsies were stained
with all three antibodies.
The immunocytokine F8-IL2 (the F8 antibody fused to human interleukin-2)
promoted significant tumor inhibition in immunocompromised mice bearing
human leukemia and promoted complete tumor eradication in combination
with cytarabine in 40% of the treated animals. Immunocompetent mice
bearing murine leukemia revealed long-lasting complete tumor eradication
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