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
RECOMBINANT ANTIBODIES AND THE PHAGE DISPLAY
TECHNOLOGY
Éva Csősz
Molecular Therapies - Lecture 7
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
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
Heavy chain:
constant region, variable region
Light chain:
constant region, variable region Hinge region
Supervariable region
Disulfide bonds
COO-
COO-
COO- COO-
NH3+ NH3+
NH3+ NH3+
V
HV
HV
LV
LCH3
CL CL
CH2 CH1
CH1
F
abregion
Fc region
The structure of antibodies
VH1 VH2 VH3 VH4 VHn DH1 DHn JH1 JH2 JH3 JHn Cµ C
C
C
Cα
JH2 DH1
VH4 C
IgG
Heavy chain
kb. 85 gene kb. 27 gene kb. 6 gene
The structure of antibody heavy chain
VL1 VL2 VL3 VL4 VLn JL1 JL2 JL3 JLn C
JL3
VL2 C kappa light chain approx. 35 kappa gene approx. 5 kappa gene
approx. 30 lambda gene approx. 4 lambda gene
The structure of antibody light chain
B cell
Antibody
Production of antibodies in B cells
Recombination Junctional diversity Somatic hipermutation
B cell
Antigene/epitope
Plazma cell Specific antibody BCR
Y Y Y Y
B cell
Clonal selection
Clonal expansion
Clonal selection and clonal expansion
Y
Y Y
Y
B cell B cell
B cell
antibody
antigene
epitope
antibody
Polyclonal antibodies
B cell
antigene
epitope
antibody
Monoclonal antibodies
Spleen cell isolation
HGPRT
antibody production
Myeloma cells
HGPRT antibody production
Mouse immunization Antigene
Fusion of spleen and myeloma cells, generation of hibridoma cells
Culturing of the hibridoma cells
antibody isolation
Y Y Y Y
Y Y
Y
Production of antiodies in hybridoma cells
Mouse antibody Human antibody
Humanized antibody / chimera antibody
Humanized antibodies
Mouse immunoglobulin gene
Human immunoglobulin gene
Human or humanized antibody production
Production of human antibodies in genetically modified mice
5 db p9
5 db p7 5 db p3
5 db p6
2700 db p8
DNS - 6.4 kb M13 bacteriophage
E. coli 900 nm
The structure of M13 phage
F-pilus
Immobilized protein / affinity matrix
Specific elution
Specific elution of immobilized phage particles
matrix
Enzyme phage display
matrix
Substrate phage display I.
matrix matrix
Substrate phage display II.
matrix
Enzyme-substrate phage display I.
matrix matrix
Enzyme-substrate phage display II.
Phagemid
Recombinant phagemid
Various sequences
Generation of phage libraries
hGH gene Protease substrate M13 gIII gene
phagemid vector
hGH gene Protease substrate M13 gIII gene
phagemid vector Generation of various
sequences
Phage library Protease substrate
sequence
Generation of protease substrate phage library
hGH receptor
matrix Protease
low pH
Protease resistent sequences
Protease sensitive sequences
Sequencing Protease
Substrate phage display –engineering of protease substrate
sequences
Intravenous injection of phage library
Phage particles bind to the vascular endothelial cell surface proteins
Biopsy
Removal of bound phages
Propagation of bound phages Identification of phage-
bound proteins/peptides
In vivo phage display – mapping vascular endothelial cells
Limfocytes
mRNA
cDNA
antibody specific primer Whole blood
(immunized donor)
antibody genes
phagemid
E. coli cells
contain 108 differnt antibody genes
Generation of antibody libraries from whole blood
Tumor cell
Killer cell (NK cell or monocyte)
Antibody against tumor cells
Fc receptor
The mechanism of antibody dependent cell mediated cytotoxicity
(ADCC)
TNFalpha
IL2 receptor alpha chain
Inhibition of organ rejection after
transplantation, especially in case of kidney
transplantations.
• Psoriasis
• Rheumatoid arthritis
• Crohn disease
• Spondilitis
Human-mouse chimera antibody Monoclonal antibody
• Adalimumab
• Infliximab
• Golimumab
• Cetrolizumab pegol
Basiliximab
Administration of therapeutic antibodies with
immunosupressant activity
Bispecific antibody (approx. 300 kDa)
IgG - scFv
(Fab – scFv)2
Forms of therapeutic antibodies
Fv
Tandem scFv Diabody Triabody Bispecific antibody F(ab’)2
Fab
scFv
S-S
scFv2 dsFv
S-S
Nanobody