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`The earliest work on humanising MAbs by CDR-grafting was
`carried out on MAbs recognising synthetic antigens, such
`as the NP or NIP antigens.
`However, examples in which a
`mouse MAb recognising lysozyme and a rat MAb recognising
`an antigen on human T-cells were humanised by CDR-grafting
`have been described by Verhoeyen et al (S) and Riechmann
`et al (6) respectively.
`The preparation of CDR-grafted
`antibody to the antigen on human T cells is also described
`in WO 89/07452 (Medical Research Council).
`
`!
`
`1
`
`In Riechmann et al/Medical Research Council it was found
`that transfer of the CDR regions alone [as defined by
`Kabat refs. (7) and (8)] was not sufficient to provide
`satisfactory antigen binding activity in the CDR-grafted
`product.
`Riechmann et al found that it was necessary to
`convert a serine residue at position 27 of the human
`sequence to the corresponding rat phenylalanine residue to
`obtain a CDR-grafted product having improved antigen
`binding activity.
`This residue at position 27 of the
`heavy chain is within the structural loop adjacent to
`CDR!.
`A further construct which additionally contained a
`human serine to rat tyrosine change at position 30 of the
`heavy chain did not have a significantly altered binding
`activity over the humanised antibody with the serine to
`phenylalanine change at position 27 alone.
`These results
`indicate that changes to residues of the human sequence
`outside the CDR regions, in particular in the structural
`loop adjacent to CDR!, may be necessary to obtain
`effective antigen binding activity for CDR-grafted
`Even
`antibodies which recognise more complex antigens.
`so the binding affinity of the best CDR-grafted antibodies
`obtained was still significantly less than the original
`MAb.
`
`Very recently Queen et al (9) have described the
`preparation of a humanised antibody that binds to the
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`The earliest work on humanising MAbs by CDR-grafting was
`carried out on MAbs recognising synthetic antigens, such
`as the NP or NIP antigens.
`However, examples in which a
`mouse MAb recognising lysozyme and a rat MAb recognising
`an antigen on human T-cells were humanised by CDR-grafting
`have been described by Verhoeyen et al (S) and Riechmann
`et al (6) respectively.
`The preparation of CDR-grafted
`antibody to the antigen on human T cells is also described
`in WO 89/07452 (Medical Research Council).
`
`!
`
`1
`
`In Riechmann et al/Medical Research Council it was found
`that transfer of the CDR regions alone [as defined by
`Kabat refs. (7) and (8)] was not sufficient to provide
`satisfactory antigen binding activity in the CDR-grafted
`product.
`Riechmann et al found that it was necessary to
`convert a serine residue at position 27 of the human
`sequence to the corresponding rat phenylalanine residue to
`obtain a CDR-grafted product having improved antigen
`binding activity.
`This residue at position 27 of the
`heavy chain is within the structural loop adjacent to
`CDR!.
`A further construct which additionally contained a
`human serine to rat tyrosine change at position 30 of the
`heavy chain did not have a significantly altered binding
`activity over the humanised antibody with the serine to
`phenylalanine change at position 27 alone.
`These results
`indicate that changes to residues of the human sequence
`outside the CDR regions, in particular in the structural
`loop adjacent to CDR!, may be necessary to obtain
`effective antigen binding activity for CDR-grafted
`Even
`antibodies which recognise more complex antigens.
`so the binding affinity of the best CDR-grafted antibodies
`obtained was still significantly less than the original
`MAb.
`
`Very recently Queen et al (9) have described the
`preparation of a humanised antibody that binds to the
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`interleukin 2 receptor, by combining the CDRs of a murine
`MAb (anti-Tac) with human immunoglobulin framework and
`constant regions.
`The . human framework regions were
`chosen to maximise ho~ology with the anti-Tac MAb
`sequence.
`In addition computer modelling was used to
`identify framework amino acid residues which were likely
`to interact with the CDRs or antigen, and mouse amino
`acids were used at these positions in the humanised
`antibody.
`
`In WO 90/07861 Queen et al propose four criteria for
`designing humanised immunoglobulins.
`The first criterion
`is to use as the human acceptor the framework from a
`particular human immunoglobulin that is unusually
`homologous to the non-human donor immunoglobulin to be
`humanised, or to use a consensus framework from many human
`antibodies.
`The second criterion is to use the donor
`amino acid rather than the acceptor if the human acceptor
`residue is unusual and the donor residue is typical for
`human sequences at a specific residue of the framework.
`The third criterion is to use the donor framework amino
`acid residue rather than the acceptor at positions
`immediately adjacent to the CDRs.
`The fourth criterion
`is to use the donor amino acid residue at framework
`positions at which the amino acid is predicted to have a
`side chain atom within about 3 A of the CDRs in a
`three-dimensional immunoglobulin model and to be capable
`of interacting with the antigen or with the CDRs of the
`It is proposed that criteria
`humanised immunoglobulin.
`two, three or four may be applied in addition or
`alternatively to criterion one, and may be applied singly
`or in any combination.
`
`WO 90/07861 describes in detail the preparation of a
`single CDR-grafted humanised antibody, a humanised
`antibody having specificity for the p55 Tac protein of the
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`The combination of all four c·riteria, as
`IL-2 receptor.
`above, were employed in designing this humanised antibody,
`the variable region frameworks of the human antibody Eu
`(7) being used as acc~ptor.
`In the resultant humanised
`antibody the donor CDRs were as defined by Kabat et al ( 7
`and 8) and in addition the mouse donor residues were used
`in place of the human acceptor residues, at positions 27,
`30, 48, 66, 67, 89, 91, 94, 103, 104, 105 and 107 in the
`heavy chain and at positions 48, 60 and 63 in the light
`frameworks.
`chain, of the variable region
`The humanised
`reported to have an affinity
`anti-Tac antibody obtained is
`for p55 of 3 x 109 M-1, about
`one-third of that of the
`murine MAb.
`
`i
`
`~
`
`We have further investigated the preparation of CDR(cid:173)
`grafted humanised antibody molecules and have identified a
`hierarchy of positions within the framework of the
`variable regions (i.e. outside both the Kabat CDRs and
`structural loops of the variable regions) at which the
`amino acid identities of the ·residues are important for
`obtaining CDR-grafted products with satisfactory binding
`affinity. This has enabled us to establish a protocol
`for obtaining satisfactory CDR-grafted products which may
`be applied very widely irrespective of the level of
`homology between the donor immunoglobulin and acceptor
`framework.
`The set of residues which we have identified
`as being of critical importance does not coincide with the
`residues identified by Queen et al (9).
`
`Summary of the Invention
`Accordingly, in a first aspect the invention provides a
`CDR-grafted antibody heavy chain having a variable region
`domain comprising acceptor framework and donor antigen
`binding regions wherein the framework comprises donor
`residues at at least one of positions 6, 23 and/or 24, 48
`and/ or 49, 71 and/or 73 , 75 and/or 76 and/or 78 and 88 and/
`or 91.
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`..
`
`In preferred embodiments, the heavy chain framework
`comprises donor residues at positions 23, 24, 49, 71, 73
`and 78 or at positions 23, 24 and 49.
`The residues at
`positions 71, 73 and 78 of the heavy chain framework are
`preferably either all acceptor or all donor residues.
`
`In particularly preferred embodiments the heavy chain
`framework additionally comprises donor residues at one,
`some or all of positions 6, 37, 48 and 94. Also it is
`particularly preferred that residues at positions of the
`heavy chain framework which are commonly conserved across
`species, i.e. positions 2, 4, 25, 36, 39, 47, 93, 103,
`104, 106 and 107, if not conserved between donor and
`acceptor, additionally comprise donor residues. Most
`preferably the heavy chain framework additionally
`comprises donor residues at positions 2, 4, 6, 25, 36, 37,
`39, 47, 48, 93, 94, 103, 104, 106 and 107.
`
`In addition the heavy chain framework optionally comprises
`donor residues at one, some or all of positions:
`1 and 3,
`72 and 76,
`69 (if 48 is different between donor and acceptor),
`38 and 46 (if 48 is the donor residue),
`80 and 20 (if 69 is the donor residue),
`67,
`82 and 18 (if 67 is the donor residue),
`91,
`88, and
`any one or more of 9, 11, 41, 87, 108, 110 and 112.
`
`In the first and other aspects of the present invention
`reference is made to CDR-grafted antibody products
`comprising acceptor framework and donor antigen binding
`regions.
`It will be appreciated that the invention is
`widely applicable to the CDR-grafting of antibodies in
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`Thus, the donor and acceptor antibodies may be
`general.
`derived from animals of the same species and even same
`antibody class or sub-class. More usually, however, the
`donor and acceptor antibodies are derived from animals of
`different species. Typically the donor antibody is a
`non-human antibody, such as a rodent MAb, and the acceptor
`antibody is a human antibody.
`
`~
`
`~
`
`In the first and other aspects of the present invention,
`the donor antigen binding region typically comprises at
`least one CDR from the donor antibody. Usually · the donor
`antigen binding region comprises at least two and
`preferably all three CDRs of each of the heavy chain
`and/or light chain variable regions.
`The CDRs may
`comprise the Kabat CDRs, the structural loop CDRs or a
`composite of the Kabat and structural loop CDRs and any
`combination of any of these.
`Preferably, the antigen
`binding regions of the CDR-grafted heavy chain variable
`domain comprise CDRs corresponding to the Kabat CDRs at
`CDR2 (residues 50-65) and CDR3 (residues 95-100) and a
`composite of the Kabat and structural loop CDRs at CDRl
`(residues 26-35).
`
`The residue designations given above. and elsewhere in the
`present application are numbered according to the Kabat
`numbering (refs. (7). and (8)].
`Thus the residue
`designations do not always correspond directly with the
`linear numbering of the amino acid residues.
`The actual
`linear amino acid sequence may contain fewer or additional
`amino acids than in the strict Kabat numbering
`corresponding to a shortening of, or insertion into, a
`structural component, whether framework or CDR, of the
`basic variable domain structure.
`For example, the· heavy
`chain variable region of the anti-Tac antibody described
`by Queen et al (9) contains a single amino acid insert
`(residue 52a) after residue 52 of CDR2 and a three amino
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`acid insert (residues 82a, 82b and 82c ) after framework
`residue 82, in the Kabat numbering.
`The correct Kabat
`numbering of residues may be determined for a given
`antibody by alignment at regions of homology of the
`sequence of the antibody with a "standard" Kabat numbered
`sequence.
`
`The invention also provides in a second aspect a CDR(cid:173)
`grafted antibody light chain having a variable region
`domain comprising acceptor framework and donor antigen
`binding regions wherein the framework comprises donor
`residues at at least one of positions 1 and/or 3 and 46
`and/or 47.
`Preferably the CDR qrafted light . chain of the
`second aspect comprises donor residues at positions 46
`and/or 47.
`
`The invention also provides in a third aspect a
`CDR-grafted antibody light chain having a variable region
`domain comprising acceptor framework and donor antigen
`binding regions wherein the framework comprises donor
`residues at at least one of positions 46, 48, 58 and 71.
`
`In a preferred embodiment of the third aspect, the
`framework comprises donor residues at all of positions 46,
`4 8 , 58 and 7 1.
`
`In particularly preferred embodiments of the second and
`third aspects, the framework additionally comprises donor
`residues at positions 36, 44, 47, 85 and 87.
`Similarly
`positions of the light chain framework which are commonly
`conserved across species, i.e. positions 2, 4, 6, 35, 49,
`62, 64-69, 98, 99, 101 and 102, if not conserved between
`donor and acceptor, additionally comprise donor residues.
`Most preferably the light chain framework additionally
`comprises donor residues at positions 2, 4, 6, 35, 36, 38,
`44, 47, 49, 62, 64-69, as, 87, 98, 99, 101 and 102.
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`In addition the framework of the second or third aspects
`opt ionally comprises donor residues at one, some or all of
`positions:
`1 and 3,
`63,
`60 (if 60 and 54 are able to form at potential saltbridge),
`70 (if 70 and 24 are able to form a potential saltbridge),
`73 and 21 (if 47 is different between donor and acceptor),
`37 and 45 (if 47 is different between donor and acceptor),
`and
`any one or more of 10 , 12, 40, 80, 103 and 105.
`
`Preferably, the antigen binding regions of the CDR-grafted
`light chain variable domain comprise CDRs corresponding t o
`the Kabat CDRs at CDR1 (residue 24-34), CDR2 (residues
`50-56) and CDR3 (residues 89-97).
`
`The invention further provides in a fourth aspect a
`CDR-grafted antibody molecule comprising at least one
`CDR-grafted heavy chain and at least one CDR-grafted light
`chain according to the first and second or first and third
`aspects of the invention.
`
`The humanised antibody molecules and chains of t he present
`invention may comprise:
`a complete antibody molecule,
`having full length heavy and light chains;
`a fragment
`thereof, such as a Fab, (Fab ') 2 or FV fragment;
`a light
`chain or heavy chain monomer or dimer; or a single chain
`antibody, e.g. a single chain FV in which heavy and light
`chain variable regions are joined by a peptide linker; or
`any other CDR-grafted molecule with the same specificity
`as the original donor antibody.
`Similarly the
`CDR-grafted heavy and light chain variable region may be
`combined with other antibody domains as appropriate.
`
`~
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`Also the heavy or light chains or humanised antibody
`molecul es of the present invention may have attached to
`them an effector or reporter molecule.
`For instance, i t
`may have a macrocycle, for chelating a heavy metal atom,
`or a toxin, such as ricin, attached to it by a covalent
`bridging structure. Alternatively, the procedures of
`recombinant DNA technology may be used to produce an
`immunoglobulin molecule in which the Fe fragment or CH3
`domain of a complete immunoglobulin molecule has been
`replaced by, or has attached thereto by peptide linkage, a
`functional non-immunoglobulin protein, such as an enzyme
`or toxin molecule.
`
`Any appropriate acceptor variable region framework
`sequences may be used having regard to class/ type of the
`donor antibody from which the antigen binding regions are
`derived.
`Preferably, the type of acceptor framework used
`is of the same/ similar class/ type as the donor antibody.
`Conveniently, the framework may be chosen to maximise/
`optimise homology with the ddnor antibody sequence
`particularly at positions close or adjacent to the CDRs.
`However, a high level of homology between donor and
`acceptor sequences is not impor t ant for application of the
`present invention.
`The present invention identifies a
`hierarchy of framework residue positions at which donor
`residues may be important or desirable for obtaining a
`CDR-grafted antibody product having satisfactory binding
`properties.
`The CDR-grafted products usually have
`binding affinities of at least 105 M-1, preferably at
`l east about 108 M-1, or especially in the range 108-1o12
`M-1.
`In principle, the present invention is applicable
`to any combination of donor and acceptor antibodies
`irrespective of the level of homology between their ·
`sequences.
`A protocol for applying the inventi on to any
`particular donor-acceptor antibody pair is given
`hereinafter.
`Examples of human frameworks which may be
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`used are KOL, NEWM, REI, EU, LAY and POM (refs. 4 and 5)
`and the like;
`for instance KOL and NEWM for the heavy
`chain and REI for the light chain and EU, LAY and POM for
`both the heavy chain and the light chain.
`
`Also the constant region domains of the products of the
`invention may be selected having regard to the pr.oposed.
`function of the antibody in particular the effector
`functions which may be required.
`For example, the
`constant region domains may be human IgA, IgE, IgG or IgM
`domains.
`In particular, IgG human constant region
`domains may be used, especially of the IgGl and IgG3
`isotypes, when the humanised antibody molecule is intended
`for therapeutic uses, and antibody effector functions are
`required. . Alternatively, IgG2 and IgG4 isotypes may be
`used when the humanised antibody molecule is intended for
`therapeutic purposes and antibody effector functions are
`not required, e.g. for simple blocking of lymphokine
`activity.
`
`However, the remainder of the antibody molecules need not
`comprise only protein sequences from immunoglobulins.
`For instance, a gene may be constructed in which a DNA
`sequence encoding part of a human immunoglobulin chain is
`fused to a DNA sequence encoding the amino acid sequence
`of a functional polypeptide such as an effector or
`reporter molecule.
`
`Preferably the CDR-grafted antibody heavy and light chain
`and antibody molecule products are produced by recombinant
`DNA technology.
`
`Thus in further aspects the invention also includes DNA
`sequences coding for the CDR-grafted heavy and light
`chains, cloning and expression vectors containing the DNA
`sequences, host cells transformed with the DNA sequences
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`and processes for producing the CDR-grafted chains and
`antibody molecules comprising expressing the DNA sequences
`in the transformed host cells.
`
`The general methods by which the vectors may be
`constructed, transfection methods and culture methods are
`well known per se and form no part of the invention.
`Such
`methods are shown, for instance, in references 10 and 11 .
`
`The DNA sequences which encode the donor amino acid
`sequence may be obtained by methods well known in the
`art.
`For example the donor coding sequences may be
`obtained by genomic cloning, or eDNA cloning from suitable
`hybridoma cell l i nes.
`Positive clones may be screened
`using appropriate probes for the heavy and light chain
`genes in question. Also PCR cloning may be used.
`
`DNA coding for acceptor, e.g. human acceptor, sequences
`may be obtained in any appropriate way.
`For example DNA
`sequences coding for preferred human acceptor frameworks
`such as KOL, REI , EU and NEWM, are widely available to
`workers in the art.
`
`The standard techniques of molecular biology may be used
`to prepare DNA sequences coding for the CDR-grafted
`products. Desired DNA sequences may be synthesi sed
`completely or in part using oligonucleotide synthesis
`techniques.
`Site-directed mutagenesis and polymerase
`chain reaction (PCR) techniques may be used as
`appropriate.
`For example oligonucleotide directed
`synthesis as described by Jones et al (ref. 20 ) may be
`used. Also oligonucleotide directed mutagenesis of a
`pre-exising variable region as, for example, described by
`Verhoeyen et al (ref . 5) or Riechmann et al (ref . 6 ) may
`be used. Also enzymatic filling in of gapped
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`oligonucleotides using T4 DNA polymerase as, for example,
`described by Queen et al (ref. 9) may be used.
`
`Any suitable host cell/vector system may be used for
`expression of the DNA sequences coding for the CDR-grafted
`heavy and light chains. Bacterial e.g. E. coli, and
`other microbial systems may be used, in particular for
`expression of antibody fragments such as FAb and (Fab')2
`fragments, and especially FV fragments and single chain
`antibody fragments e.g. single chain FVs.
`Eucaryotic
`e.g. mammalian host cell expression systems may be used
`for production of larger CDR-grafted antibody products,
`including complete antibody molecules.
`Suitable
`mammalian host cells include CHO cells and myeloma or
`hybridoma cell lines.
`
`Thus, in a further aspect the present invention provides a
`process for producing a CDR-grafted antibody product
`comprising:
`
`(a) producing in an 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.
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`The CDR-grafted product may comprise only heavy or light
`chain derived polypeptide, in which case only a heavy
`chain or light chain polypeptide c oding sequence is used
`t o transfect the host cells.
`For production of products c omprising 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 s e quences
`encoding both light chain- and heavy chain-derived
`polypeptides.
`
`The DNA in the coding sequences for the light and heavy
`chains may comprise eDNA or genomic DNA or both.
`However, it is preferred that the DNA sequence encoding
`the heavy or light chain comprises at least partially,
`genomic DNA, preferably a fusion of eDNA and genomic DNA.
`
`The present invention is applicable to antibodies of any
`appropriate specificity. Advantageously, however, the
`invention may be applied to t he 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(cid:173)
`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, stich 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
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`example, the antibodies may have specificity for any of
`Interferonso(, ~, Y or~, ILl, IL2, IL3 ,
`the following :
`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
`t he invention as hereinbefore described and defined.
`
`Protocol
`It is first of all necessary to sequence the DNA coding
`for the heavy and l ight 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 variabl e regions, of known amino acid
`sequence.
`The CDR-grafted chain is then designed
`
`t
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`It
`starting from the basis of the acceptor sequence.
`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:
`
`Heavy chain
`
`Light chain
`
`CDRl:
`CDR2:
`CDR3:
`CDR1:
`CDR2:
`CDR3:
`
`residues 26-35
`residues 50-65
`residues 95-102
`residues 24-34
`residues 50-56
`residues 89-97
`
`The positions at which donor residues are to be
`substituted for acceptor in the framework are then
`chosen as follows, first of all with respect to the
`heavy chain and subsequently with respect to the
`light chain.
`
`2.
`
`Heavy Chain
`
`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.
`
`Board Ass gned Page #61 1
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`PFIZER EX. 1095
`Page 764
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`- 18 -
`
`2.3 To further optLmise affinit y consi der choosing donor
`residues at one, some or any of:
`
`t
`
`~
`
`v.
`
`iv.
`
`i.
`1, 3
`72 , 76
`ii.
`iii. If 48 is different between donor and acceptor
`sequences, consider 69
`If at 48 the donor residue is chosen, consider
`38 and 46
`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
`9, 11, 41, 87, 108, 110, 112
`x.
`
`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.
`ii.
`
`1, 3
`63
`
`Board Assgned Page #612
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`PFIZER EX. 1095
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`
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`
`- 19 -
`
`iii .
`
`iv.
`
`60, if 60 and 54 are able to form potential
`saltbridge
`70, if 70 and 24 are able to form potential
`saltbridge
`73, and 21 if 47 is different between donor
`acceptor
`37, and 45 if 47 is different between donor
`acceptor
`vii. 10, 12, 40, 80, 103 , 105
`
`v.
`
`vi.
`
`and
`
`and
`
`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 o f 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 fJ barrel framework of the light
`
`and heavy variable domains. For H1 there was a
`discrepancy i n that the loop was f rom 26 to 32
`inclusive and for H2 the loop was 52 to 56 -and
`for L2 from 5 0 to 5 3 .
`However, with t he
`exception of Hl the CDR regions encompassed the
`loop regions and extended into the~ strand
`
`Board Ass gned Page #613
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`PFIZER EX. 1095
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`PCf/GB90/ 02017
`
`- 20 -
`
`In H1 residue 26 tends to be a
`frameworks.
`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 H1
`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-Hl.
`In order to produce
`efficient antigen binding, residue 27 also needed
`to be recruited from the donor (rat ) antibody.
`
`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 o f
`groups.
`Surface residues near CDR [all numbering as in
`Kabat et al (ref. 7) ] .
`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.
`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
`
`2.
`
`2.1
`
`2.1.1.·
`
`2 . 1. 2
`
`Board Assgned Page #614
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`PFIZER EX. 1095
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`
`- 21 -
`
`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 6 0 and 7 0 i f 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.
`Packing residues near the CDRs.
`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, 6.7 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
`86
`against 69 and in turn 20 packs against 80.
`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.
`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.
`
`2.2
`2.2.1.
`
`2.2.2.
`
`Board Assgned Page #615
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`PFIZER EX. 1095
`Page 768
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`
`2.3.
`
`2.3.1.
`
`2 .3. 2.
`
`2.4 .
`
`- 22 -
`
`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.
`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.
`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.
`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 VH 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
`
`~
`
`~
`
`~
`
`~
`
`Board Ass gned Page #616
`
`PFIZER EX. 1095
`Page 769
`
`
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`PCT/GB90/02017
`
`- 23 -
`
`region contact points vary, and the V & C regions
`are not in such close proximity as the heavy
`chain.
`Therefore the influences
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