5 antibody+ctl

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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
http://ntri.tamuk.edu/monoclonal/monoclonal.map


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Preparation the monoclonal antibody to the
http://ntri.tamuk.edu/monoclonal/monoclonal.maphttp://ntri.tamuk.edu/monoclonal/monoclonal.maphttp://ntri.tamuk.edu/monoclonal/monoclonal.maphttp://ntri.tamuk.edu/monoclonal/monoclonal.map


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Cell fusion
http://ntri.tamuk.edu/monoclonal/monoclonal.maphttp://ntri.tamuk.edu/monoclonal/monoclonal.maphttp://ntri.tamuk.edu/monoclonal/monoclonal.map


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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
http://www.biotech.ufl.edu/~hcl/%20antibody_apps.htm


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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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5 antibody+ctl

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