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`
`Plant Molecular Biology
`Plasmids [2nd edition]
`* Platelets
`Pollination Ecology
`Postimplantalion Mummalian
`Embryos
`Preparative Cantrifugulion
`Prostaglandins and Related
`Substances
`Protein Blotting
`Protein Engineering
`Protein Function
`Protuin Phosphoi'ylntion
`Protein Purification
`Applications
`i’rotain Purification Methods
`Protnin annuncing
`
`Protein Structure
`i’rotein Targeting
`Protcolytic Enzymes
`i Pulsed Field Gel
`Electrophoresis
`Radioisotopcs in Biology
`Receptor Biochemistry
`Receptor—Ligand interactions
`RNA Processing i and ii
`Signal Transduction
`Solid Pliasc Peptide
`Synthesis
`’i'rnnscriptinn Factors
`Transcription and Translation
`Tumour immunoliiulogy
`Virology
`Yeast
`
`Antibody
`engineering
`A Practical Approach
`
`Edited by
`JOHN MCCAFFERTY
`CumbriLigu Iiniiimdy ’I'nnlumlagy Lid
`The Science Park
`Mal/mum
`Cnmbrlrigushlru SUB [ill
`HENNlE llOOGENBOOM
`CESAME at Department o/Pathuiogy
`Academiscli Ziekenhuis
`Munstrichi PO Box 511110
`6202 AZ Mausliizzhi
`Tim Netherlands
`and
`DAVE CHISWELL
`Cambridge Antibody Technology Ltd,
`The Sciennn Park
`Mnllmurn
`Cambridgsshirc SGH till
`
` ——III.
`OXFORD UNIVERSITY PRESS
`Union! New York Tokyo
`
`
`
`
`
`Lassen — Exhibit 1059, p. 1
`
`Lassen - Exhibit 1059, p. 1
`
`

`

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`I‘rlnml m cm: flrwnln M'Mfl‘lmnhn Fran. LIII, Eimiu-rn, Oxuu.
`
`
`
`Preface
`
`lullunimd llw Isnlnliun nml luuduv
`Itcminlunnnl DNA mclltmls have In.
`
`
`y. wunmli
`1| nnlilnnlicx m-n'
`:iun u'l nnllluldiu in mm“ ycnra.
`lul
`
`
`~1lu Mi Mi Ihe hm: mm: uiiiucnl
`g.
`card Lind uxpnmud in mnnmu:
`in:
`csprcssiull ul unlihmljiI Ilaqnncnlx 1n lun'lvrin 11ml Ivccrl IlL'Ilmlmunrd. Mun-
`:cvcnlly. 151:: mtlnlinn nl new rmlil‘mly VillL'lJlllclllIh Inn I'm-n II.‘l[I5[II(!ll¢d by
`lhu uuminn of large rupcllnivn nl nnllhudy grim urul llh: duwlnpnlcnl ul
`"will-i111 lmwd un tun-u. iuplmge di'illlil!‘ in isnlhlc specific nnllhrhlws.
`I|1'|.-
`11.15 ibuL-II [Inmllrlul by :m imam-ml nnLlcnlunumg nl lhr kcy n:\l|l.|lc\ Lillucl-
`
`in; ntiimdy “with": and DTKTIIlEi!" lupulhci wilh mun- qn
`tin: "Miran
`
`
`i-I‘ Iln: binding pnipcrliu uf |Il|.'
`:
`liluulim. In .iLIdiliun.
`Inn-ping and
`Ilu‘
`
`“mum I11: nl Ilw lupul tuiuu in! human .inlihnily gurmlinc gcnn lm pun unr
`nudcnlmnling nl Iln‘ uhnngcs Imulrllns dining in I'm: nll'lnity mnlumtlnn un
`n llrmcr (inning.
`We leicvc lhul now is the righl limu In assemble in (nu: vulumc. lilolm‘ulli
`which alluw lhc researcher In isulale u nuw amilwdy. analyse ils [ll'l'lp'fl'llt'h-
`(nrmnl lhc righl amibndy moluuulc nr lrngnlznl. and product sull’icicnl quan-
`IiLicsm In: uxcl'nl.
`'T‘In: husk i. mum-ml in Iwn purl}: 11w Iim dciciilm flu.- gunulaliun ”ml
`ilflfllj'fii! III :nlilmdlfl um] lln: wtunu “mum unglnccring :iml pmduulllm.
`(1lflllldl I i\ dusignml In [no lln: mulu- u! 1h: alnmlatd 'Icptlluil'c' 0E phage-
`ilisl‘luy prulnmla. In [In- I‘nlltm'injt L'llnrilut Ill: ‘lilw MI. 01' Ilw swam. mg.
`
`.Ill'tnily rmlumliun.
`i< dusmhcd. Mutlimh [or lnlmdnting amihndy repul-
`mim 'nnn transgenic mice:
`u allm dmurilml in llh: [Inn in
`.m an: IllL'lh-
`
`
`ugh Inr Ihu : nlysis 0111“:
`Iainauluilslln- n! nnlilmdws Wilh i't'nfhrtl lu Ialrinily
`and sequvnu‘.
`
`Th: swim! secliun dca’rihn lhc cnglnruing ol' nalurnl and man-made
`cll'emnl lnnunons. The com-min" of rod
`unlihudlcs lo hunnm antibodies
`by cilhur (‘DR mulling or 'guidcd «tin: un' using pllugi.’ display is also
`covered. In addillun. methods lu munuluclure signiflcmn qudlllhlcs of various
`antibody-hissed molecules in bolh eukaryolic and proknrynlic syslcms urc
`uulllncll.
`Tuidilmnnl munmlnnnl unlit-mi;
`Icclnmlugy has. pmviilcsl u wuullli o:
`[raga-m I'm min-nick nmi dinwnmw applicnlinnL 'I'llu: niulhmh and luclnm-
`IllllIf-‘h dnnibcd Ilurc can hulp expand lllc vernlillly Ill nnu‘mdy-hnsed
`rengcnl! in llluw Ilruus. We have undunwuwd in Ill‘u bank in [Inimit-
`rcwnrclwrs walking wilh unlilundws in «march and dlngnmllh with :I mule
`inn: IIIc 'nuw I; Imnlugian'. .‘l! placlisud by Iln: lundcrs 'n Ill;- fluid.
`
`
`This 'rnulc'
`hi: at equal value Lu wink
`n in Iln: Ii
`Ill u! militiudy IFn-m-
`
`peullu's. The nhllllyI Iu flCIIL‘HIIB higilvnlli
`ly. higJI-nfrccilicuy human anil-
`I‘vdiu in npnmpriuiu lulnnrh will Ilclp circunmnl mm;- ul Iln- pmhlmns
`
`
`
`Lassen — Exhibit 1059, p. 2
`
`Lassen - Exhibit 1059, p. 2
`
`

`

`
`
`Pi'efncn
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`assrmialed with earlier work using mouse monoclonal nnlihudics and aid in
`[hair successful use in viva. As we draw to the and of the millunniuxn we have
`moved closer to the “magic bullet‘ first hypothesized by Paul Ehrlich at lllc
`beginning of this cenlury,
`Cambridge
`Mum/rich!
`May I996
`
`J.Mc(‘.
`H.R H.
`D..l.(_‘.
`
`vlli
`
`
`
`Contents
`
`List of Contributors
`Abbreviations
`
`1. Construction and use of antibody gene
`repertoires
`A R l’uln'. M. I, Emblem". am! Rt Murlmugh
`1. Inlrnduction
`2. Vectors for [he display of proteins on the surface of
`bacteriophage id
`3. Preparation and clnning of uniibudy DNA
`Pr: partition [if niRNA
`cDNA prepariitinn
`Primary PCR
`Assembly of acFv fragments
`Amplification and digcslirm
`Ligation um] irunslurrnntinn
`4. Growth and expression of pliaga aniiburlins
`Ruxcuu of phage
`(lruwlh and saluhlu expression
`Purificatinn of soluble nnlihmiy
`5. Selection of nniibody variants displayed on the surface 01'
`bucturinplmge
`0. Analysis nl'phage-Liarlved Imlibodiuu by ELISA
`7. ln»nnli PCR Isusmifly
`introduction
`Application
`8. (Jonclusluns
`References
`
`A l'finity maturation of antibodies using
`phage display
`Kt’l'ill Si III/mm" amt Robert E. Hawkins
`1. introduction
`(iunurul considcrulions
`
`xvii
`XXI
`
`“on
`v
`
`20
`22
`23
`Z]
`25
`27
`
`Zil
`10
`12
`32
`37
`38
`39
`
`41
`
`41
`41
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`Lassen — Exhibit 1059, p. 3
`
`Lassen - Exhibit 1059, p. 3
`
`

`

`Contents
`
`Mulagenusis
`Sch,
`ion
`Screening
`2. Mnlugenosis
`General cmisideialiuns
`Error-prone PCR
`Sitealirected mutagcnesis
`Mulagenesis using ‘spiked' PCR primers
`3. Selection oianlibodies with altered properties
`Selection at phage hy panning
`Solution capture an soluble antigen
`Oir-ralc selection
`4. Screening soloolod populations of antibodies
`Affinity screen for phage antibody clones (Kd assay)
`5. Soqnonca and fingerprint analysis
`References
`
`Human antiliotly repertoires in transgenic
`mice: manipulation and transfer of YACs
`Nicholas P, Davies, Amlm' V. l’npmt. X[angling ion. and
`Mm inmw Briiggemnmt
`1. Introduction
`2. Yeast artificial chromosomes
`Library screening
`Maintenance
`3. Modification of YACs
`Universal YAC vectors
`Sitodirectcd introduction
`Mapping site-specific integration]
`Profile of single and multiple integrations
`4. YAC lransfor into embryonic: stem cells
`Spheroplaat fusion
`Picking and analysing clones
`5. Conclusions
`References
`
`Measuring antibody affinity in solution
`1.12m Djamidi-()hmn‘nnepY Mic/ml E, Goldberg, and Bertram] Frignel
`t. (loneml considerations
`K“ does not directly rellect association or dissociation kinetics
`X
`
`41
`4.1
`4.1
`44
`44
`44
`45
`47
`4!)
`50
`51
`53
`54
`54
`57
`5K
`
`50
`
`59
`6 I
`62
`62
`h}
`54
`65
`67
`67
`7U
`7U
`73
`7.1
`74
`
`77
`
`77
`77
`
`Contents
`
`True K4 cannot he determined when the mAb or the Ag is
`immobilized in a solid-pints: assay
`rit: determination at K,. must take into account the valency of
`the mAb and of the Ag molecule
`2. Overview ot'molhods to measure affinities in solution
`Fluorescence
`ELISA- and RlA-hased methods
`3. Affinity measurements in solution by competition ELISA
`Theoretical aspects
`Rationale
`Requirements for thc determination of K“
`Determination of K4
`(‘alculations
`Determination otKu with impure antibody
`4. Affinity measurement in solution by an KIA-based mothud
`Rationale
`Requirements (or the determination 01' Kg
`Determination at K“
`5. Conclusions
`References
`
`Measuring antibody affinity using
`biosensors
`Laura]. Hcfm, Anna M. Wu, Michael Neumnit'r, and
`John If. Shiite/y
`1. Introduction
`
`2. Theoretical aspects
`Mcasuringasso
`iron and dissociation rate constants
`Limitations on measuring all'inity constants
`3. Immobilization, binding, and regeneration oftiio HlAeoro
`Immobilization step
`Regeneration step
`‘
`4. Kinetic analysis of antl-(il-IA antibodies
`Direct binding assays: cumparimn of mttrine and chimeric
`.
`TM. l2
`.
`Indirect binding assayn; comparison of marine and chimeric
`T8466
`Assays for engineered antlhody fragments
`As‘tays tor anti—idiotypic anliliudy
`5. Conclusions
`
`xi
`
`78
`73
`78
`79
`tit)
`til
`HI
`til
`82
`86
`85
`91)
`‘41
`9|
`VI
`04
`95
`96
`
`9‘)
`
`99
`till
`IUI
`HR
`103
`MM
`[OH
`I03
`108
`100
`”2
`115
`llfi
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`
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`
`
`Lassen — Exhibit 1059, p. 4
`
`Lassen - Exhibit 1059, p. 4
`
`

`

`
`Contents
`
`
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`Acknowledgements
`Rufrtrnncns
`
`Analysis of human antibody sequences
`Gt'rltlrl Waller/Int] Inn M. Tmnlinron
`1, Introduction
`2. Amplification and cloning ol‘atttibudy V genes
`(icrmlinc V segments
`Ruarrttnged Vgenes
`3. Sequencing of innnunuglubulin genes
`Sequencing printers
`Templatt.I preparation
`Sequattcittg techniques
`4. Analysis 01 antibody sequences
`Stil'twnrc pack nit-art for sequence analysis
`Edi mg. translating, and comparingscqucnccs
`Multiple alignments
`Databases
`Statistical analyses
`Roforonces
`
`Rodent to human antibodies by CDR
`grafting
`Mary M. Hendig and S ’I'arran Jane:
`1. introduction
`2. Clotting ttnd nutlttettcing mouse variable regions
`:1. Construction ofa chimeric antibody
`4. Dtmign and construction 0151 roshnpod humntt antibody
`Analysis 01 the mouse Variable regionn
`Design 01 the reshaped human antibody
`Construction of the rthflchl human antibody
`5. Preliminary OXprosuion and analysis of the reshaped humntt
`antibodies
`Acknowledgements
`References
`
`1""
`
`Contents
`
`8. Converting rodent into human antibodies
`by guided selection
`Ilt'nm't.’ R Nougat/mom. Dubwth lillt'n, unrl Antiwar]. Rottem
`l. introduction
`2. Cloning. expression. and characterization of rodent :1qu
`fragmnnts
`3. Construction oftnrgtt chain-shuffled rnpnrtoircs tn guided
`selection
`’I he tirst DNA shuine: combining muritte V“ with .1 human VL
`rcpt-stain:
`('ttmtrttt'tion or fully human ClIain-slitltllcd rupertoircs hy
`mtni‘ining the selected human VL grot- witlt a human V”
`rcpcrtoire
`4. Selecting hulf—htttttatn or ( tttttplclaly human antibodies by
`display and enrichment sit-m
`References
`
`9. Choosing and manipulating effector
`functions
`Inger Smnllie and 'I'erju E. Mit‘lmalrun
`1. Choosing effector functions
`2. Modiation of efl'nclor functions
`(.‘omplcmuttt activation and lyxin
`1-‘cyR-mediutud activities
`It. Mt‘t‘h‘ttltllfl cotnpletnrtnt activation and train in vitm
`tit
`stluclurtll thuircutcrtt‘lurar-ttmlcntcltl urination
`(nurturing thr igu \ullt‘lnmfi 1n \“iilttplulilul'll :iL'IIVLlllU'I and lysis
`4. Mnnnttfltig Anlli'. in win”
`Iht: structural “outremetm for FqR binding.
`Comparing tttc lgtj IMhElJlles in ADCC
`5. Measuring phngocytosis and respiratory burst
`ti. Optimizing effector functions
`Attknnwludgentnnts
`References
`
`lft‘)
`
`169
`
`173
`
`1111
`
`162
`155
`
`187
`
`187
`1118
`18‘)
`111‘)
`Wt)
`WU
`1‘10
`195
`1'5
`1‘15
`197
`200
`201
`201
`
`Iln
`11h
`
`119
`
`11‘)
`119
`123
`I25
`125
`120
`126
`12‘)
`1.17
`137
`137
`1315
`139
`140
`144
`
`147
`
`147
`147
`154
`155
`155
`155
`157
`
`104
`166
`168
`
`
`
`Lassen — Exhibit 1059, p. 5
`
`Lassen - Exhibit 1059, p. 5
`
`

`

`.
`”II
`
`f I
`
`-.
`.-,I:_
`-
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`
`Contents
`10. Producing antibodies in Escherichia coli:
`from PCR to fermentation
`Amlrurls Plllckllum, Alike Krrbhrr. C[mm Kn'lzhfl, Uwr Hum,
`Uwr Kuiipfer, Roi] Wemlrrmh. Lars Niclu‘r, Karl I‘mbn. mm
`0m” Rn’mrbem
`‘1. introduction
`2. Cloning of antibody variable domains
`(‘lloircolanlibody format
`.1. Expression slrnlugieu
`Ovurvicw
`Svcrvlion
`Fuuclional anlihodies from the cylnplnsm
`.
`.
`,
`,
`4. Improvmg :IRIIH‘SSIOHI [he influence nl Ihe anuencc on
`nxprms' n uml folding
`Analysis and lung-lurm solutions
`ShUrHcrm solutions
`5. Growlh and fermentation
`C'ullivnl‘ion in slnndard shake llnsks
`Cullivulion in henchlop flasks Io medium cell-densities
`Cullivuliun of IL. unli in a ll) litre [crinenler lo high
`cell-densities
`fi. Antibody purificalion
`Gunaral considcraliuns
`inlmohilizcd mclnl ion-nliinity chromatography (MAC)
`Acknowledgements
`Appendix: Abbrovinlinns for IICDC
`References
`
`11. Production of single-chain Fv monomers
`and multimers
`.
`.
`,
`,
`deul Ftlpulu, Inf/my Mern', turd Marc Whillmv
`1. Introduction
`2' Linker designs
`3. scFv gone construction from hybridornn cells
`4 FX m ,
`1.
`.
`l
`'h .
`F i
`,
`I'
`'
`' P ' won 0 5mg 81’ mn V n h‘ m'
`5. Farmontnlion, reriaturoliur1.flnd purification of an san
`protein
`Re I’erenuns
`
`2m
`
`203
`204
`2|]
`2I5
`7”
`5.5
`22|
`
`226
`226
`22”
`229
`22‘)
`23l
`235
`244
`244
`245
`248
`249
`
`2’”
`
`25]
`
`253
`254
`255
`259
`261
`260
`
`l'f
`
`'
`
`Calllelllh‘
`n
`l
`I
`12' prrassron of immunoglobulm genes In
`inninmallan Cells
`Ullislvpllrr Dubbingmu
`1- lnlroduction
`2. Transiont expression ulanlihodins in COS cells
`Choice of cell line and medium
`Choice 0‘ expression Wm"
`3. Dillydrolololo reduclnss selection in Chinese hamster
`“WW (CH0) cells
`Choice of cell lin: and medium
`DI IFR vector design
`Trunsiection 0‘ CH0 cells
`Selerlion for amplification of DHFR vector wilh melholruxme
`.
`i
`.
`.
`4. Gluturmna synlholaso selection in Cl'HHGSB hamster ovary
`(Cl 10) Rolls
`Choice ofccll lype and medium
`2/36”" dersigp
`IL
`[ans a lo”
`5- Clulumine synthetasn selection in N50 Inyeloma walls
`Cllolfl‘ Ul Cells and mcdium
`GS-vrclor dcxign
`.
`.
`.
`.
`.
`.
`.
`CE" ".mmmun
`Selcuuon for (ls-gene amplification in Nbll cells using MSX
`8. Cunnluding remarks
`References
`
`’
`13. Preparation and uses of Fab’ fragments from
`Escherichia call
`
`l’rml Caner, Maria L. lerigm'sJalm W. Park, and
`“WW" Z‘W‘W
`1.1
`do 1101
`"gsmit-w I
`High-love] mmlumiun ol' Ah fragmunls in E. mli
`'l'mching of Fat“ fragments l0 lht: pcriplasmic space of E. ml]
`2' Recovery ”l “l" l’afln‘MlS [mm 35- "”1"
`Release of slalom: FIN—5H minim-III: ham {-7. ml!
`Recovery of Fab’—Sll Imgmcnlslrom E call
`Endmoxin rcmovul
`Dclcrminalion of free Illinl content
`
`2611
`
`26"
`270
`270
`27‘
`
`273
`273
`274
`275
`27H
`
`27"
`27‘)
`3:5?)
`h
`282
`2K2
`2H3
`2M
`287
`28“
`253
`
`291
`
`NI
`191
`292
`292
`293
`293
`293
`294
`2%
`
`
`
`Lassen — Exhibit 1059, p. 6
`
`I
`
`'
`
`
`
`Lassen - Exhibit 1059, p. 6
`
`

`

` Contrmls
`
`'
`
`'.
`
`Acknowledgements
`Rnfnrnncns
`
`A1. Addresses of suppliers
`
`A2. Sequencing primers for antibody Vgrmcs
`
`A3. Sequences ofhuman germ/ins VH,V.., I”, and Ir
`segments
`Index
`
`
`3. Application of Fab’ fragments from E. coli
`
`
`Cnnstruullun o! monnspccific F(ab’)_y fragments
`Construction oi hispccifir: Half), lrngmunts
`(‘tmslruclion oi Half—PEG
`
`Cunslruution of Fab‘—inimunolipusumcs
`
`lmmnliilimlion of Fnh’ SH for use in allinity purification
`
`.'-
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`297
`297
`Jill)
`301
`303
`305
`307
`Jm
`
`.109
`
`3L5
`
`31f:
`3Z|
`
`Contributors
`
`DEDDRAH J. ALLEN
`Cnm'luidnu Antibody Technology Ltd. The Science Park. Melhnurn. Cum-
`bridgc SGS 6E1. UK.
`MARY M. lleNDIG
`Ludwig Institute for Cancer Research. (nh Floor. Glen House, Slug Place.
`London SWIE 5A], UK.
`CHRIS'I I)I'HL"R DEBBING I‘ON
`Celllcch Therapeutics Ltd. 216 Bath Rd. Slough. Berkshire SL14EN, UK.
`MANIANVF untrur.r:.\i.w.n
`[)qmrmum oi [Jul-aluminum and Signalling. Bubrahum lnstllule. Bahrahum.
`Cambridge ('32 IlA'i'. UK.
`Mill I'AHTER
`Department of Mulcrulnr Ont-nlogy, (icncnlech Inc. 460 Point San Bruno
`Boulevard. Scull: San Frann-wu. California 94080—4‘1WI. USA.
`DAVE CulswtzLL
`Cambridge Antibody 'Iechnolngy Ltd. The Science Park. Melliuurn. Cam-
`bridge 508 (ill, UK.
`Nlrllfil as l-', DAVIH
`I)£p:lltl'nunl ol‘ Bincltumhtry and Molecular Genetics. SI Mnry's Hospital
`Mvdrunl School. Nurt'ulli Place. London W2 1P6. UN.
`LISA DJAVADl-OIIANIANCE
`Uniu': dc Biochlmle Ccllulairc. lnslilut Pnslcur. 28 rue de Doctcui Roux.
`75724 Paris. cudcx 15. France.
`M. JIM EMBLE‘I‘ON
`Paterson Institute for Cancer Research. Christie Hnspilul NHS Trust. Wilm-
`slow Rd. Manchester M20 98X. UK
`DAVIE! Flt I'LII.A
`Da-pnriuu‘nl ul
`'l'rrlnur Biology and Molecular Genulics. Enzon Inc.. 40
`Kingshridgc Rd. Pisciilaway. New Jersey 08854-3998. USA.
`ll'l-LllTRAND FRIUUL’T
`Unilc' dc Biochimie Cellulniru. Institut Pasteur. 28 rue de Doctcur Roux.
`75724 Paris. cedex 15. France.
`MICHEL H.001 DuERG
`Unilé de Biochimie Ccllulairc. lnslitul Pasluur. 28 rue d: Doctcur Roux
`75724 Paris. cedex 15. France,
`
`
`
`Lassen — Exhibit 1059, p. 7
`
`Lassen - Exhibit 1059, p. 7
`
`

`

`Nicholas P Davies at al.
`
`4h.
`
`49.
`50.
`
`I’.
`
`
`47.
`timbers. N. Taylor. L. D. Harding. F. A.. ‘i'mnitsline. Mt. Higgins. K, M,.
`
`Bit-Ionann. S. R.. Kuo. C,(‘ Maslteyekli. R , Wymnrc. K.. McCabe. .I. (7.. M|II|n'/-
`0'an . It
`(J'LMnm-I ,. _
`fik'lltLS r. . [lemma-lien.
`l' Fntimlu. I). M..
`
`
`
`l‘arntark. C. L':.. Kay. H M ..tllrl|iuanl.1).{i‘I‘m Nmnn'. 36!. HFE-
`'i.
`Wagner,
`5'
`l1. Witlnmn. tr.
`I anon. T.. NL‘ttl‘cISttl. 5i
`.‘i . Kin-mum. f)..
`|'..
`
`Ilaiewslty.
`h.
`. Klan I_ Ailtl Htumeinamt. M [I'W-IJ. Aim! dtid- Mrs. 22,
`I389793
`
`Iluxley. C. Farr. C. Gennuro. M L. and Hauf. r (I994) Bitllt't‘llflnirlgy.
`I2.
`Shh-9i].
`
`Luutner-Rieske. A.. Htltnristcr. H.. Barbi. 0.. and Zachau. H. 0 (I993)
`Gum/nits. 16. 497—501
`
`Weiclthold. G M.. Huher. C . Parnes. J. R . and Zachuu. H. (10993) (immnim.
`6.51244
`
`52
`Weichltold. G. M.. Ohnlteiseri R. and Zaeliau. H 0 (I993) GL’IIIIIHiL'I
`l6.
`SUI-ll
`
`53.
`Kuhn R. M. .tntJ Ludwig. R. A. “994) Gone. MI. |25-7,
`
`. Spenser. Ft. Huge-tat. Y.. Sitnchcn. 0.. Hurku. Oi. ('onnelly. C. and Hieler.
`
`(I994). Geimmit‘x. 22. I Iii-2t).
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`4
`
`Measuring antibody affinity in
`solution
`LISA DJAVADI-OHANIANCE. MICHEL E GOLDBERG. and
`BERTRANI) FRIGUET
`
`1. General considerations
`Determination of the affinity ot'a monoclonal ttnlibody (mAb) for its antigen
`(Ag) is of considerable importance It gives a quantitative indication of how
`strong the interaction is between the Ab and the Ag. It is the basic experi-
`mental parameter in a variety of studies. such as the analysis of mAh/Ag
`binding mechanism (1 , 2) or the use of mAbs as confotinationnl probes (.3. 4).
`Hence the need for convenient and rigorous methods to determine the Win-
`ity. The affinity. KI (Ki : tin). of a mm for its antigen is defined by the
`Law of Mass Action as:
`
`
`
`K. = [Ag] x [AM/[complex]:
`where K4 is the equilibrium dissociation constant. [complex] is tlte concentra-
`tion of saturated antigen or mAh sites. [Ag] is the concentration of free anti—
`genic sites on the antigen. and [Ab] is the concentration) of tree binding sites
`on the antibody, 'I'hese concentrations are funding .n'le cmtcz'rttmtiun: in the
`solution at equilibrium. This has three important consequences. as described
`in Sections I.l-l.3.
`
`1.1 K. does not directly reflect association or dissociation
`kinetics
`K.. depicts an equilibrium property. and therefore does not reflect the speed
`at which equilibrium is reached. Yet. Kd does depend on the association and
`dissociation rate constants.
`In many cases. the relationship between the equilibrium and rate constants
`is simple. When binding is it simple one-step reaction, K. = knit/kn... where km,
`and kn" are the association and dissociation rate constants. However. when a
`significant conformational change of either the mAb or the Ag occurs upon
`association. important deviations front that simple equation can be observed
`
`
`
`Lassen — Exhibit 1059, p. 8
`
`Lassen - Exhibit 1059, p. 8
`
`

`

`Lisa Djavarli-Ohnninnce at al.
`
`(I). For such cases, measuring only k0,. and kn" and using K, = [gm/kl... would
`
`provide an erroneous estimate of the affinity.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`1.2 True K“ control be determined when the mAb or the Ag
`is immobilized in a solidphaso assay
`Several convenient methods such as ELISA or surface plasmon resonance,
`with the Ag or the mAb immobilized on the titration plate or on the ‘scnsor
`chip'
`(mgr gold covered with n catmsstttcthylaled dextran hydrogcl). are
`often used to provide affinity values. Such measurements yield rent values of
`Kd only rarely. One reason is that immobilization often results in a partial
`denaturation of tire proteinI thus modifying its binding properties (see ref 5
`for review) Secondly. Kd its defined in solutiott, with both the mAb and the
`Ag diffusing .mt! totaling freely in solution. but lit the \tIIlU-ftlluat assay rim.-
`of the partners is immobite. This can result in estimates of N. by Solid-phase
`nanny being orders of magnitude different from the real affinityl itt solution
`UI'II.‘ should fmpl‘liflilr‘lll however. that mcauutcfilcttls or an apparent bind-
`ing constant' by solid-phase assays may. for some purposes (such as compara-
`tive studies). be sufficient, In some instances. the binding constant obtained
`by solid-phase assays might even be of more significance than the Ks deter-
`mined with the Ag in solution. Thus, with respect to diffusion and rotatiort,
`an antigen on the surface of a large cuktttyotic cell may behave more like
`the Ag immobilimd in a solid-phase assay than the Ag in solution. provided
`that precautions have been taken to avoid dcnaturation of the Ag upon
`immobilization.
`
`
`
`1.3 The determination of Kd must take into account the
`valency of the mAb and of the Ag molecule
`The Law of Mass Action (K. = I/tgl x [AH/[compfrxll expresses the concen-
`trations of reagents in terms of reactive sites. that is. antibody-binding sites
`(free or saturated) and antigenic sites on the antigen molecule (free or satu»
`rated). To determine the affinity front binding experiments. one must there-
`fore determinc these values. Thus, one must measure not only the atttibody
`and antigen concentrations. but also their valencics (number of sites per
`molecule) and understand how this valency influences the experimental
`determination of the binding. This is discussed in a recent review (6).
`
`
`
`
`
`
`
`
`
`2. Overview of methods to measure affinities in
`
`solution
`
`Measuring Kd consists of mixing the mAb and Ag at various initial concen-
`trations. bringing to equilibrium, then measuring the concentrations of free
`and saturated sites at equilibrium and analysing the binding curve. The
`
`78
`
`
`
`
`4: Measuring uttlr’body affinity in solution
`experimental difficulty resides in distinguishing the free and bound states of
`either the mAb or the Ag, Several methods can be used such as:
`o equilibrium dialysis for haptens rind dialysablc antigen
`- radioimnruttoassay using precipitation with salts or other agents
`0 tiltration
`- fluorescence measurements
`- ELISA- or RlA-based methods.
`In this chapter, we discuss the practical aspects of measurement using fluor-
`escence. ELISA. or RlA and describe, in detail, the ELISA- and the RlA-
`based competition methods for measuring affinity in solution.
`2.] Fluorescence
`The use of fluorescence to determine K4 requires that either the mAb or the
`Ag be fluorescent. and that a change in fluorescence should occur upon for-
`mation of the mAb/Ag complex. The fluorescence signal used can be either
`intrinsic (e.g. tryptophan residues from the mAb and. possibly, from protein
`antigens: some prosthetic groups such as pyridoxal phosphate. NADll,
`flavins, etc.) or result from prior fluorescent labelling of the Ag or the mAb
`with a fluorochrome, The fluorescent change observed upon association may
`be one of the following:
`a wavelength shift
`a fluorescence quenching
`a fluorescence transfer
`a change in fluorescence polarization.
`We shall not discuss the practical aspects of these experiments because
`each mAb/Ag complex poses Unique problems, and hence the exact pro-
`cedure depends on the fluorocltromes used. Thus, the wavelengths of fluor-
`escence excitation and emission vary from mAb to mAb and front Ag to Ag.
`even when the same fluorochrome is studied, The procedure used also
`depends on the nature of the change observed (quenching, transfer. polariza-
`tion, shift) and on the type of fluoritneter used. Muny unlabelled mAb and
`Ag complexes do not give rise to a fluorescence change and individual
`labelling procedures must be devised for each. There is no reliable way to
`predict whether or not a given label will give rise to a measurable fluorescent
`change upon complex formation. it is therefore impossible to provide a rule
`of thumb as to which fluorochrome one should use and how exactly one
`should proceed to measure the affinity.
`We shall. however. briefly discuss some of the pitfalls that should be
`avoided when using fluorescence for affinity measurements:
`(a) Reagents used for fluorescence labelling should be highly purified
`because of the intense fluorescence signals sometimes produced by im-
`purities.
`
`79
`
`
`
`Lassen — Exhibit 1059, p. 9
`
`Lassen - Exhibit 1059, p. 9
`
`

`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Lisa DjnvrtdiOhaniance ul it].
`
`(c)
`
`(b) The sensitivity of current fluorintetcrs sets il lower limit of 10‘“ to Itl’" M
`on the Ku that can be determined. Some IIIAl“ ltnte much higher affinities
`for their Ag.
`,
`to heternet‘ttemtsly
`I.‘.Ii‘:|iill[i. ol antibody UI antigen oilett eive< res:
`lzilwlled products which retltttrc pmilieutiun. ainee imilt the number nl
`lluutnehruutes and their position on the trnileeule affect llte fluorescence
`signal.
`the binding eltnl.li:tt‘t'tv~llt.‘\. eitlier hy sterie liil'lv
`(d) 'I1te Libel mny nlfect
`drnuee or through n change in conlnrmniinn. tine. mttdilying the A}.
`this.
`should ulwrira he elieeked by peril-tinting competition experiments with
`the unlabelled molecule.
`(c) when tending the fluorescence in the tluarimetrr, one should inke- tutu
`uerotint the :ibmrhnnee ufthe solution :it the wavelength of emitatinn nr
`anti-«ion when this nttsurltrtnce is Imt negligible. i.c.
`ili‘tfl't:
`lLtlfi (ciieet
`reieued as to inner llltcr eftect]. Keel-ling lhehfl! precautions in mind.
`Iltlttlettcellt'e mcaktttementt should provide precise at Emit).I values (it.
`
`2.2 ELISA- and RIA-based methods
`A method that in Frequently meet for eetimmini: ilie altiuity iii .1 hints [or a
`innerumnteeuiur antigen iv. to Measure the moment in! tuM: hound III on M.-
`eonlcd ELISA plate utter ineuhatlt-u with dim-rent eunmntmtmns ht nint-
`In the-i4.- rxperiinents, the man u eentratinn lhlll yieidi tutti-meme“! ililili‘
`
`illf, till
`the eonted utitigeit
`ie taken in the reetpmenl
`til
`the alrtnity. 'I'Iiis
`:Imiiuneh does not. however. nlltrw detelrni
`itinn nl reul
`.ifliuity became
`eqtiitiltriunt
`it attained at
`the liiiuiilkulid in ulnee retliet th I
`in whiten:
`
`[nee Scetiun l2). Moreover. as nlready diseussed [see Section 1.3]. mating
`the antigen on to the 1.11mi: tty um.- oi the emit-entities
`:Idwnttiott ntethudn.
`may alter its ettttlormminn i5. til. which :Iiltruh the mhliim: htteruetitiri um]
`hence the nl'i'inily. Thus. although providing tut enliitiute ul the within: elii‘
`cietiey oi the binding. at a limb to the inurrtrtrflirwi antigen, nueh direct
`IEIJSA- ur KIA-based methods luii to provide Ihe teitl nfilttil)‘ [or llu: nun-w
`Ilflliflcl].
`Twit rnnin ELISA-hunt! methods him.- lteett described {see rel! ‘l' and 9}
`{or nllltlylt'lf. tlte tlsmtclalilrnidimtlwlttllufl etluiiihtinttt
`in interim! and have
`been teeerltly reviewed {it}. Beth tely on the [allowing principle. Minutes a!
`the limb u:
`it listed eoneentrntittrt untl the antigen Ill Vitryirti: mneeulmiuni
`lIfL'
`ineullttled ttttlil equilibrium is rent-lied A 'milIvahilliL' assay using Ag:-
`eitntetl [times in: used It: determine the mneemrttlirltt ol' the free (Le. not
`n‘fln‘iillml with antigen! Innbnl equilibriuhi.‘l'lte method:- difler in two mititt
`resiteett: lllc euitdttium under which equtllhriurn is
`hIi-ihed (in the plet-
`
`enee ur absentee of the muted tinligen} umi the em one under which the
`ELISA is |terlormed TIms. Friguet rr nt. :7) first ineuhale Ilte utAt't IIt‘ILI the
`80
`
`4: Measuring antibody "[[inity in solution
`31-. in <olttliutt for It Iltne nttiiieietit In tench eqetlthriutu. ullt‘l onty Ihcrt "DII5'
`
`let the euuihltrnte snlutitut ll'lltl un If .l$:\1ilute lfjdcltlllllllt‘ the cemenlrav
`
`thin 01' Itee mAli.
`‘he .nnmmt oi M: milled in enclt ucll :uttl llte intuhutnui
`time In: the ELISA are such thnt only .‘I sins-It [mutton in! Hunt 5— "mi u!
`
`the unsaturated mAh .11 equilibrium I'- "rim“.‘d It).I the e
`mi
`.iiihgen.
`'l'lth
`ensures thnl. tlttimp. the ELISA. equililuiuiit in solution it; not nignifirnntly
`
`modified. By lIliI means. the UhWIVHI etitiilihrit
`I constant corresponds to
`the teal nl‘lintty. In I'ln: other method [we [or example lei. 9} where the unli-
`Itody l'i Ill t'nnlnu With the noinb'lt: ill'lll imtnohiiizcd :tnllgen .aitttulltittettllsiy.
`
`uncontrolled kinelit' [at-Inn are likely to .tlt'ect
`the relative hinditt of the
`:mtilaody to the antigen in the liquid and solid plumes, nod no cure is taken to
`.wnltl J sltiil oi lltL' cttuiiihriunt
`in Ilte liquid pltnu: when some til
`the lie:
`mitt: becomes hound In the solid phase. This method Ihcrel‘nrc mien yield-t
`ulttlet'cstimuteet oi the real - i 'ntly. Thus. the method iii Frittuet rt iii. (7) is
`
`rccoittmenued when deleri
`ion at the real nitinity is required nnil this
`method is described in detail in Section 3.
`
`3. Affinity measurements in solution by competition
`ELISA
`3.1 Theoretical aspects
`The antibody site to antigenic site association teaction can be written as fol-
`lows:
`
`antibody + antigen H complex:
`
`with the concentration oi antibody sites. antigen sites, and complex at equi-
`lihriunt givenm|eliii.l21g|.tindl.t‘]. respectively.
`The concentration of antibody tile: lat-‘1] and the antigen sites [Ag] at equi-
`librium .1“: related to the mini nntihitdi‘ sites [Abll and the total antigen sites
`[’4in by:
`
`W71= lAbtl-[Xl
`Mal = We] - (Xi.
`K4. the dissociation constant. is defined by:
`Kd =l/1sll/1bl/[Xl-
`If [Ag.] is varied while [Ab.] is kept constant:
`K: = Mela/4M - [I‘D/[xii
`
`consequently:
`
`[ll/[Ah] = [Aid/(Mel + Ki)
`81
`
`[ll
`[2]
`
`[3]
`
`
`
`
`
`Lassen — Exhibit 1059, p. 10
`
`Lassen - Exhibit 1059, p. 10
`
`

`

`3.2 Rationale
`The method we have developed requires two steps
`
`(a) in the first step. the antibody. at a mllh‘flfll concentration. and the antigen,
`tlI. various concentrations, are incubated in min/ion until equilibrium is
`reached
`
`(b) in the second step. the concentration ol the antibody that rurriains tree at
`
`equilibrium is inettsnrcu by .1 classical utilirect ELISA in which the anti-
`gett is coated on the rniemtiti
`tun plate
`Tire atate tnutrvc rrr partially denatured uptnt uniting) ut the united antrv
`gen nntl whether or not it I: recrtgnittctl try the MM! dilletentltr Iran: the solu-
`
`ble ltl'l gen 1: not important as long an the coated antigen enn «pectin-ally and
`quantitatively trap lht: tree at body.
`
`4.- Measuring antibody rtffint'tyin solution
`Protocol 1.
`Indirect ELISA procedure
`
`
`
`Equipment and reagents
`Iltmti-
`uELISA pllla spectrophotometer or
`matar tLabsy-Iarns. Dynaleeh. SLT Lat»
`Instrumental
`o Flaw washer. Hundlwuh lypa (Titafleit.
`Dynarachl
`nafi-wnll
`Ilat bottom mictotitralimt plazas
`and plate saalara
`- 6 ml glau tubes
`- Rape-tor plpnnaa and lips to a. Epnenderl
`Multipanal
`. Intnwnpwtturuntl' mtibody
`directed
`limit mnula Immunufilflbulifll linluul tn
`nmltns minimal!" or
`ll-galactoslrtssa'
`to t!
`Irom Bin-ya, Snulhflrn Biotechnology
`Associates Inc.. Frontage)
`.Subllralo lululion 1:
`Int alkali