DATE FILED: 05/28/2010
`DOCUMENT NO: 67
`HUMANISED ANTIBODIES
`
`Field of the Invention
`
`The present invention relates to humanised antibody molecules (HAMs), to processes for their
`production using recombinant DNA technology, and to their therapeutic uses.
`
`In the present application, the term “recombinant antibody molecule” (RAM) is used to describe
`an antibody produced by an process involving the use of recombinant DNA technology, including
`any analogues of natural immunoglobulins or their fragments. The term “humanised antibody
`molecule” (HAM) 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 may comprise
`either complete variable domains fused onto constant domains ortypically comprises
`complementarity determining regions grafted onto(CDRs) which determine the binding specificity
`of the antibody molecule and which are carried on appropriate framework regions in the variable
`domains. The abbreviation “MAb” is used to indicate a monoclonal antibody There are 3 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
`
`Natural immunoglobulins have been known for many years, as have the various fragments thereof,
`such as the Fab, (Fab’)2 and Fc 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, have beenwere hindered until recently by the
`polyclonal nature of natural immunoglobulins. A significant step towards the realisation of the
`potential of immunoglobulins as therapeutic agents was the discovery of techniquesprocedures for
`the preparationproduction 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 (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. Thus, inIn practice,
`
`Carter Exhibit 2037
`Carter v. Adair
`Interference No. 105,744
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`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 major anti-idiotype component, may build up on
`use. Clearly, it would be highly desirable to diminish or abolish this undesirable KAMA 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
`generallytypically involve the use of recombinant DNA technology to manipulate DNA sequences
`encoding the polypeptide chains of the antibody molecule.
`
`Some early methods for carrying out such a procedureEarly 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 EP A 0 171
`4960120694 (Celltech Limited), EP0125023 (Genentech Inc. and City of Hope), EP-A-0 171496
`(Res. Dev. Corp. Japan), EP-A-0 173 494 (Stanford University), EP A 0 194 276and WO
`86/01533 (Celltech Limited) and WO A 8 702 671 (Int. Gen. Eng. Inc.). The. This 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. It
`also shows the production of an antibody molecule comprising the variable domains of a mouse
`MAb, the CH1 and CL domains of a human immunoglobulin, and a non immunoglobulin derived
`protein in place of the Fc portion of the human immunoglobulinSuch 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 still elicit some HAMA response,
`particularly if administered over a prolonged period [Begent 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. There are 3 CDRs (CDR1, CDR2 and CDR3) in each of the heavy and light
`chain variable regions. The present invention relates to HAMshumanised antibody molecules
`prepared according to this alternative approach, i.e. CDR-grafted HAMshumanised 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.
`
`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 respectively were
`humanised by CDR-grafting are shownhave been described by Verhoeyen et al (25) and
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`Riechmann et al (36) 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 the latter case (Riechmann et al)/Medical Research Council it was found that transfer of the
`CDR regions alone ([as defined by Kabat refs. 4(7) and 5(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
`satisfactoryimproved 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 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 were likely to interact with the CDRs or antigen,
`and mouse amino acids were used at these positions in the humanized 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
`Å 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.
`
`In recent years a number of rodent MAbs have been developed for therapeutic applications. For
`instance, OKT3 a mouse IgG2a/k MAb which recognizes an antigen in the T cell receptor CD3
`complex has been approved for use in the USA as an immunosuppressant in the treatment of acute
`allograft rejection (Chatenond et al (1986) J. Immunol., 137, 830 838, and Jeffers et al (1986)
`Transplantation, 41, 572 578). 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, builds
`up on use. Clearly, it would be highly desirable to diminish or abolish this undesirable HAMA
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`response by suitable humanisation or other recombinant DNA manipulation of these very useful
`antibody and thus enlarge their areas of use.
`WO 90/07861 describes in detail the preparation of a single CDR-grafted humanised antibody, a
`humanized 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 humanised antibody,
`the variable region frameworks of the human antibody Eu (7) being used as acceptor. In the
`resultant humanized 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 chain, of the variable region frameworks. The humanized anti-Tac antibody
`obtained is reported to have an affinity for p55 of 3 x 109 M-l, about one-third of that of the murine
`MAb.
`
`We have further investgatedinvestigated the preparation of CDR-grafted HAMshumanised
`antibody molecules and have identified residuesa hierarchy of positions within the framework of
`the variable regionregions (i.e. outside both the Kabat CDRs and structural loops of the variable
`regions) at which the amino acid identities of whichthe 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 humanacceptor framework and non human (rodent)donor
`antigen binding regions wherein the human framework comprises non human (rodent)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, 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,
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`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 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 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).
`
`Preferably, the antigen binding regions of the CDR grafted heavy chain variable domain comprise
`CDRs corresponding to the Kabat CDR at CDR2 (residues 50 65), the structural loop residues at
`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
`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 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-grafted antibody light chain having a
`variable region domain comprising humanacceptor framework and non human (rodent)donor
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`antigen binding regions wherein the human framework comprises non human (rodent)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 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, 48, 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),
`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 to the Kabat CDRs at CDR1 (residue 24-34) and, CDR2 (residues 50-56) and
`the structural loop residues at CDR3 (residues 9189-9697).
`
`The invention further provides in a fourth aspect a CDR-grafted HAMantibody 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 residue designations given above and elsewhere in the present specification are numbered
`according to the Kabat numbering (refs. 4 and 5).
`
`Preferably the CDR grafted heavy chain comprises non human (rodent) residues at positions 23
`and/or 24, 48 and/or 49 and 71 and/or 73. Preferably, the CDR grafted light chain comprises
`non human (rodent) residues at positions 46 and/or 47.
`
`Preferably the CDR graftedhumanised antibody heavymolecules and light chains and HAM are
`produced by recombinant DNA technology.The HAM of the present invention may comprise: a
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`complete antibody molecule, having full length heavy and light chains; a fragment thereof, such as
`thea Fab or, (Fab’)2 or FV fragment; a light chain or heavy chain monomer or dimer; or any othera
`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 non human (rodent)donor antibody. Similarly the CDR-grafted heavy and light chain
`variable region may be combined with other antibody domains as appropriate.
`
`Alternatively,Also the heavy or light chains or HAMhumanised antibody molecules of the present
`invention may have attached to them an effector or reporter molecule. For instance, theyit 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 Fc 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.
`
`For CDR grafted products of the invention,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 humanacceptor framework used is of the
`same/similar class/type as the donor antibody. AdvantageouslyConveniently, the framework
`ismay be chosen to maximise/optimise homology with the donor antibody sequence particularly at
`positions close or adjacent to the CDRs. It will be appreciated that in some cases that the
`non human and human amino acid residues, identified above in connection with the first and
`second aspects of the invention, may be the same and thus no change of the human framework to
`the corresponding non human framework residue is requiredHowever, a high level of homology
`between donor and acceptor sequences is not important 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-l, preferably at least about 108 M-1, or especially in the range 108-1012 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, RE1, EU, LAY and POM (refs. 4 and 5) and
`the like; for instance KOL and NEWM for the heavy chain and RE1 for the light chain and EU,
`LAY and POM for both the heavy chain and the light chain.
`
`Also humanthe constant region domains of the products of the invention may be selected having
`regard to the proposed 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
`domaindomains. In particular, IgG human constant region domains may be used, especially of the
`IgG1 and IgG3 isotypes, when the HAMhumanised 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.
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`However, the remainder of the HAMantibody molecules need not comprise only protein
`sequences from the human immunoglobulinimmunoglobulins. 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
`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 cDNA 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, RE1, 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 synthesized 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 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.
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`Thus, according toin a further aspect the present invention provides a process for producing an
`anti CD3 HAM which process comprisesa CDR-grafted antibody product comprising:
`
`(a) producing in an expression vector an operon having a DNA sequence which encodes an
`antibody heavy or light chain according to the first or second aspect of the invention;
`
`and/or
`
`(b) producing in an expression vector an operon having a DNA sequence which encodes a
`complementary antibody light or heavy chain according to the second or firstthird aspect of
`the invention;
`
`(c) transfecting a host cell with the or each vector; and
`
`(d) culturing the transfected cell line to produce the HAMCDR-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.
`
`For production of products comprising both heavy and light chains, the cell line may be transfected
`with two vectors, the first vector containingmay 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 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 cDNA 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 cDNA and genomic DNA.
`
`The present invention also includes cloning and expression vectors and transfected cell lines used
`in the process of the invention, therapeutic and diagnostic compositions comprising the variable
`domains or the HAM of the invention and uses of such compositions in therapy and diagnosis.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 in 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.
`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 ,
`,
`, or
`, IL1, IL2, IL3, or IL4, etc., TNF,
`GCSF, GMCSF, EPO, hGH, or insulin, etc.
`
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`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
`comprising 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 protocol and rationale are given without prejudice to the generality of the
`invention as hereinbefore described and defined.
`
`Protocol
`
`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 be identical at a particular position and thus no change of acceptor framework residue is
`required.
`
`As a first step donor residues acceptor residues in the CDRs. For this purpose the CDRs
`1.
`are preferably defined as follows:
`
`Heavy chain - CDR1:
`- CDR2:
`- CDR3:
`- CDR1:
`- CDR2:
`- CDR3:
`
`Light chain
`
`residues 26-35
`residues 50-65
`residues 95-102
`residues 24-34
`residues 50-56
`residues 89-97
`
`The positions at which donor residues are to be substituted for acceptor in the framework are then
`chosen as follows, first of all with respect to the heavy chain and subsequently with respect to the
`light chain.
`2.
`Heavy Chain
`
`Choose donor residues at all of positions 23, 24, 49, 71, 73 and 78 of the heavy chain or all
`2.1
`of positions 23, 24 and 49 (71, 73 and 78 are always either all donor or all acceptor).
`
`10
`
`Board Assigned Page #1232
`
`PFIZER EX. 1095
`Page 1385
`
`

`

`Check that the following have the same amino acid in donor and acceptor sequences, and if
`2.2
`not preferably choose the donor: 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.
`
`2.3
`
`To further optimise affinity consider choosing donor residues at one, some or any of:
`
`i.
`ii.
`iii.
`iv.
`v.
`vi.
`vii.
`viii.
`ix.
`x.
`
`1, 3
`72, 76
`If 48 is different between donor and acceptor sequences, consi