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`PARTS OF APPLICATION
`FILED SEPARATELY
`NOTICE OF ALLOWANCE MAILED
`
`A /Y1, D aie '" P
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`ISSUE
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`NUMBER U. I I
`WARNING: The information disclosed herein may be restricted. Unauthorized disclosure may be prohhited
`by the United States Code 'Iltle 35, Sections 122, 181 and 368. Possession outside the U'S'
`patent & Tradomark otfice is restricted to authorized employees and contractars only'
`
`(FACE)
`
`
`
`Page 1 of 220
`
`ILMN EXHIBIT 1035
`
`

`
`oo/zffiasa -';
`
`CONTENTS
`
`Pl l,i:{'lffi [\-,rffi;1;;
`,{lil il i' X$gdt
`{ :i l"I{" be.rs., i', t"i,_;
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`d'**r ?ql i
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`
`r,'.-APPROVED FOR LICENSE l-l
`lul'2zgqaui
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`(6qs
`ct lX
`)s --
`
`Date
`Received
`or
`Mailed
`
`,l
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`\\.
`-
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`11.
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`15.
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`16.
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`17.
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`18.
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`19.
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`27.
`
`31.
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`
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`Page 2 of 220
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`

`
`POStTtON
`
`CLASSIFIER
`
`EXAMINER
`TYPIST
`VERIFIER
`EORPS CORR.
`SPEC. HAND
`FILE MAINT.
`DRAFTING
`
`Date
`
`Staple lss,ue Slip Here
`
`rD NO.
`/f
`Vz*"'
`323
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`INDEX OF CLAIMS
`
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`I{\'
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`{}to
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`- 0hrouoh numbenl) Canceled
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`N .,................,............., Non-elected
`| ....................,..........,. lntsrtetence
`A ..........................,..'... Appoal
`0 ........,........................ 0bjscted
`
`(LEFT TNSIDE)
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`-
`
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`
`Page 3 of 220
`
`

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`SEARCHED
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`Sub.
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`Date
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`Page 4 of 220
`
`

`
`Cfrb
`'Ilrxttd
`
`PTOUNUWGRANT
`It
`PaperNumberl /T7-I
`
`The Commissioner of Patents
`and Trademarks
`' Has recetved on opplicotion for o potent
`for o new ond useful invention, The title
`and description of the invention ore en-
`closed. Thte requirements of law have
`been complied with, and it hos been de-
`termined that a patent on the invention
`sholl be gronted under the law.
`
`Therefore, this
`
`United States Patent
`
`Gronts to the penon or persons having
`title to this patent the right to exclude
`othercfrom making, using or selling the
`invention throughout the llnited States
`of America for the tern of seventeen
`yean from the dote of this patent, sub-
`ject to the payment of maintenance fees
`as provided by law.
`
`..4,h(
`7
`Alata( Qhr*
`^lMrtfi"-t
`
`Commisslonet o! Patenls and Tmdenu*s
`
`Arftst
`
`Pro-1584
`
`(RTGHT TNSTDE)
`
`
`
`Page 5 of 220
`
`

`
`;{."Jq
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`
`...
`
`PATENT A??LICATION SERTAL NO.
`
`U.S.. DEPARTT,IENT OF COMMERCE
`PATENT AND TRADEMARK OFFICE
`FEE RECORD SHENT
`
`:i.4* X.P *6/?'l/F4 il$fSX*S4.
`
`.1. cuj.
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`3$5. S0 fl{ i},1$-S01 , esiu
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`
`
`
`Page 6 of 220
`
`

`
`;,f\
`
`. -.1!r
`\!'S**
`
`3AB CODE LABEL
`
`ililllililililililililtililtil tilil tilil til ilil
`
`U.S. PATENT
`
`!\
`$.,*
`tr --r"\.
`,S.-'-,\CATION
`
`SERIAL NUMBER
`
`FILING DATE
`
`CLASS
`
`GROUP ABT UNIT
`
`o8/253,854
`
`06/03/e4
`Rrrr.n 6r)
`? RT.HARD A. HoucHTEN, soLANA BEA.H, cA; .rulro H. cunRvo, LA ,JoLLA, cA;
`g CLEMENCIA PINILLA, CARDIFF, CA; .ION R. APPEL, JR., CARDItr'F, CAi SILVIE
`E BLoNDELLE, LA JoLLA, cA.
`
`435
`
`L802
`
`**CONTINUING DATA*tr*** * ******** *******
`VERTFTED THIS AppLN rs A DrV OF 07/797,5s1 LL/L,/gL
`
`*,TFoREIGN/PCT APPLICATIONS'I * * * * * * * * * * *
`VERIFIED
`
`FOREIGN FILING LICENSE GRANTED 07 /tL/94
`rOTAL
`NDEPENDENT
`CLAIMS
`]LAIMS
`
`SHEETS
`DRAWING
`
`OB
`]OUNTRY
`'TATE
`
`**d.** SMATL ENTITy *****
`CTTORNEY DOCKET NO.
`
`FILING FEE
`RECEIVED
`
`CA
`
`7
`
`L1
`
`2
`
`s3s5.00
`
`IMSOOl.2DIVI
`
`DRESSLER, GOLDSMITH,
`AND MILNAMOW
`180 N. STETSON AVE.
`47OO TWO PRUDENTIAL
`cHrcAco, rL 60601
`
`SHORE, SUTKER
`
`PLAZA
`
`SYNTHESIS OF EQUIMOLAR I'{ULTIPLE OLICOIT{ER MIXTURES, ESPECIALLY OF
`oLrcoPEPTrDE ltrxruREs
`
`an
`an
`
`uJEoo
`
`H
`F
`
`This is to certify that annexed hereto is a true copv from the records of the United States
`Patent and Trademark Office of the application Which is identified above.
`By authority of the
`COMMISSIONER OF PATENTS AND TRADEMARKS
`
`Date
`
`Certifying Officer
`
`
`
`Page 7 of 220
`
`

`
`0B'a5i]85,{
`
`ffinrr;\
`/r' .trflhf ?
`w" 'iS$4
`Wrt***S
`
`IN TIIE rnrIlED qTATES PATENT Al.iD TRADEMARK OFFICE
`FileNo. rqs-001.2 prv I 34L8/6090:7)
`Anticipated Classification
`of this application:
`Oass-Subclass-
`Prior application:
`F.,:raminer-Jr"-grceq-
`Art Unit___-I992_
`TIIE COMMISSIONER OF
`PATENTS Al.{D TRADEIViARIGS
`WASHINGTON, D.C. 2tr23l
`
`I hereby certitY that this P^apet
`b b€ing acpositeO with the Unibd $des
`Postal -Ssrvice as Express Mail in on
`enveloge tddressed to: Hononblg
`Conrmisibrrr of htents and Tra<lcaurtf
`Wr*rlr1bn, D.C. .20231,. ql
`Oate Jr:ne 3, 1994
`Erpr6 Mttit tabel No'
`T859847 47 45US
`
`Sin
`
`.
`
`This is a request ;sl filing a
`( ) Continuation application,
`E) Divisional application,
`under 37 CFR 1.60, of pending prior application Serial 11o.
`074797'551
`of Richard A. Houqhten et aI.
`filed on LL/Lq/9L
`(Date)
`(Inventor)
`fs1 SYNIHESIS oF EerJIlaoIAR MUL, rIPLE,oLIGo}IER MD${.rRES, ESBESI4IL- Y-9F.
`frXilr*",
`1. (x) Encloced is a copy of fhe prior application, including the oath or declaration as originally
`filed. (&e 8 and 8a for drawing requirements.) The attached Application papers are a
`true copy of prior application Serial No. 07,/79?,551
`as originally filed
`on Nov' 19, 1991 . and no amendments referred to in the oath or declaration as
`originally filed introduced new matter. This statement is made with the knowledge that
`willful filse statements and the like so made are punishable by fine or imprisonment, or
`both, under Section 1001 of Title 18 of the United States Code and that such willful false
`statements may jeopardize the validity of the application or any patent issuing thereon.
`
`2. (x) The filing fee is calculated below:
`
`CI.AIMS.{S FILED IN TI{E PRIOR APPLICATION, LESS ANY CI-AIMS
`CAI.ICELLED BY AIVIENDMENT BELOW
`
`For
`
`Basic Fee. .
`
`Independent Claims.
`
`Total Claims..
`
`Statement of Status as
`Small Entity Reducing
`Filing Fee By Half To
`
`AI)M08&1strA092
`
`No. Filed
`
`No. E:rtra
`
`Rate
`
`Fee
`
`2 -3 =
`11 -20 --
`
`-0-
`-0-
`Total
`Filing Fee
`
`$ 710.00
`x J74.00 = $--
`x $22.00 = $--
`
`$ 710.00
`
`g 355.00
`
`
`
`Page 8 of 220
`
`

`
`ts.JllN
`
`f'',*;,*;* # A verified statement (Declaration) clairning small entity statu was filed in
`
`the prior application and small entity status lor this application is proper
`and desired"
`
`4.
`
`5.
`
`( x) A check in the amount of $ 355. L
`is enclosed"
`( x) The Commissioner is hereby authorized to charge any additional fees which
`may bi required for this,application under 37 C.F.R !$1.161.17, or credit
`any o/erpaymenf to Depcit Account No.23{920. A duplicate coPy of this
`rheet is encloeed"
`6- (x) Cancel iD this application original claims 1 through 71, inclusirre
`of the prior application before calculating the filing fee. (At least one
`original independent claim mrst be retained for filing purposes.)
`7. ( x) Amend the specification by inrcrting before the first line the sentence:
`-This is a ( ) mntinuation ( x) divisiou of application Serial
`. ffi"O 'Novenbdf 19, 1991
`No. o7n)7;55L
`& ( ) Transfer the drawingp from the prior application to this application and
`abandon said prior application as of the filing date accorded this application.
`A duplicate copy of this sheet is enclosed for filing in the prior application
`file. (May be used only if signed by penon authorized by $t.t$ and before
`pa)'rnent of bqse issue tee.)
`8a. ( ) Netn formal drawinp are encloqed.
`9. ( ) Priority of application Serial No.
`on-in
`
`' filed
`
`(Country)
`
`claimed under 35 U.S.C 119.
`
`The certified copy has been filed in prior application Serial
`filed
`No.
`10. ( x) The prior application is assigned of record to Iterex Pharrnaceutj-cals l,td. - .
`ETtnership
`11. ( x) The power of attorney in the prior application is to:
`Reg. No. 29 381
`Ed\rsard P. C'amson
`DRESSLER, GOLDSMITH, SHORE,
`SUTKER & MILNAIyIOW, LTD.
`4700 Two Prudential Plaza
`l-80 North Stetson Avenue
`Chicago, l11inois 60501-
`(a) ( x) The power appears in the original papers in the prior
`application.
`
`ADM$S.2'rrll092
`
`
`
`Page 9 of 220
`
`

`
`-\f
`
`( ) Since the power does not appear in the original-papers' a copy
`of the F wer in the prior application is enclosed
`(x ) Address all future oommunications to:
`WEISH &\<AT14LTD. ?
`135 South LaSalle Street
`Suite 1625
`Chicago, Illinois 60603
`(312) 781-9470
`(May be completed only by applicanf or the attorney or agent
`of record-)
`
`6hlt e-*.
`lo -.xl l.F{
`ffis
`, *' '1$$4
`fut***S
`
`June 3, L994
`(Date)
`
`Registration No.
`
`(x ) Attorney or agent of remrd
`( ) Filed Under $13a(a)
`
`:"'
`
`WEISH &KAn', LTD.
`135 South IaSalle Street
`Chicago, Illinois 60603
`(3r2) 787-9470
`
`ADM08&3srr\1092
`
`
`
`Page 10 of 220
`
`

`
`f:;h\
`frs&
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`SYNTEESIE OF EOUII{OLAR I'TUIJTIPLE OLIGOI{ER
`IdIX!URE8. ESPECIAI,IJY Otr OIJIGOPEPTIDE
`
`Description
`Cross-Reference to Related Application
`NTql=acontinuation-in-nart*.9,€",.?R3J.*oution
`Serial No.^ o7/7oLr658 filed May L6, t99LA that $ras a
`continuation-iri-part of application Serial Nq.
`ar{ nDO o\oqn'rr €,U
`07/6L7,O23, filed November 21, f-990i- whose disclosures
`are incorporated by reference.
`
`Technical Field
`The present invention relates to the organic
`synthesis.of oligorneric sequences of cornpounds. More
`particularly
`it relates to stepwise synthesis of
`rnultiple independent sequences, especially oligoneric
`peptide chains
`
`Background and Related Art
`Over the last several years, developments in
`peptide synthesis technology have resulted in automated
`synthesis of peptides accornplished through the use of
`solid phase synthesis methods. The solid phase
`synthesis chemistry that made this technology possible
`was first described in Merrifield et al. J. Amer. Chem.
`Soc., 85:2L49-2L54 (L963). The "Merrifield rnethod" has
`for the most part remained unchanged and is used in
`nearly all automated peptide synthesizers available
`today.
`
`fn brief, the Merrifield rnethod involves
`synthesis of a peptide chain on solid support resin
`particles. These particles typically consist of
`polystyrene cross-Iinked with divinyl benzene to form
`porous beads which are insoluble in both water and
`various organic solvents used in the synthesis pr.otocol.
`
`%'
`
`L0
`
`L5
`
`20
`
`25
`
`30
`
`35
`
`
`
`Page 11 of 220
`
`

`
`.'
`
`. :-
`
`2-
`
`The resin particles contain a fixed amount of arnino- or
`hydroxylnethyl aromatic noiety which serves as the
`linkage point for the first amino acid in the peptide'
`Attachmentofthefirstaninoacidentails
`chemically reacting its carboxyl-terninal (C-terrninal)
`end with derivatized resin to form the carboxyl-terninal
`end of the oligopeptide. The alpha-amino end of the
`amino acid is typically blocked with a t-butoxy-carbonyl
`group (t-Boc) or with a 9-fluorenylurethyloxycarbonyl
`(F-Moc) group to prevent the amino group which could
`otherwise react from participating in the coupling
`reactj.on. The side chain groups of the amino acids, if
`reacti,ve' are also blocked (or protected) by various
`benzyl-derived protecting grouPs in the form of ethers'
`thioethers, dsters, and carbamates'
`The next step and subsequent repetitive cycles
`involve deblocking the amino-terminal (N-terminal)
`resin-bound amino acid (or terrninal residue of the
`peptide chain) to remove the alpha-arnino blocking group,
`followed by chernical addition (coupling) of the next
`blocked amino acid. This process is repeated for however
`many cycles are necessary to synthesize the entire
`peptide chain of interest. After each of the coupling
`and deblocking steps, the resin-bound peptide is
`thoroughly washed to remove any residual reactants
`before proceeding to the next. The solid support
`particles facilitate removal of reagents at any given
`st,ep as the resin and resin-bound peptide can be readily
`filtered and washed while being hetd in a column or
`device with Porous oPenings.
`Synthesizedpeptidesarereleasedfromt,he
`resin by acid catalysis (typically with hydroftuoric
`acid or trifluoroacetic acid), which cleaves the peptide
`from the resin leaving an arnide or carboxyl group on its
`c-terminal amino acid. Acidolytic cleavage also serves
`
`5
`
`10
`
`l_5
`
`2A
`
`25
`
`30
`
`35
`
`
`
`Page 12 of 220
`
`

`
`3-
`
`to reraove the protecting groups from the side chains of
`the amino acids in the synthesized peptide. Finished
`peptides can then be purified by any one of a variety of
`chromatography methods.
`Thoughmostpeptidesaresynthesizedwiththe
`above described procedure using automated instruments, a
`recent advance in the solid phase nethod by R'A'
`Houghten allows for synthesis of nultiple independent
`peptides sinultaneously through uranually perforaed
`means. The trsimultaneous Multiple Peptide Synthesisrl
`(ttsltPsrt) process is described in U.S. Patent No'
`4,63L,2IL (1986); Houghten, Proc. Natl' Acad' Sci"
`82:5131-5L35 (1985); Houghten et al., Int' J' Peotide
`ProteiJr Res. , 4-2673-678 (1985) ; Houghten et dI',
`Biotechnioues, L, 6, 522-528 (1986) ' and Houghten, U'S'
`patent No. 4,63I,zLL, whose diSCloSUreS are incorporated
`by reference.
`the SMPS process enploys
`Illustratively,
`porous containers such as plastic bags to hold the solid
`support synthesis resin. A Merrifield-t'ype solid-phase
`procedure is carried out with the resin-containing bags
`grouped together appropriately at any given step for
`addition of the same, desired aurino acid residue. The
`bags are then washed, separated and regrouped for
`addition of subseErent same or different amino acid
`residues until peptides of the intended length and
`sequence have been synthesized on the separate resins
`within each resPective bag.
`That rnethod allows nultiple, but separate,
`peptides to be synthesized at one tine, since the
`peptide-linked resins are maintained in their separate
`bags throughout the process. The SMPS method has been
`used to synthesize as many as 200 separate peptides by a
`single technici.an Ln as little as two weeks, a rate
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`r(
`
`
`
`Page 13 of 220
`
`

`
`,
`
`-4
`
`vastly exceeding the output of most automated peptide
`synthesizers.
`A robotic device for autonated rnultiple
`peptide synthesis has been recently conmercialized. The
`device perfotms the seqrrential steps of nultiPle'
`separate solid phase peptide synthesis through iterative
`mechanicaL-intensive means. This instrunent can
`synthesize up t,o 95 separate peptides at one tirne' but
`is linited at present by the quantity of its peptide
`yield.
`
`Several research groups have reported the
`synthesis of synthetic conbinatorial librari.es of
`peptides. Thgse reports are discussed below'
`of int'erest is work by Geysen et dl' , which
`deals with urethods for synthesizing peptides with
`specific seqruences of arnino acids and then using those
`peptides to identify reactions with various receptors.
`Geysen et aI. rs work presupposes that one has a prior
`knowledge of the general nature of the sequences
`required for the particular receptors, so that the
`appropriate group of peptides can be synthesized. see
`U.S. Patents Nos. 4'7O8t87L and 4r833,o92i P'C'T'
`Publications Nos. WO 84103505 and WO 84/03564; Geysen et
`al.,Proc.NatI.Acad.Sci.U.S.A.,.9.!!3998-4002(].984);
`Geysen et al., Proc. NAII. Acad. Sci. U'S'A' , 82-2L78'L82
`(1985) i Geysen et al., in svnthelic Peptides as
`Antiqens, 130-149 (1986); Geysen et aI', J' Immunol'
`Etr!-, L9.2.:25s-'274 (1987); and Schoofs et a1',
`J. fmnunol . , .Ll-9.: 611-516 ( 1988 ) .
`InpublishedPCTapplicationPcT/Au8s/00]-65
`(WO e6/OOggL, I Geysen describes a method for deter:nining
`so-called rrmimotopestr. A rninotope is def ined as a
`catarner (a pollnner of precisely defined sequence forned
`by the condensation of a precise number of small
`molecules), which in at least one of its confornations
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`has a surface region with the equivalent rnolecule
`topology to the epitope of whLch it is a mimic' An
`epitope is defined as the surface of an antigenic
`molecule which is delineated by the area of interaction
`with an antibodY rnolecule.
`The mirnotopes are synthesized on a series of
`solid pollmer (e.g. pol-yethylene with a coating of
`grafted acrylic acid) rods having a dianeter of about 4
`rnm and a length of about 50 rnrn. A spacer formed by
`reaction of the e-amino group of !-Boc-tysine rnethyl
`ester and then !-Boc-alanine was added to the resins,
`followed by renoval of the !-Boc group to provide an
`amino group to be used to begin the syntheses'
`Anixtureofblockedaminoacidscontaining
`different amotrnts of each of the blocked twenty anino
`acids to be used was dissolved' in dimethy'l for::rrarnide and
`then coupled to the rods. That first coupling was
`repeated three tirnes using conventional solid phase
`synthesis techniques. Twenty arnino acid residues were
`individually next added so that twenty S-mer seqfuences
`were prepared, each having a single, known amino acid
`residue at the anino-terminus and a mixture of anino
`acid residues at each of the four other positions of the
`chain. Each of those twenty rod-linked peptides was
`then individual.ly reacted with each of the twenty amino
`acid residues to forni 400 (20 x 20) 6-mer peptides
`having the two amino-tetminal positions defined and the
`four remaining positions as mixtures' Ttro more
`positions of mixtures of anino acids were then added,
`and the ter:minal amine acetylated to for"m N-acetyl
`g-mers linked to the rods whose first two amino acid
`positions were undefined (nixtures), followed by two
`defined positions, foJ.Iowed by four undefined positions
`(rnixtures), followed by the sPacer and then the
`supporting rods.
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`6-
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`The 40o rod-linked N-acetyl 8-mer peptide
`mixture preparations nere then screened in an ELISA
`assay using a monoclonaL antibody to a desired antigenic
`protein. The 8-ners having the best binding to the
`antibody nere identified. Two sets of further 8-ners
`that contained the identified best-binding 2-mer-
`sequences within those 8-mers were prepared'
`Afirstsetcontainedrnixedaminoacidsatthe
`three C-terminal positi'ons, followed toward the
`N-te:minus, by a position containing each of the twenty
`amino acids made by twenty seParate couplings' the
`identifi,ed 2-rner seqluences, two further nixtures at the
`next two positions, and an N-terninaL acetyl group' The
`second group contained mixed arnino acids at the four
`C=terninat po'si.tions, the identifiE{ l-ltr€"I' seqluences' a
`position made by separate couplings of each of the
`twenty amino acids, mj-xed amino acids as the terminal
`residues and an N-ter"ninal acetyl group'
`Eachofthoserod-}inkedN-acetylS.rnerswas
`again screened in an ELISA with the monoclonal antibody'
`The best binding sequences for each group were
`identified, ancl thus 4-mer, best-binding sequences were
`identified.
`The above process of separately adding each of
`the anino acids on either side of identified best-
`binding sequences was repeated until an optinun binding
`seqluence was identif,ied.
`Theabovemethod,whileelegant,suffersfrom
`several disadvantages. First, owing to the snalL size
`of each rod used, relatively srnall amounts of each
`peptide is produced. second, each assay is carried out
`using the rod-linked peptides, rather than the free
`peptides in solution. Third, even though specific
`amounts of each blocked arnino acid are used to prepare
`the rnixed amino acid residues at the desired positions,
`ra
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`7
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`there is no way of ascertaining that an equimolar amount
`of each residue is tnuly present at those positions.
`In addition, Furka et al',
`(1988, 14th
`International Congress of Biochernistry, Volune 5,
`Abstract FR:013) described the synthesis of nine
`tetrapeptides eaeh of which contained a single residue
`at each of: the amino- and carboxy-tetmini and mixtures
`of three residues at each position therebetween. The
`abstract futher asserts that those authorsr experiments
`indicated that a mixture containing up to 180
`pentapeptides could be easily synthesized in a single
`run. No biologicat assays were reported'
`Recent reports (Devlin et aI', Science,
`2492404-405 lis9Ol and Scott et aI., Science,
`249-:386-390 t199Ol) have described the use of
`recornbinant DNA and bact,erial expression to create
`highly cornplex mixtures'of peptides. For example, a
`4S-nucLeotide base pair stretch of DNA was synthesized
`in which the individual nucleotide bases were varied t'o
`contain all four possible nucleotide bases (guanine,
`adenine, cytosine and thlgnidine) at every position in
`the synthesized DNA chain, except at each third position
`(3, 6' g, etc.) which contained only guanine and
`cytosine. The onission of adenine and thymidine at
`every third position in the synthesized DNA removed the
`possibility of chain teminator triplet codons ending in
`A or T, such as TAA or TGA.
`The resulting DNA sequence would then code for
`a mixture of 15-mer peptides with all conbinations of
`the 20 naturaLly occurring arnino acids at each position..
`Those investigators fused the 45 synthetic
`nucleotide sequence to a gene coding for the coat
`protein of a sinple bacteriophage and created a large
`library of these bacteriophages. Each member of the
`library contained a different 45-mer DNA fusion seqluence
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`resulted in a
`and therefore each member of the ii!t"t"
`different 15-mer peptide fused to the outer coat protein
`of its corresponding full1r' assenbled bacteriophage
`particle. screening of the recornbinant bacteriophage
`particles in a biochenical assay allowed the
`investigators to find individual peptide-coat protein
`fusions (bacteriophages) that were active in that assay
`by enrichrnent, selection and clonal isolation of the
`enriched bacteriophages that contained active peptide
`fusions. By determining the DNA sequence of the cloned
`bacteriophages, the investigators could deduce which
`peptide sequences were active in their assay'
`Thatnethodyieldedseveralpeptideseqruences
`from a mixtur6 of 107 or more recombinant,
`bacteriophages. Each of the 15-D€r peptides found
`contained the same four-arnino-acid sequence somewhere
`within its overall sequence, thereby alleged1y
`validating the assay accuracy and nrethodological
`approach.
`
`The reconrbinant DNA nethod is extremely
`powerful for screening l.arge nuhbers of peptides'
`However, it is linited in that the peptides must be
`fused to a larger protein as a result of and integral to
`the design of the method. The peptide-protein fusions
`(and corresponding bacteriophage partictes) are likely
`to be unreactive in many biochernical, biological and b
`vivo assays where the peptides roust be present in
`solution without steric hindrance or conformational
`In addition, the rnethod results in an over-
`distortion.
`representation of some sequences of peptides due to the
`inherent redundancy of the genetic code which has
`several codons per araino acid in some cases and only one
`codon per amino acid in others.
`'
`further, neither group reported data as
`Still
`being definitive for the determination of optional
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`-9
`peptide ligands for strepavidin (Devlin et aI'), or for
`the two monoclonal antibodies raised against
`myohemorythinin (snith et al.). Neither group provided
`a single specific answer comparable to the expected
`sequence.
`
`More recently, Fodor et al', Egig,ngg, 25Lt767-
`773 (1991), descriibed the solid phase synthesis of
`mixtures of peptides or nucleotides on glass microscope
`slides treated with aminopropyltriethoxysilane to
`provide amine functional groups. Predeter"rrined amino
`acids were then coupled to predefined areas of the
`slides by the use of photomasks. The photolabile
`protecting group lwoc (nitroveratryIoxycarbonyl) was
`used as the agrino-teminal protecting group
`Byusingirradiation,aphotolabileprotecting
`group and masking, an array of LO24 different peptides
`coupled to the slide was prepared in ten steps'
`Immunoreaction with a fluorescent,-labeled monoclonal
`antibody vras assayed with epifluorescence microscopy.
`This elegant method is also lirnited by the
`sma}I amount of peptide or oligonucleotide produced' by
`use of the synthesized peptide or nucleotide affixed to
`the slide, and also by the resolution of the photomasks.
`This method is also less useful where the epitope bound
`by the antibody is unknown because alL of the possible
`sequences are not PrePared-
`The prinarlr' Iirnitation of the above new
`approaches for the cLrcumvention of indivldual screening
`of nillions of individual peptides by the use of a
`cornbinatorial library is the inability of the peptides
`generated in those systems to int'eract in a rrnoraalrl
`manner with acceptor sites, analogous to natural
`interaction processes (i.e., in solution at a
`concentration relevant to the receptors, antibody
`binding sites, enzyme binding pocketsr or the like being
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`10
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`studied without the exclusion of a large percentage of
`the possible cornbinatorial lihrary). Secondarily, the
`expression vector syste-ns do not readily per:rnit the
`incorporation of the D-forns of the natural amino acids
`or the wide variety of unnatural amine acids which would
`be of interest in the study or development of such
`interactions.
`The interest in obtaining biologically active
`peptides for pharmaceutical, diagnostic and other uses
`would make desirable a procedure designed to find a
`nixture of peptides or a single peptide within a mixture
`with optiural activity for a target application'
`Screening mixtures of peptides enables the researcher to
`great}y sirnplify the search for useful therapeutic or
`diagnostic peptide compounds. Mixtures containing
`hundreds of thousands or more peptides should be readily
`screened since many biochernical, biological and small
`animal assays are sensitive enough to detect activity of
`compounds that have been diluted down to the nanogram or
`range, the concentration
`even picograur per rnilliliter
`range at which naturally occurring biological signals
`such as peptides and proteins operate'
`Alrnost all of the broad diversity of
`biologicatly relevant ligand-receptor (or affector-
`acceptor) interactions occur in the presence of a
`complex nilieu of other SubStanCes (i.e., proteins make
`up approximately 5-1O percent of plasma, e'g' albumin
`l-3 percent, antibodies 2-5 percent-salts, lipids/fats,
`etc. ) . This is true for virtualJ.y alL biologically
`active compounds since most are commonly present, and
`active, dt nanonolar and lower concentrations. These
`cornpounds are also, in most instances, produced distant
`fron their affection sites. That a srnall peptide (or
`other molecule) can readily xfindtr an acceptor system,
`bind to it, and affect a necessary bio}ogical function
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`11
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`prior to being cleared from the circulation or degraded
`suggested that a single specific peptide sequence can be
`present in a very wide diversity, and concentration, of
`other individual peptides and stil,l be recognized by its
`particular acceptor systen (antibody, cellular receptor,
`If one could devise a means to prepare and
`etc. ) .
`screen a synthetic conbinatorial tibrary of peptides,
`then the norhal exquisite selectivity of biological
`affector/acceptor systems could be used to screen
`through vast numbers of synthetic oligopeptides.
`The availability of a wide variety of clearly
`identified peptides in relatively lirnited nixtures would
`greatly facititate the search for the optirnurn peptide
`for any particular therapeutic end use application. At
`the present tirne, researchers are hampered by the
`inability to rapidly create, identify and screen large
`numbers of peptides with specific receptors. Work such
`as reported by Geysen has been valuable where the
`general nature of the required amino acid resj-due
`sequence could be previously detemined, so that the
`specific peptides of interest could be individually
`formulated. However, such techniques cannot insure that
`the optimun peptides are identified for testing.
`It would therefore be of considerable interest
`to have a urethod for the precise synthesis of mixtures
`of peptides in which individual peptide sequences can be
`specifically defined, such that a comprehensive array of
`peptides is available to researchers for the
`identification of one or more of the optinun peptides
`for reaction with receptors of interest, from which one
`can derive optimun therapeutic materiaLs for treatnent
`of various organisn dysfunctions. It would also be of
`value for such a process to have the capability to
`produce eguivalent sequences of other tlpes of
`oligomeric conpoinds.
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`L2
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`Brief Sumlnary of the Invention
`In one asPect, the invention herein
`contemplates a process that provides for the synthesis
`of conplex mixtures of step-growth oligoners, especially
`peptides, wherein each position in the oligorneric
`sequence chain contains an equinolar representation of
`reacted. bifunctional monomeric repeating unit compound,
`such as an amino acid residue, added at that step. In
`peptide synthesis, the nethod circuulents the problen of
`unequal reaction yields during addition of blocked arnino
`acids reacted as a mixture in a coupling step in the
`Merrifield solid phase synthesis procedure' Use of the
`present method also provides a reLatively much larger
`anount of coupled oligorner than previously contenplated'
`Initspreferredenbodimentrtheinvention
`contemplates the organic synthesis of cornplex equinolar
`mixtures of oligopeptide sequences on a solid support
`material. The eguimolar oligopeptide sequences consist
`essentially of chains of arnino acid residues linked
`end-to-end by peptide bonds wherein the amino acid
`residue incorporated at any one position in the chain
`can be varied, such as to contain all or a combination
`of the twenty naturally occurring amino acids and/or
`their derivatives. The inventl.on enables synthesis of
`these peptide mixtures with equal and precise
`representation of any arnino acid residues at, any
`position in the chain at which a mixture of amino acid
`residues is intended to be represented. The process can
`use any type of peptide addition chemistry and
`protocols, but preferably uses the Uerrifield solid
`phase synthesis procedure in protocoJ.s sinilar to that
`of the Houghten S!{PS Process.
`In yet another aspect, the invention cornprises
`a method for the ident,ification of one or more optinum
`peptides for reaction with a designated acceptor, such
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`13
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`that design of therapeutic naterials for treatnent of
`organism dysfunctions invoLving such receptor can be
`facilitated.
`In its broadest fotn, a process of this
`invention is defined as a process for the synthesis of a
`complex mixture pooJ. of solid support-coupled monorneric
`repeating unit compounds, wherein the nixture pool
`contains a substantially equinolar representation of the
`reacted monomeric repeating unit cornpound, such as a