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`CU\IMS RECEIVED
`DOCKET NO.
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`
`BIOEPIS EX. 1002
`Page 2
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`BIOEPIS EX. 1002
`Page 3
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`
`BIOEPIS EX. 1002
`Page 4
`
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`
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`staple additional sheet here
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`
`BIOEPIS EX. 1002
`Page 5
`
`
`
`-• •
`
`•
`
`11 ft...,.aPCrJPTr. 17 NOV .. --'"'""
`
`PCT
`
`ogf1.162oh us
`lnnex b's~. page I
`s·
`.cant's Guide - Volume II - National Chaptc
`EX0:
`ss MAIL: BB214759358Us
`MAILED:
`17 NOVEMBER 1993
`U.S. DEPAR~T~M;,EN~;'f~O~F ~CO~M~M~E~RC!!'!E~P~AT!!'!E!!'!l>,~AN~'O~T~RA""!O~E""!M""!AR~l.:~O~F~FIC~E~ATT~OR~N~'EY'~S~DOC~~l.:ET~N~U~M~BE~R;;..;.,;;;;;;,;;;,;~..;;,,;;.;.;;.._
`
`fllljU
`FOl<M rro.1 )•JO
`(REV 11-9lJ
`
`~
`
`-- -r
`
`TRANSMITTAL LETTER TO THE UNITED STATES
`DESIGNATED/ELECTED OFFICE (DO/EO/US)
`NTERNATIONAL Al'l'LICATION NO.
`PCT/US92/05126
`TITLE OF INVENTION
`METHOD FOR MAK.ING HUMANIZED ANTIBODIES
`Al'PLICANT(S) FOR DO/EO/US
`Paul J. CARTER Leonard G. PRESTA
`pplicant herewith submits to the United States Designated/Elected Office (DO/EO/US) the following items under 35 U.S.C. 371:
`I. cg This express request to immediately begin national examination procedures (35 U.S.C. 37J(f)).
`2. C!I The U.S. National Fee (35 U.S.C. 37l(c){l)) and other fees as follows:
`(2) NUMBER FILED
`(3) NUMBER EXTRA
`-20 =
`3
`23
`-3 ...
`7
`
`(4) RATE
`X $22.00
`X$74.00
`+ $230.00
`
`(5) CALCULATIONS
`$
`
`518.00
`230.00
`
`709Pl
`PRIORITY DATE CLAIMED
`14 June 1991 14.06.91
`
`'BAsrc NATXONAL FEE (37 CFR 1.492(a)(l)-(S))l
`. D For filing with EPO or JPO search report (37CFR l .492(a)(5)).....
`S830.00
`D Intemational preliminary examination fee paid to USPTO (37 CFR 1.482)
`S640.00
`................................................................................................
`No international preliminary examination fee paid to USPTO (37 CFR 1.482)
`but international search fee paid to USPTO (37 CFR I .445(a)(2))..
`$710.00
`
`Neither international preliminary examination fee (37 CFR 1.482) nor
`$950.00
`international search fee (37CFR I .445(a)(2)) paid to USPTO.......
`
`International preliminary examination fee paid to USPTO (37 CFR 1.482)
`S90.00
`and all claims satisfied provisions of PCT Articles 33(2)-33(4)...
`
`Surcharge ofSIJ0.00 for fumishing the National fee or oath or
`0 30 months from the earliest
`declaration later thanO 20
`claimed priority date (37 CFR I .492(e)).
`
`950.00
`
`TOTAL OF ABOVE CALCULATIONS = 1,. 764.00
`
`Affidavit must be filed also.
`
`SUBTOTAL
`D 20 D 30
`
`+
`
`TOTAL NATIONAL FEE $1~764.00
`
`+
`
`40.00
`
`TOTAL FEES ENCLOSED S1,,804.00
`
`a. 0 A check in the amount of S
`to cover the above fees is enclosed.
`b. ~ Please charge my Deposit Accourit No. 07-0630
`in the amount of $1,804.00
`A duplicate copy of this sheet is enclosed.
`c. ~ The Commissioner is hereby authorized to c_ha~Jl8Y additional fees which may be required, or credit any
`overpayment to Deposit Account No.
`A duplicate copy of this sheet is enclosed.
`0/-U-b::i'
`
`to cover the above fees.
`
`(January 1993)
`
`BIOEPIS EX. 1002
`Page 6
`
`
`
`-
`
`JI":.
`
`I .
`i i\~;l;,. fr\ )ri·
`
`. US
`Annex U~. Vl,,page. 2.· .
`
`·
`
`.
`.
`Applicant's Guide - Volume II - National Cl
`
`r- US
`
`.. .
`
`3.
`
`.
`A copy of the International Application as filed (35 U.S.C. 37l(c)(2))
`a. O
`is transmitted herewith (required only if not transmitted by the International bureau).
`b. ~ is not required, as the application was filed in the United States receiving Office (RO/US).
`c. D has been transmitted by the International Bureau.
`
`4. D A translation of the International Application into English (35 U.S.C. 37l(c)(2)).
`
`5.
`
`Amendments to the claims of the International Application under PCT Anicle 19 (35 U.S.C. 371(c){3))
`a D
`are transmitted herewith (required only if not transmitted by the International Bureau).
`b. D have been transmitted by the International Bureau.
`
`6. D A translation of the amendments to the claims under PCT Anicle 19 (35 U.S.C. 37l(c}(3)).
`
`7.
`
`IKl An oath or declaration of the inventor (35 U.S.C. 37I(c)(4)).
`
`8. D A translation of the annexes to the International Preliminary Examination Report under PCT Article 36
`(35 U.S.C. 371(c)(5)).
`
`Other docwnent(s) or information included:
`9. D An Information Disclosure Statement under 37 CFR 1.97 and 1.98.
`
`10. ~ An assignment document for recording.
`PLEASE MAIL THE RECORQED ASSIGNMENT DOCUMENT TO :
`a. Cl the person whose signature, name and address appears at the bottom of this page.
`b. D
`the following:
`
`11.
`
`The above checked items are being transmited:
`a. 0
`before the eighteenth ( 18) month publication.
`b. D after publication of the Article 20 communication but before twenty (20) months from the priority date.
`c. D after twenty (20) months but before twenty-two (22) months (surcharge and/or processing fee included).
`d. O after twenty-two (22) months (surcharge and /or processing fee included).
`·
`NOTE: Petition to revive (37 CFR l.137(a) or (b)) is necessary if3S U.S.C. 371 requirements submitted
`after 22 months and NO propel' dema11d for lntematio11al Preliminary Examination was made by 19 months
`from the earliest claimed priority date.
`!XI by thirty (30) months and a proper·demand for International Preliminary Examination was made by the 19th
`month from the earliest claimed priority date.
`after thirty (30) months but before thirty-two (32) months and a proper demand for International Preliminary
`Examination was made by the 19th month from the earliest claimed priority date (surcharge and/or processing
`fee included).
`g. D after thirty-two (32) months (surcharge and/or processing fee included).
`NOTE: Petition to revive (37 CFR l.137(a) or (b)) is necessary if JS U.S.C. 371 requirements submitted
`after 32 months and a proper demand for International Preliminary Examination was made by 19 months
`from the earliest claimed priority date.
`
`f. O
`
`e.
`
`12.
`
`At the time of transmittal, the time limit for amending claims under Anicle 19:
`a. IE has expired and no amendments were made.
`b. D has not yet expired.
`
`13. D Certain requirements under 35 U.S.C. 371 were previously submitted by the applicant on _ _ _ _ _ _ _ _ _
`namely:
`14. Submitted herewith ares
`Amendment
`
`Sequence Diske~te, Sequence Listing, Preliminary
`
`\
`
`NAME
`
`Janet E. Hasak
`ADDRESS Genent;ech, Inc
`460 6oiDt San Bruno Boule::n:al:'d
`
`SIONA TURI!
`
`Sout San Francisco, CA 94080-4990
`REGISTRATION NUMBER
`28,616
`
`•ormr10-JJ90(REv 11-92)
`
`Etll 6 I i.:::
`n I r1 I I ') I': n.:i-.: .:J
`
`BIOEPIS EX. 1002
`Page 7
`
`
`
`I
`
`• CERTIFICATION UNDER 37 CFR 1.10
`
`HB214759358US : Express Mail Number
`
`17 NOVEMBER 1993 : Date of Deposit
`
`I hereby certify that this request to initial national processing, including: TRAN SM ITT AL
`LETTER, PRELIMINARY AMENDMENT, SEQUENCE LISTING & DISKETTE, COMBINED
`DECLARATION & POWER OF ATTORNEY, ASSIGNMENT and COPY OF PRELIMINARY
`EXAMINATION REPORT is being deposited with the United States Postal Service "Express
`Mail Post Office to Addressee" service under 37 CFR 1.10 on the date indicated above and
`is addressed to the Commissioner of Patents and Trademarks, Washington, D.C. 20231.
`
`Carol Koehler
`
`BIOEPIS EX. 1002
`Page 8
`
`
`
`•
`
`405
`
`B0405
`
`50
`40
`30
`20
`10
`OIVMTQSHKFMSTSVGORVSITCJCASQOVHTAVAWYQQKPGHSPKLLIYSASFRYT
`I
`1111
`I
`I
`I
`11
`I l l
`1111
`I
`I
`I
`I
`I I
`I l l
`OIQMTQSPSSLSASVGORVTITCRASQOVHTAVAWYQQKPGKAPKLLIYSASFLES
`1111
`I
`I
`I
`I
`1111
`OIQMTQSPSSLSASVGORVTITCRASQOVSSYLAWYQQKPGKAPKLLIYAASSLES
`
`405
`
`B0405
`
`90
`80
`100
`70
`60
`GVPDRFTGNRSGTOFTFTISSVQAEOLAVYYCQQHYTTPPTFGGGTKLEIJCRA
`l
`I I
`I
`I I I I
`I
`I
`I
`I
`I I
`I
`I I I I
`I
`I
`I
`GVPSRFSGSRSGTDFTLTISSLQPEOFATYYCQQHYTTPPTFGQGTICVEIKRT
`1111
`I
`I
`I
`I
`I I
`I
`I
`GVPSRFSGSGSGTOFTLTISSLQPEOFATYYCQQYNSLPYTFGQGTKVEIJCRT
`
`BIOEPIS EX. 1002
`Page 9
`
`
`
`'-
`
`.. "'·
`
`t,-"
`
`,
`
`•':,
`
`•
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`•
`
`PCT!US 92/05126
`Nj1LJ&~&
`
`405
`
`HU405
`
`BUV8 III
`
`50 A
`40
`30
`20
`10
`EVQLQQSGPELVKPGASLKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGRIYPTN
`I I
`I I
`I
`I
`I
`I
`I
`I
`I I
`I I
`I I
`I I
`I
`I
`I
`I
`I
`I
`I I
`I I
`EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN
`111 1111
`I 1111
`111 1111
`I 1111
`EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENG
`---------
`
`405
`
`HU405
`
`BUV8 III
`
`90
`80 ABC~
`100ABC
`70
`60
`GYTRYOPKFQOKATITAOTSSNTAYLQVSRLTSEOTAVYYCSRWGGDGFYAMDYW
`11111111
`I
`I
`111 II
`I
`111 ti
`t
`11111111
`I
`I
`GYTRYADSVKGRFTISAOTSKNTAYLQMNSLRAEOTAVYYCSRWGGDGFYAMDVW
`II
`I
`I
`I
`I
`I
`II 111111
`It
`I
`I
`I
`I
`I
`II 111111
`SOTYYADSVKGRFTISROOSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVW
`
`405
`
`HU405
`
`110
`GQGASVTVSS
`I
`I
`I
`I
`GQGTLVTVSS
`
`HUV8 III
`
`GQGTLVTVSS
`
`BIOEPIS EX. 1002
`Page 10
`
`
`
`•
`
`Anneal hu VL or huV8 oligomers to pAKl template
`
`1. Ligate
`2. Isolate assembled oligomers
`3. Anneal to pAKl template (XhoJ-, StuJ+)
`4. Extend and ligate
`
`I
`
`Xhol
`
`1. Transform E. coli
`2. Isolate phagemid pool
`3. Enrich for hu\i and huV8 (Xho / ~ Srul-)
`4. · Sequence verify
`
`pAK2
`
`BIOEPIS EX. 1002
`Page 11
`
`
`
`~T/US 9 2 / .Q 5 1 2 6
`If r/1~ .2(J ,6
`
`100
`
`-
`
`C: g.g 80
`8e
`~'2 o::.
`-e 5 c. 60
`~=
`~8
`
`40
`
`huMAb4D5-1
`
`· huMAb4DS-8
`
`muMAb4D5
`
`I
`
`4
`
`12
`8
`[MAb4D5 variant] µg/ml
`
`16
`
`BIOEPIS EX. 1002
`Page 12
`
`
`
`If T!US 9 2 I O 5 1 2 6
`- ~//4'~"
`TUS 92/05126
`
`~;
`
`\
`
`
`
`--
`
`. . . .-·
`
`BIOEPIS EX. 1002
`Page 13
`
`BIOEPIS EX. 1002
`Page 13
`
`
`
`
`i' \(",·
`' '.
`'
`
`1r1us 921 o 512 6
`· o if J L{(c, z._ofo
`
`VL
`muxCD3
`huxCD3vl
`huKI
`
`40
`10
`20
`30
`• • • • • •
`DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKP
`**
`*
`*
`DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKP
`# #
`#
`DIQMTQSPSSLSASVGDRVTITCRASQSISNYLAWYQQKP
`
`A.AAA.A.AAA
`
`CDR-Ll
`
`muxCD3
`huxCD3vl
`huKI
`
`70
`60
`80
`50
`••••••
`DGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEO
`*
`****
`*
`*
`* **
`GKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQP
`## ·#
`#
`GKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQP
`
`CDR-L2
`
`muxCD3
`huxCD3vl
`hulCI
`
`100
`90
`•••••••
`EDIATYFCQQGNTLPWTFAGGTKLEIK
`*
`*
`**
`*
`EDFATYYCQQGNTLPWTFGQGTKVEIK
`# #
`EDFATYYCQQYNSLPWTFGQGTKVEIK
`
`CDR-L3
`
`Va
`muxCD3
`huxCD3vl
`huIII
`
`10
`40
`20
`30
`• • • • •
`EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQS
`**
`**
`*
`* ***
`*
`* *
`EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQA
`## ## # #
`EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA
`
`CDR-Hl
`
`a
`70
`60
`50
`•
`• •••••••••
`HGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAY
`muxCD3
`**
`*
`*
`*
`**** ** **
`**
`huxCD3vl PGKGLEWVALINPYKGVTTYADSVKGRFTISVDKSKNTAY
`## #### # #
`# #
`#
`huIII
`PGKGLEWVSVISGDGGSTYYADSVKGRFTISRDNSKNTLY
`
`CDR-H2
`
`muxCD3
`huxCD3vl
`huIII
`
`110
`lOOabcde
`90
`80 abc
`••••••••
`MELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVSS
`****
`**
`*
`*
`*
`LQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS
`########### #
`LQMNSLRAEDTAVYYCARGRVGYSLSGLYDYWGQGTLVTVSS
`D E T
`S
`.
`
`CDR-H3
`
`fl<JuRt- 5
`
`. , /
`
`BIOEPIS EX. 1002
`Page 14
`
`
`
`'
`
`'f ?T!US 9 2 /-0 5 1 2 6
`. 0 ~ /1 4C:i 2-0&,
`
`l'XGURB 6A
`
`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pHS2-8.0
`
`H52H4-160
`
`pHS2-8.0
`
`30
`20
`10
`QVQLQQSGPELVKPGASVKISCKTSGYTFTE
`·*** ·** **·**·*···** ********
`MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCATSGYTFTE
`20
`30
`40
`50
`10
`
`80
`70
`60
`50
`40
`YTMHWMKQSHGKSLEWIGGFNPKNGGSSHNQRFMDKATLAVOKSTSTAYM
`******·*· **·***··*·******·********· *··**********
`YTMHWMRQAPGKGLEWVAGINPKNGGTSHNQRFMDRFTISVOKSTSTAYM
`60
`70
`80
`90
`100
`
`130
`120
`110
`100
`90
`ELRSLTSEOSGIYYCARWRGLNYGFDVRYFOVWGAGTTVTVSSASTKGPS
`•• ** ·**···********************** ** ************
`QMNSLRAEDTAVYYCARWRGLNYGFDVRYFOVWGQGTLVTVSSASTKGPS
`110
`120
`130
`140
`150
`
`180
`170
`160
`150
`140
`VFPLAPSSKSTSGGTAALGCLVKOYFPEPVTVSWNSGALTSGVHTFPAVL
`****** *·*** ·************************************
`VFPLAPCSRSTSESTAALGCLVKOYFPEPVTVSWNSGALTSGVHTFPAVL
`160
`170
`180
`190
`200
`
`230
`220
`210
`200
`190
`QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCOKTH
`************** **··***** ***·********** ** * *
`QSSGLYSLSSVVTVTSSNFGTQTYTCNVOHKPSNTKVOKTVERl<CC---v
`210
`220
`230
`240
`
`240
`280
`270
`260
`250
`TCPPCPAPELLGGPSVFLFPPKPKOTLMISRTPEVTCVVVOVSHEOPEVK
`*******
`··*************************************·
`ECPPCPAPP-VAGPSVFLFPPKPKDTLMISRTPEVTCVVVOVSHEOPEVQ
`250
`260
`270
`280
`290
`
`HS2H4,-160·
`
`pH52-8.0
`
`330
`320
`310
`300
`290
`FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQOWLNGKEYKCKVS
`*******·*************·***·********·***************
`FNWYVOGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQOWLNGKEYKCKVS
`300
`310
`320
`330
`340
`
`BIOEPIS EX. 1002
`Page 15
`
`
`
`H52H4-160
`
`pHS2-8.0
`
`H52H4-l60
`
`pHS2-8.0
`
`H52H4-160
`
`pH52-8.0
`
`380
`370
`360
`350
`340
`NKALP!NPIEKTISKAKGQPREPQVYTLPPSREEMT~QVSLTCLVKGFY~(cid:173)
`**·***********·******•****************************
`NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEM'f.'ANQV~LTCLVKGFYP
`350
`360
`370
`38~
`390
`
`420
`430
`410
`400
`390
`SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
`**********************·***************************
`SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR'liQ03NVFS
`440
`400
`410
`420
`430
`
`450
`440
`CSVMHEALHNHYTQKSLSLSPGK
`***********************
`CSVMHEALHNHYTQKSLSLSPGK
`450
`460
`
`BIOEPIS EX. 1002
`Page 16
`
`
`
`l':IGURB 18
`
`H52L6-158
`
`30
`20
`10
`DVQMTQTTSSLSASLGDRVTINCRASQDINN
`*·****· ******·****** *********
`pH52-9.0 MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCRASQDINN
`20
`30
`40
`50
`10
`
`80
`70
`60
`50
`40
`H52L6-158 YLNWYQQKPNGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLDQE
`*********
`• ***************************·****·*·.
`YLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDYTLTISSLQPE
`60
`70
`100
`80
`90
`
`pH52-9.0
`
`130
`120
`110
`100
`90
`H52L6-158 DIATYFCQQGNTLPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
`*·***·*******************************************
`DFATYYCQQGNTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
`110
`120
`130
`140
`150
`
`pH52-9.0
`
`180
`170
`160
`150
`140
`H52L6-158 VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
`**************************************************
`VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
`160
`170
`180
`190
`200
`
`pH52-9.0
`
`H52L6-158
`
`pH52-9.0
`
`·210
`200
`190
`SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
`*********************************
`SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
`210
`220
`230
`
`I
`
`BIOEPIS EX. 1002
`Page 17
`
`
`
`... J
`
`-,,- --... _ ,, ·----·-~·.'."°'~_ ..
`
`I
`
`IMMUNOGLOBULIN VARIANTS
`
`:5
`
`This invention relates to methods for the preparation and use of variant antibodies and
`
`10
`
`application particularly in the fields of immunology and cancer diagnosis and therapy.
`
`Background of the Invention
`
`...... :
`-. ""''
`
`Naturally occurring antibodies (immunoglobulins) comprise two heavy chains linked
`
`15
`
`together by disulfide bonds and two light chains, one light chain being linked to each of the
`
`heavy chains by disulfide bonds. Each heavy chain has at one end a variable domain (V H)
`
`followed by a number of constant domains. Each light chain has a variable domain CVL) at one
`end and a constant domain at its other end; the constant domain of the light chain is aligned
`with the first constant domain of the tieavy chain, and the light chain variable domain is
`aligned with the variable domain of the heavy chain. Particular amino acid residues are
`
`believed to form an interface between the light and heavy chain variable domains, see e.g.
`
`Chothia et al., J. Mo/. Biol. 186:651-663 ( 1985); Novotny and Haber, Proc. Natl. A cad. Sci.
`
`20
`
`USA 82:4592-4596 {1985).
`
`The constant domains are not involved directly in binding the antibody to an antigen,
`
`25
`
`but are involved in various effector functions, such as participation of the antibody in antibody(cid:173)
`
`rr··
`
`dependent cellular cytotoxicity. The variable domains of each pair of light and heavy chains
`
`are involved directly in binding the antibody to the antigen. The do.mains of natural light and
`
`heavy chains have the same general structure, and each domain comprises four framework
`(FR} regions, whose sequences are somewhat conserved, connected by three hyper-variable
`
`30
`
`or complementarity determining regions (CDRs) (see Kabat, E. A. et al., Sequences of Proteins
`
`of lf!lmunological Interest, National Institutes of Health, Bethesda, MD, (1987)). The four
`
`framework regions largely adopt a P-sheet conformation and the CDRs form loops connecting,
`
`and in some cases forming part of, the P-sheet structure. The CDRs in each chain are held in
`close proximity by the framework regions and, with the CDRs from the other chain, contribute
`
`BIOEPIS EX. 1002
`Page 18
`
`
`
`'!US 92/ 0512 6
`
`2.
`
`to the formation of the antigen binding site.
`Widespread use has been made of monoclonal antibodies, particularly those derived
`
`from rodents including mice, however they are frequently antigenic in human clinical use. For
`
`5
`
`example, a major limitation in the clinical use of rodent monoclonal antibodies is an
`anti-globulin response during therapy (Miller, R. A. eta/., 8/ood62:988-995 (1983); Schroff,
`R. W. et al., Cancer Res. 45:879-885 (1985)).
`
`The art has attempted to overcome this problem by constructing "chimeric" antibodies
`
`in which an animal antigen-binding variable domain is coupled to a human constant domain
`
`(Cabilly eta/., U.S. patent No. 4,816,5-67; Morrison, S. L. eta/., Proc. Natl. Acad. Sci. USA
`
`10
`
`81:6851-6855 (1984); Boulianne, G. L. eta/., Nature 312:643-646 (1984); Neuberger, M. S.
`
`et al., Nature 314:268-270 {1985)). The term "chimeric" antibody is used herein to describe
`
`a polypeptide comprising at least the antigen binding portion of an antibody molecule linked
`to at least part of another protein (typically an immunoglobulin constant domain).
`
`The isotype of the human constant domain may be selected to tailor the chimeric
`antibody
`for participation
`in · antibody-dependent cellular cytotoxicity
`(ADCC) and
`
`15
`
`complement-dependent cytotoxicity (see e.g. Bruggemann, M. et al., J. Exp. Med.
`
`166:1351-1361 (1987); Riechmann, L. et al., Nature 332:323-327 (1988); Love et al.,
`
`Methods in Enzymology 178:515-527 (1989); Bindon et al., J. Exp. Med. 168:127-142
`
`(1988).
`
`20
`
`In the typical embodiment, such chimeric antibodies contain about one third rodent (or
`
`other non-human species) sequence and thus are capable of eliciting a significant anti-globulin
`
`response in humans. For example, in the case of the murine anti-CD3 antibody, OKT3, much
`
`of the resulting anti-globulin response is directed against the variable region rather than the
`constant region (Jaffers, G. J. et al., Transplantation 41 :572-578 (1986)).
`
`25
`
`In a further effort to resolve the antigen binding functions of antibodies and to minimize
`
`the use of heterologous sequences in human antibodies, Winter and colleagues (Jones, P. T.
`
`et al., Nature 321 :522-525 (1986); Riechmann, L. et al., Nature 332:323-327 (1988);
`
`Verhoeven, M. et al., Science 239:1534-1536 (1988)) have substituted rodent CDRs or CDR
`
`sequences for the corresponding segments of a human antibody. As used herein, the term
`
`30
`
`"humanized" antibody is an embodiment of chimeric antibodies wherein substantially less than
`
`an intact human variable domain has been substituted by the corresponding sequence from a
`
`non-human species. In practice, humanized antibodies are typically human antibodies in which
`some CDR residues and possibly some FR residues are substituted by residues from analogous
`
`sites in rodent antibodies.
`
`BIOEPIS EX. 1002
`Page 19
`
`
`
`•
`
`3
`
`i/US 9 2 / 0 5 1 2 6
`
`The therapeutic promise of. this approach is supported by the clinical efficacy of a
`
`humanized antibody specific for the CAMPA TH-1 antigen with two non-Hodgkin lymphoma
`
`patients, one of whom had previously developed an anti-globulin response to the parental rat
`
`antibody (Riechmann, L. et al., Nature 332:323-327 (1988); Hale, G. et al., Lancet
`
`5
`
`i: 1394-1399 ( 1988)). A murine antibody to the interleukin 2 receptor has also recently been
`
`humanized (Queen, C. et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989)) as a
`
`potential immunosuppressive reagent. Additional references related to humanization of
`
`antibodies include Co eta/., Proc. Natl. Acad. Sci. USA 88:2869-2873 (1991}; Gorman eta/.,
`
`Proc. Natl. Acad. Sci. USA 88:4181-4185 (1991 ); Daugherty et al., Nucleic Acids Research
`
`10
`
`19(9):2471-2476 (1991 ); Brown et al., Proc. Natl. Acad. Sci. USA 88:2663-2667 (1991 );
`
`Junghans et al., Cancer Research 50: 1495-1502 ( 1990).
`
`In some cases, substituting CDRs from rodent antibodies for the human CDRs in human
`
`frameworks is sufficient to transfer high antigen binding affinity (Jones, P. T. et al., Nature
`
`15
`
`321 :522-525 (1986); Verhoeven, M. eta/., Science239:1534-1536 (1988)), whereas in other
`cases "it has been necessary to · additionally replace one (Riechmann, L. et al., Nature
`332:323-327
`(1988)) or several
`(Queen, C. et al., Proc. Natl. Acad. Sci. USA
`
`86:10029-10033 (1989)) framework region (FR} residu~s. See also Co eta/., supra.
`
`For a given antibody a small number of FR residues are anticipated to be important for
`
`antigen binding. Firstly for example, certain antibodies have been shown to contain a few FR
`
`20
`
`residues which directly contact antigen in crystal structures of antibody-antigen complexes
`
`(e.g., reviewed in Davies, D.R. eta/., Ann. Rev. Biochem. 59:439-473 (1990)). Secondly,
`
`a number of FR residues have been proposed by Chothia, Lask and colleagues (Chothia, C. &
`Lesk, A. M., J. Mo/. Biol. 196:901-917 (1987); Chothia, C. et al., Nature 342:877-883
`(1989); Tramontano, A. et al., J. Mo/. Biol. 215:175-H!2 (1990)) as critically affecting the
`
`25
`
`conformation of particular CDRs and thus their contribution to antigen binding. See also
`
`Margolies eta/., Proc. Natl. Acad. Sci. USA 72:2180-2184 (1975).
`
`It is also known that, in a few instances, an antibody variable domain (either VH or VL)
`
`may contain glycosylation sites, and that this glycosylation may improve or abolish antigen
`
`binding, Pluckthun, Biotechnology 9:545-51 (1991 ); Spiegelberg eta/., Biochemistry 9:4217-
`
`30
`
`4223 ( 1970); Wallie et al., J. Exp. Med. 168: 1099-1109 ( 1988); Sox et al., Proc. Natl. A cad.
`
`Sci. USA 66:975-982 (1970); Margni et al., Ann. Rev. lmmunol. 6:535-554 (1988).
`
`Ordinarily, however, glycosylation has no influence on the antigen-binding properties of an
`
`antibody, Pluckthun, supra, (1991 ).
`
`The three-dimensional structure of immunoglobulin chains has been studied, and crystal
`
`BIOEPIS EX. 1002
`Page 20
`
`
`
`•
`
`'j,us 921 o 5 1 2 6
`
`structures for intact immunoglobulins, for a variety of immunoglobulin fragments, and for
`
`antibody-antigen complexes have been published (see e.g., Saul et al., Journal of Biological
`
`Chemistry 25:585-97 (1978); Sheriff eta/., Proc. Natl. Acad. Sci. USA 84:8075-79 (1987);
`
`Segal et al., Proc. Natl. Acad. Sci. USA 71 :4298-4302 (1974); Epp et al., Biochemistry
`
`5
`
`14(22):4943-4952 (1975); Marquart eta/., J. Mo/. Biol. 141:369-391 {1980); Furey eta/.,
`
`J. Mo/. Biol. 167:661-692 (1983); Snow and Amzel, Protein: Structure, Function, and
`
`Genetics 1 :267-279, Alan R. Liss, Inc. pubs. ( 1986); Chothia and Lesk, J. Mo/. Biol. 196:901-
`
`917 (1987); Chothia et al., Nature 342:877-883 (1989); Chothia et al., Science 233:755-58
`
`( 1986); Huber et al., Nature 264:415-420 ( 1976); Bruccoleri et al., Nature 335:564-568
`
`10
`
`(1988) and Nature 336:266 (1988); Sherman eta/., Journal of Biological Chemistry 263:4064-
`
`4074 (1988); Amzel and Poljak, Ann. Rev. Biochem. 48:961-67 (1979); Silverton et al., Proc.
`
`Natl. Acad. Sci. USA 74:5140-5144 (1977); and Gregory et al., Molecular Immunology
`
`24:821-829 (1987). It is known that the function of an antibody is dependent on its three
`
`dimensional structure, and that amino acid substitutions can change the three-dimensional
`
`15
`
`structure of an antibody, Snow and Amzel, supra.
`
`It has previously been shown that the
`
`antigen binding affinity of a humanized antibody can be increased by mutagenesis based upon
`
`molecular modelling (Riechmann, L. et al., Nature 332:323-327 ( 1988); Queen, C. et al., Proc.
`Natl. Acad. Sci. USA 86:10029-10033 (1989)).
`Humanizing an antibody with retention of high affinity for antigen and other desired
`
`20
`
`biological activities is at present difficult to achieve using currently available procedures.
`
`Methods are needed for rationalizing the selection of sites for substitution in preparing such
`
`antibodies and thereby increasing the efficiency of antibody humanization.
`
`The proto-oncogene HER2
`
`(human epidermal growth factor receptor 2) encodes a
`
`25
`
`protein tyrosine kinase (p185HER2) that is related to and somewhat homologous to the human
`epidermal growth factor receptor (see Coussens, L. et al., Science 230: 1132-1139 (1985);
`Yamamoto, T. et al., Nature 319:230-234 {1986); King, C. R. et al., Science 229:974-976
`
`( 1985))!- HER2 is also known in the field as c-erbB-2, and sometimes by the name of the rat
`
`homolog, neu. Amplification and/or overexpression of HER2 is associated with multiple human
`
`malignancies and appears to be integrally involved in progression of 25-30% of human breast
`
`30
`
`and ovarian cancers (Slamon, D. J. et al., Science 235:177-182 (1987), Slamon, D. J. et al.,
`
`Science 244: 707-71 2 ( 1 989)). Furthermore, the extent of amplification is inversely correlated
`
`with the observed median patient survival time (Slamon, supra, Science 1989).
`The murine monoclonal antibody known as muMAb405 (Fendly, B. M. et al., Cancer
`Res. 50:1550-1558 (1990)), directed against the extracellular domain (ECD) of p185HER2,
`
`BIOEPIS EX. 1002
`Page 21
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`.CT/US 92/ 0512 6
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`5
`specifically inhibits the growth of tumor cell lines overexpressing p1 s5HER2 in monolayer
`culture or in soft agar {Hudziak, R. M. et al., Malec. Cell. Biol. 9: 1165-11 72 ( 1989}; Lupu, R.
`
`et al., Science 249:1552-1555 (1990)). MuMAb405 also has the potential of enhancing
`
`tumor cell sensitivity to tumor necrosis
`
`factor, an
`
`important effector molecule
`
`in
`
`s
`
`macrophage-mediated tumor cell cytotoxicity {Hudziak, supra, 1989; Shepard, H. M. and
`
`Lewis, G.D. J. Clinical Immunology 8:333-395 (1988)). Thus muMAb405 has potential for
`clinical intervention in and imaging of carcinomas in which p195HER2 is overexpressed. The
`muMAb405 and its uses are described in PCT application WO 89/066