W089/06692
`
`PCT/US89/00051
`
`-39-
`
`21. A method as in claim 16 wherein said antibodies are monoclonal
`
`antibodies.
`
`22. A method as in claim 16 wherein the tumor cells comprise a
`carcinoma selected from human breast, renal, gastric and salivary
`gland carcinomas, or other tumor cell ·types expressing the HER2
`receptor.
`
`23. A method of treating tumor cells comprising the steps of:
`administering to a patient a
`therapeutically effective
`amount of antibodies capable of inhibiting growth factor receptor
`function; and
`administering to a patient a therapeutically effective
`amount of a cytotoxic factor.
`
`A method as in claim 23 wherein said cytotoxic factor is
`24.
`selected from the group consisting of TNF-a, TNF-P, IL-1, IFN-7 and
`IL-2.
`
`5
`
`10
`
`15
`
`20
`
`25. A method as in claim 23 wherein said cytotoxic factor is TNF-a
`
`26. A method as in claim 23 wherein said ·antibodies interrupt an
`autocrine growth cycle.
`
`25
`
`30
`
`27. A method as in claim 23 wherein said antibodies specifically
`bind a growth factor receptor.
`
`28. A method as in claim 27 wherein the growth factor receptor is
`selected from the group consisting of the EGF receptor and the HER2
`receptor.
`
`29. A method as in claim 23 wherein said antibodies specifically
`bind a growth factor.
`
`PFIZER EX. 1002
`Page 1001
`
`

`

`W089/06692
`
`PCT/US89f0005l
`
`-40-
`
`30. A method as in claim 29 wherein said growth factor is selected
`from the group consistin_g of EGF, TGF-a and TGF-./J.
`
`31. A method as in claim 23 wherein said antibodies are monoclonal
`antibodies.
`
`5
`
`32. A method as in claim 23 wherein said antibodies are conjugated
`to a cytotoxic moiety.
`
`33. A method as in claim 23 wherein said antibodies are capable of
`activating complement.
`
`34. A method as in claim 23 wherein said antibodies are capable of
`mediating antibody dependent cellular cytotoxicity.
`
`35. A method as in claim 23 wherein the tumor cells comprise a
`carcinoma selected from human breast, renal, gastric and salivary
`gland carcinomas.
`
`An assay for receptors and other proteins having increased
`36.
`tyrosine kinase activity comprising the steps of:
`(a) exposing cells suspected to be TNF-a sensitive to TNF-a;
`(b)
`isolating those cells which are TNF-a resistant;
`(c)
`screening the isolated cells for increased tyrosine kinase
`activity; and
`(d)
`isolating receptors and other proteins having increased
`tyrosine kinase activity.
`
`37. A composition suitable for administration to a patient: having
`a growth factor receptor dependent tumor comprising (a) antibodies
`capable of inhibiting growth factor receptor function, and (b) a
`cytotoxic factor.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`PFIZER EX. 1002
`Page 1002
`
`

`

`W089/06692
`
`PCT /US89/0005 I
`
`-41-
`
`38. A composition as in claim 37 wherein the cytotoxic factor is
`selected from the group consis.ting of .TNF-.a, TNF-P, IL-1, IFN--y and
`IL-2.
`
`5
`
`39. An immunotoxin as in Claim 10 wherein the cytotoxic moiety is
`ricin A chain.
`
`PFIZER EX. 1002
`Page 1003
`
`

`

`W089/06692
`
`.... ~ ...
`
`;-··1
`
`140
`
`100
`
`80
`
`60
`
`40
`
`20
`
`> a..
`a::
`>-
`t-
`_J
`
`cc
`<t
`>
`t-z
`w
`LJ ex: w
`a..
`UJ >
`t-
`<t
`
`-' LLI ex:
`
`PCT/US89/00051
`
`7/b
`Fig .1 a
`
`• NIH 3T3neo/dhfr
`o HER2-3
`o HER2-3 200
`i:J HER 2- 3400
`X HER2-3soo
`
`0
`0
`
`1
`
`Fig .1 b
`
`100
`10
`TNF-«( U/ml)
`
`1000
`
`10,000
`
`100
`
`80
`
`-'
`0
`ex:
`t-z
`0
`LJ
`u..
`0
`t-
`. ~ 60
`LJ ex:
`w
`a..
`
`L.
`
`u 20
`T"""
`U"'I
`
`• NIH3T3 neo/dhfr
`X HER2-3aoo
`0 NIH 3T3 neo /dhfr H TR
`
`0
`
`2:1
`1:1
`0·5:1
`0·25:1
`EFFECTOR: TARGET CELL RATIO
`
`4:1
`
`41111111.a -~~ - • - • • - - - - -
`
`•. -- . -
`
`PFIZER EX. 1002
`Page 1004
`
`

`

`Fig. 3
`INHIBITION OF SKBR 3 GROWTH BY ANTI-HER
`-2 MABS
`
`10000
`
`8000
`
`0
`N
`X
`
`ffi 6000
`I ~ vj I
`CD
`1:
`:::,
`z
`...J
`...J e:J 4000
`
`2000
`
`o--~
`
`MAb Supns.
`MAB SUPERNATANTS
`
`~ ~
`
`Ill myl. supn
`~ 4(8
`rm, 405 ~
`Q 3E8
`0
`3H4
`
`r:.
`" t.. r,
`
`,::
`
`~
`0
`
`~ l IC
`
`N
`
`"I:
`
`(', .. .....,;
`C: j
`-
`
`Fig.2
`
`10000
`
`• NH 3T3 neo/dhtr
`• HER 2-3800
`a ...
`fr aooo• •
`E
`D z
`:::,
`~
`w
`LL u 1.1.J
`
`6000
`
`a_
`Vl
`~
`I
`LL z
`t-
`i
`.......
`u,
`N
`
`~
`
`4000
`
`2000
`
`0o 0·001 0·01 0·1 1 10 100 1000
`[ TNF-oc)nM
`
`.._
`
`PFIZER EX. 1002
`Page 1005
`
`

`

`Fig. 4
`
`ANTI- HEPATITIS B MAb
`
`\
`
`:,_
`\
`
`,,
`;
`
`~
`0
`~
`c$
`~ N
`
`.
`
`~ ~
`
`14000
`
`13000
`
`12000
`11000 I\
`0
`N
`~ 10000
`UJ
`co
`1:
`
`9000
`~ 8000 ~
`7000
`
`_J
`LLJ
`u
`
`6000
`5000.
`
`I
`
`4000.
`
`ANTI -HERZ MAb 405
`
`0·5
`
`1-0
`
`2·0
`1 · S
`ANTIBODY CONCENTRATION
`µglml
`
`2-5
`
`3-0
`
`"'O
`
`00
`
`~ Cl)
`j
`(.h -
`
`PFIZER EX. 1002
`Page 1006
`
`

`

`.=a.a...,.~.-•
`
`.... • 1 - ..__,;_~, -
`
`._
`
`•~a ••Jo.--• ·-
`
`· . . •
`
`"'•. -· • -·· • •
`
`WO 89/06692
`
`PCT/US89/00051
`
`S/IP
`
`Fig. 6a
`
`... ,- ,.,
`
`>
`a.
`~ 100
`>-
`t:
`::d
`CD BO
`<t
`>
`t-z
`L&.J u
`a::
`L&.J a.
`1.1..J 40
`>
`t-
`<t
`.....I
`L&.J 20
`a::
`
`60
`
`0 0
`
`100
`0·05
`
`1000
`0·5
`
`10CXX) U/ml TNF-oc
`S·O µg/ml Ab
`
`Fig. 6b
`
`> a.
`~ 100
`>-
`t:
`::d
`ID 80
`<t
`>
`I-z 70
`UJ u
`a::
`w a.
`w 40
`>
`I-
`<{
`
`_J 20
`UJ a::
`
`0
`
`0
`
`100
`0·05
`
`1000
`O·S
`
`10(XX) U / ml TN F -ex
`5·0 µg/ml Ab
`
`PFIZER EX. 1002
`Page 1007
`
`

`

`W089/06692
`
`PCT/US89/00051
`
`F·ig.Sa
`
`> a.
`~ 100
`>-
`t:
`~
`~ 80
`>
`r-
`~ 60
`u
`a::
`UJ
`CL
`w 40
`>
`~
`<!
`_J
`w 20
`a::
`
`0
`
`100
`0·05
`
`1000
`O·S
`
`10000 U / ml TN F -0<
`S·O µg/ml Ab
`
`Fig. Sb
`
`_J
`
`>
`CL
`~ 100
`>-
`t::
`CD BO
`<!
`>
`r-z
`UJ 60
`u a::
`
`LLJ
`CL
`UJ 40
`>
`r-
`<!
`_J
`I.L.J 20
`a::
`
`0
`
`0
`
`100
`0·05
`
`1000
`O·S
`
`100JO U / m l TN F -0<
`5·0 µg/ml Ab
`
`PFIZER EX. 1002
`Page 1008
`
`

`

`W089/06692
`
`.•• ,.-
`
`.;-··#
`
`PCT/US89/000S1
`
`> a..
`~ 100
`>-
`C:
`:::!
`cc BO
`<t:
`>
`r-
`z
`UJ u a:::
`UJ a..
`UJ >
`.:=
`<t:
`_J
`UJ a:::
`
`201
`
`60
`
`40
`
`Fig.7
`
`I ft
`
`0
`
`100
`0·05
`
`1000
`0·5
`
`' 10(X)() U / ml TN F -o<
`S·O µg/ml MAb 405
`
`Fig .8
`
`> a..
`~ 100
`>-c:
`:::!
`~ 80
`>
`r-
`~ 60
`w a:::
`UJ a..
`u., 40
`>
`r-
`<t:
`.....J
`uJ 20
`a:::
`
`·O
`
`100
`0·05
`
`1000
`0·5
`
`10(X)() U / ml TN F -o<
`S·O µg/ml MAb
`
`PFIZER EX. 1002
`Page 1009
`
`

`

`INTERNATIONAL SEARCH REPORT
`lntematlonal Applicatl011 No PCT/US 89/00051
`' -· ,. 'tUSSIFICATION OF suaJICT MATTIII (II ..... , •• claaalflcalioll aymOOII aooly, l11dlcat1 alO I
`ACCOIClillO to lfttlmallonal Patent ClaNlftcatiofl (IPCJ Of to llotll National Claaaiflcauon and !PC
`4 C 12 P 21/00; C 12 N 15/00; C 12 N 5/00;
`IPC: A 61 K 39/395
`II. FIIL.DS SIAIICHID
`
`G 01 N 33/574;
`
`Cluaificalion S,atem I
`
`Minimum Ooc~o11 Searched '
`
`Cle1alficat1011 Symbola
`
`IPC4
`
`I A 61 K; C 12 N
`I
`
`Docume11tat1011 S-rcflacl otller Ulan Minimum OOCVlllllltatlon
`to tM &t8ftt tflat aucll Docu"*ltt .,. Included In Ule fielda S.arcNCI •
`
`Ill. DOCUMINTS CONSIDIIIID TO 81 IIIUVANT•
`Cateoorr • I
`Citation of Document. " wlUI Indication. wllere aooroimate. of tlle rele,,ant paaaaoH ••
`y
`
`I ~ant to Claim No. 11
`
`12-15
`
`y
`
`y
`
`i
`
`12-15
`
`;
`
`1-9
`
`WO, A, 85/03357 (ICRF PATENTS LTD;
`YEDA RESEARCH & DEVELOPMENT co. LTD;
`GENENTECH INC.)
`l August 1985
`see page 36, line 15 - page 40, line
`3
`--
`J. Natl. Cancer Instit., vol. 79, no. 3,
`September 1987,
`R.E. Sobol et al.: "Epidermal growth
`factor receptor expression in human
`lung carcinomas defined by a mono-
`clonal antibody", pages 403-407
`see page
`403, abstract
`--
`I
`Cancer Res., vol. 47, no. 14, July
`1987,
`u. Rodeck et al.: "Tumor growth
`modulation by a monoclonal
`antibody to the epidermal growth
`factor receptor: immunologically
`mediated and effector cell-independent
`effects", pages 3692-3696
`~/
`-T" tater document publlahad after tfle lftt-tional fllino data
`or priority date and not 1ft COftfllet with tl\1 application t111t
`ctteci to understand Ule priftCipla or tflaor, u11C1erlyi11g tl\e
`lnnfttlOII
`•x- document of oerttcular re1 .... nc,; u,, clalmad _l11Hntlo11
`cannot be co111idarad IIOffl or caftftOt Ill co11•1C1arad to
`lnYoha an inffntiq atap
`..,.. documellt of pafticular ,....,_11e,:· the dalmad lllfl11tio11
`cannot be con1iderad to l11•01Y1 an iftn11tiff 1t10 wl\a11 tl\a
`-.Cuffllflt ii combined with on, or more otl\er aucl\ docu•
`~ luctl COfflOlllatiOII NiflO 011\'IOUI to a pal'SOII aailled
`1ft "" art.
`•&• -~ fflelllbaf of tlla ume patent tam11,
`
`• Special cateoortn of cttad docvmenta: ..
`conaidareCI to be of particular ,_.,,11ca
`,
`•A• document d.timn; the oaneral aiate of Ula art wfllctl la not
`
`"!· urtiar docu~ llut pvbflallad Oft or after tlll lntaffl8tiOMI
`ftli110 ate
`9t.• document wlllcll may tllrow doubt• Oft priority cfalm(a) or
`whicl\ la cttld to Htablial\ the publication ate of anotller
`CitltiOft Of otllar 1pac;a1 ru1on (a aPldfiadl
`•o• documellt flfantno to an oral diKloa.,,., - . •llltlltlon or
`otl\ar_,
`-P- doculMllt ovllllallad prior 10 Ula llltefflatlOMI 8111141 dat9 but
`Illar Vian the onontr elate Claimed
`IV. CllllTl'1CATION
`Data of tlle Actual Co111Pletlo11 of tlll l~tloll&I Search
`2nd May 1989
`
`.
`
`lntamallonal S.arc111no Authority
`EUROPEAN PATENT OFFICE
`
`Form PCT/fSA/210 (Hcond a""1) CJaftuary 1N5)
`
`I Date of MaWno of Ulla lllteffllUOll&I S.vcti Report
`1 6. 06. 89
`~uruf...Autllor t1 tt Oftlcer
`-~.i
`,,\.
`
`~_V.ttf DER PISTTEN
`
`PFIZER EX. 1002
`Page 1010
`
`

`

`- 2 -
`
`lntematlonal A1111llcation No. PCT /US 8 9 / Q Q Q 51
`
`Ill, DOCUIIIIINTS CONalDIIIID TO &I IIIUVANT
`
`(CONTINUID FIIOIIII THI SICOND SHIIT)
`j Rele,ant to Cla,m No
`
`see page 3695
`
`Cancer Res., vol. 46, no. 11, November
`1986,
`H. Masui et al.: "Mechanism of anti(cid:173)
`tumor activity in mice for anti(cid:173)
`epidermal growth factor receptor
`monoclonal antibodies with
`·
`different isotypes", pages 5592-5598
`~ee page 5592, abstract
`
`Proc. Natl. Acad. Sci., USA, vol. 83,
`December 1986,
`J.A. Drebin et al.: "Inhibition
`of tumor growth by a monoclonal
`antibody reactive with an oncogene(cid:173)
`encoded tumor antigen", pages
`9129-9133
`see page 9129, abstract
`cited in the application
`
`Journal of Cellular Biochemistry, Growth
`Regulation of Cancer, vol. 35, no.
`4, 1987, Alan R. Liss, Inc.,
`U. Rodeck et al.: "Interactions
`between growth factor receptors
`and corresponding monoclonal
`antibodies in human tumors",
`pages 315:JCB-320:JCB, 65:GRC-
`70:GRC
`see page 65:GRC
`
`Cell, vol. 41, July 1985, MIT,
`J.A. Drebin et al.: "Down-modulation
`of an oncogene protein product and
`reversion of the transformed
`phenotype by monoclonal antibodies",
`pages 695-706
`see page 695, abstract
`cited in the application
`
`1-9
`
`1-9
`
`1-9
`
`1-9
`
`y
`
`y
`
`y
`
`y
`
`•
`
`i
`: I
`I
`!
`I I
`
`I
`I
`i
`
`..
`
`i
`
`I
`i
`I .
`I
`I
`
`Form PCT ISA:210 (aura ellHtl (Janu..., 1115)
`
`PFIZER EX. 1002
`Page 1011
`
`

`

`.. __ ~IJJ'Hlll INFORMATION CONTINUED FROM THI HCOND SHHT
`
`International Aoolication No. PCT /US 8 9 / 0 0 0 51
`
`v.(is OSSlltVATIONS WHIRi ClllTAIN CLAIMS WEltl FOUND UNSIARCHASU '
`
`1.(E Claim numbera
`
`Tlll1 lntemauonal M&rcfl '900rt l\aa not been Htablltlled In raaoect of certain cl&lrna vnder Article 17CJ) Cal for Ille f0Uowln9 rHaon,:
`."' .•.••• llecavM UleJ relate to aubject matter not requlr.d lo be -flM IIJ tlllt Allthortty, na-'J:
`* 16-35, see PCT-rule 39.l(IV); methods for treatment of the
`human or animal body by surgery or therapy, as well as diag(cid:173)
`nostic methods.
`
`i.O Claim numben ............ becauH ,,,.,. ralalt to oarta of tlle lntematlonal aoollcatlon that do not comply wltll ttlt onacnbff reQul, ..
`ments to such an uttnt lllat no meanln9h1I lntematlonal Match can be carried out.
`
`flCaly:
`
`30 c:i.n """"*"···- DecaUN t1111y .,..
`
`PCT Rule 1.4(a).
`
`;
`
`•
`
`,1 Claiffla ano .,. not cntteo 1n accordanc:11 wttft tne NCCnCS ano 1NrU aantencn Of
`
`VI.[) OSSIIIVATIONS WHIRi UNITY OF INVENTION IS LACKING •
`
`Ttlla lntamatJonal Searchlnt AuthorttJ found mulUole lltftlltlona In tflla lrumatlonal aopllcatlon H followa:
`
`1.[] A• au required additional Nardi fMa were tlfflelr paid 117 Ille aopllcant. lflla lni.matlonal March '900ft COfff'a au Marcllallle claim•
`of Ille lntematJonal aoolicatlon.
`i.D At only aome of tha raQulrad additional Marctl fMS ..,. timely paid 117 the aooUcant. tflla lnternatlonal Marctl report cowera onl1
`lfloH clalma of tlle lntematlonal appllcatlon fof wlllcll fMa · - paid, 1oeclflcaU, clal1111:
`
`a.[] Np recrulred tddlbonal March fff• _,. timely paid 117 tlle 1opllcant. ConHQuently, tfllt lnternatlonal aaarch report la '9atnctad to
`Ille lnfffttlon first IMfttloned In the c1&1ma: n It coYerad 11, dalm nu111Nf1:
`
`•.[] A• all Harcllable dairna c;uld be Harched without ,«ort jultlfyint an additional fN, tha lntamatlonal S..rcllln; AutllOtlty did not
`lftwlte paJmant of an, additional IN.
`it.marl on Pmeat
`0 TIie additional ... ,ch IN• watt accom111nlecl IIJ apollcant'a protHt.
`0 No proteat accompanied tlla pay1111m of addltlonal Marctl INL
`
`,_ PCT/ISA/rtO (1upplernental ahNt (2)) ~ tNS)
`
`PFIZER EX. 1002
`Page 1012
`
`

`

`. ,-.-
`
`.;- .-,
`
`ANNEX TO TIIE INTERNATIONAL SEARCH REPORT
`ON INTERNATIONAL PATENT APPLICATION NO.
`
`•
`Tbis MMX lists die patent family members retatinc to the patent documents cited in die abovHDelltioned intemationaJ s:eardl l'\.'POrt.
`Tbe members are as contained in the European Patent Office EDP file on 09/06/89
`Tbe European Patent Office is in no way liable for tbese partiadars wbicb are menly civen for the purpose of information.
`
`us 8900051
`.
`SA
`26543
`
`Patent ftCUIDalt
`cited in sareb report
`
`Publication
`date
`
`Pataat family
`-ber(s)
`
`. .
`
`Publication
`date
`
`..
`
`-1
`
`WO-A- 8503357
`.. - --
`
`01-08-85
`
`AU-A-
`3934085
`09-08-85
`EP-A-
`19-02-86
`0171407
`JP-T-
`12-06-86
`61501168
`----------------------------------------------------------------------
`- . -- .
`..
`- .
`
`--
`
`.
`
`-·
`
`-
`
`-·
`
`,
`l
`"' 0 .. C
`~
`
`:: For more details .ibout this IUUlex : see Official Journal of the European Patent Office. 11,io: 12/82
`
`PFIZER EX. 1002
`Page 1013
`
`

`

`., . ' . ...:. -
`
`._ .. __
`
`To'OIILO ll'(T'[ll.fCTUAl ,,_OP'tUY OIIOANIZATION
`
`PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDfR THE PATENT COOPERATION TR.EAlY (PCl)
`f (S!l larnulloa1!.l.Pa1n1 OamfkaliM:
`_CllP 11100, c,n?n1110, 7/01, ISIOO •
`
`(ll)lattnatioul P.~llcadoa N-i..tt:
`
`WO 90/07861 I
`
`(0) ~ I P.Mladoe Datt:
`
`26 July 1990 (26.07.90)
`
`Al
`
`(ll) latffadoul nu91 Datt:
`
`28 Dcormbn 1989 (28. I 2J59)
`
`PCT !US89/05857
`
`(JC!) Pneri1y 4a111:
`290,975
`3 I0.2.S2
`
`28 Dcormbn 1988 (28.12.88) us
`13 February 1989 ( 13.02.89) US
`
`(71) A,,llca•t: PROTEIN DESIGN LA BS. INC. fUSIUSI;
`3181 Poner Drive, Palo Aho. CA 94304 (US).
`
`C7l} 1-ten: QUEEN. Cary, L ; DOO Oak Crttk Ori~. Palo
`Alto. CA 94304 (USJ. SELICK. Harold. E,d..,n ; 1673
`Sunnyllope Avniuc., Belmont. CA 94002 (LIS).
`_
`
`(74)AfNf: SMITH, 'William. M.; To"""~d and T°"""stnd.
`One Marktt Pl&U. 2000 S1n11n T~ -n. San F111nci\CO,
`CA 94 !O~ (US).
`
`('Ill) OnfpatN S111tn: AT, AT(Europcan patmt). AU, BB, BE
`(European paicnt). BF (OAPI patmt). BG, BJ (OAPI
`patent), BR. CF (OAPI patmt), CG (OAPI patrnt), CH,
`CH (European patmt), CM (OAP! patent), DE. DE
`(European patent), DK. ES (European paten!), Fl, FR
`(European patent). GA (OAPI patmtl, GB. GB {Euro(cid:173)
`rc•n patent), HU, IT (European patent), JP, KP. KR.
`LK. LU, LU (European patent), MC. MG. ML (OAPI
`patent), MR (OAPI patent), MW, NL. NL (European
`paten!), NO, RO. SD, SE. SE (European pa1mt). SN
`(OAPI p11cn1), SU, TD (OAPI pa1rn1), TG (OAPI P•·
`tent).
`
`Plibllqf4
`l+itlt borrn.otioftol #'ad ,q,orr.
`Beforr tlte upirotio,. of rite riMr Ii.Mil for O""ffl4ittt rlu
`daiJru att.d /0 ~ rrpwblisW itt tJtr romt of tit~ tTOn/lf of
`o.,.ntd.,.r,,a.
`
`(S.)Tldr: CHIMERIC IMMUNOGLOBUUNS SPEOFIC FOR pSS TAC PROTEIN OF TI{E lt.,.2 RECEPTOR
`
`N=I mtthods for dnigning humanized immuno!llobulins having one or mort oomplemmtary deicnnining regions
`{CDR·s) from a donOT immunoglobuhn and a f111m('WOrt region from a human immunoglobulin comprising ri~t comparing the
`f111ml'WOrt or variable rqion amino acid sequnioc of the donor immunoglobulin to oonnponding sequence, in I collC'C'lion or
`human immuno!llobulin chains. and Klecting II the human immunog!obulin one of thr more homologous scqucnc,c,s from the
`collmion. Each humaniu:d immunO!,lobulin chain may comprise about l or mort amino acid, from the donor immunoglobulin
`in addition to the CDR'1, usually 11 leai!t one of "'hich i1 immediately 1djaocnt to a CDR in the donor immuno11lobuhn. The
`heavy and light chain, msy uch be dnigncd by using any one or all thrtt additional po1i1ion cri1eri1. Whm combined into an
`intaa antibody, the humaniud immunoglobulins or the prtRnt inW'Tltion will be 1ub$tantially non-immun01,mic in humans and
`rcuin substantially the umc affinity u the donor immunoglobulin 10 the antigen. such as a pro(rin or other compound contain(cid:173)
`ing an cpitopc.
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`CHIMERIC IMMUNOGLOBOLINS SPECIFIC POR p55 TAC PROTEIN
`OF THE IL-2 REOPTOR
`.ti:l~ or tbe invention
`.The .present inv-ention ·relates gene-ral'ly 'to ·the
`combination of recombinant ONA and ~onoclonal antibody
`technologies tor developing novel therapeutic agents and,
`more particularly, to the production of non-immunogenic
`antibodies and their uses.
`
`Bactgrouna or tbe lnYcntion
`In mammals, the immune response is mediated by two
`types of cells that interact specifically with foreign
`material,~. antigens. One of these cell types, B-cells,
`are responsible for the production of antibodies. The second
`cell class, T-cells, include a wide variety of cellular
`subsets controlling the .1n Y.lYs2 !unction o! both B-cells and
`a wide variety of other hematopoietic cells, including T(cid:173)
`cells.
`
`one way in which T-cells exert this control is
`through the production o! a lflllphokine known as interleukin-2
`(IL-2), originally named T-cell growth factor.
`IL-2's prime
`!unction appears to be the stimulation and maintenance of T(cid:173)
`cells.
`Indeed, some immunologists believe that IL-2 may be
`at the center of the entire immune response (~ , Farrar, J.,
`et al., Immupol. Bev, ~:129-166 (1982), which is
`incorporated herein by reference).
`To exert its biological effects, IL-2 interacts
`with a specific high-affinity membrane receptor (Greene, w.,
`et al., Progress in Hematology xrv, E. Brovn, Ed., Grune and
`Statton, New York (1986), at pgs. 283 ~f). The h~an IL-'(cid:173)
`receptor is a ~omplex multichain glycoprotein, vith one
`chain, knovn as the Tac peptide, being about 5Sk0 in size
`(~, Leonard, w., et al., J, Biel, Chern, ~:1872 (1985),
`which is incorporated herein by reference). A gene encoding
`this protein has been isolated, and predicts a 272 amino acid
`peptide, including a 21 amino acid signal peptide (~.
`Leonard, w., et al., Noture ~: 626 (1984)). The 219 NHJ-
`
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`terminal amino acids of the pJS Tac protein apparently
`comprise an extracellular domain(~, Leonard, W., et al.,
`science, ~:633-639 (1985), which is incorporated herein by
`reference).
`Much o! the elucidation or the human IL-2
`receptor•• structure and function is due to th• development
`of specifically reactive monoclonal antibodies.
`In
`particular, one mouse monoclonal antibody, known as anti-Tac
`(Uchiyama, et al., J, Immunol, ~:1393 (1981)) has shown
`that IL-2 receptors can be detected on T-cells, but also on
`cells or the monocyte-macrophage family, Kupffer cells or the
`liver, Langerhans• cells of the skin and, ot course,
`activ~ted T-cells.
`Importantly, resting T-cell•, B-cells or
`circulating machrophages typically do not display the IL-2
`receptor (Herrmann, et al., J, Exp, Med. l&Z:llll (1985)).
`The anti-Tac monoclonal antibody has also been used
`to define l)'Tllphocyte functions that require IL-2 interaction,
`and has been shovn to inhibit various T-cell functions,
`including the generation of cytotoxic and suppressor T
`lYl!lphocytes in cell culture. Al6w, based on studies with
`anti-Tac and other antibodies, a variety of disorders are now
`ass~ciated with improper IL-2 receptor expression by T-cells,
`in particular adult T-cell leukemia.
`Hore recently, the IL-2 receptor has beP.n shown to
`be an ideal target for novel therapeutic approaches to T-cell
`mediated diseases. It has been proposed that IL-2 receptor
`specific antibodies, such as the anti-Tac monoclonal
`antibody, can be used either alone or as an immunoconjugate
`(.e....s...., with Ricin A, isotopes and the like} to effectively
`remove cells bearing the IL-2 recep~or. These agents can,
`tor example, theoretically eliminate IL-2 receptor-expressing
`leukemic cells, certain a-cells, or activated T-cells
`involved in a disease state, yet allow the retention of
`mature normal T-cells and their precursors to ensure the
`capability of mounting a normal T-cell iZ!llllune response as
`needed.
`In general, most other T-cell specific agents can
`destroy essentially all peripheral T-cells, which limits the
`agents' therapeutic efficacy. overall, the use of
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`appropriate monoclonal ~~tihodiea E!,)ecitic tor the IL-2
`receptor may have therapeutic uti~ity Jn autoimmun• diseases,
`organ tranaplantation and any unwanted roaponae by activated
`T-cella.
`Indeed, clinical trialc have been initiated using,
`~ . -anti~"Tac antibodies (~, generally, Waldman, T., et
`al., cancer Res. ~:625 (1985) and Waldman, T., science
`l.J. 4 :727-732 (1986), both ot which are incorporated herein by
`reference).
`Unfortunately, the use of the anti-Tac and othe.·
`non-human monoclonal antibodies have certain drawbacks,
`particularly in repeated therapeutic regimens as explained
`below. House monoclonal antibodies, ~or example, do not fix
`hWDan complement vell, and lack other important
`immunoglobulin functional characteristics when used in
`humans.
`
`Perhaps more importantly, anti-Tac and other non(cid:173)
`human monoclonal antibodies contain substantial stretches of
`amino acid sequences that will be, immunogenic when injected
`into a human patient. NW!lerous studies have shown that,
`atter injection o! a foreign antibody, the immune response
`elicited by a patient against an antibody can be quite
`strong, e~sentially eliminating the antibody's therapeutic
`utility after an initial treatment. Moreover, as increasing
`numbers of different mouse or other antigenic (to humans)
`monoclonal antibodies can be expected to be developed to
`treat various diseases, after the first and second treatments
`with any different non-human antibodies, subsequent
`treatments even for unrelated therapies can be ineffective or
`even dangerous in the~selves.
`While the production of so-called "chimeric
`antibodies" (~ . mouse variable regions joined to human
`~or.1tant regions) has proven somewhat successrul, a
`si~niticant immunogenicity problem remains.
`In general, the
`production of hUlllan i11U11unoglobulins reactive with the hWllan
`IL-2 receptor, as vlth many human antigens, has been
`extremely di!!icult using typical human monoclonal antibody
`production techniques. Similarly, utilizing recoinbinant ONA
`technology to produce so-called "humanized" antibodies (~,
`
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`~ . EPO Publication No. 0239400), pro•tid•c unc•rtain
`results, in part due to unpredictable binding a!tinitiea.
`Thus, there is a need for improved forms ot hUl:lan(cid:173)
`like immunoglo~ulins, Jauch .aa those specitic·~or the1luman
`11~2 receptor, tnat are substantially non-immunogenic in
`humans, yet easily ~nd economically produced in a manner
`suitable for therapeutic formulation and other uaea. The
`present invention fulfill• these and other needs.
`
`svrnmnrv or tbe Ipyeotloo
`The present invention provides novel compositions
`useful, tor example, in the treatment o! T-cell mediated
`human disorders, the compositions containing human-like
`immunoglcbulins specifically capable of blocking the binding
`er human IL-2 to its receptor and/or capable of binding to
`·the p55 Tac protein en human IL-2 receptors. The
`immunoglobulins can have two pairs or light chain/heavy chain
`complexes, typically at least one pair having chains
`comprising mouse complementarity deten:ining regions
`functionally joined to human framework region segments. For
`example, mouse complementarity determining regions, with er
`without additional naturally-associated mouse amine acid
`residues, can be used to produce human-like antibodies
`capable of binding to the human IL-2 receptor at affinity
`levels stronger than about 108 M" 1
`•
`The immunoglobulins, including binding fragments
`and other derivatives thereof, of the present invention may
`be produced readily by a variety ot recombinant ONA
`techniques, vith ultimate expression in transtected cells,
`preferably immortalized eukaryotic cells, such as myelcma or
`hybridoma cells. Polyn~cleotides comprising a first sequence
`coding for human-like illllllunoglobulin framework regions and a
`second sequence set cc~ing for the desired immun.>globulin
`complementarity determining regions can be produced
`synthetically or by combining arprcpriate cDNA and genomic
`ONA segments.
`The human-like immunoglobulins may be utilized
`alone in substantially pure fonn, or complexed with a
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`cytotoxic agent, auch aa a rbdionuclide, e ribosomal
`inhibiting protoin or a cytotoxic agent active at cell
`surfaces. All o! these compounds will be particularly useful
`in treating .T~cell ..JDediated ,disorders. The ·human-li·ke
`immunoglobulins or their complexes can be prepared in a
`pharmaceutically accepted dosage form, vhich vill vary
`depending on the mode of administration.
`The present invention also provides novel methods
`for designing hw:ian-like immunoglobulin chains having one or
`more complementarity determining regions (CDR'a) from a donor
`immunoglobulin and a framework region from a human
`immunoglobulin, the preferred methods comprising first
`comparing the framework or variable region amino acid
`sequence of the donor immunoglobulin to corresponding
`sequences in a collection or ~wnan immunoglobulin chains, and
`selecting as the human immunoglobulin one of the more
`homologous sequences from the collection. The human
`it1munoglobulin, or acceptor immunoglobulin, sequence is
`typically selected from a collection of at least 10 to 20
`i?:ll:lunoglobulin chain sequ~nces, and usually will have the
`highest homology to the donor il!llnunoglobulin sequence of any
`sequence in the collection. The human immunoglobulin
`framework sequence will typically have about 65 to ?O\
`homology or more to the donor immunoglobulin framework
`sequences. The donor immunoglobulin may be either a heavy
`chain or li;ht chain (or both), and the human collection will
`contain the same kind of chain. A humanized light and heavy
`chain can be used to form a complete humanized immunoglobulin
`or antibody, h~ving two light/heavy chain pairs, vith or
`without partial or full-length human constant regions an~
`other proteins.
`In another embodiment of the present invention,
`either in conjunction with the above comparison step or
`separately, additional amino acids in an acceptor
`illllllunoglobulin chain may be replaced with amino acids form
`the CDR-donor immunoglobulin chain. More specifically,
`further optional substitutions of a human framework amino
`acid of the acceptor immunoglobulin with a corresponding
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`amino acid from a donor illllllunoglobulin v!ll be made at
`position• i~ th• illllllunoglobulin• vhere:
`(a)
`th• amino acid in th• human tra~evork region
`of an accept:or.immunoglobulin is ·rare for that position and
`the corresponding amino acid in the donor immunoglobulin is
`common tor that position in ,,uman immunoglobulin sequences,
`-or
`
`the amino acid is immediately adjacent to one
`(b)
`of the CDR's: or
`(c) the amino acid is predicted to be vithin about
`JA of the CDR's in a three-dimen~ional illllllunoqlobulin model
`and capable o! interacting vith the antigen or vith the CDR's
`of the humanized i111111unoglobulin.
`T?- ~ hu1"!lanized immunoglobulin chain vill typically
`comprise at least about J amino acids from the donor
`·immunoglobulin in addition to the CDR's, usually at least one
`of vhich is illllllediately adjacant to a CDR in the donor
`immunoglobulin. The heavy and light chains may each be
`designed by using any one or all three of the position
`criteria.
`
`When co~~ined into an intact antibody, the
`humanized light and heavy chains of the rresent invention
`vill be substantially non-immunogenic in humans and r~tain
`sub~tantially the same affinity as the donor iz:ununoglobulin
`to th~ antigen (such as a protein or other compound
`contail"ing an epitope). These affinity levels can vary from
`about 10 1 H" 1 or higher, and may be vithin about 4 fold of the
`donor illllllunoglobulin's original affinity to the antigen.
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`BRIEF OESCJUPrION OF THE FIGUJU:S
`Figure 1. Comparison of sequences ot anti-Tac
`heavy chain (upper lines) and Eu heavy chain (lower lines).
`The l.-letter·eode 'for ·ami-no acids is used. Tha first amino
`acid on each line ia numbered at the le!t.
`Identical amino
`acids in the tvo sequences are connected by lines. The 3
`CORs are underlined. Other amino acid positions for which
`the anti-Tac amino acid rather than the Eu amino acid was
`used in the humanized anti-Tac heavy chain are denoted by
`an•
`
`Figure 2. Comparison o! sequences of anti-Tac
`light chain (upper lines) and Eu light chain (lov~7 lines) •
`The single-letter code for amino acids is used. The first
`amino acid on each line is numbered at the left.
`Identical
`amino acids in the tvo sequences are connected by lines. The
`3 CDRs are underlined. Other amino acid positions for which
`the anti-Tac amino acid rather than the Eu amino acid was
`used in the humanized anti-Tac heavy chain are denoted by
`an•
`
`Figure 3. Nucleotide sequence of the gene for the
`hwnanized anti-Tac heavy chain variabl~ region gene. The
`translated amino acid sequence for the part o! the gene
`encoding protein is shown underneath the nucleotide sequence.
`The nucleotides TCTAGA at the beginning and end of the gene
`are Xba I sites. The mature heavy chain sequence begins vith
`amino acid 120 Q.
`Figure 4. Nucleotide sequence of the gene for the
`humanized anti-Tac light chain variable region gene. The
`translated amino acid sequence tor the part of the gene
`encoding protein is shown underneath the nucleotide sequence.
`The nucleotides TCTAGA at the beginning and end of the gene
`are Xba I sites. The mature light chain sequence begins vith
`amino acid t21 O.
`Figure S. A. Sequencea of the tour
`oligonucleotides used to synthesize the humanized anti-Tac
`heavy chain gene, printed 5' to 3'. B. Relative positions
`of the oligonucleotides. The arrows point in the 3 1
`direction for each oligonucleotide.
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`(A) Sequence• of th• tour
`Figure 6.
`oligonu~l.otides used to synthesize the humanized anti-Tac
`light chain gene, printed 5 1 to J'.
`(B) Relative positions
`of the oligonuc~ao.Udes.
`-The arrow• -poin:t -in the 3'
`direction for each nligonucleotide. The position of a Hind
`III site in the overlap of JFD2 and JFDl is shown.
`Figure 7. Schematic diagram of th• plasmid
`pHuGTACl used to express the humanized anti-Tac heavy chain.
`Relevant restriction sites are shovn, and coding regions of
`the heavy chain are displayed as boxes. The direction of
`transcription from the immunoglobulin (lg) promoter is shown
`by an arrow. ER• heavy chain enhancer, Hyg • hygromycin
`resistance gene.
`Fig _·re 8. Schematic diagram of the plasmid pHuLTAC
`used to express the humanized anti-Tac light chain. Relevant
`restriction sites are shown, and coding regions of the light
`chain are displayed as boxes. The direction of transcription
`from the Ig promoter is shown by an arrow.
`Figure 9. Fluorocytometry ct .HUT-102 and Jurkat
`cells stained with anti-Tac antibody or humanized anti-Tac
`antibody followed respectively by fluorescein-conjugated goat
`anti-mouse lg antibody or goat anti-human lg antibody, as
`labeled.
`In each panel, the dotted curve shovs