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`First Named Inventor
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`John R. Adair et al.
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`BEST AVAILABLE COPY PCT/GB90/0l017
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`HtJKANISED ANTIBODIES
`
`F~eld of the Invention
`The present 1nvention relates ~o h~ised antibody
`molecules, to processes for their production ua1ng
`recombLnant DNA technoloqy, and to theLr therapeutic usee.
`
`The term •hnmaoiaed antibody molecule" ia aaed to describe
`a molecule havinq an antigen binding site derived from an
`~oqlobulin from a non-human species, and remoininq
`~oglobul~-derived parts of the molecule being der1ved
`from a human i=munoglobulin.
`The antigen binding s1te
`typically comprises complementarity determining regions
`(CDRs) which determine the binding specificity of the
`antibody molecule and which are carried on appropriate
`framework regions in the variable domains.
`There are J
`CDRs (CDR!, 'CDR2 and CDR3) in each of the heavy and liqht
`chain variable domains.
`
`In the description, reference is made to a number of
`publications by number.
`The publications are listed in
`numerical order at the end of the description.
`
`Background of the Invention
`Natural jmmunoglohulins have been known for many years, as
`have the various fragments thereof, such as the Fab,
`(Fab')2 and Fe fragments, which can be derived by
`enzymatic cleavage. Natural immunoglobulins comprise a
`generally Y-shaped molecule havinq ·an antiqe.n-bi.ndi.nq site
`towards the end of each upper arm.
`The remainder of the
`structure, and particularly the stem of the Y, mediates
`the effector functions associated with immunoqlobulins.
`
`Natural immunoqlobulins have been us.ed in assay, diaqnosis
`and, to a more limited extent, therapy.
`However, such
`uses, especially in therapy, were hindered until recently
`by the polyclonal nature of natural immunoglobulins.
`A
`significant step towards the realisation of the potential
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`W091/09967
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`PCr/GB90/02017
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`of t D"'nmoqlohu.U.na aa therapeutic agenta waa the ~covery
`of proce<lu.re.a for the producti.on of monoclonal. antibodies
`(~a) of defined apecifici.ty (1).
`
`HoWever, moat MAbs are produced by hybridomaa whi.ch are
`fasiona of rodent spleen cella with rodent myeloma
`cella.
`They are therefore essentia11y rodent proteins.
`There are very f~ reports of the production of human MAba.
`
`Since most available HAbs are of rodent oriq:i.n, they are
`naturally antigenic i.n humans and tbua can gi.ve rise to an
`undesirable i mmnne response termed the BAMA (Duman
`Anti-Mouse Antibody) response.
`Therefore, the use of
`rodent MAbs as therapeutic agents in humans is inherently
`l~ted by the fact that the human subject will mount an
`Lmmunological response to the MAb and will either remove
`it entirely or at least reduce its effectiveness.
`Xn
`practice, M.Abs of rodent origin may not be used in
`patients for more than one or a few treatments as a BAMA
`response soon develops rendering the MAb ineffective as
`well as giving rise to· undesirable reactions.
`For
`instance, OKT3 a mouse IgG2a/k MAb which recognises an
`antigen in the T-cell receptor-CD3 complex has been
`.. .
`approved for use in many co~ntries· throughout the worrd ·
`as an immunosuppressant in the treat=ent of acute
`allograft rejection (Chatenoud et al (2) and Jeffe~s et al
`However, i.n view of the rodent nature of thi.s and
`( 3) ] •
`other such KAbs, a significant BAMA response which may
`include a majo.r anti-idiotype component, may build up on
`uae.
`c~~arly, it would be highly desirable to djmjn;sh
`or abolish this undesirable BAHA response and thus enlarge
`the areas of use of these very useful antibodies.
`
`Proposals have therefore been made to render non-human
`MAbs lesa antigenic in hWDAns.
`Such techniques can be
`geiJerieally termed •hnman.i sation• techniques.
`These
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`technJ.quea typical.ly .involve the use of recombinant DNA
`technology to manipulate DNA sequences encoding the
`polypeptide chaine of the antibody molecule.
`
`Early methods for hnmani sing HAba involved production of
`chimeric antibodies in which an antigen bindinq site
`comprising the complete variable domains of one antibody
`is linked to constant domains derived from another
`antLbody. Methods for carry~g out such chimerisat~on
`procedures are described in BP0120694 (Celltech L~ted),
`EP012502l (Genentech me. and City of Hope), BP-A-0 171496
`(Res. Dev. Corp. Japan), EP-A-0 173 494 (Stanford
`University), and WO 86/01533 (Celltech Limited) •. This
`latter Celltech application (WO 86/01533) ~scloses a
`process for preparinq an antibody molecule havinq the
`variable domains from a mouse MAb and the constant domains
`from a human immunoglobulin.
`Such humaniaed chimeric
`antibodies, however, still ·c~ntain a siqnificant
`proportion of non-human amino acid sequence, i.e. the
`complete non-human variable domains, and thus may still
`elicit some RAMA response, particularly if administered
`over a prolonged period (Bagent at al (ref. 4)].
`
`In an alternative approach, described in EP-A-0239400
`(Winter), the complementarity determining regions (CDRs)
`of a mouse MAb have been grafted onto the framework
`regions of the variable domain• o£ a human L=munoglobulin
`by site directed mutagenesis usLng long oligonucleotides.
`The present invention relates to humanised antibody
`molecules prepared according to this alternative approach,
`i.e. CDR-grafted humariised aptibody molecules.
`Such
`CDR-grafted humanised antibodies are much less likely to
`give rise to a HAMA response than humanised chimeri~
`antibodies in view of the much loWer proportion of
`non-human amino acid sequence which they contain •
`
`. '
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`The earliest work on bnment si.nq NAbs by CDR-qra£ti.ng was
`carried out on HAbs reeoqni.si.nq synthetJ.c antigens, such
`as the NP or Nl:P antigens .
`However, ex•mples J.n whi.cb a
`mouse HAb recoqnisi.nq lysozyme and a rat ~ recoqnisi.ng
`an antigen on huma.a T-cells were httman~sed by CDR-qra£tJ.ng
`have been described by Verhoeyen et al ( 5) and Riechmenn
`et al (6) respectively.
`The preparation of CDR-grafted
`antibody to the antigen on bwaa.a 'f' cell.s is also described
`in WO 89/07452 (Me~al Research CouncL!).
`
`rn RJ.ecbmana et ~/Medic&! Research Council it was found
`that transfer of the CDR regions alone (as defi.ned by
`Kabat refs. (7) and (8)} was not sufficient to provide
`satisfactory antigen bi.nding activi.ty in the CDR-qrafted
`product.
`Riecbm•nn et al found that it was necessary to
`convert a serine residue at position 27 of the human
`sequence to the corresponding rat phenylalanine residue to
`obtain a CDR-grafted product havinq improved antiqen
`binding activity.
`This residue at position 27 of the
`heavy chain is within the structur&l loop adjacent to
`CDR!.
`A further construct which additionally contained a
`human serine to rat tyrosine chanqe at position 30 of the
`heavy chain did not have a significantly altered binding
`activity over the hnmanised antibody witb the ser.ine to
`phenylalanine change at position 27 alone.
`These results
`indicate that changes t .o residues of the human sequence
`outside the CDR regions, in particular in the structural
`l.oop adjacent to CDRl, may be necessary to obtain
`effective antigen bindinq activity for CDR-grafted
`Even
`antibodies which recognise more complex antigens .
`ao . tbe b;nd1ng affinity of the .best CDR-grafted antibodies
`obtained was still. aiqnifi.cantly leas than the original
`MAb.
`
`Very recently Queen et al (9) have described the
`preparation of a hnmaniaed antibody that binds to the
`
`\
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`J.nter~aulc.in 2 receptor, by combLn.i.ng tha CDRa of a marine
`MAb ( &.llti-Tac) with hWDAD
`i mmgaoglobul.in framework and
`constant regions.
`The -human framework regions were
`chosen to maximise h~logy with the anti-Tac HAb
`sequence.
`In addition computer model~ing was used to
`identify framework amino acid residues which ware lLkely
`to interact with the CDRa or antigen, and mouse amino
`acids ware used at these positions in the hmaaaiaed
`Alltihody.
`
`In WO 90/07861 Quean at al propose four criteria for
`desiqning humaniaed immunoqlobulins.
`The first criterion
`is to use as the human acceptor the framework from a
`particu~ar .human Lmmunoqlobulin that is unusu~ly
`·homologous to the non-human · donor immunoglobulin to be
`humanised, or to use a consensus framework from many human
`antibodies.
`The second criterion is to uae the donor
`amino acid rather than the acceptor if the human acceptor
`r .esidue is unusual. and the donor residue is typical. for
`human sequences at a specific residue of the framework .
`The third criterion ia to use the donor framework amLno
`acid residue rather than the acceptor at positions
`immediately adjacent to the CDRs.
`The fourth criterion
`is to use the donor amino acid residua at framework
`positions at whl.ch the a.m.lno acid is .predicted to. have a ·
`si.de chai.n atom withi.n about 3 A of the CORa in a'
`three-d.imenaionu immunoglobulin model and to be . capab~e
`of interacting with the antigen or with the CDRs of the
`bnmanj sed .immunoqlobulin.
`It is proposed that criteria
`two, three or four may be applied in addition or
`alternati.vely to criterion one·, and may be appli.ed singly
`,or in any combination.
`
`WO 90/078·61 describes i.n detai.~ the preparati.on of a
`sinqle CDR-grafted humaniaad antibody, a humanised
`antibody havinq specificity for the p55 Tac proteLn of the
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`PCT/CB90/0l017
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`The c:ambina.ti.on of al.1 four criteri..a, as
`IL-2 receptor.
`above, were employed J.n desiqn.i.Dq th.ia humani sed a..uti.bociy,
`the variable reqion frameworks of the human antibody Eu
`(7) be.inq used 48 ac:c:e~tptor.
`l:n the resultant hnmeniae<l
`antibody the donor CD~ a were as defined by Kabat et al ( 7
`and 8) and .in add.ition the mouse donor residues were used
`in place of the human acceptor re~idues, at position• 27,
`30, 4a, 66, 67, 89, 91, 94, 103, 104, lOS and 101 in the
`heavy cha..i.n and at positions 48, 60 and 63 in the liqht
`cha.i.n, of the variable reqion frameworbl.
`The hu.m.&ni.sed
`anti-'l'ac antibody obtained i.a reported to have an affJ..ni.ty
`for pSS of 3 x 109 Mll, about one-third of that of the
`murine HAb.
`
`We have further i.nvesti.qated the preparation of CDR(cid:173)
`grafted humani.sed antibody molecules and have identified a
`Mera.rchy of positions wi.thi.n the framework of the
`variable reqions (i.e. outside both th~ Kabat CORa and
`st~ctural loops of the variable reqi.ons) at whlch the
`ami.no acid identities of the ·residues are important for
`obtaining CDR-grafted products with satisfactory bindinq
`affi.n.ity.
`This ha.s enabled us to establish a protocol
`for obtaininq satisfactory CDR-qrafted products which may
`be applied very widely .irrespective of the l.evel of
`homoloqy between the donor immunoqlobu1.in and acceptor
`framework.
`The set of residues which we have identified
`as beinq of critical importance does not coincide with the
`residues identified by Queen et al. (9).
`
`Snmmacy of the Invention
`Accordingl.y, in a first aspect the invention provides a
`CDR-grafted antibody heavy chain havinq a variabl.e reqion
`domain comprising acceptor framework and donor antiqen
`bindinq regions wherein the framework comprises donor
`residues at at least one of positions 6, 23 and/or 24, 48
`and/or 49, 71 and/or 73, 75 and/or 76 and/or 78 and 88 and/
`or- 91.
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`ID pre£erred embcKU menta 1 the heavy C~ £ram.ework
`campr1aea donor res1dnes at poa1tiona 23, 24, 49, 71, 73
`and 78 or at pos.itionli 23, 24 and 49.
`The residues at
`positions 71, 73 and 78 of tbe heavy chain framework are
`preferably either all acceptor or all donor residues.
`
`In particularly pre£erred embodiments the heavy ch~
`framework additionally compr1aea d~nor residue• at one,
`soma or all of positions 6, 37, 48 and gc·. Also it .is
`particularly preferred that residnea at positions of the
`heavy chain framework wh.ic~ are commonly conserved across
`species, i.e. positions 2, 4, 25, 36, 39, 47, 93, 103,
`104, 106 and 107, if not conserved between donor and
`acceptor, additionally comprise donor reaidues. Moat
`preferably the heavy chain framework additionally
`comprises donor residues at positions 2, 4, 6, 25, 36, 37,
`39, 47, 48, 93, 94, 103, 104, 106 and 107.
`
`In addition the heavy chain fr&JD.ework optionally comprises
`donor residues at one, some or all of posit ions:
`1 and 3,
`72 and 76,
`69 (if 48 is different between donor and acceptor),
`•' 38 and 46 (if 48 is the donor residue),
`. 80 and 20 {if 69 is the donor residue),
`67,
`82 and 18 (if 67 is the donor residue),
`91,
`88, and
`any one or more of 9, 11, 41, 87, 108, 110 and 112.
`
`In the firat and other aspects of the present invention
`reference is made to CDR-grafted antLbody products
`comprising acceptor framework and donor antiqe~ binding
`regions.
`It will be appreciated that the invention is
`widely applicable to the CDR-qraftinq of antibodies in
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`geueral..
`Thus, the donor and acceptor an~ea may be
`derived £rom antmala of the same species and eveu same
`anti.bod.y cl.aas or . aub-claaa. More uaua.ll.y, however, the
`donor and acceptor an~~·· are derived from anim•ls o£
`different species. Typically the donor antLbody ia a
`non-huma..n antibody, such aa a rodent MAb, and the acceptor
`anti.body J.a a hum&D antibody.
`
`In the first ADd other aspects of the present invention,
`the donor antigen binding region typical.l.y comprises at
`least one CDR from the donor antibody. Osual.ly · the donor
`antigen binding reqion comprises at l.eaat two and
`preferabl.y all three CDRs of each of the heavy chain
`and/or light chaLn variable regions.
`The CORa may
`comprise the Kabat CDR•, the structural. ·loop CORa or a
`composite of the Kabat and structural. loop CDRs and any
`combination of an~ of these.
`Preferabl.y, the antigen
`binding regions of the CDR-grafted heavy chain variable
`domain comprise CORa corresponding to the Kabat CORa at
`COR2 (residues 50-65) ~d CORJ (residues 95-100) and a
`composite of the Kabat and structural. l.oop CORa at COR!
`(residues 26-35).
`
`The residue designations given above. and el.setoi'here .in ~he
`present appl.i.cation are numbered according to the Kabat
`nu.mberi.nq [refs. ( 7) and ( 8 J ] •
`Thus the residue · ·.
`designations do not al.waya correspond di.rectl.y with the
`l.inear numbering of the amino acid residues.
`The actual
`linear amLno acid sequence may contain fewer or additional
`a.mizlo acids than i.n the strict Kabat numbering
`· corresponding to a shortening of, or insertion into, a
`structural component, whether framework or CDR, of the
`~aaic variable domain · stru~ture.
`For exampl.e, the· heavy
`ch~ variabl.e region of the anti-Tac antLbody described
`by Queen et al. (9) contains a singl.e amino acid insert
`(·residue 52a) after residue 52 of CDR2 and a three amino
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`acid insert (residues 82a, 82b and 82c) uter framework
`residue 82, .in the Kabat numberinq.
`The correct Kabat
`numberLng of resLdues may be determ1ned for a gLven
`antibody by al.J.qumant :at regions of homology of the
`sequence of the antibody with a •standard• Kabat numbered
`sequence.
`
`The LllventJ.on also provLdes in a second aspect a CDR(cid:173)
`grafted antibody light chain having a variable region
`domain comprising acceptor framework and donor antigen
`binding regions wherein the framework comprises donor
`residues at at least one of positions l and/or 3 . and 46
`and/or 47.
`Preferably the CDR grafted light - ~hain of the
`second aspect comprises donor residues at positJ.ons 46
`and/or 47.
`
`The invention also provJ.des in a thLrd aspect a
`CDR-grafted antibody light chain havLilg a variable region
`domain camprising acceptor framewark and donor antigen
`bLnding reqions wherein· the frame~ork comprises donor
`residues at at least one of positions 46, 48, 58 and 71.
`
`In a preferred embodLment of the third aspect, the
`framework comprises donor residues at all of positions 46,
`48, 58 and 71.
`
`In particularly preferred embodiments of the second and
`third aspects, the framework additionally comprises donor
`residuaa at positions 36, 44, 47, 85 and 87.
`S~larly
`positions of the 1ight chain framework which are commonly
`conserved across species, i.e. positions 2, 4, 6, 35, 49,
`62, 64-69, 98, 99, 101 and 102, if ·not conserved between
`donor and acceptor, additionally comprise donor residues.
`Most preferably ·the light chain framework a~tionaLly
`comprises donor residues at positions 2, 4, -6, 35, 36, 3a,
`4 4, 4 1 , 4 9 , 6 2 , 6.4-6 9 , a 5 , a 1 , 9 8, 9 9 , 1 o 1 and 1 o 2 •
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`Xu a~tion the f~amework of the eeeoDd or third aspects
`opti.ona11y comprises donor reai.dues at one, same or ~ of
`poai.tJ.ons a
`1 and 3,
`63,
`60 (i£ 60 and 5& are able to foz:m at potential. sal.thridqe},
`70 (i£ 70 and 2& are able to form a potential saltbri.dqe),
`73 and 21 (i.f 47 i.e different between donor and acceptor),
`37 and .45 (i.f 47 ia di.fferent between donor and acceptor),
`and
`any one or more of 10, 12, 40, 80, 103 and 105.
`
`Preferably, the antigen bindi.nq reqi.ona of the CDR-qrafted
`light chain variable domain comprise CDRa corresponding to
`the Kabat CDRs at CORl (residue 24-34), CDR2 (residues
`S0-56} and CDR3 (residues. 89-97).
`
`The invention furthe~ provides in a fourth aspect a
`CDR-grafted antibody molecule comprising at least one
`CDR-grafted heavy cha~ and at least one CDR-grafted light
`chain · accordinq to the first and second or first and third
`aspects of the invention.
`
`The humanised ant~ody molecules and chains of the present
`invent.ion may comprise:
`a c.omplete ant_Lbody molecul~,
`having full lenqth heavy and light chains1
`a fragment
`thereof, such aa a Fah, ( Fab .' ) 2 or FV fragment i
`a l.ight
`chai.n or heavy chain monomer or. daeri or a single chain
`antibody, e.g. a single chain FV in which heavy and light
`chain variable regions are joined by a peptide lLnker; or
`any other COR-grafted molecule w.ith the same specificity
`as the original donor antibody.
`S1milarly the
`CDR-grafted heavy and light chain variable region may be
`combined with other antibody_ domajns as appropriate.
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`Also the heavy or light chai.ns or human; sed aritJ.body
`molecules o~ the present invention may have attached to
`them an effector or reporter mole¢ule.
`For instance, it
`may have a macrQCYcle( for chelating a heavy metal atom,
`or a toxtD, such as ricin, attached to it by a covalent
`bridging structure. . Alternatively, the procedures o£
`recombinant DHA technology may be used to produce an
`fmmJnoglobulin molecule in which the Fe fragment or CBJ
`domaLa of a complete Lmmunoglobulin molecule. has been
`replaced by, or baa attached thereto by peptide linkage, a
`functional non-Lmmnnoglobulin protein, such as an enzyme
`or toxin molecule.
`
`Any appropriate acceptor variable region framework
`sequences may be u,ed havinq regard to class/type of the
`donor antibody from which the antiqen binding regions are
`derived.
`Preferably, the type of acceptor framework used
`is of the same/similar class/type as the donor antibody.
`Conveniently, the framework may be chosen to maximise/
`optJ.mise homology with the douor . antibody sequence.
`particularly at positions close or adjacen.t to ·the CORa.
`However, a hiqh level of homoloqy between donor · and
`acceptor sequences is not important for application of the
`prese~t invention.
`The present invention identifies a
`hierarchy of framework residue positions at which donor
`residues may be important or des~ab~e for obtaining a
`CDR-grafted antibody product having satisfactory binding
`properties.
`The CDR-grafted products usual~y have
`binding affinities of at least to5 Hrl, preferably at
`least about 10a K-1, or especially in the ranqe · to8-tol2
`In. principle, the present invention is applicable
`Mrl.
`to any combination of donor and acceptor antibodies
`i.rrespecti ve of ·the level of homology between the·ir
`.
`.
`sequences.
`A protocol for applying the invention to any
`particular donor-acceptor antibody pair is given
`hereinafter.
`Examples of. human frameworklf wh.ich may be
`
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`used are KOL, HmiH, RBX, BtJ, .LA:f and POH (ref a. 4 and S)
`and the ~ike J
`for i.nsta.nee KOL and NBWH for the heavy
`cha.in a.Jid RBI for the 1ight chain and BU, LA% and POH for
`· both the heavy chain and the 1iqht c~n.
`
`Also the constant raqion domains of the products of the
`iDvention may be selected having regard to the proposed
`:function of the antlllody in parti~u1ar the effector
`functions whi.ch may be required.
`Por exampl.e, the
`constant reg~on dom•tns may be human IgA, IgB, IqG or IgM
`domajna.
`In p~cular, IgG human constant region .
`doma.Ula may be used, . eapeciall.y of the IgGl and IgG3
`i.sotypes, when the hnmanised an~ody molecu1e is i.ntended
`for therapeutic uses, and ant~ody effector functi.ons are
`requi.red. . JUternati.vely, IgG2 and IqG4 iaotypea may be
`.used when the humani.sed antibody mol.ecul.e is intended for
`therapeuti.c purposes and antibody effector functions are
`not required , e.g. for aimpl.e bl.ocki.ng of l.ymphokine
`actJ.vity.
`
`However, the remainder of the antibody molecu1ea need not
`compri.se only protei.n sequences from i.mmunoglobu~ins .
`For i.natance , a gene. may be constructed . .in whJ.cb ·a DNA
`sequence encoding part of a human i.mmunoqlobul.Ln cbaJ.n i.s
`fused to a DNA sequence encodLng the amino acid sequence
`of a functional pol.ypeptJ.de such as an e ·ffector or
`. reporter molecul.e.
`
`Preferably the CDR~grafted antibody heavy and l.i.ght" chain
`and antibody molecule products axe produced by recombi.nant
`DNA tecbnol.ogy.
`
`Thus in further aspects the inventJ.on al.so includes DNA
`sequences cocU.ng for the CDR-grafted heavy and ligb~
`chaJ.ns, cl.oni.ng and expression vectors contai.ning the DNA
`sequences , host cells transformed with the DNA sequences
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`and processes £or producing the CDR-grafted chains and
`antJ.body moJ:ecules comprising expresaU1g the DNA sequences
`.in the transformed boat cella .
`
`The geaera.J. methO<U by wb.icb the vectors may be
`constructed, trana£ec:tioa methods and cuiture methods are
`well known per se and foDil ao part o£ the i.aventioa.
`such
`methods are shown, £or instance, ·in references 10 and 11.
`
`The DNA sequences which encode the donor amino acid
`sequence may be obtained by methods wall known U1 the
`art .
`For example the donor coding sequences may be
`obtained by qenomic cloninq, or eDNA cloninq fr~ suitable
`hybridoma cell lines. Positive clones may be screened
`using appropriate probes for the heavy and light chain
`genes in question .
`Also PCR clon.inq may be used.
`
`DNA coding for acceptor, e.g. human acceptor, sequences
`may be obtained in any appropriate way.
`For example DNA
`sequences coding for preferred human acceptor frameworks
`such a s KOL,. REI, EO and NEWH, are widely available to
`workers .in the art.
`
`The standard techniques of molecular biology may be used
`to ·prepare DNA sequences codi.riq for the CDR-grafted
`prod~cta. Deai.red DNA sequences may be synthesised
`completely or in part using oligonucleotide synthesis
`techniques.
`Site-directed mutagenesis and polymerase
`chain reaction (PCR) techniques may be used as
`For example oligonucleotide directed
`appropriate .
`synthesis as described by · Jones et a.l (ref. 20) may be
`used. Aiao oliqonncleotide directed mutagenesis o-f a.
`pre-ex..ising variable reqion a.s, for example, described by
`Verhoeyen et al (ref. S) or Riechmann et al (ref. 6) may
`be used. Also enzymatic filling in of gapped
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`ol..iqonuc~eoti.des using 'r4 DNA po~yme.rase aa, for ex•m:p~e,
`described by Queen et a~ (ref. 9) may be used.
`
`Any suitable boat cell/vector system may be used for
`expressU,n of the DNA sequences codinq for the CDR-qra£ted
`heavy and ~.iqht chaha.
`Bacter.ia~ e.q. B. coli., and
`other ~robLal ayat._. may be used, in particu1ar tor
`expreaa.ion of ant~y fragments such as FAb and ( Pab' ) 2
`fraqmenta, and eapecia~y PV fragments and sinqle chain
`anti.body fraqments e.g. sinq~e chain PVa.
`Eucaryoti.c
`e.q. mamm•l.ian host cell express.ion systems may be used ·
`for productJ.on of larger CDR-grafted antibody products,
`includ.inq complete antibody molecules.
`Suitable
`mammalian boat cella include CBO cella and myeloma or
`hybridoma cell linea.
`
`Thus, in a further aspect the present invention prov.idea a
`process for producing a CDR-grafted antibody product
`com:priainq:
`
`(a) producinq in an expression vector an operon havLng a
`DNA sequence which encodes an antibody heavy chain
`according to the first aspect of the invention1
`
`and/or
`
`(b) producinq in an expression vector an operon having a
`DNA sequence which encodes a complementary antibody
`Light cha.in according to the second or third aspect
`of thci i.Jwent.ion; .
`
`(c)
`
`transfectinq a h~st cell wi~h the or each vector; and
`
`(d) culturinq the transfected cell line to produce the
`CDR-grafted antibody product.
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`~· CDR-grafted product may comprise on1y heavy or light
`cha.i.n derived polypeptide, in whi.ch caee only a heavy
`cha.in or light chain polypept . .ide cod.J.ng sequence is used
`to tranafect tho boat cells.
`Por production of products compris.ing both heavy and light
`chaine, tho call line may be tranefected with two vectors,
`the fi.rat vector may contain &Q operon encoding a light
`chain-derived polypeptide and the aecond vector containing
`an operon encod.i.ng a heavy cba.in-derived polypep.ti.de.
`Preferably, the vect~rs are identical, except in so far as
`tho coding sequences and selectable marker• are concerned,
`eo as to· enQure as far as possible that each polypeptide
`chain i.s equally expressed. A1ternati.vely, a single
`vector may be used, the vector .including the sequences
`e ncoding both light chain- and heavy chain-derived
`polypeptides.
`
`The DNA in the coding sequences for the light and heavy
`chains may comprise eDNA or genomic DNA or both.
`However, it is preferred that the DNA sequence encoding
`the heavy or light chain comprises at least partially,
`genom.ic DNA, preferably a fusion of eDNA and genom.ic DNA.
`
`The present invention i.s applicable to antibodies of any
`appropriate speci.fi.city. Advantageously, however, the
`inventi.on may be applied to the humanisation of non-human
`antibodiee which are used for in vivo therapy· or
`diagnosis.
`Thus the antibodies may be site-specific
`anti.bodiea such as tumour-specific or cell surface(cid:173)
`specific antibodies, suitable for use in in vivo therapy
`Examples of cell
`or diaqnosi.s, e.g. tumour ~g.ing.
`surface-specific antibodies are ant.i-T cell antibodies,
`-such as anti-CD3, and CD4 and adhesion molecules, sdch as
`CRl, ICAH a.nd BLAH.
`The antibodJ.ea may have specificity
`for interleukina (including lymphokinea, growth factQrs
`and stimulating factors), hormones and other biologically
`active compoUnds, and receptors for any of these.
`For
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`example, the ant:ibodi.ea may have specJ.f.ici.ty for any of
`the followi.nqa
`or .IL4, etc. , TRP, GCSP, GMCSP, 2PO, hGH, or i.nsuli.n, etc •
`
`:Inte.rferonao(, p, Y or~, .ILl, :IL2, rr.l,
`
`The the present i.nvantion Alao includes therapeutic and
`diagnostic compositions comprising: the CDR-qrafted
`products of