HUMANISED ANTIBODIES

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) and/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 (1) 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 diagnosis. A generally applicable protocol is disclosed for obtaining CDR-grafted antibodies.

Description: HUMANISED ANTIBODIES 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 i munoglobulin 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 (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 7 )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, were 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 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 (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 giving rise to undesirable reactions. For instance, 0KT3 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 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 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 University), and WO 86/01533 (Celltech Limited). 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. 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 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. 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. 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 (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 al/Medical Research Council it was found that transfer of the CDR regions alone [as defined by Kabat refs. (7) and (8) ] was not su ficient 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 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 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 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 humanised antibody, the variable region frameworks of the human antibody Eu (7) being used as acceptor. 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 chain, of the variable region frameworks. The humanised anti-Tac antibody obtained is reported to have an affinity for p55 of 3 x 10 9 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 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. 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 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) . 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 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 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), 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 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 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 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. 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 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 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 10 * ** * M ** ****, preferably at least about 10*** M~-- , or especially in the range 10^-lθl2 M" * *-. 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 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 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 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, 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 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 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. Thus, in a further aspect the present invention provides a process for producing a CDR-grafted antibody 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 complementary 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. 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 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 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^, β, tf or0 , IL1, 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 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. 1. As a first step donor residues are substituted for . acceptor residues in the CDRs. For this purpose the CDRs are preferably defined as follows: 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 2.1 Choose donor residues at all of positions 23, 24, 49, 71, 73 and 78 of the heavy chain or all of positions 23, 24 and 49 (71, 73 and 78 are always either all donor or all acceptor) . 2.2 Check that the following have the same amino acid in donor and acceptor sequences, and if 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. 1, 3 ii. 72, 76 iii. If 48 is different between donor and acceptor sequences, consider 69 iv. If at 48 the donor residue is chosen, consider 38 and 46 v. If at 69 the donor residue is chosen, consider 80 and then 20 vi. 67 vii. If at 67 the donor residue is chosen, consider 82 and then 18 viii. 91 ix. 88 x. 9, 11, 41, 87, 108, 110, 112 3. Light Chain 3.1 Choose donor at 46, 48, 58 and 71 3.2 Check that the following have the same amino acid in donor and acceptor sequences, if not preferably choose donor: 2, 4, 6, 35, 38, 44, 47, 49, 62, 64-69 inclusive, 85, 87, 98, 99, 101 and 102 3.3 To further optimise affinity consider choosing donor residues at one, some or any of: i. 1, 3 ii. 63 iii. 60, if 60 and 54 are able to form potential saltbridge iv. 70, if 70 and 24 are able to form potential saltbridge v. 73, and 21 if 47 is different between donor and acceptor vi. 37, and 45 if 47 is different between donor and acceptor vii. 10, 12, 40, 80, 103, 105 Rationale In order to transfer the binding site of an antibody into a different acceptor framework, a number of factors need to be considered. 1. The extent of the CDRs The CDRs (Complementary Determining Regions) were defined by Wu and Kabat (refs. 4 and 5) on the basis of an analysis of the variability of different regions of antibody variable regions. Three regions per domain were recognised. In the light chain the sequences are 24-34, 50-56, 89-97 (numbering according to Kabat (ref. 4), Eu Index) inclusive and in the heavy chain the sequences are 31-35, 50-65 and 95-102 inclusive. When antibody structures became available it became apparent that these CDR regions corresponded in the main to loop regions which extended from the barrel framework of the light and heavy variable domains. For HI there was a discrepancy in that the loop was from 26 to 32 inclusive and for H2 the loop was 52 to 56-and for L2 from 50 to 53. However, with the exception of HI the CDR regions encompassed the loop regions and extended into the r) strand frameworks. In HI residue 26 tends to be a serine and 27 a phenylalanine or tyrosine, residue 29 is a phenylalanine in most cases. Residues 28 and 30 which are surface residues exposed to solvent might be involved in antigen-binding. A prudent definition of the HI CDR therefore would include residues 26-35 to include both the loop region and the hypervariable residues 33-35. It is of interest to note the example of Riechmann et al (ref. 3), who used the residue 31-35 choice for CDR-H1. In order to produce efficient antigen binding, residue 27 also needed to be recruited from the donor (rat) antibody. 2. Non-CDR residues which contribute to antigen binding By examination of available X-ray structures we have identified a number of residues which may have an effect on net antigen binding and which can be demonstrated by experiment. These residues can be sub-divided into a number of groups. 2.1 Surface residues near CDR [all numbering as in Kabat et al (ref. 7)]. 2.1.1. Heavy Chain - Key residues are 23, 71 and 73. Other residues which may contribute to a lesser extent are 1, 3 and 76. Finally 25 is usually conserved but the murine residue should be used if there is a difference. 2.1.2 Light Chain - Many residues close to the CDRs, e.g. 63, 65, 67 and 69 are conserved. If . conserved none of the surface residues in the light chain are likely to have a major effect. However, if the murine residue at these positions is unusual, then it would be of benefit to analyse the likely contribution more closely. Other residues which may also contribute to binding are 1 and 3, and also 60 and 70 if the residues at these positions and at 54 and 24 respectively are potentially able to form a salt bridge i.e. 60 + 54; 70 + 24. 2.2 Packing residues near the CDRs. 2.2.1. Heavy Chain - Key residues are 24, 49 and 78. Other key residues would be 36 if not a tryptophan, 94 if not an arginine, 104 and 106 if not glycines and 107 if not a threonine. Residues which may make a further contribution to stable packing of the heavy chain and hence improved affinity are 2, 4, 6, 38, 46, 67 and 69. 67 packs against the CDR residue 63 and this pair could be either both mouse or both human. Finally, residues which contribute to packing in this region but from a longer range are 18, 20, 80, 82 and 86. 82 packs against 67 and in turn 18 packs against 82. 80 packs against 69 and in turn 20 packs against 80. 86 forms an H bond network with 38 and 46. Many of the mouse-human differences appear minor e.g. Leu-Ile, but could have an minor impact on correct packing which could translate into altered positioning of the CDRs. 2.2.2. Light Chain - Key residues are 48, 58 and 71. Other key residues would be 6 if not glutamine, 35 if not tryptophan, 62 if not phenylalanine or tryosine, 64, 66, 68, 99 and 101 if not glycines and 102 if not a threonine. Residues which make a further contribution are 2, 4, 37, 45 and* 47. Finally residues 73 and 21 and 19 may make long distance packing contributions of a minor nature. 2.3. Residues at the variable domain interface between heavy and light chains - In both the light and heavy chains most of the non-CDR interface residues are conserved. If a conserved residue is replaced by a residue of different character, e.g. size or charge, it should be considered for retention as the murine residue. 2.3.1. Heavy Chain - Residues which need to be considered are 37 if the residue is not a valine but is of larger side chain volume or has a charge or polarity. Other residues are 39 if not a glutamine, 45 if not a leucine, 47 if not a tryptophan, 91 if not a phenylalanine or tyrosine, 93 if not an alanine and 103 if not a tryptophan. Residue 89 is also at the interface but is not in a position where the side chain could be of great impact. 2.3.2. Light Chain - Residues which need to be considered are 36, if not a tyrosine, 38 if not a glutamine, 44 if not a proline, 46, 49 if not a tyrosine, residue 85, residue 87 if not a tyrosine and 98 if not a phenylalanine. 2.4. Variable-Constant region interface - The elbow angle between variable and constant regions may be affected by alterations in packing of key residues in the variable region against the constant region which may affect the position of VL and Vg with respect to one another. Therefore it is worth noting the residues likely to be in contact with the constant region. In the heavy chain the surface residues potentially in contact with the variable region are conserved between mouse and human antibodies therefore the variable region contact residues may influence the V-C interaction. In the light chain the amino acids found at a number of the constant region contact points vary, and the V & C regions are not in such close proximity as the heavy chain. Therefore the influences of the light chain V-C interface may be minor. 2.4.1. Heavy Chain - Contact residues are 7, 11, 41, 87, 108, 110, 112. 2.4.2. Light Chain - In the light chain potentially contacting residues are 10, 12, 40, 80, 83, 103 and 105. The above analysis coupled with our considerable practical experimental experience in the CDR-grafting of a number of different antibodies have lead us to the protocol given above. The present invention is now described, by way of example only, with reference to the accompanying Figures 1 - 13. Brief Description of the Figures Figure 1 shows DNA and amino acid sequences of the OKT3 light chain; Figure 2 shows DNA and amino acid sequences of the OKT3 heavy chain; Figure 3 shows the alignment of the OKT3 light variable region amino acid sequence with that of the light variable region of the human antibody REI; Figure 4 shows the alignment of the OKT3 heavy variable region amino acid sequence with that of the heavy variable region of the human antibody KOL; Figure 5 shows the heavy variable region amino acid sequences of 0KT3, KOL and various corresponding CDR grafts; Figure 6 shows the light variable region amino acid sequences of 0KT3, REI and various corresponding CDR grafts; Figure 7 shows a graph of binding assay results for various grafted 0KT3 antibodies' Figure 8 shows a graph of blocking assay results for various grafted 0KT3 antibodies; Figure 9 shows a similar graph of blocking assay results; Figure 10 shows similar graphs for both binding assay and blocking assay results; Figure 11 shows further similar graphs for both binding assay and blocking assay results; Figure 12 shows a graph of competition assay results for a minimally grafted OKT3 antibody compared with the OKT3 murine reference standard, and Figure 13 shows a similar graph of competition assay results comparing a fully grafted 0KT3 antibody with the murine reference standard. DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION EXAMPLE 1 CDR-GRAFTING OF OKT3 MATERIAL AND METHODS 1. INCOMING CELLS Hybridoma cells producing antibody OKT3 were provided by Ortho (seedlot 4882.1) and were grown up in antibiotic free Dulbecco's Modified Eagles Medium (DMEM) supplemented with glutamine and 5% foetal calf serum, and divided to provide both an overgrown supernatant for evaluation and cells for extraction of RNA. The overgrown supernatant was shown to contain 250 ug/mL murine IgG2a/kappa antibody. The supernatant was negative for murine lambda light chain and IgGl, IgG2b, IgG3, IgA and IgM heavy chain. 20mL of supernatant was assayed to confirm that the antibody present was 0KT3. 2. MOLECULAR BIOLOGY PROCEDURES Basic molecular biology procedures were as described in Maniatis et al (ref. 9) with, in some cases, minor modifications. DNA sequencing was performed as described in Sanger et al (ref. 11) and the Amersham International Pic sequencing handbook. Site directed mutagenesis was as described in Kramer et al (ref. 12) and the Anglian Biotechnology Ltd. handbook. COS cell expression and metabolic labelling studies were as described in Whittle et al (ref. 13) 3. RESEARCH ASSAYS 3.1. ASSEMBLY ASSAYS Assembly assays were performed on supernatants from transfected COS cells to determine the .amount of intact IgG present. 3.1.1. COS CELLS TRANSFECTED WITH MOUSE OKT3 GENES The assembly assay for intact mouse IgG in COS cell supernatants was an ELISA with the following format: 96 well microtitre plates were coated with F(ab')2 goat anti-mouse IgG Fc. The plates were washed in water and samples added for 1 hour at room temperature. The plates were washed and F(ab')2 goat anti-mouse IgG F(ab')2 (HRPO conjugated) was then added. Substrate was added to reveal the reaction. UPC10, a mouse IgG2a myeloma, was used as a standard. 3.1.2. COS AND CHO CELLS TRANSFECTED WITH CHIMERIC OR CDR-GRAFTED 0KT3 GENES The assembly assay for chimeric or CDR-grafted antibody in COS cell supernatants was an ELISA with the following format: 96 well microtitre plates were coated with F(ab')2 goat anti-human IgG Fc. The plates were washed and samples added and incubated for 1 hour at room temperature. The plates were washed and monoclonal mouse anti-human kappa chain was added for 1 hour at room temperature. The plates were washed and F(ab')2goat anti-mouse IgG Fc (HRPO conjugated) was added. Enzyme substrate was added to reveal the reaction. Chimeric B72.3 (IgG4) (ref. 13) was used as a standard. The use of a monoclonal anti-kappa chain in this assay allows grafted antibodies to be read from the chimeric standard. 3.2. ASSAY FOR ANTIGEN BINDING ACTIVITY Material from COS cell supernatants was assayed for OKT3 antigen binding activity onto CD3 positive cells in a direct assay. The procedure was as follows: HUT 78 cells (human T cell line, CD3 positive) were maintained in culture. Monolayers of HUT 78 cells were prepared onto 96 well ELISA plates using poly-L-lysine and glutaraldehyde. Samples were added to the monolayers for 1 hour at room temperature. The plates were washed gently using PBS. F(ab')2 goat anti-human IgG Fc (HRPO conjugated) or F(ab')2 goat anti-mouse IgG Fc (HRPO conjugated) was added as appropriate for humanised or mouse samples. Substrate was added to reveal the reaction. The negative control for the cell-based assay was chimeric B72.3. The positive control was mouse Orthomune 0KT3 or chimeric 0KT3, when available. This cell-based assay was difficult to perform, and an alternative assay was developed for CDR-grafted 0KT3 which was more sensitive and easier to carry out. In this system CDR-grafted OKT3 produced by COS cells was tested for its ability to bind to the CD3-positive HPB-ALL (human peripheral blood acute lymphocytic leukemia) cell line. It was also tested for its ability to block the binding of murine OKT3 to these cells. Binding was measured by the following procedure: HPB-ALL cells were harvested from tissue culture. Cells were incubated at 4°C for 1 hour with various dilutions of test antibody, positive control antibody, or negative control antibody. The cells were washed once and incubated at 4°C for 1 hour with an FITC-labelled goat anti-human IgG (Fc- specific, mouse absorbed) . The cells were washed twice and analysed by cytofluorography. Chimeric 0KT3 was used as a positive control for direct binding. Cells incubated with mock- transfected COS cell supernatant, followed by the FITC-labelled goat anti-human IgG, provided the negative control. To test the ability of CDR-grafted 0KT3 to block murine 0KT3 binding, the HPB-ALL cells were incubated at 4°C for 1 hour with various dilutions of test antibody or control antibody. A fixed saturating amount of FITC 0KT3 was added. The samples were incubated for 1 hour at 4°C, washed twice and analysed by cytofluorography. FITC-labelled 0KT3 was used as a positive control to determine maximum binding. Unlabelled murine 0KT3 served as a reference standard for blocking. Negative controls were unstained cells with or without mock-transfeeted cell supernatant. The ability of the CDR-grafted OKT3 light chain to bind CD3-positive cells and block the binding of murine OKT3 was initially tested in combination with the chimeric 0KT3 heavy chain. The chimeric OKT3 heavy chain is composed of the murine 0KT3 variable region and the human IgG4 constant region. The chimeric heavy chain gene is expressed in the same expression vector used for the CDR-grafted genes. The CDR-grafted light chain expression vector and the chimeric heavy chain expression vector were co-transfected into COS cells. The fully chimeric OKT3 antibody (chimeric light chain and chimeric heavy chain) was found to be fully capable of binding to CD3 positive cells and blocking the binding of murine 0KT3 to these cells. 3.3 DETERMINATION OF RELATIVE BINDING AFFINITY The relative binding affinities of CDR-grafted anti-CD3 monoclonal antibodies were determined by competition binding (ref. 6) using the HPB-ALL human T cell line as a source of CD3 antigen, and fluorescein-conjugated murine 0KT3 (F1-0KT3) of known binding affinity as a tracer antibody. The binding affinity of F1-OKT3 tracer antibody was determined by a direct binding assay in which increasing amounts of F1-0KT3 were incubated with HPB-ALL (5xl0 5 ) in PBS with 5% foetal calf serum for 60 min. at 4°C. Cells were washed, and the fluorescence intensity was determined on a FACScan flow cytometer calibrated with quantitative microbead standards (Flow Cytometry Standards, Research Triangle Park, NC). Fluorescence intensity per antibody molecule (F/P ratio) was determined by using microbeads which have a predetermined number of mouse IgG antibody binding sites (Simply Cellular beads. Flow Cytometry Standards). F/P equals the fluorescence intensity of beads saturated with F1-0KT3 divided by the number of binding sites per bead. The amount of bound and free F1-0KT3 was calculated from the mean fluorescence intensity per cell, and the ratio of bound/free was plotted against the number of moles of antibody bound. A linear fit was used to determine the affinity of binding (absolute value of the slope) . For competitive binding, increasing amounts of competitor antibody were added to a sub-saturating dose of F1-OKT3 and incubated with 5xl0 5 HPB-ALL in 200 mi of PBS with 5% foetal calf serum, for 60 min at 4°C. The fluorescence intensities of the cells were measured on a FACScan flow cytometer calibrated with quantitative microbead standards. The concentrations of bound and free F1-0KT3 were calculated. The affinities of competing anti- bodies were calculated from the equation [X]-[0KT3] = (1/Kx) - (1/Ka), where Ka is the affinity of murine 0KT3, Kx is the affinity of competitor X, [ ] is the concentration of competitor antibody at which bound/free binding is R/2, and R is the maximal bound/free binding. 4. cDNA LIBRARY CONSTRUCTION 4.1. mRNA PREPARATION AND cDNA SYNTHESIS OKT3 producing cells were grown as described above and 1.2 x 10**- 1 cells harvested and mRNA extracted using the guanidinium/LiCl extraction procedure. cDNA was prepared by priming from Oligo-dT to generate full length cDNA. The cDNA was methylated and EcoRI linkers added for cloning. 4.2. LIBRARY CONSTRUCTION The cDNA library was ligated to pSP65 vector DNA which had been EcoRI cut and the 5' phosphate groups removed by calf intestinal phosphatase (EcoRI/CIP) . The ligation was used to transform high transformation efficiency Escherichia coli (E.coli) HB101. A cDNA library was prepared. 3600 colonies were screened for the light chain and 10000 colonies were screened for the heavy chain. 5. SCREENING E.coli colonies positive for either heavy or light chain probes were identified by oligonucleotide screening using the oligonucleotides: 5' TCCAGATGTTAACTGCTCAC for the light chain, which is complementary to a sequence in the mouse kappa constant region, and 5' CAGGGGCCAGTGGATGGATAGAC for the heavy chain which is complementary to a sequence in the mouse IgG2a constant CHI domain region. 12 light chain and 9 heavy chain clones were identified and taken for second round screening. Positive clones from the second round of screening were grown up and DNA prepared. The sizes of the gene inserts were estimated by gel electrophoresis and inserts of a size capable of containing a full length cDNA were subcloned into Ml3 for DNA sequencing. 6. DNA SEQUENCING Clones representing four size classes for both heavy and light chains were obtained in M13. DNA sequence for the 5' untranslated regions, signal sequences, variable regions and 3' untranslated regions of full length cDNAs [Figures 1(a) and 2(a)] were obtained and the corresponding amino acid sequences predicted [(Figures 1(b) and 2(b)]. In Figure 1(a) the untranslated DNA regions are shown in uppercase, and in both Figures 1 and 2 the signal sequences are underlined. 7. CONSTRUCTION OF cDNA EXPRESSION VECTORS Celltech expression vectors are based on the plasmid pEE6hCMV (ref. 14). A polylinker for the insertion of genes to be expressed has been introduced after the major immediate early promoter/enhancer of the human Cytomegalovirus (hCMV) . Marker genes for selection of the plasmid in transfected eukaryotic cells can be inserted as BamHI cassettes in the unique BamHI site of pEE6 hCMV; for instance, the neo marker to provide pEE6 hCMV neo. It is usual practice to insert the neo and gpt markers prior to insertion of the gene of interest, whereas the GS marker is inserted last because of the presence of internal EcoRI sites in the cassette. The selectable markers are expressed from the SV40 late promoter which also provides an origin of replication so that the vectors can be used for expression in the COS cell transient expression system. The mouse sequences were excised from the M13 based vectors described above as EcoRI fragments and cloned into either pEE6-hCMV-neo for the heavy chain and into EE6-hCMV-gpt for the light chain to yield vectors pJAl36 and pJA135 respectively. 8. EXPRESSION OF cDNAS IN COS CELLS Plasmids pJA135 and pJAl36 were co-transfected into COS cells and supernatant from the transient expression experiment was shown to contain assembled antibody which bound to T-cell enriched lymphocytes. Metabolic labelling experiments using ~-~-S methionine showed expression and assembly of heavy and light chains. 9. CONSTRUCTION OF CHIMERIC GENES Construction of chimeric genes followed a previously described strategy [Whittle et al (ref. 13)]. A restriction site near the 3' end of the variable domain sequence is identified and used to attach an oligonucleotide adapter coding for the remainder of the mouse variable region and a suitable restriction site for attachment to the constant region of choice. 9.1. LIGHT CHAIN GENE CONSTRUCTION The mouse light chain cDNA sequence contains an Aval site near the 3' end of the variable region [Fig. 1(a)]. The majority of the sequence of the variable region was isolated as a 396 bp. EcoRl-Aval fragment. An oligonucleotide adapter was designed to replace the remainder of the 3' region of the variable region from the Aval site and to include the 5' residues of the human constant region up to and including a unique Narl site which had been previously engineered into the constant region. A Hindlll site was introduced to act as a marker for insertion of the linker. The linker was ligated to the VL fragment and the 413 bp EcoRl-Narl adapted fragment was purified from the ligation mixture. The constant region was isolated as an Narl-BamHl fragment from an Ml3 clone NW361 and was ligated with the variable region DNA into an EcoRI/BamHI/CIP pSP65 treated vector in a three way reaction to yield plasmid JA143. Clones were isolated after transformation into E.coli and the linker and junction sequences were confirmed by the presence of the Hindlll site and by DNA sequencing. 9.2 LIGHT CHAIN GENE CONSTRUCTION - VERSION 2 The construction of the first chimeric light chain gene produces a fusion of mouse and human amino acid sequences at the variable-constant region junction. In the case of the OKT3 light chain the amino acids at the chimera junction are: Leu-Glu-Ile-Asn-Arg/ -/Thr-Val-Ala -Ala VARIABLE CONSTANT This arrangement of sequence introduces a potential site for Asparagine (Asn) linked (N-linked) glycosylation at the V-C junction. Therefore, a second version of the chimeric light chain oligonucleotide adapter was designed in which the threonine (Thr) , the first amino acid of the human constant region, was replaced with the equivalent amino acid from the mouse constant region, Alanine (Ala) . - 34 - An internal Hindlll site was not included in this adapter, to differentiate the two chimeric light chain genes. The variable region fragment was isolated as a 376 bp EcoRl-Aval fragment. The oligonucleotide linker was ligated to Narl cut pNW361 and then the adapted 396bp constant region was isolated after recutting the modified pNW361 with EcoRI. The variable region fragment and the modified constant region fragment were ligated directly into EcoRl/ClP treated pEEδhCMVneo to yield pJA137. Initially all clones examined had the insert in the incorrect orientation. Therefore, the insert was re-isolated and recloned to turn the insert round and yield plasmid pJAl41. Several clones with the insert in the correct orientation were obtained and the adapter sequence of one was confirmed by DNA sequencing 9.3. HEAVY CHAIN GENE CONSTRUCTION 9.3.1. CHOICE OF HEAVY CHAIN GENE ISOTYPE The constant region isotype chosen for the heavy chain was human IgG4. 9.3.2. GENE CONSTRUCTION The heavy chain cDNA sequence showed a Banl site near the 3' end of the variable region [Fig. 2(a)] The majority of the sequence of the variable region was isolated as a 426bp. EcoRl/ClP/Banl fragment. An oligonucleotide adapter was designated to replace the remainder of the 3' region of the variable region from the Banl site up to and including a unique Hindlll site which had been previously engineered into the first two amino acids of the constant region. The linker was ligated to the Vg fragment and the EcoRl-Hindlll adapted fragment was purified from the ligation mixture. The variable region was ligated to the constant region by cutting pJA91 with EcoRI and Hindlll removing the intron fragment and replacing it with the Vg to yield pJAl42. Clones were isolated after transformation into E.coli JM101 and the linker* and junction sequences were confirmed by DNA sequencing. (N.B. The Hindlll site is lost on cloning) . 10. CONSTRUCTION OF CHIMERIC EXPRESSION VECTORS 10.1. neo AND gpt VECTORS The chimeric light chain (version 1) was removed from pJAl43 as an EcoRI fragment and cloned into EcoRl/ClP treated pEE6hCMVneo expression vector to yield pJA145. Clones with the insert in the correct orientation were identified by restriction mapping. The chimeric light chain (version 2) was constructed as described above. The chimeric heavy chain gene was isolated from pJA142 as a 2.5Kbp EcoRl/BamHl fragment and cloned into the EcoRl/Bcll/ClP treated vector fragment of a derivative of pEE6hCMVgpt to yield plasmid PJA144. 10.2. GS SEPARATE VECTORS GS versions of pJAl41 and pJA144 were constructed by replacing the neo and gpt cassettes by a BamHl/Sall/ClP treatment of the plasmids, isolation of the vector fragment and ligation to a GS-containing fragment from the plasmid pR049 to yield the light chain vector pJA179 and the heavy chain vector pJAlδO. 10.3. GS SINGLE VECTOR CONSTRUCTION Single vector constructions containing the cL (chimeric light), cH (chimeric heavy) and GS genes on one plasmid in the order cL-cH-GS, or cH-cL-GS and with transcription of the genes being head to tail e.g. cL>cH>GS were constructed. These plasmids were made by treating pJAl79 or pJA180 with BamHl/ClP and ligating in a Bglll/Hindlll hCMV promoter cassette along with either the Hindi11/BamHI fragment from pJAl41 into pJA180 to give the cH-cL-GS plasmid pJA182 or the Hindlll/BamHl fragment from pJAl44 into pJA179 to give the cL-cH-GS plasmid pJA181. 11. EXPRESSION OF CHIMERIC GENES 11.1. EXPRESSION IN COS CELLS The chimeric antibody plasmid pJA145 (cL) and pJAl44 (cH) were co-transfected into COS cells and supernatant from the transient expression experiment was shown to contain assembled antibody which bound to the HUT 78 human T-cell line. Metabolic labelling experiments using 35g methionine showed expression and assembly of heavy and light chains. However the light chain mobility seen on reduced gels suggested that the potential glycosylation site was being glycosylated. Expression in COS cells in the presence of tunicamycin showed a reduction in size of the light chain to that shown for control chimeric antibodies and the OKT3 mouse light chain. Therefore JA141 was constructed and expressed. In this case the light chain did not show an aberrant mobility or a size shift in the presence or absence of tunicamycin. This second version of the chimeric light chain, when expressed in association with chimeric heavy (cH) chain, produced antibody which showed good binding to HUT 78 cells. In both cases antigen binding was equivalent to that of the mouse antibody. 11.2 EXPRESSION IN CHINESE HAMSTER OVARY (CHO) CELLS Stable cell lines have been prepared from plasmids PJA141/pJA144 and from pJA179 pJA180, pJAl81 and pJAl82 by transfection into CHO cells. 12. CDR-GRAFTING The approach taken was to try to introduce sufficient mouse residues into a human variable region framework to generate antigen binding activity comparable to the mouse and chimeric antibodies. 12.1. VARIABLE REGION ANALYSIS From an examination of a small database of structures of antibodies and antigen-antibody complexes it is clear that only a small number of antibody residues make direct contact with antigen. Other residues may contribute to antigen binding by positioning the contact residues in favourable configurations and also by inducing a stable packing of the individual variable domains and stable interaction of the light and heavy chain variable domains. The residues chosen for transfer can be identified in a number of ways: (a) By examination of antibody X-ray crystal structures the antigen binding surface can be predominantly located on a series of loops, three per domain, which extend from the B-barrel framework. (b) By analysis of antibody variable domain sequences regions of hypervariability [termed the Complementarity Determining Regions (CDRs) by Wu and Kabat (ref.- 5)] can be identified. In the most but not all cases these CDRs correspond to, but extend a short way beyond, the loop regions noted above. (c) Residues not identified by (a) and (b) may contribute to antigen binding directly or indirectly by affecting antigen binding site topology, or by inducing a stable packing of the individual variable domains and stabilising the inter-variable domain interaction. These residues may be identified either by superimposing the sequences for a given antibody on a known structure and looking at key residues for their contribution, or by sequence alignment analysis and noting "idiosyncratic" residues followed by examination of their structural location and likely effects. 12.1.1. LIGHT CHAIN Figure 3 shows an alignment of sequences for the human framework region REI and the 0KT3 light variable region. The structural loops (LOOP) and CDRs (KABAT) believed to correspond to the antigen binding region are marked. Also marked are a number of other residues which may also contribute to antigen binding as described in 13.1(c). Above the sequence in Figure 3 the residue type indicates the spatial location of each residue side chain, derived by examination of resolved structures from X-ray crystallography analysis. The key to this residue type designation is as follows: N - near to CDR (From X-ray Structures) P - Packing B - Buried Non-Packing S - Surface E - Exposed I - Interface * - Interface - - Packing/Part Exposed ? - Non-CDR Residues which may require to be left as Mouse sequence. Residues underlined in Figure 3 are amino acids. REI was chosen as the human framework because the light chain is a kappa chain and the kappa variable regions show higher homology with the mouse sequences than a lambda light variable region, e.g. KOL (see below). REI was chosen in preference to another kappa light chain because the X-ray structure of the light chain has been , determined so that a structural examination of individual residues could be made. 12.1.2. HEAVY CHAIN Similarly Figure 4 shows an alignment of sequences for the human framework region KOL and the 0KT3 heavy variable region. The structural loops and CDRs believed to correspond to the antigen binding region are marked. Also marked are a number of other residues which may also contribute to antigen binding as described in 12.1(c). The residue type key and other indicators used in Figure 4 are the same as those used in Figure 3. KOL was chosen as the heavy chain framework because the X-ray structure has been determined to a better resolution than, for example, NEWM and also the sequence alignment of 0KT3 heavy variable region showed a slightly better homology to KOL than to NEWM. 12.2. DESIGN OF VARIABLE GENES The variable region domains were designed with mouse variable region optimal codon usage [Grantham and Perrin (ref. 15)] and used the B72.3 signal sequences [Whittle et al (ref. 13)]. The sequences were designed to be attached to the constant region in the same way as for the chimeric genes described above. Some constructs contained the "Kozak consensus sequence" [Kozak (ref. 16)] directly linked to the 5' of the signal sequence in the gene. This sequence motif is believed to have a beneficial role in translation initiation in eukaryotes. 12.3. GENE CONSTRUCTION To build the variable regions, various strategies are available. The sequence may be assembled by using oligonucleotides in a manner similar to Jones et al (ref. 17) or by simultaneously replacing all of the CDRs or loop regions by oligonucleotide directed site specific mutagenesis in a manner similar to Verhoeyen et al (ref. 2). Both strategies were used and a list of constructions is set out in Tables 1 and 2 and Figures 4 and 5. It was noted in several cases that the mutagenesis approach led to deletions and rearrangements in the gene being remodelled, while the success of the assembly approach was very sensitive to the quality of the oligonucleotides. 13. CONSTRUCTION OF EXPRESSION VECTORS Genes were isolated from M13 or SP65 based intermediate vectors and cloned into pEE6hCMVneo for the light chains and pEE6hCMVgpt for the heavy chains in a manner similar to that for the chimeric genes as described above. TABLE 1 CDR-GRAFTED GENE CONSTRUCTS CODE MOUSE SEQUENCE METHOD OF KOZAK CONTENT CONSTRUCTION SEQUENCE LIGHT CHAIN ALL HUMAN FRAMEWORK REI 121 26-32, 50-56, 91-96 inclusive SDM and gene assembly + n.d. 121A 26-32, 50-56, 91-96 inclusive Partial gene assembly n.d. + +1, 3, 46, 47 121B 26-32, 50-56, 91 -96 inclusive Partial gene assembly n.d. + + 46, 47 221 24-24, 50-56, 91- -96 inclusive Partial gene assembly + + 221A 24-34, 50-56, 91--96 inclusive Partial gene assembly + + +1, 3, 46, 47 221B 24-34, 50-56, 91- 96 inclusive Partial gene assembly + + +1, 3 221C 24-34, 50-56, 91- 96 inclusive Partial gene assembly + + HEAVY CHAIN ALL HUMAN FRAMEWORK KOL 121 26-32, 50-56, 95-100B inclusive Gene assembly n.d. + 131 26-32, 50-58, 95-100B inclusive Gene assembly n.d. + 141 26-32, 50-65, 95-100B inclusive Partial gene assembly + n.d. 321 26-35, 50-56, 95-100B inclusive Partial gene assembly + n.d. 331 26-35, 50-58, 95-100B inclusive Partial gene assembly + Gene assembly + 341 26-35, 50-65, 95-100B inclusive SDM + Partial gene assembly + 341A 26-35, 50-65, 95-100B inclusive Gene assembly n.d. +6, 23, 24, 48, 49, 71, 73, 76, 78, 88, 91 (+63 - human) 341B 26-35, 50-65, 95-100B inclusive Gene assembly n.d. + + 48, 49, 71, 73, 76, 78, 88, 91 (+63 + human) KEY n.d not done SDM Site directed mutagenesis Gene assembly Variable region assembled entirely from oligonucleotides Partial gene Variable region assembled by combination of restriction assembly fragments either from other genes originally created by SDM and gene assembly or by oligonucleotide assembly of part of the variable region and reconstruction with restriction fragments from other genes originally created by SDM and gene assembly 14. EXPRESSION OF CDR-GRAFTED GENES 14.1. PRODUCTION OF ANTIBODY CONSISTING OF GRAFTED LIGHT (gL) CHAINS WITH MOUSE HEAVY (mH) OR CHIMERIC HEAVY (cH) CHAINS All gL chains, in association with mH or cH produced reasonable amounts of antibody. Insertion of the Kozak consensus sequence at a position 5' to the ATG (kgL constructs) however, led to a 2-5 fold improvement in net expression. Over an extended series of experiments expression levels were raised from approximately 200ng/ml to approximately 500 ng/ l for kgL/cH or kgL/mH combinations. When direct binding to antigen on HUT 78 cells was measured, a construct designed to include mouse sequence based on loop length (gL121) did not lead to active antibody in association with mH or cH. A construct designed to include mouse sequence based on Kabat CDRs (gL221) demonstrated some weak binding in association with mH or cH. However, when framework residues 1, 3, 46, 47 were changed from the human to the murine 0KT3 equivalents based on the arguments outlined in Section 12.1 antigen binding was demonstrated when both of the new constructs, which were termed 121A and 221A were co-expressed with cH. When the effects of these residues were examined in more detail, it appears that residues 1 and 3 are not major contributing residues as the product of the gL221B gene shows little detectable binding activity in association with cH. The light chain product of gL221C, in which mouse sequences are present at 46 and 47, shows good binding activity in association with cH. 14.2 PRODUCTION OF ANTIBODY CONSISTING OF GRAFTED HEAVY (gH) CHAINS WITH MOUSE LIGHT (mL) OR CHIMERIC LIGHT (cL) CHAINS Expression of the gH genes proved to be more difficult to achieve than for gL. First, inclusion of the Kozak sequence appeared to have no marked effect on expression of gH genes. Expression appears to be slightly improved but not to the same degree as seen for the grafted light chain. Also, it proved difficult to demonstrate production of expected quantities of material when the loop choice (amino acid 26-32) for CDR1 is used, e.g. gHl21, 131, 141 and no conclusions can be drawn about these constructs. Moreover, co-expression of the gH341 gene with cL or mL has been variable and has tended to produce lower amounts of antibody than the cH/cL or mH/mL combinations. The alterations to gH341 to produce gH34lA and gH341B lead to improved levels of expression. This may be due either to a general increase in the fraction of mouse sequence in the variable region, or to the alteration at position 63 where the residue is returned to the human amino acid Valine (Val) from Phenylalanine (Phe) to avoid possible internal packing problems with the rest of the human framework. This arrangement also occurs in gH331 and gH321. When gH321 or gH331 were expressed in association with cL, antibody was produced but antibody binding activity was not detected. When the more conservative gH341 gene was used antigen binding could be detected in association with cL or mL, but the activity was only marginally above the background level. When further mouse residues were substituted based on the arguments in 12.1, antigen binding could be clearly demonstrated for the antibody produced when kgH341A and kgH341B were expressed in association with cL. 14.3 PRODUCTION OF FULLY CDR-GRAFTED ANTIBODY The kgL221A gene was co-expressed with kgH341, kgH341A or kgH34lB. For the combination kgH221A/kgH341 very little material was produced in a normal COS cell expression. For the combinations kgL22lA/kgH34lA or kgH221A/kgH341B amounts of antibody similar to gL/cH was produced. In several experiments no antigen binding activity could be detected with kgH221A/gH341 or kgH221A/kgH341 combinations, although expression levels were very low. Antigen binding was detected when kgL22lA/kgH34lA or kgH22lA/kgH341B combinations were expressed. In the case of the antibody produced from the kgL221A/kgH341A combination the antigen binding was very similar to that of the chimeric antibody. An analysis of the above results is given below. 15. DISCUSSION OF CDR-GRAFTING RESULTS In the design of the fully humanised antibody the aim was to transfer the minimum number of mouse amino acids that would confer antigen binding onto a human antibody framework. 15.1. LIGHT CHAIN 15.1.1. EXTENT OF THE CDRs For the light chain the regions defining the loops known from structural studies of other antibodies to contain the antigen contacting residues, and those hypervariable sequences defined by Kabat et al (refs. 4 and 5) as Complementarity Determining Regions (CDRs) are equivalent for CDR2. For CDR1 the hypervariable region extends from residues 24-34 inclusive while the structural loop extends from 26-32 inclusive. In the case of OKT3 there is only one amino acid difference between the two options, at amino acid 24, where the mouse sequence is a serine and the human framework REI has glutamine. For CDR3 the loop extends from residues 91-96 inclusive while the Kabat hypervariability extends from residues 89-97 inclusive. For 0KT3 amino acids 89, 90 and 97 are the same between OKT3 and REI (Fig. 3). When constructs based on the loop choice for CDR1 (gLl21) and the Kabat choice (gL221) were made and co-expressed with mH or cH no evidence for antigen binding activity could be found for gL121, but trace activity could be detected for the gL221, suggesting that a single extra mouse residue in the grafted variable region could have some detectable effect. Both gene constructs were reasonably well expressed in the transient expression system. 15.1.2. FRAMEWORK RESIDUES The remaining framework residues were then further examined, in particular amino acids known from X-ray analysis of other antibodies to be close to the CDRs and also those amino acids which in OKT3 showed differences from the consensus framework for the mouse subgroup (subgroup VI) to which 0KT3 shows most homology. Four positions 1, 3, 46 and 47 were identified and their possible contribution was examined by substituting the mouse amino acid for the human amino acid at each position. Therefore gL221A (gL221 + D1Q, Q3V, L46R, L47W, see Figure 3 and Table 1) was made, cloned in EE6hCMVneo and co-expressed with cH (pJA144). The resultant antibody was well expressed and showed good binding activity. When the related genes gL221B (gL221 + D1Q, Q3V) and gL221C (gL221 + L46R, L47W) were made and similarly tested, while both genes produced antibody when co-expressed with cH, only the gL221C/cH combination showed good antigen binding. When the gL12lA (gL121 + D1Q, Q3V, L46R, L47W) gene was made and co-expressed with cH, antibody was produced which also bound to antigen. 15.2. HEAVY CHAIN 15.2.1. EXTENT OF THE CDRs For the heavy chain the loop and hypervariability analyses agree only in CDR3. For CDR1 the loop region extends from residues 26-32 inclusive whereas the Kabat CDR extends from residues 31-35 inclusive. For CDR2 the loop region is from 50-58 inclusive while the hypervariable region covers amino acids 50-65 inclusive. Therefore humanised heavy chains were constructed using the framework from antibody KOL and with various combinations of these CDR choices, including a shorter choice for CDR2 of 50-56 inclusive as there was some uncertainty as to the definition of the end point for the CDR2 loop around residues 56 to 58. The genes were co-expressed with mL or cL initially. In the case of the gH genes with loop choices for CDR1 e.g. gH121, gH131, gHl41 very little antibody was produced in the culture supernatants. As no free light chain was detected it was presumed that the antibody was being made and assembled inside the cell but that the heavy chain was aberrant in some way, possibly incorrectly folded, and therefore the antibody was being degraded internally. In some experiments trace amounts of antibody could be detected in ----S labelling studies. As no net antibody was produced, analysis of these constructs was not pursued further. When, however, a combination of the loop choice and the Kabat choice for CDRl was tested (mouse amino acids 26-35 inclusive) and in which residues 31 (Ser to Arg), 33 (Ala to Thr), and 35 (Tyr to His) were changed from the human residues to the mouse residue and compared to the first series, antibody was produced for gH321, kgH331 and kgH341 when co-expressed with cL. Expression was generally low and could not be markedly improved by the insertion of the Kozak consensus sequence 5' to the ATG of the signal sequence of the gene, as distinct from the case of the gL genes where such insertion led to a 2-5 fold increase in net antibody production. However, only in the case of gH341/mL or kgH341/cL could marginal antigen binding activity be demonstrated. When the kgH341 gene was co-expressed with kgL22lA, the net yield of antibody was too low to give a signal above the background level in the antigen binding assay. 15.2.2. FRAMEWORK RESIDUES As in the case of the light chain the heavy chain frameworks were re-examined. Possibly because of the lower initial homology between the mouse and human heavy variable domains compared to the light chains, more amino acid positions proved to be of interest. Two genes kgH34lA and kgH34lB were constructed, with 11 or 8 human residues respectively substituted by mouse residues compared to gH341, and with the CDR2 residue 63 returned to the human amino acid potentially to improve domain packing. Both showed antigen binding when combined with cL or kgL22lA, the kgH34lA gene with all 11 changes appearing to be the superior choice. 15.3 INTERIM CONCLUSIONS It has been demonstrated, therefore, for 0KT3 that to transfer antigen binding ability to the humanised antibody, mouse residues outside the CDR regions defined by the Kabat hypervariability or structural loop choices are required for both the light and heavy chains. Fewer extra residues are needed for the light chain, possibly due to the higher initial homology between the mouse and human kappa variable regions. Of the changes seven (1 and 3 from the light chain and 6, 23, 71, 73 and 76 from the heavy chain) are predicted from a knowledge of other antibody structures to be either partly exposed or on the antibody surface. It has been shown here that residues 1 and 3 in the light chain are not absolutely required to be the mouse sequence; and for the heavy chain the gH341B heavy chain in combination with the 221A light chain generated only weak binding activity. Therefore the presence of the 6, 23 and 24 changes are important to maintain a binding affinity similar to that of the murine antibody. It was important, therefore, to further study the individual contribution of othe other 8 mouse residues of the kgH34lA gene compared to kgH341. 16. FURTHER CDR-GRAFTING EXPERIMENTS Additional CDR-grafted heavy chain genes were prepared substantially as described above. With reference to Table 2 the further heavy chain genes were based upon the gh341 (plasmid pJA178) and gH341A (plasmid pJA185) with either mouse OKT3 or human KOL residues at 6, 23, 24, 48, 49, 63, 71, 73, 76, 78, 88 and 91, as indicated. The CDR- grafted light chain genes used in these further experiments were gL221, gL22lA, gL22lB and gL22lC as described above. TABLE 2 OKT3 HEAVY CHAIN CDR GRAFTS 1. gH341 and derivatives 2. gL221 and derivatives MURINE RESIDUES ARE UNDERLINED The CDR-grafted heavy and light chain genes were co-expressed in COS cells either with one another in various combinations but also with the corresponding murine and chimeric heavy and light chain genes substantially as described above. The resultant antibody products were then assayed in binding and blocking assays with HPB-ALL cells as described above. The results of the assays for various grafted heavy chains co-expressed with the gL221C light chain are given in Figures 7 and 8 (for the JA184, JA185, JA197 and JA198 constructs - see Table 2), in Figure 9 (for the JA183, JA184, JA185 and JA197 constructs) in Figure 10 (for the chimeric, JA185, JA199, JA204, JA205, JA207, JA208 and JA209 constructs) and in Figure 11 (for the JA183, JA184, JA185, JA198, JA203, JA205 and JA206 constructs). The basic grafted product without any human to murine changes in the variable frameworks, i.e. gL221 co-expressed with gh341 (JA178), and also the "fully grafted" product, having most human to murine changes in the grafted heavy chain framework, i.e. gL22lC co-expressed with gh341A (JA185), were assayed for relative binding affinity in a competition assay against murine OKT3 reference standard, using HPB-ALL cells. The assay used was as described above in section 3.3. The results obtained are given in Figure 12 for the basic grafted product and in Figure 13 for the fully grafted product. These results indicate that the basic grafted product has neglibible binding ability as compared with the OKT3 murine reference standard; whereas the "fully grafted" product has a binding ability very similar to that of the 0KT3 murine reference standard. The binding and blocking assay results indicate the following: The JA198 and JA207 constructs appear to have the best binding characteristics and similar binding- abilities, both substantially the same as the chimeric and fully grafted gH34lA products. This indicates that positions 88 and 91 and position 76 are not highly critical for maintaining the OKT3 binding ability; whereas at least some of positions 6, 23, 24, 48, 49, 71, 73 and 78 are more important. This is borne out by the finding that the JA209 and JA199, although of similar binding ability to one another, are of lower binding ability than the JA198 and JA20 constructs. This indicates the importance of having mouse residues at positions 71, 73 and 78, which are either completely or partially human in the JA199 and JA209 constructs respectively. Moreover, on comparing the results obtained for the JA205 and JA183 constructs it is seen that there is a decrease in binding going from the JA205 to the JA183 constructs. This indicates the importance of retaining a mouse residue at position 23, the only position changed between JA205 and JA183. These and other results lead us to the conclusion that of the 11 mouse framework residues used in the gH341A (JA185) construct, it is important to retain mouse residues at all of positions 6, 23, 24, 48 and 49, and possibly for maximum binding affinity at 71, 73 and 78. Similar Experiments were carried out to CDR-graft a number of the rodent antibodies including antibodies having specificity for CD4 (0KT4), ICAM-1 (R6-5), TAG72 (B72.3), and TNFa<(61E71, 101.4, hTNFl, hTNF2 and hTNF3) . EXAMPLE 2 CDR-GRAFTING OF A MURINE ANTI-CD4 T CELL RECEPTOR ANTIBODY, OKT4A Anti OKT4A CDR-grafted heavy and light chain genes were prepared, expressed and tested substantially as described above in Example 1 for CDR-grafted OKT3. The CDR grafting of OKT4A is described in detail in Ortho patent application PCT/GB 90 of even date herewith entitled "Humanised Antibodies". The disclosure of this Ortho patent application PCT/GB 90 is incorporated herein by reference. A number of CDR-grafted OKT4 antibodies have been prepared. Presently the CDR-grafted OKT4A of choice is the combination of the grafted light chain LCDR2 and the grafted heavy chain HCDR10. THE LIGHT CHAIN The human acceptor framework used for the grafted light chains was REI. The preferred LCDR2 light chain has human to mouse changes at positions 33, 34, 38, 49 and 89 in addition to the structural loop CDRs. Of these changed positions, positions 33, 34 and 89 fall within the preferred extended CDRs of the present invention (positions 33 and 34 in CDRl and position 89 in CDR3). The human to murine changes at positions 38 and 49 corresponds to positions at which the amino acid residues are preferably donor murine amino acid residues in accordance with the present invention. A comparison of the amino acid sequences of the donor murine light chain variable domain and the REI human acceptor light chain variable further reveals that the murine and human residues are identical at all of positions 46, 48 and 71 and at all of positions 2, 4, 6, 35, 36, 44, 47, 62, 64-69, 85, 87, 98, 99 and 101 and 102. However the amino acid residue at position 58 in LCDR2 is the human REI framework residue not the mouse OKT4 residue as would be preferred in accordance with the present invention. THE HEAVY CHAIN The human acceptor framework used for the grafted heavy chains was KOL. The preferred CDR graft HCDR10 heavy chain has human to mouse changes at positions 24, 35, 57, 58, 60, 88 and 91 in addition to the structural loop CDRs. Of these positions, positions 35 (CDRl) and positions 57, 58 and 60 (CDR2) fall within the preferred extended CDRs of the present invention. Also the human to mouse change at position 24 corresponds to a position at which the amino acid residue is a donor murine residue in accordance with the present invention. Moreover, the human to mouse changes at positions 88 and 91 correspond to positions at which the amino acid residues are optionally donor murine residues. Moreover, a comparison of the murine OKT4A and human KOL heavy chain variable amino acid sequences reveals that the murine and human residues are identical at all of positions 23, 49, 71, 73 and 78 and at all of positions 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107. Thus the OKT4A CDR-grafted heavy chain HCDR10 corresponds to a particularly preferred embodiment according to the present invention. EXAMPLE 3 CDR-GRAFTING OF AN ANTI-MUCIN SPECIFIC MURINE ANTIBODY, B72.3 The cloning of the genes coding for the anti-mucin specific murine monoclonal antibody B72.3 and the preparation of B72.3 mouse-human chimeric antibodies has been described previously (ref. 13 and WO 89/01783). CDR-grafted versions of B72.3 were prepared as follows, (a) B72.3 Light Chain CDR-grafting of this light chain was accomplished by direct transfer of the murine CDRs into the framework of the human light chain REI. The regions transferred were: CDR Number Residues 1 24-34 2 50-56 3 90-96 The activity of the resulting grafted light chain was assessed by co-expression in COS cells, of genes for the combinations: Supernatants were assayed for antibody concentration and for the ability to bind to microtitre plates coated with ucin. The results obtained indicated that, in combination with the B72.3 cH chain, B72.3 cL and B72.3 gL had similar binding properties. Comparison of the murine B72.3 and REI light chain amino acid sequences reveals that the residues are identical at positions 46, 58 and 71 but are different at position 48. Thus changing the human residue to the donor mouse residue at position 48 may further improve the binding characteristics of the CDR-grafted light chain, (B72.3 gL) in accordance with the present invention. (b) B72.3 heavy chain i. Choice of framework At the outset it was necessary to make a choice of human framework. Simply put, the question was as follows: Was it necessary to use the framework regions from an antibody whose crystal structure was known or could the choice be made on some other criteria? For B72.3 heavy chain, it was reasoned that, while knowledge of structure was important, transfer of the CDRs from mouse to human frameworks might be facilitated if the overall homology between the donor and receptor frameworks was maximised. Comparison of the B72.3 heavy chain sequence with those in Kabat (ref. 4) for human heavy chains showed clearly that B72.3 had poor homology for KOL and NEWM (for which crystal structures are available) but was very homologous to the heavy chain for EU. On this basis, EU was chosen for the CDR-grafting and the following residues transferred as CDRs. CDR Number Residues 1 27-36 2 50-63 3 93-102 Also it was noticed that the FR4 region of EU was unlike that of any other human (or mouse) antibody. Consequently, in the grafted heavy chain genes this was also changed to produce a "consensus" human sequence. (Preliminary experiments showed that grafted heavy chain genes containing the EU FR4 sequence expressed very poorly in transient expression systems.) ii * Results with grafted heavy chain genes Expression of grafted heavy chain genes containing all human framework regions with either gL or cL genes produced a grafted antibody with little ability to bind to mucin. The grafted antibody had about 1% the activity of the chimeric antibody. In these experiments, however, it was noted that the activity of the grafted antibody could be increased to,^ 10% of B72.3 by exposure to pHs of 2-3.5. This observation provided a clue as to how the activity of the grafted antibody could be improved without acid treatment. It was postulated that acid exposure brought about the protonation of an acidic residue (pKa of aspartic acid = 3.86 and of glutamine acid = 4.25) which in turn caused a change in structure of the CDR loops, or allowed better access of antigen. From comparison of the sequences of B72.3 (ref. 13) and EU (refs. 4 and 5), it was clear that, in going from the mouse to human frameworks, only two positions.had been changed in such a way that acidic residues had been introduced. These positions are at residues 73 and 81, where K to E and Q to E changes had been made, respectively. Which of these positions might be important was determined by examining the crystal structure of the KOL antibody. In KOL heavy chain, position 81 is far removed from either of the CDR loops. Position 73, however, is close to both CDRs 1 and 3 of the heavy chain and, in this position it was possible to envisage that a K to E change in this region could have a detrimental effect on antigen binding. iii. Framework changes in B72.3 gH gene On the basis of the above analysis, E73 was mutated to a lysine (K) . It was found that this change had a dramatic effect on the ability of the grafted Ab to bind to mucin. Further the ability of the grafted B72.3 produced by the mutated gH/gL combination to bind to mucin was similar to that of the B72.3 chimeric antibody. iv. Other framework changes In the course of the above experiments, other changes were made in the heavy chain framework regions. Within the accuracy of the assays used, none of the changes, either alone or together, appeared beneficial. v. Other All assays used measured the ability of the grafted Ab to bind to mucin and, as a whole, indicated that the single framework change at position 73 is sufficient to generate an antibody with similar binding properties to B72.3. Comparison of the B72.3 murine and EU heavy chain sequences reveals that the mouse and human residues are identical at positions 23, 24, 71 and 78. Thus the mutated CDR-grafted B72.3 heavy chain corresponds to a preferred embodiment of the present invention. EXAMPLE 4 CDR-GRAFTING OF A MURINE ANTI-ICAM-1 MONOCLONAL ANTIBODY A murine antibody, R6-5-D6 (EP 0314863) having specificity for Intercellular Adhesion Molecule 1 (ICAM-1) was CDR-grafted substantially as described above in previous examples. This work is described in greater detail in co-pending application, British Patent Application No. 9009549.8, the disclosure of which is incorporated herein by reference. The human EU framework was used as the acceptor framework for both heavy and light chains. The CDR-grafted antibody currently of choice is provided by co-expression of grafted light chain gL22lA and grafted heavy chain gH34lD which has a binding affinity for ICAM 1 of about 75% of that of the corresponding mouse-human chimeric antibody. LIGHT CHAIN gL22lA has murine CDRs at positions 24-34 (CDRl), 50-56 (CDR2) and 89-97 (CDR3). In addition several framework residues are also the murine amino acid. These residues were chosen after consideration of the possible contribution of these residues to domain packing and stability of the conformation of the antigen binding region. The residues which have been retained as mouse are at positions 2, 3, 48 (?), 60, 84, 85 and 87. Comparison of the murine anti-ICAM 1 and human EU light chain amino acid sequences reveals that the murine and human residues are identical at positions 46, 58 and 71. HEAVY CHAIN gH34lD has murine CDRs at positions 26-35 (CDRl), 50-56 (CDR2) and94-100B (CDR3). In addition murine residues were used in gH341D at positions 24, 48, 69, 71, 73, 80, 88 and 91. Comparison of the murine anti-ICAM 1 and human EU heavy chain amino acid sequences are identical at positions 23, 49 and 78. EXAMPLE 5 CDR-Grafting of murine anti-TNFa antibodies A number of murine anti-TNFa monoclonal antibodies were CDR-grafted substantially as described above in previous examples. These antibodies include the murine monoclonal antibodies designated 61 E71, hTNFl, hTNF3 and 101.4 A brief summary of the CDR-grafting of each of these antibodies is given below. 61E71 A similar analysis as described above (Example 1, Section 12.1.) was done for 61E71 and for the heavy chain 10 residues were identified at 23, 24, 48, 49, 68, 69, 71, 73, 75 and 88 as residues to potentially retain as murine. The human frameworks chosen for CDR-grafting of this antibody, and the hTNF3 and 101.4 antibodies were REI for the light chain and KOL for the heavy chain. Three genes were built, the first of which contained 23, 24, 48, 49, 71 and 73 [gH341(6)] as murine residues. The second gene also had 75 and 88 as murine residues [gH341(8)] while the third gene additionally had 68, 69, 75 and 88 as murine residues [gH341(10)]. Each was co-expressed with gL221, the minimum grafted light chain (CDRs only). The gL221/gH341(6) and gL221/gH341(8) antibodies both bound as well to TNF as murine 61E71. The gL221/gH341(10) antibody did not express and this combination was not taken further. Subsequently the gL221/gH341(6) antibody was assessed in an L929 cell competition assay in which the antibody competes against the TNF receptor on L929 cells for binding to TNF in solution. In this assay the gL221/gH341(6) antibody was approximately 10% as active as murine 61E71. hTNFl hTNFl is a monoclonal antibody which recognises an epitope on human TNF- . The EU human framework was used for CDR-grafting of both the heavy and light variable domains. Heavy Chain In the CDR-grafted heavy chain (ghTNFl) mouse CDRs were used at positions 26-35 (CDRl), 50-65 (CDR2) and 95-102 (CDR3). Mouse residues were also used in the frameworks at positions 48, 67, 69, 71, 73, 76, 89, 91, 94 and 108. Comparison of the TNF1 mouse and EU human heavy chain residues reveals that these are identical at positions 23, 24, 29 and 78. Light Chain In the CDR-grafted light chain (gLhTNFl) mouse CDRs wre used at positions 24-34 (CDRl), 50-56 (CDR2) and 89-97 (CDR3). In addition mouse residues were used in the frameworks at positions 3, 42, 48, 49, 83, 106 and 108. Comparison of the hTNFl mouse and EU human light chain residues reveals that these are identical at positions 46, 58 and 71. The grafted hTNFl heavy chain was co-expressed with the chimeric light chain and the binding ability of the product compared with that of the chimeric light chain/chimeric heavy chain product in a TNF binding assay. The grafted heavy chain product appeared to have binding ability for TNF slightly better than the fully chimeric product. Similarly, a grafted heavy chain/grafted light chain product was co-expressed and compared with the fully chimeric product and found to have closely similar binding properties to the latter product. hTNF3 hTNF3 recognises an epitope on human TNF-( .« The sequence of hTNF3 shows only 21 differences compared to 61E71 in the light and heavy chain variable regions, 10 in the light chain (2 in the CDRs at positions 50, 96 and 8 in the framework at 1, 19, 40, 45, 46, 76, 103 and 106) and 11 in the heavy chain (3 in the CDR regions at positions 52, 60 and 95 and 8 in the framework at 1, 10, 38, 40, 67, 73, 87 and 105). The light and heavy chains of the 61E71 and hTNF3 chimeric antibodies can be exchanged without loss of activity in the direct binding assay. However 61E71 is an order of magnitude less able to compete with the TNF receptor on L929 cells for TNF-a compared to hTNF3. Based on the 61E71 CDR grafting data gL221 and gH341(+23, 24, 48, 49 71 and 73 as mouse) genes have been built for hTNF3 and tested and the resultant grafted antibody binds well to TNF-a, but competes very poorly in the L929 assay. It is possible that in this case also the framework residues identified for OKT3 programme may improve the competitive binding ability of this antibody. 101.4 101.4 is a further murine monoclonal antibody able to recognise human TNF-a. The heavy chain of this antibody shows good homology to KOL and so the CDR-grafting has been based on REI for the light chain and KOL for the heavy chain. Several grafted heavy chain genes have been constructed with conservative choices for the CDR's (gH341) and which have one or a small number of non-CDR residues at positions 73, 78 or 77-79 inclusive, as the mouse amino acids. These have been co-expressed with cL or gL221. In all cases binding to TNF equivalent to the chimeric antibody is seen and when co-expressed with cL the resultant antibodies are able to compete well in the L929 assay. However, with gL221 the resultant antibodies are at least an order of magnitude less able to compete for TNF against the TNF receptor on L929 cells. Mouse residues at other positions in the heavy chain, for example, at 23 and 24 together or at 76 have been demonstrated to provide no improvement to the competitive ability of the grafted antibody in the L929 assay. A number of other antibodies including antibodies having specificity for interleukins e.g. ILl and cancer markers such as carcinoembryonic antigen (CEA) e.g. the monoclonal antibody A5B7 (ref. 21), have been successfully CDR-grafted according to the present invention. It will be appreciated that the foregoing examples are given by way of illustration only and are not intended to limit the scope of the claimed invention. Changes and modifications may be made to the methods described whilst still falling within the spirit and scope of the invention. References 1. Kohler & Milstein, Nature, 26_5, 295-497, 1975. 2. Chatenoud et al, (1986), J. Immunol. 137, 830-838. 3. Jeffers et al, (1986), Transplantation, Al , 572-578. 4. Begent et al, Br. J. Cancer 62: 487 (1990). 5. Verhoeyen et al. Science, 239, 1534-1536, 1988. 6. Riechmann et al. Nature, 332, 323-324, 1988. 7. Kabat, E.A., Wu, T.T., Reid-Miller, M. , Perry, H.M., Gottesman, K.S., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA. 8. Wu, T.T., and Kabat, E.A., 1970, J. Exp. Med. 132 211-250. 9. Queen et al, (1989), Proc. Natl. Acad. Sci. USA, 8 , 10029-10033 and WO 90/07861 10. Maniatis et al. Molecular Cloning, Cold Spring Harbor, New York, 1989. 11. Primrose and Old, Principles of Gene Manipulation, Blackwell, Oxford, 1980. 12. Sanger, F., Nicklen, S., Coulson, A.R. , 1977, Proc. Natl. Acad. Sci. USA, 74 5463 13. Kramer, W. , Drutsa, V., Jansen, H.-W., Kramer, B., Plugfelder, M. , Fritz, H.-J., 1984, Nucl. Acids Res. 11, 9441 14. Whittle, N., Adair, J. , Lloyd, J.C., Jenkins, E., Devine, J. , Schlom, J. , Raubitshek, A., Colcher, D., Bod er, M. , 1987, Protein Engineering 1 , 499. 15. Sikder, S.S., Akolkar, P.N., Kaledas, P.M., Morrison, S.L., Kabat, E.A., 1985, J. Immunol. 135, 4215. 16. Wallick, S.C., Kabat, E.A., Morrison, S.L., 1988, J. Exp. Med. Ij58, 1099 17. Bebbington, C.R., Published International Patent Application WO 89/01036. 18. Granthan and Perrin 1986, Immunology Today 1_, 160. 19. Kozak, M., 1987, J. Mol. Biol. 196, 947. 20. Jones, T.P., Dear, P.H., Foote, J. , Neuberger, M.S., Winter, G., 1986, Nature, 321, 522 21. Harwood et al, Br. J. Cancer, 54_, 75-82 (1986).

Claims: CHARTERED PATENT AGENTS EUROPEAN PATENT ATTORNEYS TRADE MARK AGENTS TELEPHONE 071242 MI2 43 BLOOMSBURY SQUARE TELEX 2β72θt FACSIMILE 07140S4166 LONDON • WC1A 2RA 0718318501 AND AT MUMCH The International Unit, The Patent Office, 1. REQUEST FOR RECTIFICATION UNDER PCT RULE 91 .1 (f ) Dear Sirs, Re: International Patent Application No. PCT/GB90/02017? Celltech Limited et al. I refer to your Invitation issued on 14th January 1991. The requir Authorisations and Formal Drawings will be filed in -due course. In checking the application, it has become apparent that there a three mistakes in the Request Form. Firstly, Secondly, .. Thirdly, for reasons which are not apparent, an old version of t Request Form (PCT/RO/101 of July 1987) was used instead of the mo up-to-date version. As a result of this, some PCT states were n designated although it was the Applicant's intention that a possible states should have been designated. As evidence of thi I attach a copy of the information sheet which was given to me hand by the Applicant's Patent Manager on the date the applicati was filed. It can be seen that this clearly indicates that a territories should have been designated. I also enclose evidence that the out-of-date Request Form was us inadvertently. At the same time as the present application w filed, I also filed two other PCT applications, Nos. PCT/GB90/020 and PCT/GB90/02018. I enclose copies of the Request Forms for the cases which, as you can see, are the most up-to-date versions of t forms. I therefore request that the Request Form be amended by addi thereto the designations of Canada and Spain as national applicatio and Greece, Spain and Denmark as designated states within the E designation. I note that it will not be necessary to pay any ext fees in respect of these inadvertently omitted designations. In order to effect all these corrections, I enclose a retyped, u to-date (at the date of filing) Request Form and request that th be substituted for the present, out-of-date Request Form. Yours truly. MERCER, Christopher Paul Authorised Representative.

Inventors: ADAIR JOHN ROBERT, ATHWAL DILJEET SINGH, EMTAGE JOHN SPENCER, ADAIR, JOHN, ROBERT, ATHWAL, DILJEET, SINGH, EMTAGE, JOHN, SPENCER
Applicants: CELLTECH LTD, CELLTECH LIMITED
Application year: 1990
Main classification: C12N5
IPC codes: A61K38, A61K39, A61K49, A61P37, C07K14, C07K16, C07K19, C12N5, C12N15, C12P21, C12R1, G01N33