Development of a highly potent bispecific antibody format targeting the noveltumor-specific antigen

128 Pages·2014·6.99 MB·English

by  Julia Holland

D
EVELOPMENT OF A HIGHLY POTENT

BISPECIFIC ANTIBODY FORMAT

TARGETING THE NOVEL
- CLDN18.2
TUMOR SPECIFIC ANTIGEN

DISSERTATION ZUR ERLANGUNG DES GRADES
„DOKTOR DER NATURWISSENSCHAFTEN“

AM FACHBEREICH BIOLOGIE
DER

VORGELEGT VON

JULIA HOLLAND

GEBOREN IN MAINZ

BRÜGGEN, DER 11. NOVEMBER 2014
MITGLIEDER DER PROMOTIONSKOMMISSION

Dekan:

Gutachter:

Gutachter:

Tag der mündlichen Prüfung: 22.01.2014

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EHRENWÖRTLICHE ERKLÄRUNG

Hiermit versichere ich, dass ich die vorliegende Dissertation selbständig und ohne unerlaubte Hilfe
verfasst und keine anderen als die angegebenen Hilfsmittel und Quellen benutzt habe. Ich habe we-
der anderweitig versucht, eine Dissertation einzureichen oder eine Doktorprüfung durchzuführen,
noch habe ich diese Dissertation oder Teile derselben einer anderen Prüfungskommission vorgelegt.

Brüggen, der 11. November 2014

________________________________
Julia Holland
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SUMMARY
SUMMARY
Due to multiple immune evasion mechanisms of cancer cells, novel therapy approaches are required to
overcome the limitations of existing immunotherapies. Bispecific antibodies are potent anti-cancer
drugs, which redirect effector T cells for specific tumor cell lysis, thus enabling the patient’s immune
system to fight cancer cells.
The antibody format used in this proof of concept study–bispecific ideal monoclonal antibodies termed
BiMAB–is a tailor-made recombinant protein, which consists of two fused scFv antibodies recognizing
different antigens. Both are arranged in tandem on a single peptide chain and the individual variable
binding domains are separated by special non-immunogenic linkers. The format is comprised of a scFv
targeting CLDN18.2–a gastric cancer tumor associated antigen (TAA) –while the second specificity
binds the CD3 epsilon (CD3ε) subunit of the T cell receptor (TCR) on T cells.
For the first time, we compared in our IMAB362-based BiMAB setting, four different anti-CD3-scFvs,
respectively derived from the mAbs TR66, CLB-T3, as well as the humanized and the murine variant of
UCHT1. In addition, we investigated the impact of an N- versus a C-terminal location of the IMAB362-
derived scFv and the anti-CD3-scFvs. Thus, nine CLDN18.2 specific BiMAB proteins were generated, of
which all showed a remarkably high cytotoxicity towards CLDN18.2-positive tumor cells.
Because of its promising effectiveness, 1BiMAB emerged as the BiMAB prototype. The selectivity of
1BiMAB for its TAA and CD3ε, with affinities in the nanomolar range, has been confirmed by in vitro
assays. Its dual binding depends on the design of an N-terminally positioned IMAB362 scFv and the
consecutive C-terminally positioned TR66 scFv.
1BiMAB provoked a concentration and target cell dependent T cell activation, proliferation, and upreg-
ulation of the cytolytic protein Granzyme B, as well as the consequent elimination of target cells.
Our results demonstrate that 1BiMAB is able to activate T cells independent of elements that are usu-
ally involved in the T cell recognition program, like antigen presentation, MHC restriction, and co-stim-
ulatory effector molecules. In the first in vivo studies using a subcutaneous xenogeneic tumor mouse
model in immune incompetent NSG1 mice, we could prove a significant therapeutic effect of 1BiMAB
with partial or complete tumor elimination.
The initial in vitro RIBOMAB experiments correspondingly showed encouraging results. The electro-
poration of 1BiMAB IVT-RNA into target or effector cells was feasible, while the functionality of trans-
lated 1BiMAB was proven by induced T cell activation and target cell lysis.
Accordingly, we could show that the in vitro RIBOMAB approach was applicable for all nine BiMABs,
which proves the RIBOMAB concept.
Thus, the CLDN18.2-BiMAB strategy offers great potential for the treatment of cancer. In the future,
administered either as protein or as IVT-RNA, the BiMAB format will contribute towards finding solu-
tions to raise and sustain tumor-specific cellular responses elicited by engaged and activated endoge-
nous T cells. This will potentially enable us to overcome immune evasion mechanisms of tumor cells,
consequently supporting current solid gastric cancer therapies.

1 NOD scid gamma (NSG):NOD.Cg-Prkdscid IL2rgtm1Wjl/SzJ (Jackson laboratory, Bar Harbour, ME, USA)
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INDEX

TABLE OF CONTENT
MITGLIEDER DER PROMOTIONSKOMMISSION II
EHRENWÖRTLICHE ERKLÄRUNG III
SUMMARY V
TABLE OF CONTENT VI
LIST OF TABLES IX
LIST OF FIGURES X
1 INTRODUCTION – TARGETED CANCER IMMUNOTHERAPY 0
1.1 Claudin-18 splice variant2–a biomarker for gastric cancer .............................................. 1
1.1.1 CLDN18.2–Target structure and tissue expression pattern ............................................. 1
1.2 Clinical validation of IMAB362 in gastric cancers targeting CLDN18.2 ............................ 3
1.3 Bispecific Antibodies and T cell-based cancer immunotherapy ....................................... 5
1.3.1 Concept of the bispecific-single-chain antibody format .................................................. 5
1.3.2 Differences in peptide- and bs-scAbs-mediated induction of T cell activation ................ 7
1.3.3 Mode of action of bs-scAbs ............................................................................................. 9
1.3.4 Advantages of the bs-scAbs over the bs-IgG format ..................................................... 10
1.3.5 Bs-scAbs in clinical trials ............................................................................................... 11
1.4 A new strategy for drug application– RIBOMABs– IVT-RNAs encoding BiMABs .......... 14
1.5 Objectives of doctoral dissertation ............................................................................... 15
2 MATERIALS & METHODS 16
2.1 Materials....................................................................................................................... 16
2.2 Hardware ...................................................................................................................... 16
2.2.1 Consumables ................................................................................................................ 17
2.3 Chemicals and reagents ................................................................................................ 17
2.3.1 Restriction enzymes ..................................................................................................... 17
2.3.2 Kits ............................................................................................................................... 17
2.3.3 Antibodies and enzyme-conjugates .............................................................................. 18
2.3.4 Media ........................................................................................................................... 19
2.3.5 Buffer ........................................................................................................................... 19
2.4 Eukaryotic cell lines ...................................................................................................... 21
2.5 Molecular biological methods ....................................................................................... 21
2.5.1 Oligonucleotide primer ................................................................................................. 21
2.5.2 Protein expression plasmids ......................................................................................... 22
2.5.3 IVT-RNA template plasmids ......................................................................................... 22
2.5.4 Pharmacologically optimized synthetic mRNA ............................................................. 22
2.5.5 DNA sequencing ........................................................................................................... 24
2.5.6 Sequence origin, design of BiMAB constructs, and cloning into expression vectors ...... 24

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INDEX
2.5.7 Design of the BiMAB and the elements required for a BiMAB protein assembly ........... 24
2.5.8 RT PCR ......................................................................................................................... 26
2.5.9 In vitro transcribed RNA synthesis ................................................................................ 27
2.6 Cell biological methods ................................................................................................ 27
2.6.1 Microscopic analysis ..................................................................................................... 27
2.6.2 Determination of cell density by Neubauer chamber .................................................... 27
2.6.3 Ficoll isolation of peripheral blood mononuclear cells (PBMCs) from buffy coats .......... 27
2.6.4 Effector cell preparation ............................................................................................... 28
2.6.5 Coincubation of effector and target cells ...................................................................... 29
2.6.6 Luciferase-based cytotoxicity assay using BiMAB proteins ........................................... 29
2.6.7 IVT-RNA electroporation .............................................................................................. 30
2.6.8 Transient transfection for small scale protein production ............................................. 31
2.6.9 Stable transfection ....................................................................................................... 32
2.7 Protein biochemical methods ....................................................................................... 32
2.7.1 Purification of BiMABs by affinity chromatography via His-Tag ................................... 32
2.7.2 Small scale purification of BiMABs................................................................................ 33
2.7.3 Concentration by protein filter ..................................................................................... 33
2.7.4 SDS-PAGE and Coomassie-staining ............................................................................. 33
2.7.5 Western blot analysis .................................................................................................... 33
2.7.6 Determination of protein concentration ....................................................................... 34
2.8 Biochemical and immunological methods .................................................................... 35
2.8.1 BiMAB detection by immunofluorescence labeling of cells ........................................... 35
2.8.2 BiMAB detection by immunocytochemistry (ICC) ......................................................... 35
2.8.3 Flow cytometry direct/indirect staining of cell surface markers .................................... 35
2.9 In vivo models ............................................................................................................... 38
2.9.1 Mouse strains ............................................................................................................... 38
2.9.2 Adequate animal housing ............................................................................................. 38
2.9.3 Anesthesia and sacrifice of mice ................................................................................... 39
2.9.4 Blood retrieval .............................................................................................................. 39
2.9.5 Organ dissection ........................................................................................................... 39
2.9.6 Analysis of engraftment of human T lymphocytes ........................................................ 39
2.9.7 Adoptive transfer of effector cells ................................................................................. 40
2.9.8 Engraftment of tumor cells ........................................................................................... 40
2.9.9 Human xenograft model ............................................................................................... 40
2.9.10 Determination of therapy influence on body weight ..................................................... 41
2.9.11 Application techniques ................................................................................................. 41
2.10 Statistical methods ....................................................................................................... 41
3 RESULTS–BIMAB THE PROOF OF CONCEPT 42
3.1 Successive cloning of BiMAB plasmids ......................................................................... 42
3.1.1 Design .......................................................................................................................... 42
3.1.2 Cloning ......................................................................................................................... 42
3.2 BiMAB proteins are expressed by mammalian cells ...................................................... 44
3.2.1 Immuno-cyto-chemical and Western blot detection of purified BiMABs produced by
HEK293 (T) cells ........................................................................................................... 44
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INDEX
3.2.2 Quantification of BiMAB using an IMAB362 specific ELISA ........................................... 45
3.3 In vitro studies-Selection of promising BiMABs ............................................................. 47
3.3.1 All CLDN18.2-specific BiMABs induced a potent target cell lysis .................................. 47
3.3.2 Functional protein characterization of 5 selected BiMABs ............................................ 49
3.3.3 Potent induction of T cell activation by selected BiMABs .............................................. 50
3.3.4 Potent induction of target cell lysis by all five BiMABs .................................................. 52
3.4 In vitro studies– Production and characterization of most potent candidate–1BiMAB .. 54
3.4.1 1BiMAB producer clones identified via ELISA ............................................................... 54
3.4.2 Feasibility of 1BiMAB protein purification and analysis ................................................. 54
3.4.3 Purification of 1.3 mg 1BiMAB protein per liter supernatant ......................................... 55
3.4.4 Purified 1BiMAB protein–functional characterization ................................................... 56
3.4.5 Evidence of 1BiMAB-mediated T cell activation and cytolytic function ......................... 59
3.5 In Vivo– 1BiMAB treatment reduces tumor size in a xenogeneic tumor mouse model. . 68
3.5.1 Ex vivo functionality test of T cells previously engrafted in NSG mice ........................... 68
3.5.2 Prolonged survival due to 1BiMAB treatment in a xenogeneic mouse model ............... 68
3.5.3 Tumor size reduction is reliant on the quality of human T cell and tumor cell
engraftment in xenogeneic tumor mouse model ......................................................... 70
3.6 Summary of the in vitro and in vivo characterization of 1BiMAB proteins ..................... 73
3.7 RIBOMABs –IVT-RNA-based bispecific antibodies ....................................................... 74
3.7.1 BioAnalyzer quality assurance of BiMAB IVT-RNA and concentration estimation of
cellular expressed 1BiMAB protein .............................................................................. 74
3.7.2 Characterization and proof of concept using IVT-RNA candidate 1BiMAB .................... 75
3.7.3 Proof of BiMAB IVT-RNA concept testing all CLDN18.2-specific BiMABs ..................... 80
3.7.4 Summary–Proof Of Concept for RIBOMABs ................................................................. 81
4 DISCUSSION 82
4.1 The bispecific antibody approach "BiMAB" in cancer immunotherapy .......................... 82
4.2 Modular design for an expandable BiMAB cloning platform ......................................... 82
4.3 Properties of CLDN18.2-specific BiMABs...................................................................... 83
4.3.1 Evaluation of BiMAB production efficiencies ................................................................ 83
4.3.2 Binding specificities and monovalent binding affinities ................................................ 84
4.3.3 Safety profiles and specificities of all BiMABs ............................................................... 86
4.3.4 Selection of the most effective BiMAB in cancer cell elimination .................................. 87
4.4 Evaluation of 1BiMAB ................................................................................................... 89
4.4.1 Setup differences in bs-scAb formats ........................................................................... 89
4.4.2 In vitro performance and aspects of the mode of action................................................ 90
4.4.3 Efficacy of 1BiMAB in vivo ............................................................................................. 92
4.5 Summary of 1BiMAB evaluation ................................................................................... 95
4.6 Perspectives– 1BiMAB in the drug development process .............................................. 95
4.7 In vitro transcribed messenger RNA as therapeutic approach ....................................... 97
4.8 Proof Of Concept for RIBOMABs–IVT-RNA-based bispecific antibodies ....................... 98
4.9 Perspectives–challenges and opportunities in the delivery of RIBOMABs ..................... 99
4.9.1 Limitations of durable IVT-RNA translation via cellular response mechanisms ........... 100

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INDEX
5 REFERENCES 102
6 APPENDIX 112
6.1 Information on used BiMAB protein elements ............................................................ 112
6.2 Information on BiMAB proteins .................................................................................. 112
6.3 Abbreviations ............................................................................................................. 113
7 ACKNOWLEDGEMENTS 114
8 CURRICULUM VITAE JULIA HOLLAND 115

LIST OF TABLES
Table 1: Tumor associated antigens targeted by bs-td-scAb ............................................................ 11
Table 2: Hardware ............................................................................................................................ 16
Table 3: Kits ................................................................................................................................... 18
Table 4: Antibodies and enzyme-conjugates .................................................................................... 18
Table 5: Cell lines .............................................................................................................................. 21
Table 6: Oligonucleotide primer ....................................................................................................... 21
Table 7: Used plasmids ..................................................................................................................... 24
Table 8: Codon usage ....................................................................................................................... 24
Table 9: CD3 variable domain sequence origin and internal nomenclature ....................................... 25
Table 10: Parameters used for the electroporation of different cell types......................................... 30
Table 11: BiMAB concentrations determined via ELISA .................................................................... 45
Table 12: The statistical significances with respect to the lysis efficiency comparing 1BiMAB with
every other BiMAB ..................................................................................................... 49
Table 13: 1BiMAB-mediated lysis is significantly higher compared to all four remaining BiMABs6 ... 53
Table 14: Protein parameter calculated by ProtParam tool .............................................................. 55
Table 15: Tumor cell lines used and their origin ................................................................................ 57
Table 16: Significant differences in median tumor size ..................................................................... 70
Table 17: Tumor free survival after 1BiMAB therapy ......................................................................... 72
Table 18: Linker amino acid sequences used in BiMAB formats ...................................................... 112
Table 19: Secretion signal of BiMABs ............................................................................................. 112
Table 20: BiMAB protein size .......................................................................................................... 112
Table 21: List of abbreviations ........................................................................................................ 113

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INDEX
LIST OF FIGURES
Figure 1: Estimated new cancer cases and deaths worldwide for leading cancer sites in 2008. ........... 1
Figure 2: Compilation of CLDN18.2 characteristics ............................................................................. 2
Figure 3: Pharmacological advantages of Ganymed´s ideal antibodies–IMABs .................................. 4
Figure 4:Scheme of two recombinant bispecific antibody formats: Diabodies and tandem-scFvs ...... 6
Figure 5: Peptide-mediated versus bs-scAb-mediated T cell activation .............................................. 8
Figure 6: Schematic representations of bispecific single-chain-based scaffolds. .............................. 12
Figure 7: Typical structure of a mature eukaryotic synthetic mRNA ................................................. 23
Figure 8: Consecutive order of both scFvs within the BiMAB format. ............................................... 25
Figure 9: Design of the bispecific-tandem-single-chain-antibodies–BiMABs .................................... 43
Figure 10: Scheme illustrating the chronology of cloning BiMABs into expression and template
plasmids ..................................................................................................................... 44
Figure 11: BiMAB proteins produced by transfected HEK293(T) cells ............................................... 46
Figure 12: Outcome of induced cancer cell lysis depended on the used BiMAB protein .................... 48
Figure 13: All five selected BiMABs were capable of binding CLDN18.2 and CD3.............................. 50
Figure 14: BiMAB induced cancer cell lysis correlates with T cell activation ...................................... 51
Figure 15: The titration of the five selected BiMAB proteins evidenced differences in the efficiency of
target cell lysis. .......................................................................................................... 52
Figure 16: 1BiMAB protein could be purified from HEK293 supernatant via IMAC. ........................... 55
Figure 17:Binding affinities for 1BiMAB defined by saturation binding lay within the nanomolar
range .......................................................................................................................... 56
Figure 18: 1BiMAB binding, T cell activation and target cell lysis is strictly TAA dependent. ............ 58
Figure 19: 1BiMAB induced cytolytic synapse formation as visualized by microscopic imaging ........ 60
Figure 20: 1BiMAB mediates T cell proliferation dependent on the presence of the TAA CLDN18.2 61
Figure 21: 1BiMAB induces the upregulation of Granzyme B production in human T cells ................ 62
Figure 22: 1BiMAB induced T cell effector functions in a donor- and dose-dependent manner ........ 63
Figure 23: CD8 and CD4-positive effector cells contribute to 1BiMAB-mediated T cell activation .... 65
Figure 24: 1BiMAB induced cancer cell lysis engaging different subsets of effector cells .................. 66
Figure 25: Effective target cell lysis at low E:T ratios using 1BiMAB .................................................. 67
Figure 26: 1BiMAB induces effector functions of ex vivo human T cells ............................................ 69
Figure 27: 1BiMAB mediates prolonged survival in a xenogeneic tumor mouse model ..................... 71
Figure 28: Heterogeneous distribution of TAA in xenogeneic engrafted HEK293-CLDN18.2 tumors
have an impact on 1BiMAB-mediated tumor size reduction ....................................... 72
Figure 29: Pure IVT-RNA is produced by in vitro transcription........................................................... 74
Figure 30: 1BiMAB IVT-RNA induced cancer target cell lysis could be visualized by microscopic
imaging ...................................................................................................................... 75
Figure 31: 1BiMAB-mediated T cell activation and cancer target cell lysis was not restricted to
1BiMAB releasing target cells. .................................................................................... 76
Figure 32: Expression of 1BiMAB IVT-RNA induced T cell effector functions in a dose-dependent
manner ...................................................................................................................... 77
Figure 33: Efficiency of cancer target cell lysis was dependent on the effector to target ratio .......... 78
Figure 34: 1BiMAB IVT-RNA expressed by T cells showed a similar potent induction of cancer target
cells lysis and T cell activation as detected with 1BiMAB expressed by cancer target
cells. ........................................................................................................................... 79
Figure 35: All CLDN18.2-specific RIBOMABs were capable of cancer target cell lysis induction and T
cell activation ............................................................................................................. 81

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