Download Humanized antibodies

Manifestation of Novel Social Challenges of the European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Manifestation of Novel Social Challenges of the European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Éva Csősz
Molecular Therapies - Lecture 7
RECOMBINANT ANTIBODIES AND
THE PHAGE DISPLAY
TECHNOLOGY
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The aim of lecture 7 is to present the possibilities for therapeutic antibody production, to highlight the pros
and cons of the different production methods. In this lecture the production of antibodies in the body and by
different techniques like in hybridoma cells or the generation of high antibody diversity by phage display
technology will be discussed.
Chapters in lecture 7.
7.1. Introduction
VI.I.1. The structure of antibodies and their production in the body
VI.I.2. Antigen-antibody binding
7.2. The production of therapeutic antibodies
VI.II.1. The production of antibodies in hybridoma cells.
VI.II.2. Humanized antibodies
VI.II.3. Production of human antibodies
7.3. Generation of antibodies by phage display
VI.III.1. The phage display technology
VI.III.2. Generation of phage libraries
7.4. Administration of therapeutic antibodies
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The structure of antibodies
Supervariable region
NH3+
NH3+
NH3+
VH
VH
VL
NH3+
VL
CH1
CH1
CL
COO-
Light chain:
CLconstant region,
variable region
COOHinge region
CH2
Heavy chain:
constant region,
variable region
Disulfide bonds
Fab region
CH3
COO- COO-
Fc region
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The structure of antibody heavy chain
kb. 85 gene
kb. 27 gene
VH1 VH2 VH3 VH4 VHn
DH1 DHn
kb. 6 gene
JH1 JH2 JH3 JHn
Heavy chain
VH4 DH1 JH2 C 
IgG
Cµ C C C  C α
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The structure of antibody light chain
approx. 35 kappa gene
VL1 VL2 VL3 VL4 VLn
approx. 30 lambda gene
approx. 5 kappa gene
JL1 JL2 JL3 JLn
C
approx. 4 lambda gene
VL2 JL3 C
kappa light chain
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Production of antibodies in B cells
B cell
Antibody
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Clonal selection and clonal expansion
Y
Recombination
Junctional diversity
Somatic hipermutation
Y
YY
B cell
BCR
Clonal selection
Antigene/epitope
B cell
Clonal
expansion
Plazma cell
Specific antibody
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Polyclonal antibodies
Y
Y
B cell
Y
B cell
antibody
antigene
antibody
B cell
Y
epitope
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Monoclonal antibodies
B cell
antibody
antigene
epitope
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Production of antiodies in hybridoma cells
Antigene
Myeloma cells
HGPRT 
antibody production

Mouse immunization
Spleen cell isolation
HGPRT
antibody production
Fusion of spleen and myeloma cells,
generation of hibridoma cells
Culturing of the hibridoma cells
Y
Y
Y Y
Y
Y Y
antibody isolation
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Humanized antibodies
Human antibody
Mouse antibody
Humanized antibody / chimera antibody
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Production of human antibodies in genetically modified mice
Mouse immunoglobulin gene
Human immunoglobulin gene
Human or humanized antibody production
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The structure of M13 phage
M13 bacteriophage
5 db p6
DNS - 6.4 kb
5 db p9
5 db p7
2700 db p8
5 db p3
F-pilus
900 nm
E. coli
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Specific elution of immobilized phage particles
Specific elution
Immobilized protein / affinity matrix
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Enzyme phage display
matrix
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Substrate phage display I.
matrix
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Substrate phage display II.
matrix
matrix
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Enzyme-substrate phage display I.
matrix
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Enzyme-substrate phage display II.
matrix
matrix
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Generation of phage libraries
Various
sequences
Phagemid
Recombinant phagemid
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Generation of protease substrate phage library
Generation of various
sequences
Protease substrate
sequence
hGH gene Protease substrate
hGH gene
M13 gIII gene
Protease substrate M13 gIII gene
phagemid vector
phagemid vector
Phage library
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Substrate phage display –engineering of protease substrate
sequences
Protease sensitive sequences
Protease
hGH receptor
Protease
low pH
Sequencing
matrix
Protease resistent sequences
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In vivo phage display – mapping vascular endothelial cells
Phage particles bind
to the vascular
endothelial cell
surface proteins
Biopsy
Intravenous
injection of
phage library
Removal of bound
phages
Identification of phagebound proteins/peptides
Propagation of
bound phages
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Generation of antibody libraries from whole blood
Whole blood
(immunized donor)
antibody genes
Limfocytes
antibody
specific
primer
phagemid
mRNA
E. coli cells
cDNA
contain 108 differnt
antibody genes
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The mechanism of antibody dependent cell mediated cytotoxicity
(ADCC)
Antibody against tumor cells
Fc receptor
Tumor cell
Killer cell (NK
cell or
monocyte)
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Administration of therapeutic antibodies with
immunosupressant activity
Monoclonal antibody
• Adalimumab
• Infliximab
• Golimumab
• Cetrolizumab pegol
Basiliximab
Human-mouse chimera antibody
• Psoriasis
• Rheumatoid arthritis
• Crohn disease
• Spondilitis
TNFalpha
IL2 receptor
alpha chain
Inhibition of organ
rejection after
transplantation, especially
in case of kidney
transplantations.
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Forms of therapeutic antibodies
Bispecific antibody
(approx. 300 kDa)
IgG - scFv
(Fab – scFv)2
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Forms of small-sized therapeutic antibodies
Fab
scFv
F(ab’)2
Tandem scFv
Diabody
S-S
Fv
S-S
dsFv
Nanobody
scFv2
Triabody
Bispecific antibody