`
`European Patent Office
`
`@ Publication number:
`
`Office europeen des brevets
`
`0239400
`A2
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`EUROPEAN PATENT APPLICATION
`
`@ Application number: 87302620.7
`
`@ Date offillng: 26.03.87
`
`® Int.Cl.•: C 12 N 15/ 00, C 07 K 15/06,
`c 12 p 21/02
`
`@ Priority: 27.03.86 GB 8607679
`
`® Applicant: Winter, Gregory Peul, 64 Cavendish Avenue,
`Cambridge (GB)
`
`@ Date of publication of application: 30.09.87
`BuRetln87/ 40
`
`@
`
`Inventor: Winter, Gregory Paul, 64 C.Vendlah Avenue,
`Cambridge (GB)
`
`@) Designated Contracting States: AT BE CH DE ES FR GB
`GR ITLI LU NL SE
`
`@ Representative: Votler, Sidney David et al, CAR PMAELS
`& RANSFORD 43, Bloomsbury Square, Lo ndo n
`WC1A2RA (GB)
`
`® Recombinant antibodies and met hods for their production.
`@ An altered antibody is produced by replacing the com(cid:173)
`plement arity determining regions (CDRs) of a variable region
`of an lmmunoglobulin (lg) w ith the CDRs from an lg of
`different specificity, using recombinant DNA techniques. The
`gene coding sequences for producing the altered antibody
`may be produced by site-directed mutagenesis using long
`ollgonucleot ides.
`
`ACTORUM AG
`
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`
`O 1.3 CDR I ollgonuel1olld1
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`
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`
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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 f rom 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
`l ight 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 Perry , H.,
`in "Sequences of Proteins of Immunological
`Interest" , US Dept . Health and Human Services 1983) .
`The four framework regions largely adopt a ~- sheet
`conformation and the CDRs form loops connecting, and
`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 , r eference may be made to: Poljak,
`R.J ., Amzel , L.M., Avey , H.P. , Chen , B. L.,
`Phizackerly , R.P. and Saul, F. , PNAS USA, ]J_,
`3305-3310, 1973; Segal, D.M., Padlan, E. A., Cohen,
`G. H. , Rudikoff , s. , Potter , M. and Davies , D.R.,
`PNAS USA , 21.1 4298-4302, 1974; and Marquart, M.,
`Deisenhofer, J., Huber , R. and Palm, 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.
`
`EP-A- 0 088 994 (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 Ig 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 .
`
`EP- A- 1 102 634 (Takeda Chemical Industries Limited)
`describes the cloning and expression in bacterial
`host organisms 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 .
`
`EP-A-0 125 023 (Genentech Inc.) 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 production 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 80s , Eds . Voller , A., Bartlett,
`A. , and Bidwell , D., MTP Press , Lancaster , 1981) but
`also in therapy (see , for example , Ritz, J. and
`Schlossman , S . F., Blood , ~' 1-11, 1982).
`
`The recent emergence of t echniques allowing the
`stable introduction of Ig gene DNA into myeloma
`cell s (see , fo r example , Oi , V.T. , Morrison , S . L. ,
`Berzenberg , L.A. and Berg , P ., PNAS USA , ~'
`825-829 , 1983; 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., PNAS USA , ~ ' 6351-6355 , 1983) , 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 is therefore. not at all clear that it 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/GB85/00392
`(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 :
`
`i) at least part, for instance the constant
`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 ;
`iii) a protein having a known binding
`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
`Igs . However , from the three dimensional structure
`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 is
`considerable overlap.
`
`Moreover , not all of the amino-acid residues in the
`loop regions are solvent accessible and in one
`case, amino-acid residues in the framework regions
`are involved in antigen binding.(Amit, A.G . ,
`Mariuzza, R.A. , Phillips , S.E.V. and Poljak , R.J.,
`Science , 233 , 747-753 , 1986).
`
`It is also known that the variable regions of the
`two parts of an antigen binding site are held in the
`correct orientation by inter-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 involve transferring framework region
`residues as well as CDR residues .·
`
`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
`(and therefore the ends of the CDR loops) are
`relatively conserved. However, small shifts in
`packing and orientation of these ~- sheets do occur
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`(Lesk, A. M. and Chothia , c. , J. Mol. Biol ., 160,
`325-342, 1982} . 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 skill ed in the
`art , either by carrying out r outine experimentation
`or by trial and error testing to obtain a functional
`altered antibody .
`
`Preferably , the variable domains in both the heavy
`and l ight 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 regions 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 variable
`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 reduced 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 framewor k
`regions 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 t he 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 recipient of the altered antibody .
`This response 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 r egions .
`It would therefore be possible to provide a panel of
`antibodies having the same partial or complete CDR
`replacements but on a series of different framework
`regions . 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
`t he 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;
`
`if necessary , preparing a second
`b)
`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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`transforming a cell line with the first or
`c)
`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.
`
`Preferably, 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 Epstein- 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
`In particular, it is envisaged that E. Coli
`used.
`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'ae 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., 6487- 6500 , 1982;
`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- 488 (1984); Kramer ,
`w. , Schughart , K. and Fritz, w. -J., Nuc . Acids Res. ,
`.!Q_, 6475- 6485 , 1982.
`
`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 80 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.
`
`EXAMPLE 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.
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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 , 1982.)
`
`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
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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
`PstI-Hind III fragment. The nucleotide sequence was
`derived from the protein sequence using the computer
`programme ANALYSEQ (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 , 28 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 , 22/24, 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
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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
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`binding site for NP - cap or the related hapten
`NIP-Cap (3- iodo- 4- hydroxy-5-nitrophenylacetyl(cid:173)
`caproic acid) (Reth, M., Hammerling , G.J. and
`Rajewsky , K., Eur. J . Immunol. , ~ , 393 - 400 , 1978) .
`
`As the plasmid pSV-HuVNp.HE contains the .9£!: marker ,
`stably transfected myeloma cells could be selected
`in a medium containing mycophenolic acid.
`Transfectant