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Engineering antibodyEngineering antibody
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Emil von Behring, 1901,
antitoxins
Georeges Kohler and Cesar Milstein,
1984, monoclonal antibody
Susumu Tonegama,1987,
structure of Ig gene
Gerald Edelmanand Rodney Porter,
1972, structure of
antibody
Paul Ehrlich, 1908,
production ofantibody
Nobel Prize winners
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Light chain
Heavy chainDisulfide bonds
Hinge region
Structure of immunoglobulin molecule
I The structure ofAntibody
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ydrolytic fragment of Immunoglobulin
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Immunoglobulin domains
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CDR:
Complement
Determinant
Region
Ag-Ab reactions is one of a key (i.e.the
Ag) which fits into a lock (i.e. the Ab).
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hypervarible region(HVR)
(complimentaritydetermining region,CDR ) :
formation of the Agbinding site
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CDR:
Complement Determinant
Region
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First phase multiclonal antiserum
Second phase cell engineering antibody
(monoclonal antibody)
Third phase gene engineering antibody
hree phases of Antibody techniqu
II Cell engineeringantibody and geneengineering antibody
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1 multiclonal antiserum
Polyvalent
antigen heterogeneous Mixture of
antibodies
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Multiple
epitopes
Mono-clone
sensitized B cell Monoclonalantibody
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monoclonal antibodyBasic principle
Cell engineering antibody
hybridoma monoclonal antibody
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Myeloma cells
tivated, antibody-producing B cell
hybridoma
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ntigen immunize mouseCulture of myeloma cell
Immunized spleen cell myeloma cell
fusion forms hybridoma with PEG
Selective culture medium (HAT culture med
Hybridoma survive, only
Selection of the positive clone and clon
cloning proliferation and preparation of
Preparation of monoclonal antibody
TA:hypoxanthine, aminopterin, and thymidine
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Preparation the monoclonal antibody to the
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Cell fusion
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3 Gene engineeringantibodyimprovement of the antibody
properties by recombination
preparation of new antibody byphage display (phage library)
Generating human antibodies which could never be
obtained from the blood stream.By random or designed mutations, affinity or
specificity of the antigen binding can be changed,
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1 smallmolecular
antibody
3Humanizedmouse MAb
Mod
ifieda
ntibodyby
2
Mouse antibody
Chimericantibody
humanizedantibody
2 Antibodyfusion protein
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(1) Small molecular1) Small molecularantibodyntibody
1
2
3
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Constructionvector of
immunoglobulingene
Vector of Hchain
expression
Co-transfection
Pr VH CH1 Pr VL CL
Fig.5 preparation of Fab antibody molecule
A Fab antibody molecule
Vector of Lchain
expression
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Antibody-secreting
cells
VH VL
CL-S-S-
CH1
Fig.5 preparation of Fab antibody molecule
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B Fv antibody molecule
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Construction ofvector
Co-transfection
Pr VH Pr VL
Fig.6 preparation of disulfide-stabilized Fv,ds-Fv) antibody
(a) ds-Fv
Vector of Hchain
expression
Vector of Lchain
expression
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Antibody-secreting cell
VH VL-S-S- disulfide-stabilized Fv,ds-Fv
Fig.6 preparation of disulfide-stabilized Fv,ds-Fv) antibody
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VH VL
Linking DNA
(Gly4Ser)3
VH primer
PCR
VH linking DNA
Restriction enzyme,
cloning
Fig.7 construction of ScFv (single chain Fv,scFv )
Denaturation ,renaturation
VL primer
(b) Sc-Fv
VL
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III Diabody structure
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Bispecific antibody
Antigen A
-S-S-
Fab
VH
CH1
VL
-S-S-CH1
VH VL
Fv VH VL
Antigen B
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CTLCD3
TUMOR
CELL
-S-S-
VH
CH1
VL
-S-S-CH1
Antigen A Antigen B
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(2) Antibody fusion protein(2) Antibody fusion protein
combining antigen-binding regionwith bioactive protein
immune guide
immune bridge
chimeric receptor
Fc-contained fusion proteinimmune
adhensionthe function of Fc test, purification of
protein
prolonging the half tine of
protein in serum
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Fcprotein
SPAthrombin
The function of Fc test, purification
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G-CSF Fc
Fusion Protein
DNA ligation
expression
Prolonging the half time in serum
DNA li ti
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DNA ligationP-GGG -HO-CCC -
-CCC - OH
-GGG - P
-G - OH P-AATTC-
-CTTAA-P HO-G -
5
3
3
5
T4 DNA ligase
ATP
Klenow fragment
dNTP
-GAATT-OH
P - AATTC--CTTAA-P HO-TTAAG -3
3
5
5
+
P-GGG -HO-CCC -
-CCC - OH
-GGG - Pblunt ends
Klenow fragment
dNTP
Klenow fragment
E.coli DNA polymerase I
Protease5-ATCGGAAATGCCGTAGTAACG-3
3-TGC-5dNTP
DNA polymerase I :
Synthesizes DNA complementary to a DNA template in a 5 to 3 direction
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Mean bean nuclease:
remove the nucleotide from the 3 end of the linear dsDNA.
DNA ligation
P-GGG -HO-CCC -
-CCC - OH
-GGG - P
-G AATT- OH P-C--C-P HO-TTAAG -
5
3
3
5
T4 DNA ligase
ATP
-G-OH P - C--C-P HO-G-
3
3
5
5+
P-GGG -HO-CCC -
-CCC - OH
-GGG - Pblunt ends
Mean bean nuclease
5-ATCGGAAATGCCGTAGTAACG-33-TAGCCTTTACGGCATC-5
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TAT AAA TAA GCT TTAATA TTT ATT CGA AAT
G-CSF Fc
Fusion Protein
HindIIIHindIII
G-CSF
TAT AAA TA A GCT TTA
ATA TTT ATT CGA AAT
Mean bean nuclease
TAT AAA TA TTA
ATA TTT AT AAT
Stop codon
TAATAG
TGA
TAA
TAT AAA TA TTA
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TAT AAA TA TTA
ATA TTT AT AAT
CCCCGGGGLigase
TAT AAA TAC CCC GGG GCC CCG GGG TTA
ATA TTT ATG GGG CCC CGG GGC CCCAAT
Stop codon
TAA
TAG
TGA
smaI CCC GGG
TAT AAA TAC CCC G GGG TTA
ATA TTT ATG GGG CCCCAAT
LigaseFc
TAT AAA TAC CCC TAAGGGG TTA
ATA TTT ATG GGG ATTCCCCAATFc
G-CSF
Fc
TAAATT
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(3) Humanized mouse monoclonal antibody(3) Humanized mouse monoclonal antibody
humanized C regionhuman-humanized C regionhuman-
mouse chimeric antibodymouse chimeric antibody
humanized V regionhumanized V region
humanized antibodyhumanized antibody
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Humanized mouse antibody
Mouse Ab Human-Mouse chimeric Ab Modified human Ab
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chimeric antibody : Ig Ig
70-80% Ig 20-30%
--
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Pr mouse VH human CH Pr mouse VL human CL
Construction vectorofIg gene
H chain chimericvector
Co-
transfection
promotor
Engineering stratagem forHuman-mouse chimeric Ab
L chain chimericvector
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Mouse VH Mouse VL
Human CL
Human CH
Ab-secreting cell
Human-mouse chimeric engineering Ab
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MabTheraabThera for the treatment of non-Hodgkin-lymphomas
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Humanized V region of mousemonoclonal antibody
Humanized antibodyHumanized antibody
CDR grafting antibodyCDR grafting antibody
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CDR CDR
Mouse MAb Human antibody
Humanized antibody
Fig. humanized V region of mouse MAb
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Antibody repertoireAntibody repertoire
III preparation of newmonoclonal antibody byengineering
phage displayphage display
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RT-PCRRT-PCR clone all V regionclone all V region
genes of antibodygenes of antibody Expression of geneExpression of gene
fragments infragments in E.coliE.coli
phage displayphage display
The three techniques forThe three techniques for
Antibody repertoire productionAntibody repertoire production
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Liker DNA
VH VL
display of the small molecular antibody on the surface of t
Gene III orgene VIII
Coat protein
VH
VL
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The construction ofThe construction ofAntibody repertoireAntibody repertoire
Human B cell
DNA recombination
Phage antibody repertoi
Transfer into E.coli
S l ti f A tib dSelection of Antibody
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Selection of AntibodySelection of Antibody
repertoirerepertoire
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Box 1 | Use of phage display libraries to
identify peptides that home to the tumor
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IV
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VC
linica
luses
forth
e
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antiserum
Monoclonal antibody
Gene engineering antibody
antitoxin
Anti-tumor
(mouse, human-mouse
)
Anti-tumor,therapy of autoimmunity
The rise of antibody-based
therapeutics further illustrates the
substantial change in the paradigms
of pharmaceutical development,
By utilizing the body's own
capabilities as a source for a drug
rather than the chemists reagents
vessel.
A ti l i i i tiI tiI ti
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Acute infection (diphtheria, tetanus, measles, rabies, etc.),
poisoning (insects, reptiles, botulism),
prophylactic (hypogammaglobulinemia).
Heterologous
Homologous
Article passive immunizatio
Human gamma-globulins, or immunized animal serum
I antiserumI antiserum
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toxoidtoxin
Horse serum
Antitoxin
Hypersensitivity
antibody
antigen
Human Antibodies
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used to prevent and/or treat a wide
variety of diseases,
Human Antibodies
Produced in Cloned Calves
production ofunlimited quantities of
human polyclonal antibodies against any
disease agent
cloning technique to produce four calves
expressing a human chromosome fragment
coding for a range ofhuman antibodies.
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human chromosome fragment containing
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g g
the un-rearranged sequences of two
human genes (coding IgH, Ig-lambda )
microcell fusion
bovine cell linetransferred
hamster
carrier cell line
bovine cells
containing the human
chromosome
fragment
cow eggs
Fused into
resulting
embryos
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cow eggs resulting
embryos
to produce four cloned,transchromosomic calves
recipient cows
Human antibody proteinswere detected in the blood
II Monoclonal antibody and
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II Monoclonal antibody and
engineering antibody
Monoclonal antibody products in market
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2001lymphomaCD52Campath
2000lymphomaCD33Mylotarg
1998rejectionRabbit mutil-Thymogloblin
1998RSV infectionRSV F proteinSynagis1998BreastHER-2Herceptin
19981999
Inflammatorydisease and RA
TNF-Remicade
1998rejectionCD25Simulect
1997lymphomaCD20H-M chimericAbRituxan
1997rejectionCD25HumanizedZenapax
1994Heart diseasePlatelet b a H-M chimericFab
ReoPr o
1995cancer17-1AMousemonoclonal
Panorex
1986rejectionCD3Mousemonoclonal
OKT3
Production timindicationtargettypename
H-M chimericAbH-M chimericAb
Humanized
Humanized
Humanized-drug coupling
Humanized-
Antibody-label
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TUMOR
CELL
-S
-S
-
Fab
Immunotoxin( plant and bacteria )isotope( I131 , I125 , In111 )
drugEnzyme (cleavage of the drug)
-S-S-
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Murine monoclonal antibodies have been
coupledcoupledto a variety of conventional
cytotoxic agentscytotoxic agents, including antifolates,anthracyclines, Vinca alkyloids, alkylating
agents, and neocarzinostatin.
Prepolymerization of some drugsdrugs, such asdoxorubicin, prior to conjugation can achieve
higher ratios ofdrug-to-antibodydrug-to-antibody.
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Tumor cell
enzyme
Ab-drug coupling
prodrug
drug
enzyme
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-S
-S
-
Prodrug (nontoxic)
Ab-drug coupling
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Herceptin( )
HER-2
cell
Development of Novel Therapeutics
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Based on Antibody Targeted Photolysis
for the treatment of certain types of cancerfor the treatment of certain types of cancer
Photolysis is a technique in which a cell is
destroyed as a result ofphototoxinsphototoxinsbeing released from a photosensitizer uponlaser illumination.
Antibody targeted photolysis (ATPL)
enhances the localizationlocalization ofphotosensitizer through the use of a cell-
specific monoclonal antibody.
Development of Novel Therapeutics Based on Antibody
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Bright field micrograph sequence indicating the point
of rupture of the cell membrane after treatment with
a photosensitizer, and illumination with laser -light
Targeted Photolysis
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Monoclonal antibody-based immunofluorescence used to identify the
parasite Encephalitozoon hellem, one of the agents of microsporidiosis.
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Lymphatic vessels staining with a monoclonal antibody specific for D6.
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The D6 antibody is detected as the red staining.
Nuclei are stained with haematoxylin.
Stars indicate the lumen of the vessels
in non-leukocytic targets of chemokines through the studies on the D6 and CCX
CKR chemokine receptors.
D6, a high affinity receptor for at least nine inducible proinflammatory chemokines,
is predominantly expressed on lymphatic endothelial cells (LECs) and the surface
of placental chorionic villi.
Figure Lymphatic vessels staining with a monoclonal antibody specific for D6.
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Antinuclearantibodies(green, FITC-
labeled) fromsystemic lupuspatientsattacking humanepithelial cells(HEp-2),
counterstainedwith Evans' Blue(red).
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Pancreas stained
with monoclonal
antibody to NSE
Colon stained with
monoclonal
antibody to p53
protein.
Lymphopma stained with
monoclonal antibody to
CD68, macrophageBreast carcinoma
stained with
HercepTest.
Intraductal breast carcinoma stainedwith Estrogen Receptor, clone 1D5
Colon stained with
Alpha Smooth Muscle
Actin, clone 1A4.
Non-Hodgkin's Lymphoma stained
with Bcl-2 Oncoprotein, clone 124
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