抗體噬菌體展示技術(shù)課件

Methods

in

Molecukr

BioogyVOIUME

178AntibodyPhageDisplayMethousand

HolocolsEdited

byPhilippaM.OBrienRobertAitkenAntibody

Phage

DisplayMeilingXiong20180629IntroductionofAbphage

DisplayTechnologyAb

FormatsforPhage

DisplayAb

LibrariesConstruction"PhageAbSelection

Methods&Strategies”PhageAbScreeningApplicationsInvitroAffinity

Maturation”Expression&PurificationofPhageAb

FragmentsContentsIntroductionofPhage

DisplayTechnologyThe

Ffbacteriophagestructurensert

withoutasignal

sequence

Insertencoding

a

signgl

sequencePromoter

+RISignafsequenceTagMCSPromoter+RBSSlgnal

sequenceOOpVllplXG1

G2CTPromoterSignalsequenceDNA

insertglllHelper

phage

F

pilusPhagemidFf

ori;PS→nner(cyoplasmic)membraneN1N2AbRPlasmid

onouter

membranedodoasmplll

fusionOOplpvmeoeBAIGregion:intergenic

region,usuallycontains

thepacking

sequence

and

replication

origin

of

minus

and

plus

strandsMolecular

tag:

to

facilitate

library

screening

and

forprotein

analysisRestrictionenzymerecognitionsites:useful

for

DNA

recombination

and

genemanipulation;multiplecloningsites

(MCS)Coat

protein:PII

(larger

protein,less

than

5

copies,)PVIII

(more

than

5

copies,decreased

length)AmbercodonTAG:supE

strains

(glutamic

acidcodon),non-suppressorstrains(stopcodon)ProteasecleavagesitePromoter*Signalpeptides:phageproteintranslocation,crucial

for

display

levelSelectivemarker:for

selection

of

infected

host

cellsIntroductionofPhage

DisplayTechnologyThescheme

of

phagemid

vectorReplicationoriginof

plasmidPromoterSignalpeptideCoat-proteinPhagemidRestrictionenzymes

recognitionsitesMolecular

tagSelectivemarkerIG

regionNonlytic

filamentous

phage

is

the

mostoften

used

for

phage

display,primarily

the

M13and

Fdstrains.Proteins

to

be

selected

are

infused

to

all

five

coat

proteins,with

plll

and

pVIll

mostcommonly

used.plll

protein

is

essential

for

infection

ofbacteria"Helper

phage:wild-type

plll

helper

phageand

special

helper

phage"Antigen

immobilized

on

magnetic

beads,polystryrene

surfaces,or

on

columns,or

is

used

insolutionas

biotinylated

antigen

andIntroductionofPhage

DisplayTechnologyRemove

unspecific

phage

antibodies

bystringentwashesElutionAmplification

+phage

production2-4selection

roundsColony

picking

ScreeningE.coli

infectionWashBind餐Moreefficientlythan

through

conventional

hybridoma

system.

Cheaperto

produce

recombinant

antibodies

using

bacteria,ratherthan

mammalian

cell

line.Easier

to

maintain

and

grow

bacterial

cultures

for

recombinant

antibody

production.Bypassimmunizationinantibody

selection.AdvantagesofBypass

the

use

of

animal

cells

for

production

of

antibodies.Phage

Display

forProducing

the

combinatorial

library(ideallywith

108to

109members)of

functional

antibodies

togeneratealargerRecombinantrepertoire

of

antibodiesthanthoseavailablethroughAntibodyconventional

hybridoma

technology.SelectionEasy

isolation

and

expression

of

the

cloned

gene

in

a

bacterial

host.Excellent

potentialto

further

improve

binding

properties

of

the

selected

antibody

by

protein

engineeringtechniques. Capable

of

generating

antibodies

against

almost

any

desired

antigen,including

highly

conserved

or

self-antigens,conformational

variants,low

immunogenic

antigens,and

alsotoxic

components,which

is

not

possible

by

in

vivo

immunization

ofanimals.”Themost

commonly

usedformat:single-chainvariablefragment(scFv)SimplicityofcloningprocessFast

and

easy

library

generationAhighdisplayrate

(small

protein

size

~25

kDa)Lessstablethan

FabfragmentsTend

to

form

dimers

(can

be

reduced

with

linker

more

than

20amino

acids)Antibody

Formats(F)ScFym

Fab√·The

light

chain

(VL-CL)and

the

Fd-domain

(VH-CH1)of

theheavy

chain

of

an

antibody.During

bacterial

expression,these

two

chains

aresynthesizedseparately,and

secreted

into

theperiplasm

where

they

fold

to

form

heterodimers.Fab

exhibit

higher

stability

than

scFvsPossessbetterPKand

PD

qualities

than

scFvsEasier

to

convert

into

full-length

antibodiesClinicalapplications:abciximab,lucentis,cimzia.(C)(D)F(ab')?Antibody

FormatsFab■Singledomainantibody√VHH:VHdomainofcamelidantibody,heavychainsonly,√

IgNAR(newantigenreceptor):sharkantibody,heavy

chainsonly,√

UniqueCDRs■Affibodies■AnticalinsDARPins■Avimers■

Affimers■MonobodiesAntibody

FormatsNanobody(Single

DomainAntibody)Camelid

AntibodyVNARMultivalent

fragments√

Miniantibodies

arescFvsor

Fabsconnectedviaaflexiblelinkerto

self-associating

structures

such

as

helix

bundles

or

leucinezippers.√

Diabodiesare

noncovalent

dimers

of

scFvs,whichspontaneously

form

depending

on

the

linker

length

between

VHand

VL.Another

form

of

diabodies

is

two

scFvs

connected

with

a

short

linker.√

Fab-Ais

created

by

genetic

fusion

ofthe

Fab

Fd

gene

with

the

alkaline

phosphatase(PhoA)gene

and

coexpressing

the

lightchaingene.√

scFv-FcarescFvsdimerized

bythe

Fcdomain.(H)(I)ScFv-FcAntibody

Formats(G)DiabodyMiniantibody(F)(J)Fab-AscFvImmune

libraries:

first,immunizeananimalwithan

antigen

and

isolate

the

mRNA

from

B

lymphocytes(forimmunized

animals)or

peripheral

blood

B

cells

(for

immunized

donors).The

mRNA

is

then

reverse

transcribed

into

cDNA,andthe

variable

regions

of

expressed

antibodies

are

amplified

via

PCRandclonedinto

a

phage

displayvector.Advantages:√

Matured

in

vivo√

Immune

libraries

can

be

generated

from

any

animal

andeven

humans:mouse,human,chicken,rabbit,camel..√Anyspeciesthathavebeenimmunized,infected,orexposedto

an

antigen.√

Usefulin

analyzingnaturalhumoralresponses,for

example,inpatientswithautoimmunedisease,viralinfection,

neoplasticdiseases,etc.RabbitCattleInfectionsAutoimmuneTumorsChimpanzeeSharkLaboratoryAnimalsLargeAnimalsHumansNon-humanprimatesAFishChicken

CamelImmunized

Disease

related

HypotheticalAntibody

LibrariesMouseSheepMacaqueSalmonImmune

statusPrimatesImmuneNaive

natural

libraries:universalantibodylibrariesgenerated

from

B-cells

ofnonimmunized

donors

andeliminate

the

need

to

construct

new

libraries

for

each

antigen.loweraffinitiesthan

those|generated

during

in

vivo

affinity

maturationto

find

good

antibodies

against

diverse

antigens,these

librariesneedto

bevery

large.Advantages:Absolutefreedom

inantigenchoice,including

self,nonimmunogenic,and

toxic

AgsSeveralantibodiesselected

byphagedisplayfromhuman

naive

librarieshave

already

beenmRNAisisolatedfrom

B

cells

and

used

for

cDNAsynthesisNatural

MixedmRNAV-geneAntibody

LibrariesV-genes

are

amplified

and

re

assemHed

in

functional

antibody

formatGeneis

synthesizedSyntheticVgenesourceandCDR

OriginWHVLWHCH1VLCLDisease

relatedFragment

formatImmunizedClonedNaivescFvFabNaiveSemisyntheticlibraries:

Naive

semisyntheticlibrariesareusually

librariesthat

have

been

isolatedfrom

nonimmune

hostsandwhereoneorseveralCDRswereexchangedwithsyntheticpeptidesorwererandomly

mutated.Thisapproachis

awayto

achieve

high

diversity

without

requiring

alargenumberofdonors

and

cangenerate

specificities

not

normally

included

in

natural

repertoires.Advantages:Lowimmunogenicity

in

hostssinceonly

a

few

of

the

CDRs

are

artificialThese

libraries

can

cover

the

entirerepertoire

ofgerm

linesmRNAisisolatedfrom

B

cells

and

used

forcDNAsynthesisNatural

MixedmRNAV-geneAntibody

LibrariesVgenesareamplifedandre-assemHedinfunctional

antibody

formatGeneis

synthesizedSyntheticV-genesourceandCDRoriginWHVLVLCLWHCH1Disease

relatedFragmentformatImmunizedClonedNaivescFvFabNaiveSynthetic

librariesAdvantages:Theprincipleadvantage

of

naivesynthetic

libraries

oversemisyntheticlibraries

is

that

the

biophysicalparameters

and

codon

usage

of

the

framework

region

can

be

optimizedforexpressibilityand

stability.Advanced

DNA

synthesis

methodssuch

as

TRIM,slonomics,or

chip-based

DNA

photolithographyofferthe

ability

to

precisely

define

thefrequency

of

each

amino

acid

at

eachposition

with

optimized

codons.CDRscanbe

of

higher

diversity,different

in

composition

thanbiologically

occurring

CDRs,henceoffering

a

potentially

larger

paratopespace.V-genesourceandCDRoriginDisease

relatedmRNAisisolatedfrom

B

cells

and

used

forcDNAsynthesisNatural

MixedmRNAV-geneClonedGene

is

synthesizedSyntheticAntibody

LibrariesV-genes

are

amplified

and

re-assemHed

in

functional

antibody

formatVLCLWHCH1WHVLFragment

formatImmunizedNaivescFvFabConstructionofLargeNaiveFab

LibraryAn

efficient

cloning

method,in

which

restrictionfragmentsinsteadofPCR

productswere

used."VH

fragments

are

isolated

by

digestion

of

plamidDNA

purified

from

the

primary

repertoires,and

cloned

intotheacceptor

phagemidvectorcontaining

the

light-chain

(LC)repertoires.This

innovation

increases

the

size

of

the

librariesdramatically.lgM-derived

antibody

repertoire

were

used.Standard

Fab

LibraryConstructionCDNA

synthesis

from

lymphoid

tissuemRNAPCR

amplication

of

LC

geneConstructionofLibrary

PhageFig.2

Basic

Protocol

of

Fab

Library

Construction

Flow

Chart.LigationofpComb3

DNA(inserted

LCand

HC

geneLigation

of

LC

gene

intopComb3PCR

amplication

of

HCgene●

To

ensurethat

allfive

Ab

classes

are

likely

to

berepresented

and

increase

the

overall

size

ofthe

final

library,

randomhexamersareemployedin

theprimary

first-strand

CDNAsynthesisfrom

PBLmRNA.·

Component

VH

and

VL

gene

segments

are

amplified

inseparate

PCR

reactions,and

initially

cloned

into

twodifferent

vectors,pCANTAB6

and

pCANTAB3his6(see

Fig.1).·

The

latter

is

used

for

cloning

the

VL

repertoire

because

it

hastheappropriatepolylinkercloningsitesfor

thedigested

VL

fragments;the

VH

repertoire

is

cloned

intopCANTAB6·A

short

linker

from

an

existing

scFv

is

cloned

(together

with

an

irrelevant

or“dummy”VH)intothe

VLrepertoire,upstream

of

the

VL

fragments.·

The

VH

and

linker-VL

repertoires

are

then

amplified

fromtheir

vectors,and

the

scFv

construct

is

prepared

using

asimple

two-fragment

PCR

assembly

procedure.Thisconstruct

is

then

cloned

into

pCANTAB6

to

create

the

largenaivescFv

libraryscFv

Library

ConstructionLigation

of

VHfragments

intopCANTAB6Ligation

of

VLfragments

intopCANTAB3his?Ligation

ofscFv

constructs

intopCANTAB6Fig.1.Protocol

flow

chart.Ligation

of

PCR

amplified

scFvlinker+“dummy”VH

upstreamof

VL

libraryPCR

amplification

of

VH

fragmentsPCR

amplification

ofVL

fragmentsFirst

strand

cDNA

synthesisfrom

PBL

mRNAAssembly

ofscFv

constructs

andpull-throughPCRPCR

amplification

of

Linker-VLlibraryPCR

amplification

ofVHlibrary"Polyclonalantibodylibraries(PCALs)arestandardizedmixturesofantibodiesspecific

for

an

antigen

or

multi-Agtargets."They

target

multiple

epitopes

on

poly-Ags,resulting

in

high-avidity

binding

and

efficient

triggering

of

effector

functions.■PCAL

generation

usually

involves

the

recovery

of

VL

and

VH

repertoires,and

theirrandompairingas

Fabsinto

aphage-display

vector.1

The‘

library

ispositivelyandnegativelyselected.SelectedVL-VHgene

pairsarethentransferredin

massto

amammalianexpressionvector.The

constructs

arethentransfected

into

a

mammaliancelllineforexpression.B

cell-containing

tissuesRT-PCRVVL

cDNA+VH

CDNAcloninginto

phagemidvectorVFab

phage

display

library

of

vectorsbacterial

transformation,VFab-displaying

phagepositive/negative

selectionVpolyantigen-specificsublibraryofphagemidvectorsmass

transfer

of

selected

VL-VH

region

gene

pairsVpolyantigen-specific

library

of

mammalian

vectors

mammaliancelltransfection,antibodypurificationPOALPolyclonalantibody

libraryconstructionsuperinfection

with

helper

phageFig.2.Flow

chart

for

PCAL

generation.PhageAbSelectionProceduresandapplicationsDiversity

inSelectionmethodsImmobilizedAg:solid

supports,columns,BIAcore

sensor

chips

BiotinylatedAg

insolutiontoavoidconformationalchangesProkaryotic

or

mammalian

cells,fluorescenceactivated

cell

sorting,tissue

sections,in

vivo

selection,etc.Elution√Acid

solutions(HCl).Glycine

buffers;Basic

solutions,triethylaming;Chaotropic

agents;Dithiothreitol;Enzymatic

cleavage;Competition

methodsSelectionofAbsforaffinityorbindingkinetics”SelectiononcomplexAgsSelectionon

cellsBiotinylateofinterest***LipidbilayerTarget

ReceptorA

Panning

on

B

Panning

usingimmobilizedantigen

specific

elutionCUsing

DAffinitybiotinylatedantigen

purification

on

columnsEDirectpanning

oncellsF

Subtractive

G

Selection

on

H

Selection

on

I

In

vivo

selectionselection

on

cells

tissue(cryo)sections

Western

blotsJ

Pathfinder

selection

K

Selective

infective

phageantigenplll

-N1and/orN2

ligand

fusionpll[C-term.domain]

antibody

fusionBiotin/tyramid

Activated

biotin

*phageHRP內(nèi)Tissuepanningforimmunohistochemistryantibodies:antibodyselectionwithformalin-fixedparaffinembedded(FFPE)tissue."Sandwich

pair

selection,complex-specific

antibodies,anddrug

monitoring:Drugmonitoring:variousforms(freeantibodydrug,antibody-target

complex,or

both

)ofantibody

therapeutics

canbeeasilytrackedandquantified

in

PK

assays,using

anti-

idiotypeantibodies√

Complex-specificantibodies:guidedselection

method√Sandwich

pairselectionSite-specificantibodyconjugationusing

methodssuchasgeneticfusion(enzyme,orfluorescentprotein).(A)Anti-complex(IgG

HRPlabeled)DrugtargetInvitroselectionof

antibodiesforspecificapplicationsDrug(in

humanserum)Isolationofanti-haptenspecificantibodyfragmentsfromcombinatoriallibrariesHapten

targets

with

molecular

weight

below

1000

DaltonTheyshouldbeconjugatedtoa

suitable

immunogeniccarrier

protein

for

presentationTo

avoid

the

selection

of

antibodies

specific

for

the

carrier

protein

or

the

linker,we

can

use

a

method

that

utilizes

two

different

hapten

conjugates

for

alternative

rounds

ofselectionThe

library

can

be

immunized

or

naive.The

naive

library

should

be

large

but

immunized

library

should

be

construct

separately.Hapten-

specific

antibody

selection"Antigens

from

a

particular

pathogen

can

be

ofvariableimmunogenicity,with

the

antigen

that

stimulates

the

strongestresponse

being

the

immunodominant

one."To

obtain

antibodies

against

the

epitope

of

interest,a

preadsorptionpanningis

used."This

facilitates

the

molecular

cloning

of

Mab

fragments

against

non-immunodominant

Agdeterminants."The

phage

library

is

first

preabsorbed

on

the

Ag

of

interest

to

remove

phage

that

react

with

the

immunodominant

epitope.The

unbound

phagearethen

incubatedasecond

time

with

Ag

and

eluted

and

amplified

accordingto

normal

protocols.CompetitiveDeselectionEpitope-

masking

StrategyConvertdominatingspecificitytosoluble

Fabs,purityanduseto

maskepitopeEpitopemasking

panningNormal

librarypanning1.CoatnitrocellulosewithFabcapturereagent2.Blockingremainingprotein-bindingsites4.Capturephage-expressedantibodies(replicalifts)6.Isolatereactive

clone(s)5.Probefilterswithtargetmolecule(s)Capture-liftScreening

procedure3.InfectbacteriawithTarget

CTargetATargetBphageCoatcaptureantibodyIncubatewithblocking

bufferIncubatewithantigen

solutionIncubatewith

Fab-displayedsolutionElutebound

phagewithelution

bufferInfect

phageto

bacteriatoamplificationStrongly

effective

to

select

Abs

against

Ags

fromcrude

preparations.Abs

against

conformation-

sensitive

Ags

can

beselected.■MAbs

against

a

variety

of

Ag

epitopes

can

beisolated

from

a

singlelibrary.Both

pAb

and

mAb

can

beused

as

capture

Abs.Capture-sandwichELISA"It

involves

the

use

ofcatalyzedreporterenzymedeposition(CARD),which

is

a

method

of

signal

amplification.CARDuses

HRP-conjugatedsecondaryantibody,biotintyramineto

biotinylate

phage

particles

that

bind

around

thesite

of

the

HRP

activity.”These

phage

can

be

recovered

on

streptavidin-coated

magneticbeads.This

selection

strategy

can

be

sued

to

isolate

phage

Ab

against

cell

surface

markers,and

other

antigens,such

as

purified

Ags,

cell

extracts,membrane

preparations.Proximity-GuidedSelection"For

selection

of

antibodies

targeting

cell-surface

antigensA

competitive

cell-panning

approach

is

used,in

which

targetcells

(positive

cells)are

precoated

with

magnetic

beads,and

mixed

with

an

excess

of

unmodified

Ag-negativecells.This

method

is

more

efficient

than

just

several

rounds

ofnegative

selection

on

Ag-negative

cells.Undesired

cells

Desired

cellsMagneticsorting

forselection

ofantibodies

tocell-surfaceantigensMINUTESScreeningforaffinityorkineticsof

bindingScreeningforbioactivity/function:receptor

blocking

ortriggering(dimerization),virusorcytokine

neutralizationSelection

for

a

particular

function:Ab

with

agonist

orantagonist

activity

for

a

given

receptor,for

drug

discovery;Abthat

dimerizes

receptors;Ab

internalization

for

gene

transfer;

Abselectionforcell

survival

or

killing;Combini