Generation of a panel of Plasmodium falciparum apical membrane antigen 1-specific human ...

157 Pages·2016·8.19 MB·English

Generation of a panel of Plasmodium falciparum apical
membrane antigen 1-specific human monoclonal antibodies by
phage display chain shuffling

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH
Aachen University zur Erlangung des akademischen Grades
einer Doktorin der Naturwissenschaften genehmigte Dissertation

vorgelegt von
Diplom-Biologin
Melanie Seidel
aus Aachen

Berichter: Universitätsprofessor Dr. rer. nat Rainer Fischer
Honorarprofessor Dr. rer. nat. Stephan Wnendt

Tag der mündlichen Prüfung: 12.12.2016

Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar.
Summary
Plasmodium falciparum which causes malaria tropica, still remains one of the most important
causes of morbidity and mortality worldwide. Emerging resistances against anti-malarial drugs
make novel therapeutic agents and strategies necessary. Antimalarial antibodies have been
discussed as alternative therapy for otherwise non-treatable patients or prophylaxis for travelers
as it is known that antibodies substantially contribute to natural acquired immunity against
malaria. Despite the progress in the field of malaria vaccine development that has recently been
achieved with the approval of vaccine candidate RTS,S/AS01, more effective second-
generation vaccines are required. For the rational design of such vaccines, a clear understanding
of protective malaria immunity is required. The study of antimalarial monoclonal antibodies
gives important insight into the underlying mechanisms. For this purpose, a parasite growth
inhibitory human monoclonal antibody (OcheA) directed against the polymorphic
P. falciparum asexual blood-stage protein, apical membrane antigen 1 (AMA1), was subjected
to phage display mediated chain shuffling and the resulting antibodies were characterized.
Chain shuffling was chosen as a tool for in vitro affinity maturation. Light chain shuffling phage
display antibody libraries were constructed in two different phagemids, pRF and pFlx, using
cDNA derived from peripheral blood total RNA of 50 malaria immune Ghanaian donors. Phage
display bio-panning of both libraries against a mix of three diversity-covering AMA1 variants,
DiCo1-3, using two types of packaging phages, led to the enrichment of four light chains. The
shuffled antibodies were transiently expressed in the IgG1 format in Nicotiana benthamiana,
achieving up to 73-fold improved accumulation levels compared to OcheA. The specificity of
the antibodies could be confirmed by enzyme linked immunosorbent assay (ELISA) and
immunofluorescence assay (IFA) whilst the affinity to different AMA1 variants was determined
by surface plasmon resonance spectroscopy, showing a 12-fold improved affinity of the highest
affinity antibody against DiCo3. An inhibitory activity of the antibodies was confirmed using
in vitro growth inhibition assays (GIA) against four P. falciparum strains. Subsequently, two
antibodies were selected for heavy chain shuffling and the libraries were constructed in
phagemid pFlx. After two rounds of panning against DiCo 1 or DiCo1-3 an enrichment of
highly homologous heavy chains of the same V gene allele as the parental heavy chain was
observed. Several of the 11 identified homologous antibodies were transiently expressed in
N. benthamiana at comparably high accumulation levels in the range of 280 to 730 mg/kg fresh
leaf mass. A further up to 21-fold increase of affinity (to 240 pM) could be experimentally
traced back to a heavy chain CDR2 residue. For two selected antibodies, E-9 and F-17B,
specificity against several recombinant AMA1 variants was confirmed by ELISA while the
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cross-strain specific recognition of parasite expressed AMA1 was demonstrated by IFA against
five P. falciparum strains. AMA1 binding interference with all chain shuffled antibodies as well
as with anti-AMA1-antibody 4G2 suggested overlapping epitopes. By conformational epitope
mapping using representative antibody F-17B and AMA1 3D7, an α-helical 13 amino acid
epitope at the interface of AMA1 domain I and II, containing two polymorphic residues, was
identified. Strain-spanning growth inhibitory activities against seven P. falciparum strains with
minimal EC values of 0.25 mg/mL were determined by GIA. According to Loewe additivity,
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a synergistic interaction of heavy chain shuffled antibody E-9 with anti-AMA1 antibody 1E4
in GIA was suggested. The here generated panel of homologous growth inhibitory human
monoclonal antibodies is expected to prove valuable for elucidating mechanisms of growth
inhibition and protective malaria immunity. This knowledge is highly important for the targeted
development of therapeutic monoclonal antibodies and next-generation vaccines.
Moreover, the chain shuffled antibodies possess a therapeutic potential against P. falciparum
malaria, which might be improved by further targeted affinity maturation, additional
modifications, and reasonable combination with other inhibitory antibodies.

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Zusammenfassung
Plasmodium falciparum, der Erreger der Malaria tropica ist bis heute einer der bedeutendsten
Ursachen für weltweite Morbidität und Mortalität. Auftretende Resistenzen gegen Malaria-
Medikamente verlangen nach neuartigen Therapeutika und Therapiestrategien. Die Benutzung
von Anti-Malaria-Antikörpern wurde als Alternative für anderweitig nicht therapierbare
Patienten oder zur Prophylaxe von Reisenden diskutiert, da Antikörper bekanntermaßen
maßgeblich zur natürlich erworbenen Immunität gegen Malaria beitragen. Trotz des Fortschritts
im Bereich der Malaria-Vakzinforschung, welcher kürzlich mit der Zulassung des
Vakzinkandidaten RTS,S/AS01 erreicht wurde, sind effektivere Vakzine der zweiten
Generation nötig. Für das sinnvolle Design solcher Vakzine ist ein klares Verständnis der
Malaria-Immunität notwendig. Die Studie von entsprechenden monoklonalen Anti-Malaria-
Antikörpern gibt wichtige Einblicke in die zugrundeliegenden Mechanismen. Zu diesem Zweck
wurde hier ein parasitenwachstumsinhibierender humaner monoklonaler Antikörper, der gegen
das polymorphe, asexuelle Blutstadiums-Protein „Apical membrane antigen 1“ (AMA1) aus P.
falciparum gerichtet ist, einem „Phage Display Chain shuffling” unterzogen und die
resultierenden Antikörper charakterisiert. Für das „Light chain shuffling“ wurden- Phage
Display- Antikörperbibliotheken unter der Benutzung von cDNA, die aus der totalen RNA von
peripherem Blut von 50 malaria-immunen ghanaischen Donoren stammte, in zwei
verschiedenen Phagemidsystemen konstruiert. Das Phage Display „Bio-Panning“ beider
Bibliotheken wurde gegen eine Mischung dreier diversitätsabdeckender AMA1-Varianten,
DiCo1-3, unter Benutzung zweier Arten von Helferphagen durchgeführt und führte zur
Anreicherung von vier „leichten Ketten“. Die „geshuffelten“Antikörper wurden transient im
IgG1-Format in Nicotiana benthamiana exprimiert, wobei sie im Vergleich zu OcheA bis zu
73-fach erhöhte Akkumulationslevel erreichten. Die Spezifität der Antikörper konnte durch
Enzyme linked immunosorbent assay (ELISA) und Immunfluoreszenz Assays (IFA) bestätigt
werden. Die Affinitäten zu verschiedenen AMA1-Varianten wurden durch
Oberflächenplasmonresonanz-Spektroskopie bestimmt, wobei eine 12-fache Verbesserung der
Affinität des höchstaffinen Antikörpers gegen DiCo3 gezeigt wurde. Eine inhibitorische
Aktivität der Antikörper wurde in vitro mittels Wachstumsinhibitions-Assay (GIA) gegen vier
P. falciparum-Stämme bestätigt. Anschließend wurden zwei Antikörper für ein „Heavy Chain-
Shuffling“ ausgewählt und die entsprechenden Bibliotheken in pFlx konsturiert. Nach zwei
Panningrunden gegen DiCo1 oder DiCo1-3 wurde eine Anreicherung höchst homologer
schwerer Ketten, deren Allel dem V-Gen Allel der parentalen schweren Kette entsprach,
beobachtet. Einige der elf identifizierten homologen Antikörper wurden mit vergleichbar hohen
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Akkumulationsraten zwischen 280 und 730 mg/kg Blatt-Frischgewicht transient in
N. benthamiana exprimiert. Eine weitere bis zu 21-fache Erhöhung der Affinität konnte
experimentell auf einen CDR2-Rest der schweren Kette zurückgeführt werden. Zwei
Antikörper, E-9 und F-17B, wurden weitergehend charakterisiert. Die Spezifität gegen mehrere
rekombinante AMA1-Varianten wurde durch ELISA bestätigt, während die kreuzreaktive
spezifische Erkennung von parasiten-exprimiertem AMA1 gegen fünf P. falciparum- Stämme
mittels IFA demonstriert wurde. AMA1-Bindungsinterferenzen aller „chain-geshuffelten“
Antikörper als auch mit AMA1-Antikörper 4G2 deuteten auf überlappende Epitope hin. Mittels
konformationsabhängiger Epitopkartierung unter Benutzung des repräsentativen Antikörpers
F-17B und Antigen AMA1 3D7 wurde ein α- helikales, 13 Aminosäuren langes Epitop an der
Schnittstelle von AMA1 Domäne I und II identifiziert, welches zwei polymorphe Reste
aufwies. Eine stammumfassende wachstumsinhibitorische Aktivität gegen sieben verschiedene
P. falciparum- Stämme mit einer minimalen EC von 0,25 mg/mL wurde mittels GIA gezeigt.
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Laut dem Loewe-Additivitäts-Modell wurde ein Synergismus des „heavy chain- geshuffelten“
Antikörper E-9 mit AMA1- Antikörper 1E4 im GIA festgestellt. Es ist zu erwarten, dass sich
die hier generierte Reihe homologer, wachstumsinhibitorischer, humaner, monoklonaler
Antikörper als wertvoll für die Aufklärung von Mechanismen der Wachstumsinhibition und der
Malaria-Immunität erweisen wird. Dieses Wissen ist von großer Bedeutung für die
zielgerichtete Entwicklung von therapeutischen monoklonalen Antikörpern als auch Vakzinen
der nächsten Generation. Außerdem besitzen die „ketten-geshuffelten“ Antikörper ein
therapeutisches Potential gegen P. falciparum-Malaria, das durch weitere zielgerichtete
Affinitätsreifung, zusätzliche Modifikationen und sinnvolle Kombination mit weiteren
inhibitorischen Antikörpern noch verbessert werden könnte.

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Scientific publications

1. Maskus DJ, Bethke S, Seidel M, Kapelski S, Addai-Mensah O, Boes A, Edgü G,
Spiegel H, Reimann A, Fischer R, Barth S, Klockenbring T, Fendel R: Isolation,
production and characterization of fully human monoclonal antibodies directed to
Plasmodium falciparum MSP10. Malar J (2015), 14: 276

2. Addai-Mensah A, Seidel M, Amidu N, Maskus D, Kapelski S, Breuer G, Franken C,
Dabo EO, Frempong M, Schinkel H, Spiegel H, Reimann A, Klockenbring T, Barth S,
Fischer R, Fendel R: Acquired immune responses to three malaria vaccine candidates
and their relationship to invasion inhibition in two populations naturally exposed to
malaria. Malar J (2016), 15 (1): 65

3. Niesen J, Sack M, Seidel M, Fendel R, Fischer R, Stein C: The SNAP-tag technology:
a useful tool to determine affinity constants and other functional parameters of novel
antibody fragments. Bioconjugate Chem (2016), 27: 1931-41
Submitted
4. Seidel M, Addai-Mensah O, Dipah GN, Schmitz S, Spiegel H, Klockenbring T, Fischer
R, Reimann A, Barth S, Fendel R: Isolation and affinity maturation of a growth
inhibitory AMA1 specific antibody by phage display.

5. Maskus DJ, Spiegel H, Krolik M, Kapelski M, Seidel M, Bethke S, Reimann A,
Klockenbring T, Barth S, Fischer R, Fendel R: Characterization of a novel inhibitory
human monoclonal antibody directed against Plasmodium falciparum apical membrane
antigen 1

In preparation

6. Seidel M, Spiegel H, Breuer G, Klockenbring T, Fischer R, Fendel R.: Phage display
mediated heavy chain shuffling and epitope mapping of AMA1-specific inhibitory
human monoclonal antibodies.

7. Seidel M, Fendel R, Fischer R: Monoclonal Malaria antibodies
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Poster presentation

Seidel M, Addai-Mensah O, Fischer R, Klockenbring T, Barth S, Fendel R: Isolation of
Plasmodium falciparum specific monoclonal human antibodies by mini phage display. Malaria
vaccines for the world, 2013 April 22nd - 24th; CHUV, Lausanne, Switzerland.

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Table of contents
INTRODUCTION ................................................................................................... 1
I.1 Plasmodium falciparum, the parasite causing Malaria tropica .................................... 1
I.2 Malaria immunity .................................................................................................. 2
I.3 Plasmodium falciparum life cycle ............................................................................ 3
I.3.1 Merozoite invasion of erythrocytes ................................................................... 5
I.3.2 Apical membrane antigen 1 ............................................................................. 7
I.4 Antibody-based therapy for malaria ........................................................................ 8
I.5 Antibody engineering ............................................................................................. 9
I.6 Antibody phage display ........................................................................................ 10
I.7 Affinity maturation .............................................................................................. 12
I.8 Aims and Objectives ............................................................................................ 13
MATERIALS AND METHODS .............................................................................. 16
II.1 Materials ........................................................................................................... 16
II.1.1 Chemicals and consumables ........................................................................ 16
II.1.2 Buffers, solutions and media ........................................................................ 16
II.1.3 Antibodies and enzymatic substrates ............................................................ 19
II.1.4 Enzymes and reaction kits ........................................................................... 20
II.1.5 Laboratory equipment ................................................................................. 20
II.1.6 Bacterial strains, parasite strains, plants and helper phages ............................ 22
II.1.7 Oligonucleotides and plasmids ..................................................................... 23
II.1.8 Human samples and ethical approval ............................................................ 23
II.1.9 Recombinant proteins and peptides .............................................................. 24
II.1.10 Approbation for the conducted work ........................................................... 24
II.2 Methods ............................................................................................................ 25
II.2.1 Molecular Biological Methods ....................................................................... 25
II.2.1.1 Polymerase chain reaction (PCR) ........................................................... 25
II.2.1.1.1 Colony PCR .................................................................................. 25
II.2.1.1.2 Variable region gene amplification .................................................. 26
II.2.1.2 Agarose gel electrophoresis .................................................................. 26
II.2.1.2.1 Preparative agarose gel electrophoresis .......................................... 26
II.2.1.3 Site-directed mutagenesis ..................................................................... 26
II.2.1.4 Cryopreservation of bacteria ................................................................. 27
II.2.1.5 Preparation of chemically competent E.coli ............................................. 27
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II.2.1.6 Preparation of electrocompetent bacteria ............................................... 27
II.2.1.7 Enzymatic restriction of DNA ................................................................. 27
II.2.1.7.1 Fingerprinting by BstNI digestion.................................................... 28
II.2.1.8 Ligation ............................................................................................... 28
II.2.1.9 Purification of ligations ......................................................................... 28
II.2.1.10 Heat shock transformation of E.coli ..................................................... 28
II.2.1.11 Electro-transformation of bacteria........................................................ 29
II.2.1.12 Plasmid isolation ................................................................................ 29
II.2.1.13 DNA sequencing of PCR products and plasmids .................................... 29
II.2.1.14 In silico DNA and protein sequence analysis ......................................... 29
II.2.1.15 Phage display bio-panning .................................................................. 30
II.2.2 Cell culture techniques ................................................................................ 31
II.2.2.1 Plasmodium falciparum cultivation and synchronization ........................... 31
II.2.2.2 Immunofluorescence assay ................................................................... 31
II.2.2.3 Growth inhibition assay ........................................................................ 32
II.2.3 Immunological methods and protein chemistry .............................................. 33
II.2.3.1 Transient expression in N. benthamiana................................................. 33
II.2.3.2 Protein A affinity chromatography ......................................................... 33
II.2.3.3 Enzyme linked immunosorbent assay ..................................................... 33
II.2.3.3.1 Monoclonal phage ELISA ............................................................... 34
II.2.3.4 Sodium dodecylsulfate polyacrylamide gel electrophoresis ....................... 34
II.2.3.5 Coomassie Brilliant Blue staining of SDS-gels .......................................... 34
II.2.3.6 Western blotting .................................................................................. 35
II.2.3.7 Spectrophotometric quantification of protein .......................................... 35
II.2.3.8 Surface plasmon resonance spectroscopy .............................................. 35
II.2.3.9 Conformational epitope mapping ........................................................... 36
RESULTS ..................................................................................................... 37
III.1 Sequence analysis of monoclonal antibody OcheA ............................................... 37
III.2 Light chain shuffling of monoclonal antibody OcheA............................................. 38
III.2.1 Preparation of pRF-OcheA for the construction of a light chain library ............ 38
III.2.2 Construction of a kappa chain library in pRF................................................. 41
III.2.2.1 Quality control of the pRF-OcheA light chain library ............................... 42
III.2.2.2 Subcloning of the OcheA light chain library into pFlx .............................. 43
III.2.3 Quality control of the pFlx-OcheA light chain library. ..................................... 43
III.2.4 Phage display bio-panning .......................................................................... 45
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III.2.5 Screening for DiCo1-3 specific binders by monoclonal phage ELISA ................ 47
III.2.6 Sequence analysis of phage ELISA positive clones ........................................ 49
III.3 IgG expression of light chain shuffled clones in Nicotiana benthamiana ................. 52
III.3.1 Cloning of light chains and OcheAmut into pTRAkt........................................ 52
III.3.2 Production and purification of light chain shuffled antibodies ......................... 53
III.4 Confirmation of binding of light chain shuffled antibodies ..................................... 54
III.4.1 Enzyme linked immunosorbent assay .......................................................... 54
III.4.2 Surface plasmon resonance spectroscopy .................................................... 55
III.4.3 Immunofluorescence assay on P. falciparum 3D7 schizonts ........................... 58
III.5 Functional testing using parasite growth inhibition assay ...................................... 60
III.6 Epitope binning using SPR ................................................................................. 62
III.7 Heavy chain shuffling of light chain shuffling derived antibodies 17 and 17B .......... 63
III.7.1 Preparation of pFlx-17 and pRF-17B for heavy chain shuffling ....................... 63
III.7.2 Cloning of a heavy chain repertoire in pFlx-17 and pFlx-SHC-17B ................... 64
III.7.2.1 Heavy chain V gene amplification and cloning of heavy chain repertoires 64
III.7.2.2 Quality control .................................................................................... 66
III.7.3 Phage display bio-panning of heavy chain shuffling libraries .......................... 67
III.7.4 Confirmation of binding and sequence analysis of heavy chain shuffled clones 69
III.8 IgG expression of heavy chain shuffled clones in N. benthamiana ......................... 74
III.8.1 Cloning of heavy chains into pTRAkt ............................................................ 74
III.8.2 Small scale expression and affinity measurements ........................................ 75
III.8.3 Site-directed mutagenesis of heavy chain E.................................................. 76
III.8.4 Accumulation and purification of E-9 and F-17B............................................ 77
III.9 Confirmation of binding of antibodies E-9 and F-17B ............................................ 78
III.9.1 Affinity ...................................................................................................... 78
III.9.2 Enzyme linked immunosorbent assay .......................................................... 80
III.9.3 Immunofluorescence assay on P. falciparum ................................................ 82
III.9.4 Western blot ............................................................................................. 84
III.10 Functional testing using parasite growth inhibition assay .................................... 85
III.10.1 Interactions between inhibitory AMA1-antibodies E-9 and 1E4 ..................... 87
III.11 Epitope mapping ............................................................................................. 89
III.11.1 Epitope binning using surface plasmon resonance ...................................... 89
III.11.2 Conformational epitope mapping of F-17B.................................................. 89

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