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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

(2)

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

(3)

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)

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

H

V

H

V

L

V

L

CH3

CL CL

CH2 CH1

CH1

F

ab

region

Fc region

The structure of antibodies

(5)

VH1 VH2 VH3 VH4 VHn DH1 DHn JH1 JH2 JH3 JHn Cµ C

C

C

C

α

JH2 DH1

VH4 C

 IgG

Heavy chain

kb. 85 gene kb. 27 gene kb. 6 gene

The structure of antibody heavy chain

(6)

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

(7)

B cell

Antibody

Production of antibodies in B cells

(8)

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

(9)

Y

Y Y

Y

B cell B cell

B cell

antibody

antigene

epitope

antibody

Polyclonal antibodies

(10)

B cell

antigene

epitope

antibody

Monoclonal antibodies

(11)

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

(12)

Mouse antibody Human antibody

Humanized antibody / chimera antibody

Humanized antibodies

(13)

Mouse immunoglobulin gene

Human immunoglobulin gene

Human or humanized antibody production

Production of human antibodies in genetically modified mice

(14)

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

(15)

Immobilized protein / affinity matrix

Specific elution

Specific elution of immobilized phage particles

(16)

matrix

Enzyme phage display

(17)

matrix

Substrate phage display I.

(18)

matrix matrix

Substrate phage display II.

(19)

matrix

Enzyme-substrate phage display I.

(20)

matrix matrix

Enzyme-substrate phage display II.

(21)

Phagemid

Recombinant phagemid

Various sequences

Generation of phage libraries

(22)

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

(23)

hGH receptor

matrix Protease

low pH

Protease resistent sequences

Protease sensitive sequences

Sequencing Protease

Substrate phage display –engineering of protease substrate

sequences

(24)

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

(25)

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

(26)

Tumor cell

Killer cell (NK cell or monocyte)

Antibody against tumor cells

Fc receptor

The mechanism of antibody dependent cell mediated cytotoxicity

(ADCC)

(27)

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

(28)

Bispecific antibody (approx. 300 kDa)

IgG - scFv

(Fab – scFv)2

Forms of therapeutic antibodies

(29)

Fv

Tandem scFv Diabody Triabody Bispecific antibody F(ab’)2

Fab

scFv

S-S

scFv2 dsFv

S-S

Nanobody

Forms of small-sized therapeutic antibodies

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