`
`Europiiisches Patentamt
`
`European Patent Office
`
`@ Publication
`number:
`
`Office europeen des brevets
`
`0239400
`A2
`
`EUROPEAN PATENT APPLICATION
`C 12 N 15/00, C 07 K 15/06,
`® Int.Cl.•:
`c 12 p 21/02
`
`@ Application 87302620.7
`
`26.03.87
`@ Date offillng:
`
`number:
`
`@ Priority: 27.03.86 GB 8607679
`
`Winter, Gregory Peul, 64 Cavendish Avenue,
`® Applicant:
`
`Cambridge (GB)
`
`@ Date of publication
`
`30.09.87
`of application:
`BuRetln87/40
`
`@ Inventor: Winter, Gregory Paul, 64 C.Vendlah
`
`Cambridge (GB)
`
`Avenue,
`
`Votler, Sidney David et al, CARPMAELS
`@ Representative:
`& RANSFORD 43, Bloomsbury Square, London
`@) Designated Contracting States:
`
`GR ITLI LU NLSE
`
`WC1A2RA (GB)
`
`AT BE CH DE ES FR GB
`
`
`
`antibodies and methods for their production.
`
`antibody is produced by replacing the com
`
`
`
`
`
`
`
`plementarity determining regions (CDRs) of a variable region
`of an lmmunoglobulin
`different specificity, using recombinant DNA techniques. The
`
`
`
`
`
`
`
`gene coding sequences for producing the altered antibody
`
`by site-directed mutagenesis using long
`gene cloned In 11131T1119
`HuV
`HP
`ollgonucleotides.
`
`Hlndlll _
`
`llemHI
`
`® Recombinant
`@ An altered
`(lg) with the CDRs from an lg of
`may be produced
`
`1 _5· _2_,- -'-s·
`HFRI - FR2 - FR3 - FR4 t'
`
`CORI CDR2 CDR3
`
`O 1.3 CDR I ollgonuel1olld1
`
`
`
`5' CT G,TCT ,CAC,CCA,GTT ,T AC,AtC,AT A,GCC,GCT ,GAA,GGT ,GCT
`
`FR2
`
`01.3 CORI
`
`FRI
`
`S' CAT ,TGT ,CAC,TCT ,GGA,TTT ,GAG,AGC,TGA,ATT ,AT A,GTC.TGT,
`
`01.3 CDR2 ollgonuc1eollde
`
`
`
`FR3
`OL! COR2
`
`GTT ,TCC,ATC,ACC,CCA,AAT ,CAT ,TCC,AAT ,CCA,CTC
`
`Ol.3CDR2
`
`FR2
`
`DI .3 CORJ ollgonucholldl
`
`
`
`,ATA,ATC,TCT ,CTC,TCT,
`
`5' GCC,TTG,ACC,CCA,GTA,GTC,A"G,CCT
`
`FR�
`
`TGC,ACA,ATA
`
`fR3
`
`ACTORUM AG
`
`01.3 COR3
`
`1 of 41
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`BI Exhibit 1073
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`RECOMBINANT DNA PRODUCT AND METHODS
`
`The present invention relates to altered antibodies
`in which at least parts of the complementarity
`determining regions (CDRs) in the light or heavy
`chain variable domains of the antibody have been
`replaced by analogous parts of CDRs from an antibody
`of different specificity. The present invention
`also relates to methods for the production of such
`altered antibodies.
`
`Natural antibodies, or immunoglobulins, comprise two
`heavy chains linked together by disulphide bonds and
`two light chains, one light chain being linked to
`each of the heavy chains by disulphide bonds. The
`general structure of an antibody of class IgG (i.e.
`an immunoglobulin (Ig) of class gamma (G)) is shown
`schematically in Figure 1 of the accompanying
`drawings.
`
`Each heavy chain has at one end a variable domain
`followed by a number of constant domains. Each
`light chain has a variable domain at one end and a
`constant domain at its other end, the variable
`domain being aligned with the variable domain of the
`heavy chain and the constant domain being aligned
`with the first constant domain of the heavy chain.
`The constant domains in the light and heavy chains
`are not involved directly in binding the antibody to
`the antigen.
`
`The variable domains of each pair of light and heavy
`chains form the antigen binding site.
`The domains
`on the light and heavy chains have the same general
`structure and each domain comprises four framework
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`regions, whose sequences are relatively conserved,
`connected by three hypervariable or complementarity
`determining regions (CDRs� (see Kabat, E . A., Wu,
`T.T., Bilofsky, H., Reid-Miller, M. and P erry, H. ,
`in "Sequences of P roteins of Immunological
`Interest", US Dept. Health and Human Services 1983) .
`
`conformation and the CDRs form loops connecting, and
`
`The four framework regions largely adopt a � -sheet
`in some cases forming part of, the � -sheet
`
`structure. The CDRs are held in close proximity by
`the framework regions and, with the CDRs from the
`other domain, contribute to the formation of the
`antigen binding site.
`
`For a more detailed account of the structure of
`variable domains, reference may be made to: P oljak,
`R. J., Amzel, L . M., Avey, H. P . , Chen, B.L. ,
`Phizackerly, R.P . and Saul, F., P NAS USA, ]J_,
`3305-3310, 1973; Segal, D. M. , Padlan, E . A., Cohen,
`G. H. , Rudikoff, s., P otter, M. and Davies, D. R.,
`P NAS USA, 21.1 4298-4302, 1974; and Marquart, M.,
`Deisenhofer, J . , Huber, R. and P alm, w., J. Mol.
`Biol. , 141, 369-391, 1980.
`
`In recent years advances in molecular biology based
`on recombinant DNA techniques have provided
`processes for the production of a wide range of
`heterologous polypeptides by transformation of host
`cells with heterologous DNA sequences which code for
`the production of the desired products.
`
`E P -A-0 08 8 994 (Schering Corporation) proposes the
`construction of recombinant DNA vectors comprising a
`ds DNA sequence which codes for a variable domain of
`a light or a heavy chain of an I g specific for a
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`predetermined ligand. The ds DNA sequence is
`provided with initiation and termination codons at
`its 5'- and 3'- termini respectively, but lacks any
`nucleotides coding for amino acids superfluous to
`the variable domain. The ds DNA sequence is used to
`transform bacterial cells. The application does not
`contemplate variations in the sequence of the
`variable domain.
`
`E P -A-1 102 634 (Takeda Chemical Industries L imited)
`describes the cloning and expression in bacterial
`host organis ms of genes coding for the whole or a
`part of human IgE heavy chain polypeptide, but does
`not contemplate variations in the sequence of the
`polypeptide.
`
`E P-A-0 125 0 23 (Genentech I nc.) proposes the use of
`recombinant DNA techniques in bacterial cells to
`produce Ig's which are analogous to those normally
`found in vertebrate systems and to take advantage of
`the gene modification techniques proposed therein to
`construct chimeric Igs or other modified forms of Ig.
`
`The term ' chimeric antibody' is used to describe a
`protein comprising at least the antigen binding
`portion of an immunoglobulin molecule (Ig) attached
`by peptide linkage to at least part of another
`protein.
`
`It is believed that the proposals set out in the
`above Genentech application did not lead to the
`expression of any significant quantities of Ig
`polypeptide chains, nor to the production of Ig
`activity, nor to the secretion and assembly of the
`chains into the desired chimeric Igs.
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`The producti on of monoclonal antibodies was first
`disclosed by Kohler and Milstein (Kohler, G. and
`Milstein, c., Nature, 256, 495-497, 1975) . such
`monoclonal antibodies have found widespread use not
`only as diagnostic reagents (see, for example,
`' Immunology for the 8 0 s, Eds. Voller, A., Bartlett,
`A. , and Bidwell, D. , MTP P ress, Lancaster, 1981) but
`also in therapy (see, for example, Ritz, J. and
`
`Schlossman, S. F. , Blood, �' 1-11, 198 2) .
`
`The recent emergence of techniques allowing the
`stable i ntroduction of Ig gene DNA into myeloma
`cells (see, for example, Oi, V.T. , Morrison, S.L. ,
`Berzenberg, L.A. and Berg, P. , P NAS USA, �'
`8 25-8 29, 198 3; Neuberger, M.S. , EMBO J., 1,
`1373-1378, 1983; and Ochi, T., Hawley, R.G. , Hawley,
`T., Schulman, M. J. , Traunecker, A. , Kohler, G. and
`Hozumi , N. , P NAS USA, �' 6351-6355, 198 3) , has
`opened up the possibility of using in vitro
`mutagenesis and DNA transfection to construct
`recombinant Igs possessing novel properties.
`
`However, it is known that the function of an Ig
`molecule is dependent on its three dimensional
`structure, which in turn is dependent on its primary
`amino acid sequence. Thus, changing the amino acid
`sequence of an Ig may adversely affect its
`activity. Moreover, a change in the DNA sequence
`coding for the Ig may affect the ability of the cell
`containing the DNA sequence to express, secrete or
`assemble the Ig.
`
`It i s therefore. not at all clear that i t will be
`possible to produce functional altered antibodies by
`recombinant DNA techniques.
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`However, colleagues of the present Inventor have
`devised a process whereby chimeric antibodies in
`which both parts of the protein are functional can
`be secreted. The process, which is disclosed in
`International Patent Application No. PCT/GB85/0 0 392
`(Neuberger et al. and Celltech Limited), comprises:
`
`a) preparing a replicable expression vector
`including a suitable promoter operably
`linked to a DNA sequence comprising a
`first part which encodes at least the
`variable domain of the heavy or light
`chain of an Ig molecule and a second part
`which encodes at least part of a second
`protein;
`
`b)
`
`if necessary, preparing a replicable
`expression vector including a suitable
`promoter operably linked to a DNA
`sequence which encodes at least the
`variable domain of a complementary light
`or heavy chain respectively of an Ig
`molecule;
`
`c) transforming an immortalised mammalian
`cell line with the or both prepared
`vectors; and
`
`d) culturing said transformed cell line to
`produce a chimeric antibody.
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`The second part of the DNA sequence may encode:
`
`at l east part, for instance the constant
`i)
`domain of a heavy chain, of an Ig molecule of
`different species, class or subclass;
`ii) at least the active portion or all of an
`enzyme;
`a protein having a known binding
`iii)
`specificity;
`iv) a protein expressed by a known gene but
`whose sequence, function or antigenicity is not
`known; or
`
`V) a protein toxin, such as ricin.
`
`The above Neuberger application only shows the
`production of chimeric antibodies in which complete
`variable domains are coded for by the first part of
`the DNA sequence. It does not show any chimeric
`antibodies in which the sequence of the variable
`domain has been altered.
`
`The present invention, in a first aspect, provides
`an altered antibody in which at least parts of the
`CDRs in the light or heavy chain variable domains
`have been replaced by analogous parts of CDRs from
`an antibody of different specificity
`
`The determination as to what constitutes a CDR and
`what constitutes a framework region was made on the
`basis of the amino-acid sequences of a number of
`However, from the three dimensional structure
`Igs.
`of a number of Igs it is apparent that the antigen
`binding site of an Ig variable domain comprises
`three looped regions supported on sheet-like
`structures. The loop regions do not correspond
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`exactly to the CDRs, although in general there i s
`considerable overlap.
`
`Moreover, not all of the amino-acid residues in the
`loop regions are solvent accessible and in one
`case, ami no-acid residues i n the framework regions
`are involved i n antigen binding. (Amit, A. G.,
`Mariuzza, R.A., Phillips, S. E . V. and Poljak, R.J. ,
`Science, 2 33, 747-753, 198 6) .
`
`It is also known that the variable regi ons of the
`two parts of an antigen binding site are held i n the
`correct orientation by i nter-chain non-covalent
`interactions. These may involve amino-acid residues
`within the CDRs.
`
`Thus, in order to transfer the antigen binding
`capacity of one variable domain to another, it may
`not be necessary to replace all of the CDRs with the
`complete CDRs from the donor variable region. It
`may be necessary only to transfer those residues
`which are accessible from the antigen binding site,
`and this may i nvolve transferring framework region
`residues as well as CDR resi dues.·
`
`It may also be necessary to ensure that residues
`essential for inter-chain interactions are preserved
`in the acceptor variable domain.
`
`Within a domain, the packing together and
`
`orientation of the two disulphide bonded P, -sheets
`packing and orientation of these � -sheets do occur
`
`(and therefore the ends of the CDR loops) are
`relatively conserved. However, small shifts in
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`(Lesk, A.M. and Chothia, c., J. Mol. Biol. , 160,
`325-342, 198 2 }. However, the packing together and
`orientation of heavy and light chain variable
`domains is relatively conserved (Chothia, c.,
`Novotny, J., Bruccoler� , R. and Karplus, M. , J. Mol.
`
`Biol;, 186, 651-653, 1985}. These points will need
`to be borne in mind when constructing a new antigen
`biding site so as to ensure that packing and
`orientation are not altered to the deteriment of
`antigen binding capacity.
`
`It is thus clear that merely by replacing one or
`more CDRs with complementary CDRs may not always
`result in a functional altered antibody. However,
`given the explanatiDns set out above, it will be
`well within the comP.etence of the man skilled in the
`art, either by carrying out routine experimentation
`or by trial and error testing to obtain a functional
`altered antibody.
`
`P referably, the variable domains in both the heavy
`and light chains have been altered by at least
`partial CDR replacement and, if necessary, by
`partial framework region replacement and sequence
`changing. Although the CDRs may be derived from an
`antibody of the same class or even subclass as the
`antibody from which the framework regions are
`derived, it is envisaged that the CDRs will be
`derived from an antibody of different class and
`preferably from an antibody from a different
`species.
`
`Thus, it is envisaged, for instance, that the CDRs
`from a mouse antibody could be grafted onto the
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`framework r egions of a human antibody. This
`arrangement will be of particular use in the
`therapeutic use of monoclonal antibodies.
`
`At present, when a mouse monoclonal antibody or even
`a chimeric antibody comprising a complete mouse
`variable domain is injected into a human, the human
`body' s immune system recognises the mouse var iable
`domain as foreign and produces an immune response
`thereto. Thus, on subsequent injections of the
`mouse antibody or chimeric antibody into the human,
`its effectiveness is considerably r educed by the
`action of the body' s immune system against the
`foreign antibody. In the altered antibody of the
`present invention, only the CDRs of the antibody
`will be foreign to the body, and this should
`minimise side effects if used for human therapy.
`Although, for example, human and mouse framework
`r egions have characteristic sequences, there seem
`to be no characteristic features which distinguish
`human from mouse CDRs. Thus, an antibody comprised
`of mouse CDRs in a human framework may well be no
`more foreign to the body than a genuine human
`antibody.
`
`Even with the altered antibodies of the present
`invention, there is likely to be an anti-idiotypic
`response by the r ecipient of the altered antibody.
`This r esponse is directed to the antibody binding
`region of the altered antibody, It is believed that
`at least some anti-idiotype antibodies are directed
`at sites bridging the CDRs and the framework regions.
`It would therefore be possible to provide a panel of
`antibodies having the same partial or complete CDR
`r eplacements but on a series of different framework
`r egions. Thus, once a first altered antibody became
`therapeutically ineffective, due to an anti-idiotype
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`response, a second altered antibody from the series
`could be used, and so on, to overcome the effect of
`the anti-idiotype response. Thus, the useful life
`of the antigen-binding capacity of the altered
`antibodies could be extended.
`
`Preferably, the altered antibody has the structure
`of a natural antibody or a fragment thereof. Thus,
`the altered antibody may comprise a complete
`antibody, an (Fab' )2 fragment, an Fab fragment, a
`light chain dimer or a heavy chain dimer.
`Alternatively, the altered antibody may be a
`chimeric antibody of the type described in the
`Neuberger application referred to above. The
`production of such an altered chimeric antibody can
`be carried out using the methods described below
`used in conjunction with the methods described in
`the Neuberger application.
`
`The present invention, in a second aspect, comprises
`a method for producing such an altered antibody
`comprising:
`
`a} preparing a first replicable expression
`vector including a suitable promoter operably linked
`to a DNA sequence which encodes at least a variable
`domain of an Ig heavy or light chain, the variable
`domain comprising framework regions from a first
`antibody and CDRs comprising at least parts of the
`CDRs from a second antibody of different specificity;
`
`b) if necessary, preparing a second
`replicable expression vector including a suitable
`promoter operably linked to a DNA sequence which
`encodes at least the variable domain of a
`complementary Ig light or heavy chain respectively;
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`c) transforming a cell line with the first or
`both prepared vectors; and
`
`d) culturing said transformed cell line to
`produce said altered antibody.
`
`The present invention also includes vectors used to
`transform the cell line, vectors used in producing
`the transforming vectors, cell lines transformed
`with the transforming vectors, cell lines tranformed
`with preparative vectors, and methods for their
`production.
`
`P referably, the cell line which is transformed to
`produce the altered antibody is an immortalised
`mammalian cell line, which is advantageously of
`lymphoid origin, such as a myeloma, hybridoma,
`trioma or quadroma cell line. The cell line may
`also comprise a normal lymphoid cell, such as a
`B-cell, which has been immortalised by
`transformation with a virus, such as the E pstein-Barr
`virus. Most preferably, the immortalised cell line
`is a myeloma cell line or a derivative thereof.
`
`Although the cell line used to produce the altered
`antibody is preferably a mammalian cell line, any
`other suitable cell line, such as a bacterial cell
`line or a yeast cell line, may alternatively be
`used.
`In particular, it is envisaged that E. Coli
`derived bacterial strains could be used.
`
`It is known that some immortalised lymphoid cell
`lines, such as myeloma cell lines, in their normal
`state secrete isolated Ig light or heavy chains. If
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`such a cell line is transformed with the vector
`prepared in step a) of the process of the invention,
`it will not be necessary to carry out step b) of the
`process, provided that the normally secreted chain
`is complementary to the variable domain of the Ig
`chain encoded by the vector prepared in step a) .
`
`However, where the immortalised cell line does not
`secrete or does not secrete a complementary chain,
`it will be necessary to carry out step b). This
`step may be carried out by further manipulating the
`
`vector produced in step a) so that this vector
`
`encodes not only the variable domain of an altered
`antibody light or heavy chain, but also the
`complementary variable domain.
`Alternatively, step b) is carried out by
`preparing a second vector which is used to transform
`the immortalised cell line. This alternative leads
`to easier construct preparation, but may be less
`preferred than the first alternative in that it may
`not lead to as efficient production of antibody.
`
`The techniques by which such vectors can be produced
`and used to transform the immortalised cell lines
`are well known in the art, and do not form any part
`of the invention.
`
`In the case where the immortalised cell line
`secretes a complementary light or heavy chain, the
`transformed cell line may be produced for example by
`transforming a suitable bacterial cell with the
`vector and then fusing the bacterial cell with the
`immortalised cell line by spheroplast fusion.
`Alternatively, the DNA may be directly introduced
`into the immortalised cell line by electroporation.
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`The DNA sequence encoding the altered variable
`domain may be prepared by oligonucleotide
`synthesis. This requires that at least the
`framework region sequence of the acceptor antibody
`and at least the CDRs sequences of the donor
`antibody are known or can be readily determined.
`Although determining these sequences, the synthesis
`of the DNA from oligonucleotides and the preparation
`of suitable vectors is to some extent laborious, it
`involves the use of known techniques which can
`readily be carried out by a person skilled in the
`art in light of the teaching given here.
`
`If it was desired to repeat this strategy to insert
`a different antigen binding site, it would only
`require the synthesis of oligonucleotides encoding
`the CDRs, as the framework oligonucleotides can be
`re-used.
`
`A convenient variant of this technique would involve
`making a symthetic gene lacking the CDRs in which
`the four framework regions are fused together with
`suitable restriction sites at the junctions. Double
`stranded synthetic CDR cassettes with sticky ends
`could then be ligated at the junctions of the
`framework regions. A protocol for achieving this
`variant is shown diagrammatically in Figure 6 of the
`accompanying drawings.
`
`Alternatively, the DNA sequence encoding the altered
`variable domain may be prepared by primer directed
`oligonucleotide site-directed mutagenesis. This
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`'
`technique' in esse��e i�volves hybridising an
`oligonucleoti
`e coding for a desired mutation with a
`single strand of DNA containing the region to be
`mutated and using the single strand as a template
`for extension of the oligonulcleotide to produce a
`strand containing the mutation. This technique, in
`various forms, is described by : Zoller, M.J. and
`Smith, M. , Nuc. Acids Res. , .!.Q., 648 7-650 0 , 198 2;
`Norris, K. , Norris F . , Christiansen, L . and Fiil,
`N. , Nuc. Acids Res. , 11, 5103-5112, 1983; Zoller,
`M.J. and Smith, M., DNA, l' 479-48 8 (1984); Kramer,
`w., Schughart, K. and Fritz, w.-J., Nuc. Acids Res.,
`.!Q_, 6475-6485, 198 2 .
`
`'a
`
`For various reasons, this technique in its simplest
`form does not always produce a high frequency of
`mutation. An improved technique for introducing
`both single and multiple mutations in an Ml3 based
`vector, has been described by Carter et al. (Carter,
`P . , Bedouelle H. and Winter, G. , Nuc. Acids Res. ,
`]d, 4431-4443, 1985)
`
`Using a long oligonucleotide, it has proved possible
`to introduce many changes simultaneously (as in
`Carter et al. , loc. cit . ) and thus single
`oligonucleotides, each encoding a CDR, can be used
`to introduce the three CDRs from a second antibody
`into the framework regions of a first antibody. Not
`only is this technique less laborious than total
`gene synthesis, but it represents a particularly
`convenient way of expressing a variable domain of
`required specificity, as it can be simpler than
`tailoring an entire VH domain for insertion into an
`expression plasmid.
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`The oligonucleotides used for site-directed
`mutagenesis may be prepared by oligonucleotide
`synthesis or may be isolated from DNA coding for the
`variable domain of the second antibody by use of
`suitable restriction enzymes. Such long
`oligonucleotides will generally be at least 30 bases
`long and may be up to or over 8 0 bases in length.
`
`The techniques set out above may also be used, where
`necessary, to produce the vector of part (b) of the
`process.
`
`The method of the present invention is envisaged as
`being of particular use in "humanising" non-human
`monoclonal antibodies. Thu?, for instance, a mouse
`
`monoclonal antibody against a particular human
`
`,
`
`cancer cell may be produced by techniques well known
`in the art. The CDRs from the mouse monoclonal
`antibody may then be partially or totally grafted
`into the framework regions of a human monoclonal
`antibody, which is then produced in quantity by a
`suitable cell line. The product is thus a
`specifically targetted, essentially human antibody
`which will recognise the cancer cells, but will not
`itself be recognised to any significant degree, by a
`human' s immune system, until the anti-idiotype
`response eventually becomes apparent. Thus, the
`method and product of the present invention will be
`of particular use in the clinical environment.
`
`The pr�sent invention is now described, by way of
`example only, with reference to the accompanying
`drawings, in which:
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`Figure 1 is a schematic diagram showing the
`structure �f an IgG molecule;
`
`.
`
`.
`
`Figure 2 shows the amino acid sequence of the VH
`domain of NEWM in comparison with the v8 domain of
`the BI-8 antibody;
`
`Figure 3 shows the amino acid and nucleotide
`sequence of the HuVNP gene;
`
`Figure 4 shows a comparison of the results for
`HUVNp-IgE and MoVNp-IgE in binding inhibition
`assays;
`
`Figure 5 shows the structure of three
`oligonucleotides used for site directed mutagenesis;
`Figure 6 shows a protocol for the construction of
`CDR replacements by insertion of CDR cassettes into
`a vector containing four framework regions fused
`together;
`
`Figure 7 shows the sequence of the variable domain
`of antibody Dl. 3 and the gene coding therefor; and
`
`Figure 8 shows a protocol for the cloning of the
`Dl.3 variable domain gene.
`
`E XAMP LE 1
`
`This example shows the production of an altered
`antibody in which the variable domain of the heavy
`chains comprises the framework regions of a human
`heavy chain and the CDRs from a mouse heavy chain.
`
`17 of 41
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`The framework regions were derived from the human
`myeloma heavy chain NEWM, the crystallographic
`structure of which is known (see Poljak et al. , loc.
`cit. and Reth, M., Hammerling, G.J. and Rajewsky;
`K. , EMBO J. , l' 629-634, 198 2. )
`
`The CDRs were derived from the mouse monoclonal · ·
`
`antibody Bl-8 (see Reth et al. , loc. cit.), which
`binds the hapten NP-cap (4-hydroxy-3-nitrophenyl
`
`acetyl-caproic acid: KNP -CAP=l. 2 JM).
`
`A gene encoding a variable domain HuVNp, comprising
`the Bl-8 CDRs and the NEWM framework regions, was
`constructed by gene synthesis as follows.
`
`The amino acid sequence of the Vtt domain of NEWM is
`shown in Figure 2, wherein it is compared to the
`amino acid sequence of the VH domain of the Bl-8
`antibody. The sequence is divided into framework
`regions and CDRs according to Kabat et al. (loc.
`cit.). Conserved residues are marked with a line.
`
`The amino acid and nucleotide sequence of the HuVNP
`gene, in which the CDRs from the Bl-8 antibody
`alternate with the framework regions of the NEWM
`antibody, is shown in Figure 3. The HuVNP gene was
`derived by replacing sections of the MoVNP gene i-n
`the vector pSV-VNP (see Neuberger, M.S., Williams,
`G.T. , Mitchell, E.B. , Jouhal, s., Flanagan, J.G. and
`Rabbitts, T.H. , Nature, 314, 268-270, 1985) by a
`synthetic fragment encoding the HuVNP domain. Thus
`the 5' and 3' non-coding sequences, the leader
`sequence, the L-V intron, five N-terminal and four
`
`18 of 41
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`C-terminal amino acids are from the MoVNP gene and
`the r�st of the coding sequence is from the
`synthetic HuVNP fragment.
`
`The oligonucleotides from which the HuVNP fragment
`was assembled are aligned below the corresponding
`portion of the HuVNP gene. For convenience in
`cloning, the ends of oligonucleotides 25 and 26b
`form a Hind II site followed by a Bind III site, and
`the sequences of the 25/26b oligonucleotides
`therefore differ from the HuVNP gene.
`
`The HuVNP synthetic fragment was built as a
`P stI-Hind III fragment. The nucleotide sequence was
`derived from the protein sequence using the computer
`programme ANALYSE Q (Staden, R., Nuc. Acids. Res. ,
`12, 521-538, 1984) with optimal codon usage taken
`from the sequences of mouse constant domain genes.
`The oligonucleotides Cl to 26b, 2 8 in total) vary in
`size from 14 to 59 residues and were made on a
`Biosearch SAM or an Applied Biosystems machine, and
`purified on SM-urea polyacrylamide gels (see Sanger,
`
`F. and Coulson, A. , FEBS Lett., �' 107-110, 1978).
`
`The oligonucleotides were assembled in eight single
`stranded blocks (A-D) containing oligonucleotides
`
`(1, 3 , 5, 7) (Block A), ( 2 , 4, 6, 8) (block A' ),
`[9, ll,13a, 13b) (Block B), [lOa, lOb,12/14) (block
`
`B' ) , [ 15, 17] (block c) , [ 16, 18) (block c' ) , [ 19,
`
`21, 23, 25) (block D) and (20, 2 2 /24, 26a, 26b)
`(block D' ).
`
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`In a typical assembly, for example of block A, 50
`pmole of oligonucleotides 1,3,5 and 7 were
`phosphorylated at the 5' end with T4 polynucleotide
`kinase and mixed together with 5 pmole of the
`terminal oligonucleotide [l] which had been
`phosphorylated with 5 pCi [�-32p] ATP (Amersham 3000
`Ci/rnrnole). These oligonucleotides were annealed by
`heating to 80°C and cooling over 30 minutes to room
`temperature, with unkinased oligonucleotides 2, 4
`
`and 6 as splints, in 150 pl of 50 mM Tris.Cl, pH
`7.5, 10 mM MgCl2. For the ligation, ATP (1 mM) and
`DTT (lOmM) were added with 50 U T4 DNA ligase
`(Anglian Biotechnology Ltd.) and incubated for 30
`minutes at room temperature. EDTA was added to 10
`mM, the sample was extracted with phenol,
`precipitated from ethanol, dissolved in 20.Jll water
`and boiled for 1 minute with an equal volume of
`formamide dyes. The sample was loaded onto and run
`on a 0.3 mm SM-urea 10% polyacrylamide gel. A band
`of the expected size was detected by autoradiography
`and eluted by soaking.
`
`Two full length single strands were assembled from
`blocks A to D and A' to D' using splint
`oligonucleotides. Thus blocks A to D were annealed
`
`and ligated in 30 y1 as set out in the previous
`
`paragraph using 100 pmole of oligonucleotides lOa,
`16 and 20 as splints. Blocks A' to D' were ligated
`using oligonucleotides 7, 13b and 17 as splints.
`
`After phenol/ether extraction, block A-D was
`annealed with block A'-D', small amounts were cloned
`in the vector Ml3mpl8 (Yanish-Perron, c., Vieira, J.
`and Messing, J., Gene, ]l, 103-119, 1985) cut with
`PstI and Hind III, and the gene sequenced by the
`
`20 of 41
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`dideoxy techni�ue (Sanger, F., Nicklen, s. and
`Coulson, A.R., PNAS USA, 74, 5463-5467, 1979).
`
`The MoVNP gene was transferred as a Hind III - BarnHI
`fragment from the vector pSV-VNP (Neuberger et al.,
`loc. cit.) to the vector Ml3mp8 (Messing, J. and
`Vieira, J., Gene, 11_, 269-276, 1982). To facilitate
`the replacement of MoVNP coding sequences by the
`synthetic HuVNP fragment, three Hind II sites were
`removed from the 5' non-coding sequence by site
`directed mutagenesis, and a new Hind II site was
`subsequently introduced near the end of the fourth
`framework region (FR4 in Figure 2). By cutting the
`vector with PstI and Hind II, most of the VNP
`fragment can be inserted as a PstI-Hind II
`fragment. The sequence at the Hind II site was
`corrected to NEWM FR4 by site directed mutagenesis.
`
`The Hind III - Barn HI fragment, now carrying the
`HuVNP gene, was excised from Ml3 and cloned back
`into pSV-VNP to replace the MoVNP gene and produce a
`vector pSV-HuVNP·
`Finally, the genes for the heavy
`chain constant domains of human Ig E (Flanagan, J.G.
`and Rabbitts, T.H., EMBO J., .!1 655-660, 1982) were
`introduced as a Barn HI fragment to give the vector
`pSV-HuVNP· HE. This was transfected into the
`myeloma line J558 L by spheroplast fusion.
`
`The sequence of the HuVNP gene in pSV-HuVNP· HE was
`checked by recloning the Hind III-Barn HI fragment
`back into Ml3mp8 (Messing et al., loc. cit.). J558L
`myeloma cells.secrete lambda 1 light chains which
`have been shown to associate with heavy chains
`containing the MoVNP variable domain to create a
`
`'
`
`.
`
`21 of 41
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`binding site for NP -cap or the related hapten
`NIP-Cap (3-iodo-4-hydroxy-5-nitrophenylacetyl
`caproic acid) (Reth, M., Hammerling, G.J. and
`Rajewsky, K. , Eur. J. Immunol., �, 393-40 0 , 1978 ) .
`
`As the plasmid pSV-HuVNp.HE contains the .9£!: marker,
`stably transfected myeloma cells could be selected
`in a medium containing mycophenolic acid.
`Transfectants secreted an antibody (HuVNp-IgE) with
`heavy chains comprising a HuVNP variable domain
`(i.e. a "humanised" mouse variable region) and
`
`human O constant domains, and lambda 1 light chains
`
`from the J558L myeloma cells.
`
`The culture supernatants of several gpt+ clones were
`assayed by radioimmunoassay and found to contain
`NIP-cap binding antibody. The antibody secreted by
`one such clone was purified from culture supernatant
`by affinity chromatography on NIP-cap Sepharose
`(Sepharose is a registered trade mark) . A
`
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