`
`•
`
`DOCKET NO.: CARP-0057
`
`PATENT
`
`immunoglobulin, wherein the sequence of
`
`the acceptor
`
`immunoglobulin heavy chain variable
`
`region framework
`
`consensus sequence of human immunoglobulin heavy chain
`
`variable
`
`region
`
`frameworks.
`
`40. First and second
`
`encoding heavy and light chain variable
`
`a humanized
`
`immunoglobulin having complementarity
`
`regions
`
`(CDRs) from a donor
`
`immunoglobulin and
`
`and light chain
`
`variable region frameworks from human ace ptor immunoglobulin
`
`heavy and light chains, which humanized mmunoglobulin
`
`specifically
`
`within about
`
`an affinity constant
`
`immunoglobulin, wherein said
`
`humanized immunoglob
`
`comprises one or more amino
`
`acids from the donor
`
`heavy chain
`
`framework
`
`outside the Kabat and Chothia
`
`wherein
`
`the donor amino
`
`acids substitute for
`
`in the acceptor
`
`immunoglobulin heavy chain f amework, and each of these said
`
`donor amino acids :
`
`( I ) is adjacent to
`
`the donor immunoglobulin sequence,
`
`or
`
`(II} contains an
`
`within a distance of 6 ANGSTROM of a
`
`CDR in said humanized mmunoglobulin.
`
`- 6 -
`
`Board Assigned Page #973
`
`BIOEPIS EX. 1595
`Page 1126
`
`
`
`•
`
`•
`
`DOCKET NO.: CARP-0057
`
`PATENT
`
`41. A humanized immunoglobulin having complementa ity
`
`determining regions (CDRs)
`
`from
`
`a donor
`
`immunoglobuli
`
`heavy and light chain variableregion
`
`frameworks from
`
`acceptor immunoglobulin heavy and light chain framewo
`
`which
`
`humanized immunoglobul in specifically binds to an
`
`with an
`
`affinity constant of at least 107 M- 1 and no
`
`four - fold
`
`that of the donor immunoglobulin,
`
`about
`
`sequence
`
`of the humanized
`
`i mmunoglobul i n
`
`n variable region
`
`framework is at least 65% identical
`
`sequence of the donor
`
`immunoglobulin heavy chain variable
`
`framework and
`
`comprises at least 70
`
`acceptor human
`
`acid sequence .
`
`identical
`
`to an
`
`variable region amino
`
`42. A
`
`h
`
`noglobulin according to claim 41
`
`whi ch is an antibody
`
`two light chain/heavy chain
`
`dimers.
`
`43.
`
`immunoglobulin having complementarity
`
`from
`
`a donor
`
`immunoglobulin and
`
`heavy and l i ght
`
`ariable region
`
`frameworks
`
`from acceptor
`
`immunoglobulin
`
`and
`
`light chain frameworks, which
`
`humanized i mmunoglo ulin spec ifically binds to an antigen with
`
`an affinity
`
`of at
`
`least about 10 8 W 1 and no
`
`- 7 -
`
`Board Assigned Page #97 4
`
`BIOEPIS EX. 1595
`Page 1127
`
`
`
`•
`
`•
`
`DOCKET NO.: CARP-0057
`
`TENT
`
`greater
`
`than about four-fold tha t of the donor immunog
`
`wherein the sequence of
`
`the acceptor
`
`immunoglobul '
`
`heavy
`
`chain variable region framework
`
`is a
`
`consensus
`
`of
`
`human immunoglobulin heavy chain variable region
`
`ameworks.
`
`44. A pharmaceutical composition co
`
`humanized immunoglobulin of claim 41 in a ph rmaceutically
`
`acceptable carrier.
`
`45 . A
`
`immunoglobulin of clai
`
`int roducing DNA
`
`seg
`
`humanized immunoglobulin
`
`heavy and light chains
`
`and
`
`expressing
`
`the DNA
`
`segments
`
`the cell
`
`to produce
`
`the
`
`humanized immunoglobulin.
`
`46 . A
`
`producing a humanized
`
`immunoglobulin,
`
`g the steps of:
`
`(1)
`
`comparing
`
`a donor immunoglobulin heavy
`
`chain variable regio
`
`against a collection of sequences of
`
`human heavy chain v riabl e regions;
`
`(2)
`
`selecting
`
`variable region from the
`
`collection of hum
`
`heavy chain variable
`
`regions
`
`to provide
`
`an acceptor heavy chai n variable region, wherein the selec ted
`
`- 8
`
`-
`
`Board Assigned Page #975
`
`BIOEPIS EX. 1595
`Page 1128
`
`
`
`•
`
`•
`
`DOCKET NO .: CARP-0057
`
`PATENT
`
`variable region framework is at least 65%
`
`identical
`
`to
`
`donor
`
`immunoglobul i n heavy chain variable region
`
`framework;
`
`(3)
`
`synthesizing a DNA segment encoding a
`
`heavy
`
`chain variable region ,
`
`comprising CDRs from
`
`immunoglobulin heavy chain variable r egion
`
`a variable
`
`r egion framework from the selected acceptor
`
`region;
`
`(4)
`
`introducing
`
`the DNA
`
`segment
`
`the humanized
`
`immunoglobulin heavy
`
`segment encoding
`
`variable region into
`
`and
`
`a DNA
`
`light c hain
`
`(5)
`
`expressing
`
`the
`
`in
`
`t he cell to produce the
`
`humanized immunoglobul i
`
`47. A method
`
`producing a humanized
`
`immunoglobulin , comprisin
`
`the steps of:
`
`(1)
`
`comparing
`
`a donor immunoglobulin light
`
`chain variable region
`
`a collection of sequences of
`
`human light chain var·able regions;
`
`(2)
`
`selecting
`
`variable region from the
`
`collection of human
`
`chain variable
`
`regions
`
`to provide
`
`an acceptor light c ain variable region , wherein the selected
`
`- 9 -
`
`Board Assigned Page #976
`
`BIOEPIS EX. 1595
`Page 1129
`
`
`
`•
`
`•
`
`DOCKET NO.: CARP-0057
`
`PATENT
`
`variable region framework is at least 65%
`
`identical
`
`to
`
`donor
`
`immunoglobulin
`
`light chain variable region framewo
`
`(3)
`
`synthesizing a DNA segment encoding a humanized
`
`chain variable region,
`
`compri s ing CDRs from the
`
`immunogl obulin ligh t chain variable region and
`
`variable
`
`region framework from the selected acceptor
`
`variable
`
`region;
`
`(4)
`
`introdu cing
`
`the DNA
`
`segment
`
`the humanized
`
`immunoglobulin light chain variable
`
`and
`
`a DNA
`
`segment encoding
`
`heavy chain
`
`variable
`
`( 5)
`
`humanized i mmuno
`
`ce ll; and
`
`DNA
`
`in
`
`the cell to produce the
`
`48.
`
`umanized
`
`noglobulin hav i ng complementarity
`
`d etermining regions (CDRs)
`
`a donor
`
`immunoglobul in and
`
`heavy and ligh t chain
`
`le r egion
`
`frame works
`
`from acceptor
`
`immunoglobulin heavy
`
`light chain f rameworks, which
`
`humanized immunoglobu li
`
`specifically binds to a n antigen with
`
`an aff inity constant
`
`about
`
`fou r -fold of that of the
`
`donor immunoglobu lin ,
`
`the s equence of the acceptor
`
`immunoglobulin heavy
`
`variable
`
`region
`
`framework
`
`is a
`
`conse nsus sequence
`
`human immunoglobulin heavy chain
`
`variable region fr
`
`- 10 -
`
`Board Assigned Page #977
`
`BIOEPIS EX. 1595
`Page 1130
`
`
`
`•
`
`•
`
`DOCKET NO.: CARP-0057
`
`PATENT
`
`REMARKS
`
`Newly added claims 32-40 have been copied from claims
`
`in Queen et al., U.S. Patent No. 5,693,761. Claims 41-48 have
`been copied from claims in Queen et al., u.s. Patent No.
`
`5,693,762. Copies of both patents are enclosed. Applicants are
`
`i n compliance with 35 USC §135(b } since both Queen patents were
`
`issued on December 2, 1997.
`
`Respectfully submitted,
`
`, ~AM/~~rr~~
`
`Vad.1s J.\. '~~tin
`Registration No. 19,386
`
`WOODCOCK WASHBURN KURTZ
`MACKIEWICZ & NORRIS LLP
`One Liberty Place - 46th Floor
`Philadelphia, PA 19103
`(215} 568-3100
`
`- 11 -
`
`Board Assigned Page #978
`
`BIOEPIS EX. 1595
`Page 1131
`
`
`
`DATE FILED: 05/28/2010
`
`e t al.
`SN: 081146,206
`Filed November 17, 1998
`
`Ptor:. Narl. Acad. Sci. USA
`Vol. 86, pp. 10029-10033, December 1989
`lmmunolo.S¥ • ··•
`
`...
`
`•'
`·":
`
`A humanized antibody that binds to the interleukin 2 receptor
`(chimeric anlibody/antlbody llffinity/autoimmane d~)
`
`CARY QuEEN*, WILLIAM P. ScHNEIDER*, HAROLD E. SEL!CK .. t, PHILIP W. PAYNE\
`NICHOLAS F. LANDOLFI*' )AM~S F. DUNCAN*~. NEVENKA M. AVDALQVIC*' MICHAEL LE.V!TT§,
`.RICHARD P. JUNGHANS1l, AND THOMAS A. WALDMANN'il
`•Protein l>esi&n Labs. 3181 Porter Dril'c. Palo Aho. CA 9430t; t~anment ofC.:II Biolo,y. S1anford University. Stanford. CA 94305: and ~Mct~bolism
`Branch, Na.lioR>I DACer lustiMe, Nationallnstilutes of Health, Bethesda. MD :10892
`
`partial or complete remission in three or nine patients with
`Tac-expressing adult T-cell leukemia (14). However, as a
`murine monoclonal antibody, anti-Tac elicits a slrong human
`antibody response against itself. as does OKTl (15). This
`response would prevent its long-term use in treating autoim·
`m11ne conditions or suppressing organ transplant rejection.
`The immune response against a murine monoclonal anti(cid:173)
`body may potentially be reduced by transforming it into a
`chimeric antibody. Such antibodies, produced by methods of
`genetic engineerine. combine the variable (V) region binding
`domain of a mouse (or ral) antibody with human antibody
`constant (C) regions (]6-18). Hence. a chimeric antibody
`retains the binding specificity of the original mouse antibody
`but contains less amino acid sequence foreign to the human
`immune system. Chimeric antibodies have been produced
`against a number or tumor-associated antigens (19-21). Jn
`some but not all cases, the chimeric antibodies have mediated
`human complement-dependent cytotoxicity (CDC) or anti·
`body-dependent cellular cytotoxicity (AOCC) more efficient·
`ly than t he mouse antibodies (21}.
`When the murine antibody OKTI is used in human pa·
`tienls. much of the resulting antibody response is directed
`against the V region of OKT3 rather than the C region (15).
`Hence. chimeric antibodies in which the V region is still
`nonhuman may not have sufficient therapeutic advantages
`oYer mouse antibodies. To furt her reduce the immunogenic·
`ity of murine antibodies. Winter and colleatues constructed
`..humanized" antibodies in which only the minimum neces·
`sary p:lrts of the mouse antibody, the complementarity·
`determining regions (CORs), were combined with human V
`region frameworks and human C regions (22- 25). We report
`here the construction of chimeric and humanized anti-Tac
`antibodies.D For the humanized antibody, sequence homo!·
`ogy and molecular modelin& were used to select a combina·
`lion of mouse and human sequence elements that would
`reduce immunogenicity while retaining high binding affinity.
`
`MATERIAlS AND METHODS
`Coostrudlon of Plasmids. eDNA cloning wlls by the
`method of Gubler and Hoffman (26), and sequencing was by
`the dideoxy method (27). The plasmid pVKl (Fig. lA) was
`constructed from the following fragments: an appr~ximately
`45~base-pair (bp) BamHl- EcoRl fragment from the plas-
`
`A.bbreviatiD~s: tL-2R, interleukin Z receplDr; CDR, complcmentar·
`ity~etennining region; CDC, complemenl-dcpendenl cytotollicity;
`ADCC, antibody..Oepentlent cellular cytotoxicity; V. variable; J,
`joining: C, constant.
`·
`tPrescnt addr~s: Biospan, 440 Chesapeake Dtin, Redwood City,
`CA 94063.
`;Present address: Beckman Instruments. 1050 Page Mill RDad, Palo
`Alto, CA 94304.
`liThe sequences reponetl in this paper have been deposited in lite
`GenBank data base (accession nos. M28250 a~d M28251).
`.
`Carter Exhibit 2023
`Carter v. Adair
`
`111029
`
`Board Assigned Page #979
`
`n
`y
`·e
`R
`·d
`:e
`.e
`·d
`0
`R
`n
`l)
`>e
`0
`:s
`y
`
`18
`m
`)f
`\3
`!r
`C·
`ttl
`
`II.
`
`0,
`
`:II
`
`s.
`
`} .
`
`&
`
`·9)
`
`&
`
`:r.
`
`:o, . l i.
`
`·1.
`ll,
`
`(/.
`
`:3)
`
`<Jr
`'l·
`
`d.
`
`&
`
`'
`ti.
`
`of
`·y.
`
`·t/.
`
`Contributed by Thomas A . Waldmann, August JO, 1989
`
`The anti·Tac monoclonal antibody is known
`ABSTRACT
`to bind to lhep55 chain or the human interleukin2 receptor and
`to inhibit proliferation of T cells by blocking inter1eukin 1.
`binding. However, usc of anti-Tac as an immunosupprt:SSanl
`dl'llg would be Impaired by the human immtme responsf
`· agalnsl this murine antibody. We have tbe~rore coostrudcd a
`"bumanized" antibody by tombining the complementariiY·
`tlctermining regions (CORs) of the anli·Tllt antibody with
`human l'ramcwork and cons~.ant ~gions. The human rrame(cid:173)
`worl< regions were choseu to maximize homology with the
`anti·Tac antibody sequence. In addition, a computer model of
`murine anti-Tae was used to identify several amino acids
`which, while outside lbe CORs, are likely to interact with the
`CDRs or antigen. These mouse amino acids were also ret.ained
`In the humanized antibody. The humanized anli· Tac antibody
`bas an affinity for pSS of3 x 109 M- t,about l/3thatofmurine
`anti-Tac.
`·
`
`The cellular receptor for the lymphokine interleukin 2 (IL-2)
`plays an important role in regulation of the immune response
`(reviewed in ref. 1). The complete lL-2 receptor (IL-2R)
`consists of at least two IL-2-binding peptide chains: the p55
`orTac peptide (2, 3), and the recently discovered p75 peptide
`(4, 5). ldentification and characterization or the p55 peptide
`were facilitated by the development of a monoclonal anti·
`body, antt-Tac, which binds to human p55 (2). The p55
`peptide was found to be expressed on the surface ofT cells
`activated by an antigen or mitogen but not on resting T-cells.
`Treatment of human T cells with anti-Tac antibody s1rongly
`inhibits their proliferative response to antigen or to IL-2 by
`preventing IL-2 binding (3, 6).
`These results suggested that anti-] Lr2R antilxtdies would
`be immunosuppressive when administered in vivo. Indeed,
`injection of an anli·lL-2R antibody into mice and rats greatly
`prolonged survival of heart allografts (7, 8). Anti-JL-2R was
`also effective in rats against experimental graft-versus-host
`disease (9). In animal models of autoimmune disease, an
`anti·IL-2R antibody alleviated insulitis in nonobese·diabetic
`mice and lupus nephritis in NZB x NZW mice (10). Anti·Tac
`itself was highly effective in prolonging survival of kidney
`allografts in cynomolgus monkeys (11).
`In human palients, the specificity of anti-Tac for activated
`T cells might give it an advantage as an immunosuppressive
`agent over OKT3 (monoclonal anti-CD3), which is effective
`in treating kidney transplant r~ection (12), but which sup(cid:173)
`presses the entire peripheral T-eell population. Jn fac:t, in
`phase I clinical trials for kidney transplantation, prophylactic
`administration ofanti-Tac significantly reduced1he incidence
`af early rejection episo~es, without associated toxicity (13).
`Funhermore, treatment with anti·Tac induced temporary
`
`The publicalion costs of !his article were defrayed in part by p2S<: charge
`payment. This anicle must therefore be hen:by tna~lced "miv~tlistmtn.t"
`in accordance with lB U.S.C §1734 solely to indieale !his fact,
`
`i
`
`u. I
`
`BIOEPIS EX. 1595
`Page 1132
`
`
`
`I
`I
`
`10030
`
`Immunology: Queen et at.
`
`Proc. Na t/. Acad. Sci. USA 86 (1989)
`
`A
`
`B
`
`o(
`
`.xoal
`
`Gpl
`
`pmnar
`
`:o;,a\
`
`f¥><id!Z• __9
`v
`c
`l J
`fxtancl
`Con•Juro. l
`
`Hybri6zo
`rev. primer
`
`. /
`
`/ '
`
`I
`Exlend
`and cut
`""
`s~ .-!f.
`~--~:~========~~~~~\
`Xbal
`J
`V
`
`f1c . I. lA l Sch~:mati~ diusmm uf the pla~mKb pVKI und pL Tac.
`Lighl chain cxuns me shuwn :1s huxcs. An arruw indicates the
`direction of tmnscripliun from the K promutcr. E 11 • heavy ctlain
`enhancer. Nut drawn 1o scale. llll Sch.:n,;uic di~b'Tam or the m(thml
`used 10 excise the V- J region. SD. ~plicc donor sequence: rev.
`primo:r. reverse primer.
`
`mid pSV2gpt C28) containing the amp :md ;:pr genes: an
`ltiOO·bp £mR I-Bg/ ll fragment from pKcatH (29} containing
`the heavy ch;~in cnh~mccr and K promoter; Md a 15()()-bp
`l:'coRl-Xh11 I fragment conlaining the hum:m C. region (30).
`Similarly. pVyl w<~s constructed starting from a 4850-bp
`BamHl-Ec.,Rl rrasment of I he plasmid pSV2hph (a gift of A.
`Smith. A. Miyajimu. and D. Strehlow. Slanford University).
`which is analogous to pSV2gpt except that the J!Pf gene is
`replaced by the l!y~: gene (31). This fragment was combined
`with the EwRI-Bg/11 fragment rrom pKcatH and a 2800-bp
`llindlll-PI'II II fragmenl containing the human -yl constant
`region. isolated from a phage kindly provided by L. Hood
`02). In each case. the fragments were combined by standard
`methods (ref. 33. pp. 390-401), with an Xbn I linker insened
`between the K promoter rragment and the 5' end of the C
`region fragment.
`Construction or Chimeric Genes. £mRI fragments contain(cid:173)
`ing the anti-Tac Iicht and heavy chain cDNAs were sepa(cid:173)
`rately insened into the £mRI site of the phage M13mpllD,
`a variant or M lJmpll (34) in which the EcoRI and Xba I sites
`of the polylinker were filled in and joined. T he resulting
`phage. in which the 5' ends of the cDNAs abutted the Xba I
`site, were respectively denoted M13Land Ml3H. The V-J (J,
`joining) segments of the cON As, followed by splice donor
`signals. were precisely excised from these phage, using a
`double-priming scheme (Fig. 18). For the light chain •• ~e
`following primer was synthesized (Applied BiosysteJ :s
`model 380B DNA synthesizer) and purified by gel elecuo(cid:173)
`phoresis: 5' -CCAGAA TTCT AG AAAAGTGT ACTT AC·
`GTITCAGCTCCAGCTIGGTCCC-3'. From the 3' end, the
`first 22 residues of the primer arc the same as the last 22
`residues of the J~5 segment (noncoding strand). The next 16
`nucleotides are the same as the sequence that follows JK5 in
`
`mouse genomic DNA and therefore includes a splice donor
`signal. The tinallO nucleotides of the oligonucleotide include
`an Xba I site.
`We hybridized this oligonucleotide to M13L and extended
`it with the·Kienow fragment of DNA polymerase. The DNA
`was heat-denatured. hybridized with an excess of the "re·
`verse primer" 5'-AACAGCTATGACCATG·3', again ex·
`tended with Klenow DNA polymerase, and digested with
`Xba I. The digested DNA was run on a gel, and an approx(cid:173)
`imately 4~bp fragment was excised and insened into the
`Xba I site of pVKl. Sequencing showed that the fragment
`consisted of the V-J region of the light chain eDNA followed
`by the splice donor "tail," as expected (Fig.lB), and pLTac,
`a clone with the appropriate orientation, was chosen. In an
`analogous fashion. the heavy chain V-J segment, followed by
`the mouse JH2 splice donor sequence, was excised from
`M13Handinsened into theXba I site ofpVyl to yield pGTac.
`Computer Analysis. Sequences were manipulated and ho·
`mology searches were performed with the MicroOenie Se·
`quence Analysis Software (Beckman). The molecular model
`of the anti-Tac V region was constructed with the ENCAD
`program (35) and examined with I he MIDAS program (36) on
`an IRIS 4D-120 graphics workstation (Silicon Graphics).
`Construction of Genes for Humaniud Antibody. Nucleolide
`sequences were selected that encoded the protein sequences
`of the humanited light and heavy chain V regions including
`signal peptide~ (Result.v), generally utilizing codons found In
`the mouse anti·Tac sequence. These nucleotide sequences
`also included the same splice donor signals used in the
`chimeric genes and an Xba I site at each end. For the heavy
`chain V region. four overlapping 120- to 130-nucleotide-long
`oligonucleotides were synthesized that encompassed the
`entire sequence on alternating strands. The oligonucleotides
`were phosphorylated with polynucleotide kinase, annealed,
`extended with T4 DNA polymerase. c•Jt with Xba 1, and
`ligated into the Xba I site of pUCl~ (34). using standard
`reaction conditions. An insert with the correct sequence was
`recloned in pVy1. The humanized light chain V region was
`construc1etl similarly.
`Transfections. For each antibody constructed, the light
`chain plasmid was first transfected into Sp2/0 mouse my·
`eloma cells (A TIC CRL 15811 by electroporation (Bio-Rad
`Gene Pulser) and cells were selected for RPI eKpression (28).
`Clone$ secreting a maximal amount or light chain. as deter·
`mined by ELISA. were transfected with the heavy chain
`plasmid and cells were selected for hygromycin B resistance
`(31). Clones secreting a maximal amount of complete anti·
`body were detected by ELISA. The clones were used for
`preparation of chimeric and humanized antibodies.
`Antibody Purlfic:ation. Medium from confluent cells was
`passed over a column of staphylococcal protein A-Sepharose
`CL-48 (Pharmacia), and antibody was eluted with 3 M
`MgCJ2• Antibody was further purified by ion-exchange chro·
`matography on BakerBond ABx (1. T. Baker). Final anti·
`body concentration was determined. assuming that 1 mg/ml
`has an A~ll3 of 1.4. Anti-Tac antibody itself was purified as
`described (2).
`Affinity Measurements. Affinities were determined by com·
`petition binding. HuT-102 human T·lymphoma cells (ATIC
`TIB 162) were used as source or p55 Tac antigen. Increasing
`amounts of competitor antibody (anti-Tac, chimeric, or hu·
`manized) were added to 1.5 ng of radioiodinated (Pierce
`l odo-Beads) tracer anti-Tac antibody (2 1-~Ci/flg; 1 Ci = 37
`GBq) and incubated with 4 x 10$ HuT cells in 0.2 ml of
`binding butTer (RPMI1040 medium with 10% fetal calf serum,
`human lgG at 100 ,ug/ml, 0.1% sodium azide) for 3 hrat room
`temperature. Cells were washed and pelleled, and their
`radioactivities were measured, and the concentrations of
`bound and free tracer antibody were calculated. The affinitY
`of mouse anti-Tac was determined by Scatchard plot analy·
`
`Board Assigned Page #98l.l
`
`BIOEPIS EX. 1595
`Page 1133
`
`
`
`I
`Qr
`ft
`
`!d
`A
`·e·
`X·
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`
`Immunology: Queen et al.
`
`Proc. Nat/. Mad. Sci. USA 86 ( 1989)
`
`10031
`
`sis, using antt-Jac itself as the competitor. Then the affinities
`of the clllmeric and humanized antibodies were each calcu(cid:173)
`(anti-Tac] = (1/Kx) (cid:173)
`lated according to the fonnula (XI -
`(1/ K.), where Ka is the affinity of anti-Tac (9 x 109 M-1), K.
`is the affinity of the competitor X, [ ) indicates the concen(cid:173)
`tration of competitor antibody at which bound/free tracer
`binding is Ro/2, and R0 is maximal bound/free tracer binding
`(37).
`
`RESULTS
`Cloning or Light and Heavy Chain eDNA. A eDNA library
`in AgtlO was prepared from anti-Tac hybridoma ceUs and
`screened with oligonucleotide probes for the mouse K and y2a
`constant regions. The eDNA inserts from four K-positive and
`four y2a-positive phage were subcloned in Ml3mp19. Partial
`s~quencing showed that two of the K isolates had one
`sequence, and the other two had another sequence. In one
`pair, a V ~gene segment was joined to the J.2 segment out of
`its reading frame. In addition, the conserved cysteine at
`position 23 was absent from this V segment, and the se·
`quences of the two isolates differed slightly. Presumably,
`these clones were the result of an aberrant joining event in
`one K allele, which continued to undergo somatic mutation
`after the formation of the hybridoma.
`The V-J segments of the other pair of K clones were
`sequenced completely and were identical. This light chain
`uses the J.5 segment. Partial sequencing of the four y2a
`clones showed they were all from the same gene. The V-J
`segments of two were sequenced completely and were iden(cid:173)
`tical. This heavy chain uses the JH2 segment and is of
`subgroup II (38). The DNA sequences have been deposited
`with GenBank; II the deduced protein sequences are shown in
`Fig. 2. As both alleles of the K light chain were accounted for
`and only one heavy chain sequence was detected, we tenta·
`tively assigned these sequences to the anti· Tac antibody
`genes.
`Construction of Chimeric Genes. Plasmid vectors were
`prepared for the construction and expression of chimeric light
`and heavy chain genes. The plasmid pVKl (Fig. lA) contains
`the human genomic C. segment. including 336 bp of the
`preceding intron and the poly(A) signal. It also contains the
`promoter sequence from the MOPC 41 K gene and the heavy
`chain enhancer sequence, which synergize to form a very
`strong transcriptional unit (29). There is a unique Xba I site
`between the promoter and the intron. A similar plasmid,
`pVyl, was prepared by using the human C.,l region in place
`of the c .. region. In that case, the region inserted between the
`Xba I and BamHl sites extended from about 210 bp 5' ofthe
`CHl exon to beyond the CH3 exon.
`Our strategy was to insert the V-J region from the anti-Tac
`K eDNA, followed by a splice donor signal, at the Xba I site
`
`v ..
`
`of p V ocl to ·construct the plasmid pL Tac. Doing sc created a
`chimeric K gene with a short synthetic intron between the
`mouse V-J and human c. segments (Fig. lA). For this
`purpose, we used a form o f double primer-directed mutagen·
`esis (Materials and Methods; Fig. 18). Similarly, the V- J
`region from the anti-Tac y2a heavy chain eDNA, followed by
`a splice donor signal. was inserted into theXba I siteofpVyl.
`The resulting plasmid, pGTac, contained a chimeric heavy
`chain gene, with a synthetic intron between the mouse V-J
`and human Cyl segments.
`Construclion oC a Humanized Anll-Tac Antibody. [n select·
`ing a human antibody to provide the variable region frame(cid:173)
`work for the humanized anti-Tac an tibv:ty, we reasoned that
`the·ow.t.b.o1I_ipfogQIJ~J!l£.bPIJl3n antibody".\\-as to the origin-al'
`~tr~TaG:"antibo_fJY.~t~~.:tesslikel'i:"ffould,W.mbin.ing"the anti·'
`:rac-:toR:s:V:itll':tlie:human framework·be ·to introduce dis-:·'
`toi1ions.Jn(o 'the CDI~s·. The anti· Tac heavy chain sequence
`was therefore compared by computer with all the human
`heavy chain sequences in the National Biomedical Research
`Foundation Protein ldentification Resource (release 15). The
`heavy chain V region of the Eu antibody (of human heavy
`·chain subgroup I; ref. 38) was 57% identical to the anti-Tac
`heavy chain V region (Fig. 2B); all other complete VH regions
`in the data bank were 30-52% identical. However. no one
`human light chain V region was especially homologous to the
`anti·Tac light chain. We therefore chose to use the Eu Iighi
`chain (of human light chain subgroup I; ref. 38) together with
`tlie Eu heavy chain to supply the framework sequences for
`the humanized a nti body. The CDRs in the humanized anti·
`body were of course chosen to be identical to the anti· Tac
`CDRs (Fig. 2).
`A computer program was used to construct a plausible
`molecular model of the anti·Tac V domain (fig. 3). based on
`homology to other antibody V domains with known crystal
`structure and on energy minimization. Graphic manipulation
`shows that a number of amino acid residues outside of the
`CDRs are in fact close enough to them to either innuence
`their conformation or interact directly with antigen. When
`these residues differ between the anti· Tac and Eu antibodies,
`the residue in the humanized antibody was chosen to be the
`anti· Tac residue rather than the Eu residue. This choice was
`made for residues 27, 30, 48, 67, 68, 98, and 106 in the
`humanized heavy chain, and for 47 and 59 in the humanized
`light chain (Figs. 2 and 3; amino acids shown in blue in Fig.
`3), although we now consider the tight chain residue 59.
`which was chosen on the basis of an earlier model, to be
`doubtful. In this way, we hoped to better preserve the precise
`structure of the CDRs at the cost of possibly making the
`humanized antibody slightly less "human."
`Different human light or heavy chain V regions exhibit
`strong amino acid homology outside of the CDRs. within the
`framework regions. However, a given V region will usually
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`FIG. 2. Amino acid sequences ofthc humanized
`aoti·Tac light (A) and heavy (B) chains. The se·
`queoces of the Eu antibody light and heavy chains
`(upper lines) are shown aligned above lhe mouse
`anti-Tac light and heavy chain sequences (lower
`lines). with a I indicating identity of amino acids.
`The three CDRs in elteh chain are underlined, and
`the other mouse amino acids used in the humanized
`..!.f!libi?'IY.l!,I'C doub.le underlined. Hern:e, the human·
`ized sequences are the same as the upper (Eu)
`sequences. except where the amino acid is under(cid:173)
`lined or double underlined.
`
`Board Assigned Page #9/j 1
`
`BIOEPIS EX. 1595
`Page 1134
`
`
`
`10032
`
`Immunology: Queen et al.
`
`Proc. Natl. A cad. Sci. USA 86 ( /989)
`
`FtG. 3. Model oft he mo~se anti-Tac untibody V region. generated with the ENCIID program and displayed with the MIDAS program. Amino
`acids in the CDRs are shown in red; amino acids poh:ntially interJcting with the CDRs are shown in blue; other mouse amino acids used in the
`humanized antibody are shown in yellow. as described in the text. Thus. all amino acids transferred It om the anti-Tac sequence to lhe humanized
`antibody ar~ shown in red, blue, or yellow. Re~idue I i~ lhe lir~t amino acid of V .; residue 301 is the first amino acid of VH.
`
`contain exceptional amino acid~. alypical of other human V
`regions. al sever-.11 framework positions. The Eu antibody
`contains such unusual residues at positions corresponding to
`93. 95. 98. 106. 107. 108. and llO of the humanized heavy
`chain and 47 and 62 of the light ch;tin (Fig. 2). as determined
`by visual compari!;on or. the Eu heavy and light chain V
`n:gions with other human V rcl!ions of subgroup I (38). The
`Eu antibody contains sc:vero~l other unusual residues. but at
`the listed positions,the murine anti·Tac antibody actually has
`a residue much more typical of human sequences than does
`Eu. At these positi~ns. we therefore chose to use the anti-Tac
`residue rather than the Eu residue in lhc: humanized antibody,
`to make the antibody more generically human. Some of these
`residues had already been selected because of their proximity
`to the CDRs. as described above (the remaining ones are
`shown in yellow in Fig. 3).
`These criteria allowed the selection of all amino adds in the
`humanized antibody V regions as coming from either anti-Tac
`or Eu (Fig. 2). DNA segl)'lents encoding the desired heavy
`and light chain amino acid sequences were synthesized.
`These DNA segments also encoded lypical imr.->unoglobulin
`s ignal sequences for processing and secretion, and they
`contained splice donor signals at their 3' end. The light and
`heavy chain segments were cloned, respectively. in p V K1 and
`pV')'1 to form the plasmid~ pHuLTac and pHuGTac.
`PToperlies or Chimeric lpld Humanized Antibodies. Sp2/0
`cells, a nonproducing mou,se myeloma line, were transfected
`sequentially with pLTac and pGTac (chimeric genes) or with
`pHuL Tac and pHuGTac ·(humanized genes). Cell clones
`were selected first for a ntibiotic resistance and then for
`maximal antibody secretion, which reached 3 p.g/1f/' cells per
`24 hr. Sl nuclease mapping of RNA extracted from the cells
`lransfected with pLTac ana pGTac showed that the synthetic
`introns between the V and C regions (Fig. lA) were correctly
`spliced (data not shown). Antibody was purified from the
`
`culture medium of cells producing the chimeric or humanized
`antibody. When analyzed by reducing SDS/ palyacrylamide
`gel electrophoresis, the antibodies showed only two bands,
`having the expected molecular weights 50.000 and 25.000.
`Flow cytometry showed that the chimeric and humanized
`antibodies bound to Hut-102 and CRII.2 cells. two human
`T-celllincs lhat express the p55 chain of the IL-2R. but not
`to CEM and other cell lines that do not express the IL-2R. To
`determine the binding affinity of the chimeric and humanized
`antibodies. their ability to compete with labeled mouse
`anti-Tac for binding to Hut-102 cells was determined. The
`affinity of chimeric anti-Tac was indistinguishable from that
`or anti-Tac (data not shown), as expected from the fact that
`their entire V regions are identical. The affinity of humanized
`anti-Tac for membrane-bound p55 was 3 x 109 M -t, about
`1/3 the measured aflinity of 9 x 109 M- 1 of anti·Tac itself
`(Fig. 4).
`
`DISCUSSION
`Because monoclonal antibodies can be produced that are
`highly specific for a wide variety of cellular targets, antibody
`therapy holds great promise for the treatment of cancer,
`autoimmune conditions. and other diseases. However, this
`promise has not been widely realized, largely because most
`monoclonal antibodies, which are of mouse origin, are im- .
`munogenic when used in human patients and are ineffective
`at recruiting human immune effector functions such as CDC
`and ADCC. A panial solution to this problem is the use of
`chimeric antibodies (16), which combine the V region binding
`domains of mouse antibodies with human antibody C regions.
`(nitially. chimeric antibodies were c