`

`FIG. 4A
`Light Chain Sequences
`
`1 N901L
`
`2 KOL
`
`3 N901L/KOL
`
`10
`70
`20
`30
`40
`60
`50
`---------+---------+--- ------+---------+ ---------+----- ----+-- -------+
`:DVLMTQTPLSLPVSLGDQASISC RSSQIIIHSDGNTY-LE WFLQKPGQSPKLLIY KVSNRFS GVPDRFSG
`
`:QSVLTQPPSASG-TPGQRVTISC SGTSSNIGS----STVN WYQQLPGMAPKLLIY RDAMRPS GVPDRFSG
`I •
`I
`I
`I
`:QVLMTQTPSSLPVTLGQQASISC RSSQIIIHSDGNTY-LE WFLQKPGQSPKLLIY KVSNRFS GVPDRFSG
`
`seq)
`
`4 KV2F$HUMAN
`[most identical
`5 N901L/KV2F
`[CDR grafted]
`6 KV4B$HUMAN
`[most identical surf)
`7 N901L/KV4B
`[Resurfaced)
`
`:DVVMTQSPLSLPVTLGQPASISC RSSQSLVYSDGNTY-LN WFQORPGQSPRRLIY KVSNRDS GVPDRFSG
`*
`•
`I
`I
`II
`:DVLMTQSPLSLPVTLGQPASISC RSSQIIIHSDGNTY-LE WFQQRPGQSPRLLIY KVSNRFS GVPDRFSG
`
`:DIVMTQSPDSLAVSLGERATINC KSSQSVLYSSNNKNYLA WYQOKPGQPPKLLIY WASTRES GVPDRFSG
`*
`I
`I
`:DVLMTQTPDSLPVSLGDRASISC RSSQIIIHSDGNTY-LE WFLQKPGQSPKLLIY KVSNRFS GVPDRFSG
`[
`L1
`L2
`
`~
`co
`
`1 N901L
`
`2 KOL
`
`3 N901L/KOL
`
`80
`100
`90
`110
`---------+---------+------ ---+------- --+-----
`:SGSGTDFTLMISRVEAEDLGVYYC FQGSH--VPHT FGGGTKLEI-
`
`(SEQ ID NO: 25)
`
`:SKSGASASLAIGGLQSEDETDYYC AAWDVSLNAYV FGTGTKVTVL
`*
`I
`I
`I
`:SGSGTSFTLAISRVEAEDEGVYYC FQGSH--VPHT FGGGTKLEI-
`
`( 44)
`
`(SEQ ID NO: 26)
`
`(104)
`
`(SEQ ID NO: 27)
`
`4 KV2F$HUMAN
`[most identical seq]
`5 N901L/KV2F
`[CDR grafted)
`6 KV4B$HUMAN
`[most identical surf)
`7 N901L/KV4B
`[Resurfaced]
`
`:SGSGTDFTLKISRVEAEDVGVYYC MQGTH--WSWT FGQGTKVEIK.
`•
`I
`I
`:SGSGTDFTLKISRVEAEDVGVYYC FQGSH--VPHT FGGGTKVEI-
`
`( 87)
`
`(SEQ ID NO,: 28)
`
`(101)
`
`(SEQ ID NO: 29)
`
`:SGSGTDFTLTISSLQAEDVAVYYC QOYDT---IPT FGGGTKVEIK
`
`( 71)
`
`(SEQ ID NO: 30)
`
`:SGSGTDFTLMISRVEAEDLGVYYC FQGSH--VPHT FGGGTKLEI-
`L3
`I
`[
`
`(109)
`
`(SEQ ID NO: 31)
`
`PFIZER EX. 1502
`Page 502
`
`

`

`

`

`EP 0 592 108 A1
`EP 0 592 106 A1
`
`y
`
`z
`
`X
`
`
`
`FIG. 5
`
`221
`
`PFIZER EX. 1502
`
`Page 504
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`PFIZER EX. 1502
`Page 504
`
`

`

`

`

`EP 0 592106 A1
`
`Knowleclge base
`1. Brookhaven databank
`l. Antibody sequence database
`3. Antibody structure database
`4. Loop database
`I
`
`I Analysis I
`I
`I Framework
`I construction
`
`Modelling
`protocol for
`each CDR
`
`I
`
`Database
`construction
`
`I
`
`I
`
`Database &
`ob-initio
`construction
`I
`
`I
`
`I
`
`ob-inilio
`construction
`
`canonical
`coustructioo
`
`I
`
`I
`
`Ana!vsis and validation
`1. Energy screening
`l. Filtering
`3. Torsional clustering
`4. Comparison with canonical structures
`
`FIG. 7
`
`I
`Loop
`insertion
`
`Energy
`minimisation
`I
`FINAL
`MODEL
`
`223
`
`PFIZER EX. 1502
`Page 506
`
`

`

`EP 0 592106 A1
`
`L2 ~ ~ .~
`cf>
`L3 ~ ~
`/
`
`Hl
`
`~ .~·
`
`3D6
`
`36-71
`
`D1.3
`
`Gloop-2
`
`FIG. 8
`
`224
`
`PFIZER EX. 1502
`Page 507
`
`

`

`
`
`.
`
`(.!)
`
`lJ._
`
`EP 0 592 106 A1
`EP 0 592108A1
`
`225
`
`PFIZER EX. 1502
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`Page 508
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`PFIZER EX. 1502
`Page 508
`
`

`

`1A6012950PE
`EP 0 592106 A1
`
`CD
`(j) .
`
`(!)
`lJ...
`
`
`
`
`
`226
`
`PFIZER EX. 1502
`
`Page 509
`
`PFIZER EX. 1502
`Page 509
`
`

`

`EP 0 592 106 A1
`EP 0 592106 A1
`
`u
`m .
`
`
`
`(!)
`I.J..
`
`227
`227
`
`PFIZER EX. 1502
`
`Page 510
`
`PFIZER EX. 1502
`Page 510
`
`

`

`EP 0 592 106 A1
`EP 0 592106 A1
`
`0
`(j)
`
`0 -l.L
`
`
`
`228
`
`PFIZER EX. 1502
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`Page 51 1
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`

`

`

`

`

`

`

`

`EP 0 620 276 A1
`
`51
`
`GAA'I'TCCCAA ACACAA.Aotg QDt;tttspag tqcoqotttl cpgctts;tg
`t;toAtt;oqtg SC't!:jAQt;nt OAtOtesogo qqocaaattg ttCtC4CCC8
`101 qtctccaqca ateatqtctq catctccagg ggagaagqtc accatgacct
`151 gcagtgccag ctcaaqtqta aqttacatga actgqtacea gcagaaqtca
`201 qgcacctc:cc ccaaaagatg qatttatgac acatccaaac tggcttctgg
`.251 aqtcectgct cacttcagqg qcaqtqgqtc tgggacctct tactctctca
`301 caatcaqcgq catqgaggct qaaqatqctg ccacttatta ctgccagcag
`351
`tggagtaqta acccattcac gttc9gctcg gqqacaaaqt tggaaataaa
`401 ccgggctgat actqcaecaa etqtatccat ctteecacca tccagtqaqc
`451 agttaacatc tggagqtgce tcagtcgtqt gcttcttqaa caacttetac
`SOl eeeaaagaea tcaatgtcaa gtggaaqatt gatggcagtg aaegaeaaaa
`551
`tggcgtcetq aacagttgga ctgatcagga cagcaaaqac agcacctaca
`601 gcatgagcag caccctcacg ttgaccaagg acgagtatga acgacataac
`6~1 agctatacct gtgaggccac tcacaagaca tcaacttcac ccattgtcaa
`701 gagcttcaac aggaatgagt gtTACAGACA AACCTCCTCA GACCCCACCA
`7 51 CCACCTCCCA CCTC:CATCCT. ATCTTCCCTT CTAAGGTCTT CGACGCTTCC
`801 CCACAAGC.GC: tTAC.C:ACTCT TGC:CCTGCTC tAAACCTCCT CCCAC:CTCCT
`851 TCTC:CTCCTC: CTCC:CTTTCC 'I"''CCCTTTTA TCATGCTAAT ATTTCCACAA
`901 AATATTCAAT AAAGTCAGTC T'I"''CCCTTCA unuuu AAA
`Fig. 1 (a)
`
`1 rmrovoxrsr ILISAsyxxs RGQIVLTQSP AIMSAS~£1( VTMTCSASSS
`51 VSYMNWYQQK SGTSPKRWIY DTSKLASGVP Al!FRCSCSCT SYSLTISGM:£
`101 AEDAATYYCQ QWSSNPF'IFG SGTKL!:II:IRA OTAPTVSIFP PSSEQLTSGG
`151 ASVVCFLNNF YPl<OIIIVltWK IOGSERQNGV LNSh"TI)QDSK DSTYSMSSTL
`201 TLTKDEYERH NSYTCEATHK TSTSPIVl<SF NRNEC•
`Fig.1(b)
`
`2
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`EP 0 620 276 A1
`
`Field of the Invention
`
`5
`
`The present invention relates to humanised antibody molecules, to processes for their production using
`recombinant DNA technology, and to their therapeutic uses.
`The term "humanised antibody molecule" in used to describe a molecule having an antigen binding site
`derived from an immunoglobulin from a non-human species, and remaining immunoglobulin-derived parts of
`the molecule being derived from a human immunoglobulin. The antigen binding site 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 3
`10 CDRs (CDR1, CDR2 and CDR3) in each of the heavy and light 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
`
`15
`
`20
`
`Natural immunoglobulins 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 im(cid:173)
`munoglobulins comprise a generally Y-shaped molecule having an antigen-binding 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 immunoglobulins.
`Natural immunoglobulins have been used in assay, diagnosis 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 of immunoglobulins as therapeu(cid:173)
`tic agents was the discovery of procedures for the production of monoclonal antibodies (MAbs) of defined
`25 specificity (1 ).
`However, most MAbs are produced by hybridomas which are fusions of rodent spleen cells with rodent
`myeloma cells. They are therefore essentially rodent proteins. There are very few reports of the production
`of human MAbs.
`Since most available MAbs are of rodent origin, they are naturally antigenic in humans and thus can
`30 give rise to an undesirable immune response termed the HAMA (Human Anti-Mouse Antibody) response.
`Therefore, the use of rodent MAbs as therapeutic agents in humans is inherently limited by the fact that the
`human subject will mount an immunological response to the MAb and will either remove it entirely or at
`least reduce its effectiveness. In practice, MAbs of rodent origin may not be used in patients for more than
`one or a few treatments as a HAMA response soon develops rendering the MAb ineffective as well as
`35 giving rise to undesirable reactions. For instance, OKT3 a mouse lgG2alk MAb which recognises an antigen
`in the T -cell receptor-CD3 complex has been approved for use in many countries throughout the world as
`an immunosuppressant in the treatment of acute allograft rejection [Chatenoud et al (2) and Jeffers et al (3)(cid:173)
`]. However, in view of the rodent nature of this and other such MAbs, a significant HAMA response which
`may include a major anti-idiotype component, may build up on use. Clearly, it would be highly desirable to
`40 diminish or abolish this undesirable HAMA response and thus enlarge the areas of use of these very useful
`antibodies.
`Proposals have therefore been made to render non-human MAbs less antigenic in humans. Such
`techniques can be generically termed "humanisation" techniques. These techniques typically involve the
`use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the
`45 antibody molecule.
`Early methods for humanising MAbs involved production of chimeric antibodies in which an antigen
`binding site comprising the complete variable domains of one antibody is linked to constant domains
`derived from another antibody. Methods for carrying out such chimerisation procedures are described in
`EP0120694 (Celltech Limited), EP0125023 (Genentech Inc. and City of Hope), EP-A-0 171496 (Res. Dev.
`50 Corp. Japan), EP-A-0 173 494 (Stanford University), and WO 86/01533 (Celltech Limited). This latter
`CeiHech application (WO 86/01533) discloses a process for preparing an antibody molecule having the
`variable domains from a mouse MAb and the constant domains from a human immunoglobulin. Such
`humanized chimeric antibodies, however, still contain a significant proportion of non-human amino acid
`sequence, i.e. the complete non-human variable domains, and thus may still elicit some HAMA response,
`55 particularly if administered over a prolonged period [Bagent et 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 domains of a
`human immunoglobulin by site directed mutagenesis using long oligonucleotides. The present invention
`
`3
`
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`

`EP 0 620 276 A1
`
`5
`
`10
`
`15
`
`relates to humanized antibody molecules prepared according to this alternative approach, i.e. CDR-grafted
`humanised antibody molecules. Such CDR-grafted humanized antibodies are much less likely to give rise to
`a HAMA response than humanised chimeric antibodies in view of the much lower proportion of non-human
`amino acid sequence which they contain.
`The earliest work on humanizing MAbs by CDR-grafting was carried out on MAbs _recognizing synthetic
`antigens, such as the NP or NIP antigens. However, examples in which a mouse MAb recognizing lysozyme
`and a rat MAb recognising an antigen on human T -cells were humanised by CDR-grafting have been
`described by Verhoeyen et al (5) and Riechmann et al (6) respectively. The preparation of CDR-grafted
`antibody to the antigen on human T cells is also described in WO 89/07452 (Medical Research Council).
`In Riechmann et ai!Medical Research Council it was found that transfer of the CDR regions alone [as
`defined by Kabat refs. (7) and (8)] was not sufficient to provide satisfactory antigen binding activity in the
`CDR-grafted product. Riechmann 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
`having improved antigen binding activity. This residue at position 27 of the heavy chain is within the
`structural loop adjacent to CDR1. A further construct which additionally contained a human serine to rat
`tyrosine change at position 30 of the heavy chain did not have a significantly altered binding activity over
`the humanised antibody with the serine to phenylalanine change at position 27 alone. These results indicate
`that changes to residues of the human sequence outside the CDR regions, in particular in the structural
`loop adjacent to CDR1, may be necessary to obtain effective antigen binding activity for CDR-grafted
`20 antibodies which recognise more complex antigens. Even so the binding affinity of the best CDR-grafted
`antibodies obtained was still significantly less than the original MAb.
`Very recently Queen et al (9) have described the preparation of a humanised antibody that binds to the
`interleukin 2 receptor, by"COmbining the CDRs of a murine MAb (anti-Tac) with human immunoglobulin
`framework and constant regions. The human framework regions were chosen to maximise homology with
`the anti-Tac MAb sequence. In addition computer modelling was used to identify framework amino acid
`residues which wore likely to interact with the CDRs or antigen, and mouse amino acids were used at these
`positions in the humanised antibody.
`In WO 90/07861 Queen et al propose four criteria for designing humanised immunoglobulins. The first
`criterion is to use as the human acceptor the framework from a particular human immunoglobulin that is
`30 unusually 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 use the donor amino acid rather than
`the acceptor if the human acceptor residue is unusual and the donor residue is typical for human
`sequences at a specific residue of the framework. The third criterion is to use the donor framework amino
`acid residue rather than the acceptor at positions immediately adjacent to the CDRs. The fourth criterion is
`to use the donor amino acid residue at framework positions at which the amino acid is predicted to have a
`side chain atom within about 3 A of the CDRs in a three-dimensional immunoglobulin model and to be
`capable of interacting with the antigen or with the CDRs of the humanised immunoglobulin. It is proposed
`that criteria two, three or four may be applied in addition or alternatively to criterion one, and may be
`applied singly or in any combination.
`WO 90/07861 describes in detail the preparation of a single CDR-grafted humanised antibody, a
`humanised antibody having specificity for the p55 Tac protein of the IL-2 receptor. The combination of all
`four criteria, as above, were employed in designing this humanized antibody, the variable region frame(cid:173)
`works of the human antibody Eu (7) being used as acceptor. In ttie resultant humanised antibody the donor
`CDRs were as defined by Kabat et al (7 and 8) and in addition the mouse donor residues were used in
`45 place of the human acceptor residues, at positions 27, 30, 48, 66, 67, 89, 91, 94, 103, 104, 105 and 107 in
`the heavy chain and at positions 48, 60 and 63 in the light chain, of the variable region frameworks. The
`humanised anti-Tac antibody obtained is reported to have an affinity for p55 of 3 x 109 M-1, about one-third
`of that of the murine MAb.
`We have further investigated the preparation of CDR-grafted humanised antibody molecules and have
`identified a hierarchy of positions within the framework of the variable regions (i.e. outside both the Kabat
`CDRs and structural loops of the variable regions) at which the amino acid identities of the residues are
`important for obtaining CDR-grafted products with satisfactory binding affinity. This has enabled us to
`establish a protocol for obtaining satisfactory CDR-grafted products which may be applied very widely
`irrespective of the level of homology between the donor immunoglobulin and acceptor framework. The set
`55 of residues which we have identified as being of critical importance does not coincide with the residues
`identified by Queen et al (9).
`
`40
`
`25
`
`35
`
`50
`
`4
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`

`EP 0 620 276 A1
`
`Summary of the Invention
`
`5
`
`10
`
`Accordingly, in a first aspect the invention provides a CDR-grafted antibody heavy 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 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.
`In preferred embodiments, the heavy chain framework comprises donor residues at positions 23, 24, 49,
`71, 73 and 78 or at positions 23, 24 and 49. The residues at positions 71, 73 and 78 of the heavy chain
`framework are preferably either all acceptor or all donor residues.
`In particularly preferred embodiments the heavy chain framework additionally comprises donor residues
`at one, some or all of positions 6, 37, 48 and 94. Also it is particularly preferred that residues at positions of
`the heavy chain framework which 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
`residues. Most preferably the heavy chain framework additionally comprises donor residues at positions 2,
`15 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.
`In addition the heavy chain framework optionally comprises donor residues at one, some or all of
`positions:
`1 and 3,
`72 and 76,
`20 69 Of 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),
`25 91,
`sa, and
`any one or more of 9, 11, 41, 87, 108, 110 and 112.
`In the first and other aspects of the present invention reference is made to CDR-grafted antibody
`products comprising acceptor framework and donor antigen binding regions. It will be appreciated that the
`invention is widely applicable to the CDR-grafting of antibodies in general. Thus, the donor and acceptor
`antibodies may be derived from animals of the same species and even same antibody class or sub-class.
`More usually, however, the donor and acceptor antibodies are derived from animals of different species.
`Typically the donor antibody is a non-human antibody, such as a rodent MAb, and the acceptor antibody is
`a human antibody.
`In the first and other aspects of the present invention, the donor antigen binding region typically
`comprises at least one CDR from the donor antibody. Usually the donor antigen binding region comprises
`at least two and preferably all three CDRs of each of the heavy chain and/or light chain variable regions.
`The CDRs may comprise the Kabat CDRs, the structural loop CDRs or a composite of the Kabat and
`structural loop CDRs and any combination of any of these. Preferably, the antigen binding regions of the
`4C CDR-grafted heavy chain variable domain comprise CDRs corresponding to the Kabat CDRs at CDR2
`(residues 50-65) and CDR3 (residues 95-100) and a composite of the Kabat and structural loop CDRs at
`CDR1 (residues 26-35).
`The residue designations given above and elsewhere in the present application are numbered accord(cid:173)
`ing to the Kabat numbering [refs. (7) and (8)]. Thus the residue designations do not always correspond
`45 directly with the linear numbering of the amino acid residues. The actual linear amino acid sequence may
`contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of,
`or insertion into, a structural component, whether framework or CDR: of the basic variable domain structure.
`For example, the heavy chain variable region of the anti-Tac antibody described by Queen et al (9) contains
`a single amino acid insert (residue 52a) after residue 52 of CDR2 and a three amino acid insert (residues
`50 82a, 82b and 82c) after framework residue 82, in the Kabat numbering. The correct Kabat numbering of
`residues may be determined for a given antibody by alignment at regions of homology of the sequence of
`the antibody with a "standard" Kabat numbered sequence.
`The invention also provides in a second aspect a CDR-grafted antibody light chain having a variable
`region domain comprising acceptor framework and donor antigen . binding regions wherein the framework
`55 comprises donor residues at at least one of positions 1 and/or 3 and 46 and/or 47. Preferably the CDR
`grafted light chain of the second aspect comprises donor residues at positions 46 and/or 47.
`The invention also provides in a third aspect a CDR-grafted antibody light chain having a variable region
`domain comprising acceptor framework and donor antigen binding regions wherein the framework com-
`
`30
`
`35
`
`5
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`EP 0 620 276 A1
`
`5
`
`10
`
`20
`
`prises donor residues at at least one of positions 46, 48, 58 and 71.
`In a preferred embodiment of the third aspect, the framework comprises donor residues at all of
`positions 46, 46, 58 and 71.
`In particularly preferred embodiments of the second and third aspects, the framework additionally
`comprises donor residues at positions 36, 44, 47, 85 and 87. Similarly positions of the light 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 additionally comprises donor residues at positions 2, 4, 6, 35, 36, 38, 44, 47, 49,
`62, 64-69, 85, 87, 98, 99, 101 and 102.
`In addition the framework of the second or third aspects optionally comprises donor residues at one,
`some or all of positions:
`1 and 3,
`63,
`60 (if 60 and 54 are able to form at potential saltbridge),
`75 70 (if 70 and 24 are able to form a potential sa!tbridge),
`73 and 21 (if 47 is different between donor and acceptor),
`37 and 45 (if 47 is different between donor and acceptor),
`and
`any one or more of 10, 12, 40, 80, 103 and 105.
`Preferably, the antigen binding regions of the CDR-grafted light chain variable domain comprise CDRs
`corresponding to the Kabat CDRs at CDR1 (residue 24-34), CDR2 (residues 5Q-56) and CDR3 (residues 89-
`97).
`The invention further provides in a fourth aspect a CDR-grafted antibody molecule comprising at least
`one CDR-grafted heavy chain and at least one CDR-grafted light chain according to the first and second or
`first and third aspects of the invention.
`The humanised antibody molecules and chains of the present invention may comprise: a complete
`antibody molecule, having full length heavy and light chains; a fragment thereof, such as a Fab, (Fab')2 or
`FV fragment; a light chain or heavy chain monomer or dimer; or a single chain antibody, e.g. a single chain
`FV in which heavy and light chain variable regions are joined by a peptide linker; or any other CDR-grafted
`30 molecule with the same specificity as the original donor antibody. Similarly the CDR-grafted heavy and light
`chain variable region may be combined with other antibody domains as appropriate.
`Also the heavy or light chains or humanised antibody molecules of the present invention may have
`·attached to them an effector or reporter molecule. For instance, it may have a macrocycle, for chelating a
`heavy metal atom, or a toxin, such as ricin, attached to it by a covalent bridging structure. Alternatively, the
`35 procedures of recombinant DNA technology may be used to produce an immunoglobulin molecule in which
`the Fe fragment or CH3 domain of a complete immunoglobulin molecule has been replaced by, or has
`attached thereto by peptide linkage, a functional non-immunoglobulin protein, such as an enzyme or toxin
`molecule.
`Any appropriate acceptor variable region framework sequences may be used having regard to
`40 class/type of the donor antibody from which the antigen 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/optimise homology with the donor antibody sequence particularly at
`positions close or adjacent to the CDRs. However, a high level of homology between donor and acceptor
`sequences is not important for application of the present invention. The present invention identifies a
`45 hierarchy of framework residue positions at which donor residues may be important or desirable for
`obtaining a CDR-grafted antibody product having satisfactory binding properties. The CDR-grafted products
`usually have binding. affinities of at least 10S M-1, preferably at least about 108 M-1, or especially in the
`range 10S-1012 M-1. In principle, the present invention is applicable to any combination of donor and
`acceptor antibodies irrespective of the level of homology between their sequences. A protocol for applying
`the invention to any particular donor-acceptor antibody pair is given hereinafter. Examples of human
`frameworks which may be used are KOL, NEWM, REI, EU, LAY and POM (refs. 4 and 5) and the like; for
`instance KOL and NEWM for the heavy chain and REI for the light chain and EU, LAY and POM for both
`the heavy chain and the light chain.
`Also the constant region domains of the products of the invention may be selected having regard to the
`55 proposed function of the antibody in particular the effector functions which may be required. For example,
`the constant region domains may be human lgA, lgE, lgG or lgM domains. In particular, lgG human
`constant region domains may be used, especially of the lgG1 and lgG3 isotypes, when the humanised
`antibody molecule is intended for therapeutic uses, and antibody effector .functions are required. Alter-
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`natively, lgG2 and lgG4 isotypes may be used when the humanised antibody molecule is intended for
`therapeutic purposes and antibody effector functions are not required, e.g. for simple blocking of lym(cid:173)
`phokine activity.
`However, the remainder of the antibody molecules need not comprise only protein sequences from
`immunoglobulins. For instance, a gene may be constructed in which a DNA sequence encoding part of a
`human immunoglobulin chain is· fused to a DNA sequence encoding the amino acid sequence of a
`functional polypeptide such as an effector or reporter molecule.
`Preferably the CDR-grafted antibody heavy and light chain and antibody molecule products are
`produced by recombinant DNA technology.
`Thus in further aspects the invention also includes DNA sequences coding for the CDR-grafted heavy
`and light chains, cloning and expression vectors containing the DNA sequences, host cells transformed with
`the DNA sequences and processes for producing the CDR-grafted chains and antibody molecules
`comprising expressing· the DNA sequences in the transformed host cells.
`The general methods by which the vectors may be constructed, transfection methods and culture
`15 methods are well known per se and form no part of the invention. Such methods are shown, for instance, in
`references 1 0 and 11 .
`The DNA sequences which encode the donor amino acid sequence may be obtained by methods well
`known in the art. For example the donor coding sequences may be obtained by genomic cloning, or eDNA
`cloning from suitable hybridoma cell lines. Positive clones may be screened using appropriate probes for
`the heavy and light chain genes in question. Also PCR cloning 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 as KOL, REI, EU and
`NEWM, are widely available to workers in the art.
`The standard techniques of molecular biology may be used to prepare DNA sequences coding for the
`25 CDR-grafted products. Desired 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 appropriate. For example oligonucleotide directed synthesis as described by
`Jones et al (ref. 20) may be used. Also oligonucleotide directed mutagenesis of a pre-exising variable
`region as, for example, described by Verhoeyen et al (ref. 5) or Riechmann et al (ref. 6) may be used. Also
`30 enzymatic filling in of gapped oligonucleotides using T4 DNA polymerase M.tor example, described by
`Queen et al (ref. 9) may be used.
`Any suitable host cell/vector system may be used for expression of the DNA sequences coding for the
`CDR-grafted heavy and light chains. Bacterial e.g. E. coli, and other microbial systems may be used, in
`particular for expression of antibody fragments such as FAb and (Fab')2 fragments, and especially FV
`fragments and single chain antibody fragments e.g. single chain FVs. Eucaryotic e.g. mammalian host cell
`expression systems may be used for production of larger CDR-grafted antibody products, including
`complete antibody molecules. Suitable mammalian host cells include CHO cells and myeloma or hybridoma
`cell lines.
`Thus, in a further aspect the present invention provides a process for producing a CDR-grafted antibody
`40 product comprising:
`(a) producing in an expression vector an operon having a DNA sequence which encodes an antibody
`heavy chain according to the first aspect of the invention;
`and/or
`(b) producing in an expression vector an operon having a DNA sequence which encodes a complemen-
`tary antibody light chain according to the second or third aspect of the invention;
`(c) transfecting a host cell with the or each vector; and
`(d) culturing the transfected cell line to produce the CDR-grafted antibody product.
`The CDR-grafted product may comprise only heavy or light chain derived polypeptide, in which case
`only a heavy chain or light chain polypeptide coding sequence is used to transfect the host cells ..
`50 For production of products comprising both heavy and light chains, the cell line may be transfected with two
`vectors, the first vector may contain an operon encoding a light chain-derived polypeptide and the second
`vector containing an operon encoding a heavy chain-derived polypeptide. Preferably, the vectors are
`identical, except in so far as the coding sequences and selectable markers are concerned, so as to ensure
`as far as possible that each polypeptide chain is equally expressed. Alternatively, a single vector may be
`55 used. the vector including the sequences encoding 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, genomic DNA, preferably a fusion of eDNA and genomic DNA.
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`The present invention is applicable to antibodies of any appropriate specificity. Advantageously,
`however, the invention may be applied to the humanisation of non-human antibodies which are used for!!:!
`vivo therapy or diagnosis. Thus the antibodies may be site-specific antibodies such as tumour-specific or
`cell surface-specific antibodies, suitable for use in in vivo therapy or diagnosis, e.g. tumour imaging.
`5 Examples of cell surface-specific antibodies are anti-T cell antibodies, such as anti-CD3, and CD4 and
`adhesion molecules, such as CR3, ICAM and ELAM. The antibodies may have specificity for interleukins
`(including lymphokines, growth factors and stimulating factors), hormones and other biologically active
`compounds, and receptors for any of these. For example, the antibodies may have specificity for any of the
`following: Interferons a, fj, "' oro, IL 1, IL2, IL3, or IL4, etc., TNF, GCSF, GMCSF, EPO, hGH, or insulin, etc.
`The the present invention also includes therapeutic and diagnostic compositions comprising the CDR-
`grafted products of the invention and uses of such compositions in therapy and diagnosis.
`Accordingly in a further aspect the invention provides a therapeutic or diagnostic composition compris(cid:173)
`ing a CDR-grafted antibody heavy or light chain or molecule according to previous aspects of the invention
`in combination with a pharmaceutically acceptable carrier, diluent or excipient.
`Accordingly also the invention provides a method of therapy or diagnosis comprising administering an
`effective amount of a CDR-grafted antibody heavy or light chain or molecule according to previous aspects
`of the invention to a human or animal subject.
`A preferred protocol for obtaining CDR-grafted antibody heavy and light chains in accordance with the
`present invention is set out below together with the rationale by which we have derived this protocol. This
`20 protocol and rationale are given without prejudice to the generality of the invention as hereinbefore
`described and defined.
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`Protocol
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`Ught chain
`
`It is first of all necessary to sequence the DNA coding for the heavy and light chain variable regions of
`the donor antibody, to determine their amino acid sequences. It is also necessary to choose appropriate
`acceptor heavy and light chain variable regions, of known amino acid sequence. The CDR-grafted chain is
`then designed starting from the basis of the acceptor sequence. It will be appreciated that in some cases
`the donor and acceptor amino acid residues may b