1. 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 /1/A

2. RECOMBINANT ANTIBODIES AND THE PHAGE DISPLAY TECHNOLOGY
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 /1/A
Éva Csősz
Molecular Therapies - Lecture 7
RECOMBINANT ANTIBODIES AND THE PHAGE DISPLAY TECHNOLOGY

3. VI.I.1. The structure of antibodies and their production in the body
TÁMOP /1/A
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

4. The structure of antibodies
TÁMOP /1/A
The structure of antibodies
Supervariable region
NH3+
NH3+
NH3+
NH3+ VH VH VL VL
Fab region
CH1
CH1
Light chain: constant region, variable region CL CL
COO-
COO-
Figure 1. The structure of antibodies.
Hinge region
CH2
Heavy chain: constant region, variable region
Disulfide bonds
Fc region
CH3
COO-
COO- 4

5. The structure of antibody heavy chain
TÁMOP /1/A
The structure of antibody heavy chain
kb. 85 gene
kb. 27 gene
kb. 6 gene
VH1
VH2
VH3
VH4
VHn
DH1
DHn
JH1
JH2
JH3
JHn Cµ C C
C 
C α
Heavy chain
VH4
DH1
JH2
C 
IgG
Figure 2. The structure of antibody heavy chain. Each heavy chain is a combination of V (variable), J ( junction), D (diversity) and C (constant) genes. 5

6. The structure of antibody light chain
TÁMOP /1/A
The structure of antibody light chain
approx. 35 kappa gene
approx. 5 kappa gene
VL1
VL2
VL3
VL4
VLn
JL1
JL2
JL3
JLn C
approx. 30 lambda gene
approx. 4 lambda gene
VL2
JL3 C
kappa light chain
Figure 3. The structure of antibody light chain. Each light chain is a combination of V (variable), J ( junction) and C (constant) genes. 6

7. Production of antibodies in B cells
TÁMOP /1/A
Production of antibodies in B cells
B cell
Antibody
Figure 4. Production of antibodies by B cells. 7

8. Somatic hipermutation
TÁMOP /1/A
Clonal selection and clonal expansion Y
Recombination
Junctional diversity
Somatic hipermutation
B cell
BCR
Clonal selection
Antigene/epitope
Figure 5. The clonal selection and clonal expansion provides the appearance of a high number of specific antibody producing B cells and plasma cells in a very short period of time.
B cell
Clonal expansion
Specific antibody
Plazma cell 8

9. Y Y Y Y Polyclonal antibodies B cell B cell antibody antigene epitope
TÁMOP /1/A
Polyclonal antibodies Y Y Y
B cell
Figure 6. Polyclonal antibodies – a combination of immunoglobulins originated from different B cells recognising different epitopes of the antigene.
B cell
antibody Y
antigene
epitope
antibody
B cell 9

10. Monoclonal antibodies
TÁMOP /1/A
Monoclonal antibodies
B cell
Figure 7. Monoclonal antibodies – immunogloulins originated from one type of B cells recognizing one epitope.
antibody
antigene
epitope 10

11. Production of antiodies in hybridoma cells
TÁMOP /1/A
Production of antiodies in hybridoma cells
Antigene
Myeloma cells
HGPRT 
antibody production 
Mouse immunization
Fusion of spleen and myeloma cells, generation of hibridoma cells
Spleen cell isolation
HGPRT
antibody production
Culturing of the hibridoma cells
Figure 8. Production of antiodies in hybridoma cells. Y Y Y Y
antibody isolation Y Y Y 11

12. Humanized antibodies Human antibody Mouse antibody
TÁMOP /1/A
Humanized antibodies
Figure 9. Humanized antibodies.
Human antibody
Mouse antibody
Humanized antibody / chimera antibody 12

13. Production of human antibodies in genetically modified mice
TÁMOP /1/A
Production of human antibodies in genetically modified mice
Mouse immunoglobulin gene
Figure 10. Production of human antibodies in genetically modified mice.
Human immunoglobulin gene
Human or humanized antibody production 13

14. The structure of M13 phage
TÁMOP /1/A
The structure of M13 phage
M13 bacteriophage
5 db p6
5 db p9
DNS kb
E. coli
5 db p3
5 db p7
2700 db p8
F-pilus
900 nm
Figure 11. The structure of M13 phage. 14

15. Specific elution of immobilized phage particles
TÁMOP /1/A
Specific elution of immobilized phage particles
Specific elution
Figure 12. Specific elution of immobilized phage particles.
Immobilized protein / affinity matrix 15

16. Enzyme phage display matrix Figure 13. Enzyme phage display. 16
TÁMOP /1/A
Enzyme phage display
Figure 13. Enzyme phage display.
matrix 16

17. Substrate phage display I.
TÁMOP /1/A
Substrate phage display I.
Figure 14. Substrate phage display I.
matrix 17

18. Substrate phage display II.
TÁMOP /1/A
Substrate phage display II.
Figure 15. Substrate phage display II.
matrix
matrix 18

19. Enzyme-substrate phage display I.
TÁMOP /1/A
Enzyme-substrate phage display I.
matrix
Figure 16. Enzyme-substrate phage display I. 19

20. Enzyme-substrate phage display II.
TÁMOP /1/A
Enzyme-substrate phage display II.
Figure 17. Enzyme-substrate phage display II.
matrix
matrix 20

21. Generation of phage libraries
TÁMOP /1/A
Generation of phage libraries
Various sequences
Phagemid
Figure 18. Generation of phage libraries.
Recombinant phagemid 21

22. Generation of protease substrate phage library
TÁMOP /1/A
Generation of protease substrate phage library
Generation of various sequences
Protease substrate sequence
hGH gene
Protease substrate
M13 gIII gene
hGH gene
Protease substrate
M13 gIII gene
phagemid vector
Figure 19. Generation of protease substrate phage library.
phagemid vector
Phage library 22

23. Substrate phage display –engineering of protease substrate sequences
TÁMOP /1/A
Substrate phage display –engineering of protease substrate sequences
Protease sensitive sequences
Protease
hGH receptor
Figure 20. Substrate phage display –engineering of protease substrate sequences.
Protease
Sequencing
matrix
low pH
Protease resistent sequences 23

24. In vivo phage display – mapping vascular endothelial cells
TÁMOP /1/A
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
Figure 21. In vivo phage display – mapping vascular endothelial cells.
Identification of phage- bound proteins/peptides
Propagation of bound phages 24

25. contain 108 differnt antibody genes
TÁMOP /1/A
Generation of antibody libraries from whole blood
Whole blood
(immunized donor)
antibody genes
Limfocytes
antibody specific primer
phagemid
Figure 22. Generation of antibody libraries from whole blood.
mRNA
E. coli cells
cDNA
contain 108 differnt antibody genes 25

26. The mechanism of antibody dependent cell mediated cytotoxicity (ADCC)
TÁMOP /1/A
The mechanism of antibody dependent cell mediated cytotoxicity (ADCC) Y Y Y
Antibody against tumor cells Y Y Y
Fc receptor Y
Figure 23. The mechanism of antibody dependent cell mediated cytotoxicity (ADCC).
Tumor cell
Killer cell (NK cell or monocyte) 26

27. TÁMOP /1/A
Administration of therapeutic antibodies with immunosupressant activity
Monoclonal antibody
Adalimumab
Infliximab
Golimumab
Cetrolizumab pegol
Psoriasis
Rheumatoid arthritis
Crohn disease
Spondilitis
TNFalpha
Figure 24. Administration of therapeutic antibodies with immunosupressant activity.
Inhibition of organ rejection after transplantation, especially in case of kidney transplantations.
IL2 receptor
alpha chain
Basiliximab
Human-mouse chimera antibody 27

28. Forms of therapeutic antibodies
TÁMOP /1/A
Forms of therapeutic antibodies
Bispecific antibody
(approx. 300 kDa)
Figure 25. Forms of therapeutic antibodies.
IgG - scFv
(Fab – scFv)2 28

29. Forms of small-sized therapeutic antibodies
TÁMOP /1/A
Forms of small-sized therapeutic antibodies
Fab
F(ab’)2
scFv
Tandem scFv
Diabody
Triabody
Bispecific antibody
Figure 26. Forms of small-sized therapeutic antibodies.
S-S Fv
S-S
dsFv
scFv2
Nanobody 29