(TWICE AMENDED) {An) A humanized amibo<ly wnicn lacks immunogeni,5it{compared to
`106.
`a non·human parent antibody upon repeated administration to a numan pa11ent1in order to treat a
`/
`chronic disease in tnar patient[ and! wnereio ~n~ numanized ~nt1l)Ody /prises _a co~sens~s
`. tiuman variaote domain of a numan neavy cna1n 1mmunoglobulin subg~up, wnere1n amino acid
`' residues forming Complementarity Determining Regions (CDRs) thef~ot comprise non·human
`
`/ antibOdy amino acid res~ues, and further [comprising) comprjsef a Framework Region (FA)
`57
`
`substitution where me substituted FA residue: (a) noncovalenttyJ.lds antigen Chreetly; (b) !.!!!_~
`with a CDA; (c) [comprises) introduces a glycosylation site which affecis ltle antigen binding or
`aftinity of the antiOOdy; or (d) participates in the VL·zi1'er1ace t>y affecting the pro,Jt1mity or
`orientation of the V ~ and V" regions with respect to one another.
`-- --· -··.
`/
`(AMENDED) The numanized antibody o claim l 06 comprising· a non-human FR residue
`107.
`which noncovalenuy binds antigen direcdy.
`
`(AMENDED) Tne hymaoji7d an oay of claim 106 comprising a non-human FR residue
`
`
`108.
`which interacts with a CDR.
`
`109.
`
`(AMENDED) TM hymaojzed antil)Ody of claim 106 compnsmg a non-human FR residue
`I
`
`0 (/" whdl (compOSH) 7 , ,_ ' °n Sile wh<I> altecis the antigen bind;ng o<.affin;1y ot "''
`_) D antibOdy.
`
`(AMENDED) Jihe oumanjzeg anlibody of claim 106 comprising a non-nu man FR residue
`11 o.
`which participates inifue vl°v" interlace by affecting me proximity or orientation of me VL·V" regions
`I
`
`wilh respect /e another.
`
`(A1NDEO) A humanized antit>ody comprising a consensus human variable domain of
`1l1.
`human v ubgrO\Jp Ill. wherein amino acia res1aues forming Complementarity Determining Regions
`(CDRs thereof comprise non-numan amil>Ody amino acid residues. and turtner comprising a
`
`Fram work Region {FR) substitution where the substituted FA residue: (a) noncovalently binas
`
`ant en directly; (b} interacts with a CDR; {e) (comprises} inrcoauces a glycosytation site which
`at ects the antigen bindtng or affinity of the antibody; or (d) panicipates in the V,·V" intefface lly
`affecting the proiumity or oriemation of the V, and V" regions with respect to one another.
`
`7
`
`576 of 1033
`
`BI Exhibit 1002
`
`

`

`(Reiteratea) The humanized anlil>Od~1m 111 wh1cn lacks immunogenicity comparea
`
`112.
`to a non-human parent antil>OC!y upon re7e ed aaminlstrat1on to a human patient in order to treat
`a chronic disease 1n that patient
`
`(AMENDEDJ A humanized variant of a non-numan parent antibooy wnich Dinos an antigen
`113.
`witl"I t>ener affinity U'lan the parent antibody and comprises a consensus numan variaole domain ot
`a human neavy chain immunog10Dulin subgroup wherein amino acia residues forming
`Complementarity Determining Regions (CORs) thereof comprise non-human an11booy amino acio
`residues. and furtner [eompri.sing] comgrises a FrameworK Region (FR) substitution where the
`substituted FA residue: (a) noncovalenuy binds antigen direclf}'; (b) interacts with a CDR; (c)
`[comprises} introduces a glycosylalion site Which affects the antigen Oinding or affinity of me
`antibody; or (d) participates in 1he V,-V,, interface by affecting the proximiry or orientation of tne V._
`and v .. regions witn respect to one anomer.
`
`(AMENDED) The humanized variant of claim 1 13 which binds the antigen at least about
`l 14.
`3-roia more tigh1ly man the parent antibody binds antigen.
`
`Please add the fo!lowjog c1a1ms to me above-identified application:
`- 115. (NEW) A humanized antit>oay heavy chain variable domain compnsing non· human
`Complementarity Determining Region (COA) amino acid residues wtiicn Dind amigen incorporated
`into a human antit>oay variable domain, and further comprising an amino ac1a substitution at a site
`selec1ed from tne group consisting of: 24H, 73H. 76H, 78H, and 93H. utiliZing !he numbering system
`set forth in Kabat
`
`(NEW) The humanized variable aomain of claim 115 wherein U"le subStJMed residue is the
`116.
`res1aue found at the corresponding 1ocalion ol tl"le non-human antibOdy from which the non-numan
`CDR amino acid residues are obtainea.
`
`(NEW) The numani2ed vanaDle domain or claim 115 wherein no human Framewo<K Region
`117.
`(FR) residue omer man tnose set forth in the group nas been sutistituted.
`
`(NEW) The humanized varia.Dle aomain of c1aim 115 wherein the human antibody vanabte
`1 18.
`domain is a consensus human variaDle oomain.
`
`8
`
`577 of 1033
`
`BI Exhibit 1002
`
`

`

`(NEW) The humanized variable domain of claim 115 wnerein the residue at site 24H has
`119.
`been suDs!ituted.
`
`(NEW) The numanized variable domain of claim 115 wtlerein ltle residue at site 73H has
`120.
`Deen substituted.
`
`(NEW) The numanized variable domain of claim 115 wherein the residue at sit.e 76H has
`121 .
`been subsrituted.
`
`122.
`
`(NEW) The humanized vanabJe domain of claim 115 wherein ma residue at site 78H has
`been substituted.
`
`123.
`
`(NEW) Tne humanized variable domain of claim 115 wnerein the residue at site 93H tias
`
`been substituted.
`
`(NEW) The humaniZ.ed variable domain of claim 115 which fuMer comprises an amino acid
`124.
`substitution at site 71H.
`
`(NEW) Ttle humanizea variable domain ol claim 115 wnicn funner comprises amino acid
`125.
`substitutions at sites 71 Hand 73H.
`
`(NEW) Tne numanized vanable domain of claim 115 which funner compri~es amino acid
`126.
`substitutions at snes 71 H, 73H and 78H.
`
`127.
`
`(NEW) An antibody compnsing the humanized variable domain of claim 115.
`
`(NEW) A humanized variant of a non-human parent antibody which Dinas an antigen.
`128.
`wherein ltle humant2e0 variant comprises Comptementatfty De1ermining Region (CDA) amino acid
`residues of lhe non-human parent an11body incorporatea into a human antibody variable domain,
`and further comprises a Framework Region (FA) suoslitulion where tne suos1itu1ed FR residue: (a)
`noncovalently Dinds antigen directly; (b) interacts with a COR; or (c} participates in the V"-V"
`interface by atfec11ng the pro~m1ty or orientation of tne v ... and \(, regions wittl respect to one
`anonier. and wMrein the humanized variant binas the antigen more tightly than the parem antibOdy
`
`b1 !'It~ o'J .
`
`9
`
`578 of 1033
`
`BI Exhibit 1002
`
`

`

`•
`
`Patent Docket P0709Pl
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`In re Application of
`
`Paul J. Carter et al.
`
`Serial No.: 08/146,206
`
`Filed: November 17, 1993
`
`Group Art Unit: 1642
`
`Examiner: J. Reeves
`
`'999
`MATRIX Ci.J r0:v°1li
`ENTER
`
`For: METHOD FOR MAKING HUMANIZED
`ANTIBODIES
`
`"!" ..... ,
`
`COMMUNICATION
`
`Assistant Commissioner of Patents
`Washington, D.C. 20231
`
`RECEIVED
`FER - ' f999
`I~ TECH CENTER 1600/2900
`Sir:
`Further to the Supplemental Amendment fax-filed on January 15, 1999, please."~nd encloseel:f)fipr.Ltv
`documents USSN 07 /290, 97 5 and USSN 07 /310,252 for the "POL Patents" as promised on page 11 of
`
`'~
`3 •I
`
`.·
`
`that amendment.
`
`Applicants further submit herewith a Supplemental Information Disclosure Statement. In this respect
`
`Applicants bring to the Examiner's attention a Celltech press release entitled: °Celltech Antibody
`
`Technology Platform Further Strengthened Through New Patents in US and Europe." (Exhibit A
`
`attached) This press release refers to on allowed US "Adair" patent application. Applicants believe
`this US Adair patent application corresponds to W091 /09967 (of record) and EP 460, 167 Bl (copy
`attached).
`
`Should the Examiner have questions concerning this communication, she is invited to call the
`
`undersigned.
`
`G
`
`By:-+-+--"""--"'-"=~---­
`Wenay M. Lee
`Reg. No. 40,378
`
`Date: January(iL 1999
`
`l DNA Way
`So. San Francisco, CA 94080-4990
`Phone: (650) 225-1994
`Fax: (650) 952-9881
`
`579 of 1033
`
`BI Exhibit 1002
`
`

`

`_, ..
`
`• ,,.
`
`Celltech Antibody Technology Platform Further Strengthened Through New Patents in US and Europe
`'
`SLOUGH, U.K., Sept. 26 /PRNewswire/ ·- Celltech announced today that the U.S.
`Patent Office has allowed one of its key patent applications covering engineered
`human antibodies. The grant of this "Adair" patent will occur in early 1997 and
`will expire in 2014. This broad product patent covers a key approach to the
`construction of new human antibodies which is essential in order to achieve full
`therapeutic activity.
`It covers all antibodies which have been constructed
`using this approach. A corresponding patP.nt has already been granted in Europe,
`although it is anticipated that the financial benefit to Celltech from the U.S.
`patent will be more significant in the near term because of the numbers of
`antibodies in late-stage development in the U.S.
`
`The •Adair" patent is an important new element in Celltech's technology
`platform, and complements previous Celltech patents in the field of antibody
`engineering.
`It covers all of Celltech's own antibodies currently in clinical
`development, thus substantially extending their period of patent protection. In
`addition the patent covers a range of antibodies under development by other
`companies. This would result in royalty revenues should these products reach
`the market.
`
`There are already a number of process patents covering the manufacture of
`engineered antibodies including those granted to Celltech, Genentech, the
`Medical Research Council and Protein Design Laboratories. Celltech has
`agreements in place with Genentech and the Medical Research Council relating to
`the commercial exploitation of some of these patents. Celltech pursues the
`strategy of licensing its existing antibody patents to any interested party for
`products which are not directly competitive with Celltech's own products. This
`policy will be pursued with the new "Adair" patent and all licensees who have
`directly licensed pre-existing patents from Celltech (in particular the "Boss"
`antibody engineering patents) will be offered favorable terms for the "Adair"
`patent.
`
`Commenting on the news today, Dr. Peter Fellner, CEO, said, "Celltech has
`built a very valuable platform technology in the field of antibody engineering
`and the grant of this patent will further strengthen our position. We expect a
`continued growth in royalty revenues from our licensed patents ·which will make a
`significant contribution to the profitability of the company. The potential in
`this area can be seen from the growing success of AeoPro(TM) (lilly/Centocor).
`Royalties on the sales of this product are paid to both Genentech and Celltech."
`SOURCE Celltech Therapeutics Ltd.
`
`/NOTE TO EDITORS:
`
`1. The Adair product patent covers any antibody in which the antigen binding
`regions from a donor antibody have been tra~sferred to the framework of a human
`antibody, and specifies certain requirements in specific amino acid residues
`within the product which are necessary to recover full antigen binqing activity
`of the newly created antibody.
`
`2. Antibodies are natural proteins which bind tightly and specifically to
`antigens. This binding property is particularly important in providing a
`defense mechanism against infectious organisms such as bacteria and viruses.
`For some time, scientists have been able to produce antibodies in the
`
`9/26(96
`
`EXHIBIT A
`
`1
`
`580 of 1033
`
`BI Exhibit 1002
`
`

`

`. ~,. .
`
`"
`
`laboratory and their availability has had a profound impact on diagnostic
`In contrast, they have had little impact on therapeutic medicine.
`medicine.
`The reason for this is that the first antibodies were derived from animal
`sources. When these animal antibodies were injected into humans they induced · a
`significant immune response which led to either adverse reactions or a rapid
`loss of therapeutic efficacy. More recently techniques have been developed to
`produce engineered human antibodies which are virtually identical to natural
`human antibodies. The main advantage of these antibodies is that they do not
`cause a significant immune response in man and they are very well tolerated.
`Because of their good tolerance, their binding properties are being used in a
`wide variety of therapeutic applications in areas such as blockade of receptor
`functions in heart disease, neutralization of cytokine in rheumatoid arthritis
`and killing of cancer cells./
`/CONTACT: Dr. David Bloxham, Chief Executive of CelltechTherapeutics
`Ltd., or Peter Allen, Finance Director of Celltech Group pie, 0-1753-534655;
`or Jon Coles of Brunswick, 0-171-404-5959; or Rich Tamrnero of Noonan/Russo
`Communications, Inc., 212-696-4455 ext. 222, e-mail: news@noonanrusso.com/
`08:52 EDT
`
`0624 09/26/96 08:52 EDT HT
`:TICKER: CEL.GB
`:SUBJECT: BIOT PTNT ENGL USA
`Copyright (c) 1996 PR Newswire
`Received by NewsEDGE/LAN: 9/26/96 6:50 AM
`
`9/26/96
`
`2
`
`581 of 1033
`
`BI Exhibit 1002
`
`

`

`•
`
`a/290975
`
`,,,,
`·1z9Q,97S
`
`8~/0585~
`;~u;;;;;,j'_~_·_-: __ 1_s ______ __..r_·_·ec __ ·_.u_· ___________ l~Q-A_~_,_a_~_;T __ J_~_·T __ .... l"'"'" ____ ·~_·_A ___ ~-1-:~,~
`
`••COMTI•UI•' DATA•••••••••••••••••••••
`VE.RlflED
`
`'2.8 DEC 1989
`REC'D
`WIPO PCT
`
`••rOIE16M/PCT AP,LlCATIONS••••••••••••
`VE.Alf JED
`
`PRIORITY DOCUMENT
`
`fOAECGH flLlNG LIC EN S E GRANTED 01/24/89
`
`••••• S"ALL ENTlTT •••••
`
`Ovu O•o
`'• ·::~ t'':iC,112 o<An•IUOn• ~•t 0yH Ono
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`v ... ,. . . . ,,. ~";. ... ..... , ~ . ·--
`--' 1L L 1 A"'
`' . • .
`TOWNSEND AND TOWN SEH'
`SfEUA AT STREET TOWER1 O~E
`SAN FRA~CISC01 CA
`94105
`
`I\ • S" I T H
`
`5 ~
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`0
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`AS
`FILED
`·:-. ·1~·-; ,. • .
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`S TATE OA S M£ETS
`COUNTAV ORWCS.
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`TOTA L.
`Cl..AIMS
`
`,.ll.!NQ '"CE
`t NO CP.
`Cl..AI MS A"CEIVCO
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`ATTOA N CY"S
`OOCKET NO.
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`llllA A1C. ET P l A 2 A
`
`NO~EL IL-2 RECEPTCQ-SPECIFIC HU"AH l"~UN0 G l08ULINS
`
`j
`!::::
`~
`
`This ij!-~to · certify that annex~d hereto
`is·a ~true copy from the records of the
`United States ~~tent and T~ademark Of fi
`of the application as originally filed
`which is identified above.
`
`By authority · of the
`COMMI~SIO,~R PATENTS
`
`AND TRADEMARKS
`
`e~ .. ~ I
`
`Certify in · Officer
`
`JAN 1990
`
`582 of 1033
`
`BI Exhibit 1002
`
`

`

`•
`
`..,
`
`PATE~T APPLTCATION SERIAL NO.
`
`Oli290975
`
`~~~~~~~~~
`
`U.S . DE?AR'l'.'l-!E!-IT OF CO~ERCE
`PATE~T AND TllADE~RK Off ICE
`fE£ RECORD ~~EET
`
`G lUll
`
`01/0~/89 2909?5
`
`20-H;)O 110 20t
`
`PT0- 1556
`(5{87)
`
`..
`
`583 of 1033
`
`BI Exhibit 1002
`
`

`

`• •
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`10.
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`2S
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`I I
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`I
`I
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`118 23-8
`
`NOVEL lL-2 SECEPIOR-SPECifIC HUMAN 1Mffi)NOGLQ8ULINS
`
`Field o f the Invention
`The present invention relates generally to the
`combination of recombinant DNA and monoclonal antibody
`technologies for developing novel therapeutic agents and,
`more particularly, to the proauction of non-imrnunogenic
`antibodies specific for the human interleukin-2 receptor and
`their uses.
`
`Background of the Invention
`In mammals, the immune response is mediated by two
`types of cells that interact specifically with for~ - 3~
`material, ~. antigens. one of these c ell types, B-cells,
`are responsible for the production of antibodies. The second
`cell class, T-cells, incl~de a wide variety of cellular
`subsets controlling the ill vivo function of both a-cells and
`a wide variety of other hematopoietic cells, including T(cid:173)
`cells.
`
`One way in which T-cells axert this control is
`through the production of a lymphokine known as interleukin-2
`(IL-2), originally r.amed T-cell growth factor.
`IL-2's prime
`function appears to be the stimulation and mainten~nce of T(cid:173)
`cells.
`Indeed, ~ome immunologists believe that IL-2 may be
`at the center of the entire ir:unune respo~se (see, :arrar, J.,
`et al., Immunol. Rev .
`.§.1:123-166 (1982) , which is
`incorporated herein by reference).
`To exert its biological effects, IL-2 interacts
`with a speciric high-affinity ~embrane receptor (Greene, W.,
`et al., Progress in Hematology XIV, E. Brown, Ed., Grune and
`Statton, New York (1986), at pgs. 28J ff). The human IL-2
`receptor is a complex roultichain glycoprotein, with one
`chai~, known as the Tac peptide, being about SSkD in size
`{~, Leor.ard, w., et al., J. Biol. Chem. 260:1872 (1985),
`which is incorporated herein bf reference). A gene encoding
`this protein has been isolated, and predicts a 272 amino acid
`peptide, including a 21 amino acid signal peptide (~,
`
`584 of 1033
`
`BI Exhibit 1002
`
`

`

`2
`
`~ature l.ll: 626 (1984)). The 219 NH~­
`Leonard, W., et al . ,
`terminal amino acids of the p55 Tac protein apparently
`comprise an extracellular domair. (see, Leonard, W., et al.,
`Science, 230:633-639 (1985), which is incorporated he~ein by
`reference).
`Much of the elucidation of the human IL-2
`receptor's structure and function is due t~ the development
`of specifically reactive monoclonal antibodies.
`In
`particular, one mouse monoclonal antibody, known as a~ti-Tac
`(Uchiyama, et al. , J. !mmunol. 11..§.:1393 (1981)) has showo
`that IL-2 receptors can be deter.ted on T-cells, but also on
`cells of t~e monocyte-macrophage family, Kupffer cells of the
`liver , L<lngerhons' cells of the skin and, of course.
`activated T-cells.
`Importantly, resting T-cell~. B-cells or
`circulating machrophages typically do not displrt the IL-2
`receptor (Herrmann, et al., J. Exp. Med. 162:1111 (1985)).
`The anti - Tac monoclonal antibody has also been used
`to define lymphocyte functions that require ·rL-2 interaction,
`and has been shown to inhibit v~rious T - cell functions,
`including the generation of cytotoxic and suppresso~ T
`lymphocytes in cell culture. Also, based on studies with
`anti-Tac and other antibodies, a variety of disorders a~e now
`associated with imprope~ IL-2 receptor expression by T-cells,
`in particular adult T-cell leukemia.
`Hore recently, the IL-2 receptor has been shown to
`be a n ideal target fo~ 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 ue used either alone or as an ir.ununoconjugate
`(~, vith Ricin A, isotopes and the like) to ef:ec~ively
`remove cells bearing the IL-2 receptor. These agen~s can,
`for example, theoretically eliminate IL-2 receptor-expr~ssing
`leukemic cells, certain B-cells, or activated T-cells
`involved in a disease state, yet allo~ the reten~ i on of
`mature normal T-cells and their precursors to ensure the
`capability of mounting a normal T-cell immune response as
`needed .
`In general, most other T-cell specific age~ts can
`destroy essentially all peripheral T-cells, which li~its the
`
`5
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`20
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`.•
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`585 of 1033
`
`BI Exhibit 1002
`
`

`

`• -·
`
`J
`
`agents' therapeutic efficacy. Overall, the use of
`appropriate monoclonal antibodies specific for the IL-2
`receptor may have therapeutic utility in autoimmune diseases,
`organ transplantation and any un~~nted response by activated
`T-cells.
`Indeed, clinical trials have been initiated using,
`S ~. anti-Tac ~ntibodies (~, generally, Waldman, T., et
`al., Cancer Re~ . .1_2:625 (1985) and Waldman, T., Science
`232:727 - 732 (1986), both of wh:ch are inco~porated herein by
`reference).
`Unfortunately, the use of the anti-Tac and other
`non-~uman rnonoclonal antibodies have certain drawbacks,
`particularly in repeated therapeutic regimens as explained
`below. House monoclonal antibodies , for example, do not fix
`human complement well, and lack other important
`immunoglobulin functional characteristics when used in
`humans.
`
`IO
`
`15
`
`ferhaps more importantly, anti-Tac and other non(cid:173)
`human monoclonal antibodies contain substantial stretches of
`am.\.no acid sequences that will be immunogenic when injected
`into a human patien~ . Numerous studies ha~e shown that,
`after injec tion of a foreign antibody, the immune response
`elicited by a patient against an antibody can be quite
`strong, essentially eliminating the antibody ' s therapeutic
`utility after an initial treatment . Moreover, as inc~easing
`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 t~eat~ents
`with any different non-human antibodies, subsequent
`treatments even ~or unrelated therapies can be ineffective or
`even dangerous in themselves.
`While the production of so-called " chimeric
`antibodies" (~, mouse variable regions joined to human
`constant regions) has proven somewhat successful, a
`significant illllllunogenicity problem remains.
`In general, the
`production of human imrnunoglobulins reactive vith the human
`IL-2 receptor, as with many human antigens, has been
`extremely difficult using typical human monoclonal antibody
`production te~hniques. Similarly, utilizing recombinant DNA
`
`20
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`586 of 1033
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`BI Exhibit 1002
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`3.0.
`
`35
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`4
`
`technoloc;y to· produce so-called "humanized" antibodies (~.
`~. EPO Publication No. 02~9400). provides cnce rtain
`results, in part due to unpredictable binding affinitiP3.
`Thus, there is a need for improv~d !onns of human(cid:173)
`like immunoglobulins specific for the human IL-2 receptor
`that are substantially ncn-immunogenic in humans, yet easily
`and economically produced in a manner suitable for
`therapeutic formulation and other uses. The present
`invention fulfills the~e and other needs.
`
`Summary of the Invention
`The present invention provides novel compositions
`useful, for ~xample, in the treatment of T -cell mediateci
`human diso~ders, the compositions containing human-like
`immunoglobulins specifically capable of blockins the binding
`of human IL-2 to its receptor and/oc capable of binding to
`the pSS Tac protein on human IL-2 receptors. The
`im.munoglobulins can have two pairs of lighc chain/heavy c hain
`complexes, typically at least one pair having chains
`corn;Jrising mouse complementarity determining regions
`functionally joined to human fr~mework region seginents. For
`exomple, mouse c~mplementarity determining regioos, with or
`without additiona l naturally-associated mouse amino acid
`residues, can be used to produce human-like antibodies
`
`•
`
`ca?able of binding to tile human IL-2 rece~tor at affinity
`levels strcnger than about 108 M-1
`The illUllunoglobulins, including bindi~g fragme nts
`and other derivatives thP-reof, of the present invention nay
`be prcduced readily by a variety of recol!l.binant DNA
`techniques, with ultimate expression in transfected cells,
`preferably iwnortalized eukaryotic cells, such as myelo~a or
`hybridoma cells. Polynucleotides comprising a first sequ:nce
`codi11g for human-1 ike immunoqlobul in framework regions and a
`second sequence set coding for the desired immunoglobulin
`complementarity determining regions can be produced
`synthetically or by combining appropriate cDNA and ge~omic
`ONA segments.
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`The human-like immunoqlobulins may be utilized
`alone in substantially pure form, or complexed with a
`cytotoxic agent, such as a radionuclide, a ribosomal
`inhibiting protein or a cytotoxic agent .acti·re at cell
`surfaces. All of these compounds will be partic~larly useful
`in treating T-cell mediated disorders. The human-like
`immunoglobulins or their complexes can be prepared in a
`pharmaceutically accepted dosage form, which will vary
`depending on the mode of administration.
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`BRIEF DESCRIPTION OF THE FIGURES
`Figure 1. Comparison of sequences of anti-Tac
`heavy cha:n (upper lines) and Eu heavy chain (lower lines).
`The 1- letter code for amino acids is used. The first amino
`acid on each line is numbered at the left.
`Identical amino
`acids in the two sequences are connected by lines. The J
`CORs arc 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 '*
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`Figure 2. Comparison of s2quences of anti-Tac
`light chain (u~per lines) and Eu light chain · (lower 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 t wo sequences ar~ connected by lines. The
`3 CDRs are underlined. Other amino a~id positions for which
`the anti-Tac amino acid rather than the Eu amino acid was
`used in the huma~ized anti-Tac heavy chain are denoted by
`an *
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`figure J. Nucleotide sequence of the gene for the
`humanized anti-Tac heavy chain variable region gene. The
`translated amino acid sequence for the part of the gene
`encoding protein is shown underneath the nucleotide sequence .
`The nucleotides TCTAGA at tte beginning and end of the gene
`are Xba I sites. The mature heavy chain sequence begins with
`amino acid #20 Q.
`Figure 4 . Nucleotide sequence of the gene for the
`humaniz~d anti-Tac light chain variable region gene. The
`translated amino acid sequence for the part of the gene
`encoding prote:n is shown underneath the nucleotide sequence.
`The nucleotides TCfAGA at th~ beginning and end of the gene
`are Xba I sites. The mature light chain sequence begins with
`amino acid #21 D.
`Figure 5. A. Sequences of the fou r
`oligonucleotides used to synthesize the humanized anti- Tac
`heavy chain gene, printed 5 ' to J'. B. Relative positions
`of the oligonucleotides. The arrows point in the 3 1
`direction for each oligonucleotide.
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`(A) Sequences of the four
`Figure 6.
`oligonucleotides used to synchesi2e the humanized anti-Tac
`light chain gene, printed 5' to J'.
`(B) Relative positions
`of the oligonucleotides. The arrows point in the J'
`direction for each oligonucleo~ide. The position of a Hind
`lII site in the overlap of JFD2 and J:OJ is s~own.
`Figure 7. Schematic diagram of the plasmid
`pHuGTACl used to express the hu~anized anti-Tac heavy chain.
`Relevant r~striction sites are shown, and coding regions of
`the hea·.ry chain are displayed as boxes. The direction of
`transcription from t!:~ immunoglo!:lulin (Ig) promoter is shown
`E~ = heavy chain enhancer, Hyg = hygromycin
`by an arrow .
`resistance gene.
`Figure 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 transc~iption
`from the Ig promoter is shown by an arrow.
`Figure 9. Fluorocytometry of HUT-102 and Jurkat
`cells stained vith anti-Tac antibody or humanized anti-Tac
`antibody followed respectively by fluorescein - conjugated goat
`3nti-mouse Ig antibody or goat anti - human Ig antibody, as
`labeled.
`In each panel, the dotted curve shows the results
`when the first antibody was omitted, and tr ~ solid curve the
`results when first and second (conjugated) antibodies we:-e
`included as described.
`Figu:-e 10.
`(A) Flnorocytometry of HUT- 102 cells
`stained with 0-40 ng of anti-Tac as indicated, then with
`biotinylated anti-Tac, and then with phycoerythrin-conjugated
`avidin.
`(B) Fluorocytometry of HUT-102 cells s~ained with
`the indicated antibody, then ~ith biotinyl~ted an~i-Tac, and
`then with phycoerythrin-conjugated avidin.
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`DETAILED DESCRIPTION OF TH: Ih"VENTION
`In accordance with the present invention, human-
`1 ike immunoglobulins specifically reactive with the IL-2
`receptor on human T- cells are provid~d. These
`ill\J:lunoglobulins, which have binding affinities of at least
`about 105 H·t, and preferably 109
`1 to 1010 H. 1 or strange:::,
`} (
`are capable of, !L:..SL., blocking the binding cf IL-2 to human
`IL-2 receptors. The human- like imrnunoglobulins will have ~
`human-like framework and can have compl~mentarity determining
`regions (CDR's) from an immunoglobulin, typically a mouse
`irn.-•moglobnlin, specific.ally reactive with a n epitope on pSS
`Tac protein. The immuno~lobulins of the present invention,
`which ca~ be produced economically in large quantities, ~ind
`use, for example, in the treatment of T- cell mediated
`disorde:::s in human patients by a variety of techniques.
`The basic antibody structural unit is known to
`comprise a tetramer . Each tetramer is .:omposed of two iden(cid:173)
`tical pairs of polypeptide chains, each pair having one
`•: light" (about 25kD) and one "heavy" cr,ain (about S0- 70kD).
`The NHz-terminus of each chain begins a variable region of
`about 100 to 110 or more amino acids primarily responsible
`for antigen recognition. The. COOH terminus of each chain
`defines a constant region primarily responsible for effec~or
`function.
`
`Light chains are classified as either kappa or
`lambda. Heavy chains are classified (and subclassified) as
`gamma, ~u, alpha, delta, or epsilon, and defir.e the
`antibody's isotype as IgG, IgM, IgA, IgD and IgE,
`respectively. Within light and heavy c~ains, the variable
`and constant regions are joined by a "J'' cegion of about 12
`or more amino acids, with the heavy chain also including a
`"0" region of about 12 more amino acids.
`(See, generallv,
`Fundament~l Immunology, Paul, w., Ed., Chapter 7, pgs. 131-
`166, Raven Press , N.Y . (1~84), whic~ is incorporated he~ein
`by reference..}
`The variable regions of each light/heavy chain ?air
`form the antibody binding site. The chains all exhibit ~~e
`same general structure of relatively conser"ed framework
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`regions joined by three hypervariable regions, also cali~d
`CDR's (see, "Seq\lences of Proteins of Immunological
`Interest," Kabat, E., et al., U.S. Department of Health and
`Hu~an Services, (1983); and Cholthia and Lesk, J. Mol. Biol.,
`196:901-917 {1987), which are incorporated herein by
`reference). The CDR's from the two chains of each pair are
`aligned by the fra~ework regions, enabling binding ~o a
`specific epitope.
`As used herein, the term "immunoglobulin" refers to
`a protein consisting of one or more polypeptides
`substan~ially encoded by immunoglobulin genes. The
`recognized immunoglobulin genes include the kappa, lambda,
`alpha, gamma, delta, epsilon and mu constant region genes, as
`~ell as the myriad immunoglobulin variable region g~nes . The
`immunoglobulins may exist in a variety of forn:.s besides
`antibodies; including, for example, Fv, Fab, and f \ab) 2 , as
`well as in single chains (g_,_g_._, Huston, et al., Proc. Nat.
`Aca d. Sci. U.S.A., 85:5879- 5883 {1988) and Bird, et al.,
`Sci2r.c~, 242:423-~26 (1988), which are incorporated herein by
`reference).
`(See, aenerally, Hood, et al., "Immunology",
`Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood,
`Nature, 323:15- 16 (1986), which are incorporated herein by
`reference).
`Chimeric antibodies are anticodies whose light and
`heavy chain genes have been constructed, typically by genetic
`engineering, free immunoglobulin gene segment~ belonging to
`different species. For example, the variable (V) segments of
`the genes from a mouse monoclonal antibody may be joined tu
`human constant (C) segments, such as 1 1 and IJ· A typical
`therapeutic chioeric antibody is thus a hybrid protein
`consisting of the V or antigen- binding domain from a mouse
`antibody and the C or effector domain from a human antibody
`(~, A.T . C.C. Accession No. CRL 9688 secretes an anti-~ac
`chimeric antibody), although other mammalian species may be
`used.
`
`As used herein, the term "framework regiou" refers
`to those portions of immunoglobulin light and heavy c~ain
`variable regions that are relatively conserved (i.e., other
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`than the CDR's) among different inununoglobulins in a single
`S?ecies, as defined by Kabat, et al., Q.Q. £1..t. As used
`h~rein, a "human-like framework region" is a framework region
`that in each existing chain comprises at least about 70 ~r
`more acino acid residues, typically 75 to as or more
`r esidues , identical to those in human irrununoglobulins.
`As used herein , the term "human-like
`inununoglobulin" refers to an immunoglobulin comprising a
`human-like framework and in which any constant region present
`is substantially homologous to a hu~an inununoglobulin
`constant region, ~ . at l~ast about 85-90%, p=eferably
`about 95\ ide ntical. Hence, all parts of a human-like
`immunoglobulin, except possibly the CDR ' s, are substantially
`homologous to corresponding parts of one or more native h