`APPLICATION SERIAL NO. 11/284,261
`
`PATENT
`
`REMARKS
`
`Claims 24 and 25 were pending. All pending claims were rejected in the Non-Final
`
`Rejection. In view of the foregoing amendments and arguments that follow, Applicants
`
`respectfully request withdrawal of all rejections upon reconsideration.
`
`Applicants acknowledge with appreciation the Office's withdrawal of the objections to
`
`claim 24 under 35 U.S.C. 112, second paragraph, as being indefinite.
`
`Rejection Under 35 U.S.C. § 112, First Paragraph
`
`Claim 24 was amended with the RCE filing and was again rejected as allegedly being
`
`indefinite. The Office alleges that the specification is enabling for a humanized antibody
`
`comprising a heavy chain variable domain and a light chain variable domain, with all 6 CDRs,
`
`and does not provide enablement for a humanized antibody heavy chain variable domain alone.
`
`The Office is clearly disregarding the fact that the CDR-grafted chains can be combined with
`
`other chains, as disclosed in the specification, including chimeric and mouse chains. Thus, it is
`
`not necessary for the claims to recite both chains. Applicants traverse this rejection but have
`
`amended claim 24 to recite a humanized antibody comprising a heavy chain variable domain.
`
`Applicants respectfully submit that this rejection has been overcome.
`
`Rejection Under 35 U.S.C.102(e)
`
`Claims 24 and 25 were rejected under 35 U.S.C. 102(e) as allegedly being anticipated by
`
`Queen, et al US Patent 5,585,089 the '"089 patent". Claim 25 has been cancelled. Applicants
`
`traverse this rejection as it applies to claim 24.
`
`With all due respect, the Office has apparently misread the claims. Initially, the office
`
`states that the claims recite that the framework region comprises a non-human amino acid
`
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`DOCKET NO.: CARPOOOl-112
`APPLICATION SERIAL NO. 11/284,261
`
`PATENT
`
`substitution at a residue selected from 23, 24, 49, 71, 73, and 78, and combinations thereof. The
`
`Office then asserts that the '"089 patent" teaches an antibody comprising a humanized heavy
`
`chain variable domain comprising human residues at positions except the CDRs, and the
`
`following framework positions-27, 93, 95, 98, 107-109, 11, 30, 67, 48, and 68. The Office
`
`concludes, thus, that all the remaining framework positions are the human antibody allegedly
`
`taught in the '"089 patent". The Office then states that, since the claims recite substitution to
`
`human residues in the heavy chain in residues 23, 24, 49, 71, 73, and 78, and the '"089 patent"
`
`teaches human residues in all of those positions, the claim limitations are met. As the Office
`
`previously acknowledged, however, these residues are non-human residues in claim 24. The
`
`"'089 patent", thus, does not anticipate the Applicants invention.
`
`Applicants respectfully request that this rejection be withdrawn.
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`DOCKET NO.: CARPOOOl-112
`APPLICATION SERIAL NO. 11/284,261
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`PATENT
`
`CONCLUSION
`
`Applicants respectfully submit that claim 24 is allowable and early allowance of the
`
`same.
`
`If a telephonic conversation with Applicants' attorney would help expedite the
`
`prosecution of the above-identified application, the Examiner is urged to call the undersigned at
`
`215-665-5593.
`
`Respectfully submitted,
`
`/Doreen Y atko Trujillo/
`
`Doreen Yatko Trujillo
`Registration No. 35,719
`
`Date: September 9, 2009
`
`COZEN O'CONNOR, P.C.
`1900 Market Street, 4lh Floor
`Philadelphia, PA 19103-3508
`Telephone: (215) 665-5593
`Facsimile: (2 15) 701-2005
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`Per
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`DATE FILED: 05/28/2010
`wo~LoOOGUM~l}!IANQ~ 30
`
`International Bureau
`
`INU!RNATIONAL APPLICATION PUBLISHED UNDER TilE PATENT COOPERATION TREATY (PC'l)
`wo 91/09967
`
`(11) IDtematiooal Publication Number:
`
`A1
`
`( 43) Intemational Publication Date:
`
`11 July 1991 (11.07.91)
`
`(51) IDtemational Patent aassificatiou S :
`C12P 21/08, C12N 15/13
`A61K 391395, C07K 15/06
`Cl2N S/10, 15/62
`
`..
`
`(21) International Application Number:
`
`PCT/GB90/02017
`
`(22) Intmational Filing Date:
`
`21 December !990 (21.12.90)
`
`(74) Agent: MERCER. Christopher, Paul; Carpmaels & Rans(cid:173)
`ford, 43 Bloomsbury Square, L<>ndon WClA 2RA (GB).
`
`(30) Priority data :
`8928874.0
`
`21 December 1989 (21.12.89) GB
`
`(71)Applicaut (for all designated States except US): CELLTECH
`LIMITED (GB/GB]; 216 Bath Road, Slough, Berkshire
`SLl 4EN (GB).
`
`(81) Designated States: AT, AT (European patent), AU, BB, BE
`(European patent), BF (OAPI patent), BG, BJ (OAPI
`patent), BR. CF (OAPI patent), CG (OAPI patent), CH,
`CH (European patent), CM (OAPI patent), DE, DE
`(EUC¢pean patent), DK, Fl, FR (European patent), GA
`(OAPI patent), GB, GB (European patent), GR, HU, IT
`(European patent), JP, KP, KR, LK, LU, LU (European
`patent), MC, MG, ML (OAPI patent), MR (OAPI pa(cid:173)
`tent), MW, NL, NL (European patent), NO, RO, SO,
`SE, SE (European patent), SN (OAPI patent), SU, TD
`(OAPI patent), TG (OAPI patent), US.
`
`(72) lnYeators; and
`(75) lnYentors/Applicants (for US only) : ADAIR, John, Robert
`(GB/GB]; 23 Gwrge Road, Stokenehurch, High Wy(cid:173)
`combe, Buckingbamshire HPI4 3RN (GB). ATHWAL, .
`Diljeet, Singh (GB/GBJ; Flat 35, Knollys House, Tavis- Publislted
`With international search report.
`1ock Square, London WCl (GB). EMTAGE, John, Spen-
`Before the expiration of tl1e time 1imit for amending the
`cer [GB/GB]; 49 Temple Mill Island, Temple, Mar.Iow,
`claims and to be republished in the event of the r£ceipt of
`Buckinghamshire SL7 ISQ (GB).
`amendmencs.
`With a requeSt for recJifu:aJion undu Rule 9/.f(j).
`
`(54) Title: HUMANISED ANTIBODIES
`
`(57) Abstract
`
`CDR-grafted antibody heavy and light chains comprise acceptor framework and donor antigen binding regions, the heavy
`.chains comprising donor residues at at least one of positions (6, 23) a:nd/or (24, 48) and/or (49, 71) and/or (73, 75) and/or (76)
`and/or (78) and (88) and/or (91). The CDR-grafted light chains comprise donor residues at at least one of positions (I) and/or
`(3) and (46) and/or (47) or at at least one of positons (46, 48, 58) and (71). The CDR-grafted antibodies are preferably humanised
`antibodies, having non human, e.g. rodent, donor and human acceptor frameworks, and may be used for in vivo therapy and di-
`. agnosis. A generaUy applicable protocol is disclosed for obtaining CDR-grafted antibodies .
`
`. . ,
`
`Carter Exhibit 2005
`Carter v. Adair
`Interlerence No. 105,744
`
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCf o n the front pages of pamphlets publishing international
`applications under the PCf.
`
`AT
`AU
`8 8
`BE
`BP
`8G
`BJ
`BR
`C A
`CF
`CG
`CH
`CJ
`C M
`cs
`D£
`DK
`
`Ausu:ia
`AusLralia
`Bar bad as
`Belgium
`Burkina Faso
`Bul&aria
`Benin
`Bra:t.il
`Canada
`Cen1ral Arrican Republic
`Congo
`SwilurlaruJ
`C6u: d'lvolrc
`Cameroon
`Czoc:hoslovakia
`Ocrma.ny
`Ot:nmark
`
`ES
`PI
`FR
`GA
`CB
`C N
`CR
`HU
`IT
`JP
`KP
`
`KR
`u
`LK
`LU
`MC
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Guinea
`Greece
`Hungary
`haly
`Japan
`Dcmoc;ratic P1.:oplc's Republic
`of Korea
`Republic: of Korea
`Ucchu:nstc:in
`Sri Lllnka
`LuJCCmbourg
`Monaco
`
`MG
`ML
`MN
`MR
`MW
`NL
`NO
`PL
`RO
`so
`SE
`SN
`su
`TO
`TC
`us
`
`Madaga:sc:ar
`Mali
`Mongolia
`Mauritania
`Malawi
`Netherlands
`Norway
`Poland
`Romania
`Sudan
`Sweden
`Scncpl
`Soviet Union
`Chrul
`Toea
`Unitctl Stau:. of America
`
`t
`
`)
`
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`HUMANISED ANTIBODIES
`
`PCf/GB90/02017
`
`Field of the Invention
`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" is 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
`CDRs (CDRl, 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
`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 immunoglobulins 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
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`of immunoglobulins as therapeutic agents was the discovery
`of procedures for the production of monoclonal antibodies
`(MAbs) of defined 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 give rise to an
`undesirable immune response termed the HAMA ( Hum.an
`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 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 countries throughout the world
`as an immunosuppressant in the treatment of acute
`allograft rejection [Chatenoud et al (2) and Jeffers et al
`(3 ) ]. However, in view of the rodent nature of this and
`other such MAbs, a significant HAMA response which may
`include a maj~r anti-idiotype component, may build up on
`use.
`Cl~arly, it would be highly desirable to 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
`
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`techniques typically involve the use of recombinant DNA
`technology to manipulate DNA sequences encoding the
`polypeptide chains of the 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. Corp. Japan), EP-A-0 173 494 (Stanford
`This
`University ) , and WO 86 / 01533 (Celltech Limited).
`latter Celltech 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 humanised 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 st ill
`elicit some HAMA response, particularly if administered
`over a prolonged period [Begent et al (ref. 4 ) ].
`
`In an alternat.ive 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 oligonucle otides .
`The present invention relates to humanised antibody
`molecules prepared according to this alternative approach,
`i.e. CDR-grafted humanised antibody molecules.
`Such
`CDR-grafted humanised 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 .
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`The earliest work on humanising MAbs by CDR-grafting was
`carried out on MAbs recognising synthetic antigens, such
`as the NP or NIP antigens.
`However, examples in which a
`mouse MAb recognising lysozyme and a rat MAb recognising
`an antigen on human T-cells were humanised by CDR-grafting
`have been described by Verhoeyen et al (S) 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).
`
`!
`
`1
`
`In Riechmann et al/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
`CDR!.
`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 CDR!, may be necessary to obtain
`effective antigen binding activity for CDR-grafted
`Even
`antibodies which recognise more complex antigens.
`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
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`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 ho~ology with the anti-Tac MAb
`sequence.
`In addition computer modelling was used to
`identify framework amino acid residues which were 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 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
`It is proposed that criteria
`humanised immunoglobulin.
`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
`
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`The combination of all four c·riteria, as
`IL-2 receptor.
`above, were employed in designing this humanised antibody,
`the variable region frameworks of the human antibody Eu
`(7) being used as acc~ptor.
`In the 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 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
`frameworks.
`chain, of the variable region
`The humanised
`reported to have an affinity
`anti-Tac antibody obtained is
`for p55 of 3 x 109 M-1, about
`one-third of that of the
`murine MAb.
`
`i
`
`~
`
`We have further investigated the preparation of CDR(cid:173)
`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 of residues which we have identified
`as being of critical importance does not coincide with the
`residues identified by Queen et al (9).
`
`Summary of the Invention
`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.
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`..
`
`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, 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,
`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 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
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`Thus, the donor and acceptor antibodies may be
`general.
`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 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 CDRl
`(residues 26-35).
`
`The residue designations given above. and elsewhere in the
`present application are numbered according to the Kabat
`numbering (refs. (7). and (8)].
`Thus the residue
`designations do not always correspond 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
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`acid insert (residues 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(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 1 and/or 3 and 46
`and/or 47.
`Preferably the CDR qrafted 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 comprises 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,
`4 8 , 58 and 7 1.
`
`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, as, 87, 98, 99, 101 and 102.
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`In addition the framework of the second or third aspects
`opt ionally 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),
`70 (if 70 and 24 are able to form a potential saltbridge),
`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 t o
`the Kabat CDRs at CDR1 (residue 24-34), CDR2 (residues
`50-56) 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 t he 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 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.
`
`~
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`Also the heavy or light chains or humanised antibody
`molecul es of the present invention may have attached to
`them an effector or reporter molecule.
`For instance, i t
`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 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 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 ddnor antibody sequence
`particularly at positions close or adjacent to the CDRs.
`However, a high level of homology between donor and
`acceptor sequences is not impor t ant for application of the
`present invention.
`The present invention identifies a
`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 105 M-1, preferably at
`l east about 108 M-1, or especially in the range 108-1o12
`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 inventi on to any
`particular donor-acceptor antibody pair is given
`hereinafter.
`Examples of human frameworks which may be
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`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 pr.oposed.
`function of the antibody in particular the effector
`functions which may be required.
`For example, the
`constant region domains may be human IgA, IgE, IgG or IgM
`domains.
`In particular, IgG human constant region
`domains may be used, especially of the IgGl and IgG3
`isotypes, when the humanised antibody molecule is intended
`for therapeutic uses, and antibody effector functions are
`required. . Alternatively, IgG2 and IgG4 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 lymphokine
`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
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`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 methods are
`well known per se and form no part of the invention.
`Such
`methods are shown, for instance, in references 10 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 l i nes.
`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 CDR-grafted
`products. Desired DNA sequences may be synthesi sed
`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 enzymatic filling in of gapped
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`oligonucleotides using T4 DNA polymerase as, for 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 product
`comprising:
`
`(a) producing in an expr