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  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
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  • C07—ORGANIC CHEMISTRY
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  • C07—ORGANIC CHEMISTRY
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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  • C07—ORGANIC CHEMISTRY
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
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  • C07K2319/00—Fusion polypeptide
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
  • C12N2310/111—Antisense spanning the whole gene, or a large part of it
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
  • C12N2310/124—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes based on group I or II introns
  • C12N2310/1241—Tetrahymena
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

• the present invention relates to polypeptides which comprise two or more component polypeptides or peptides, methods for making them and DNA constructs for the use in this making.
• it relates to the provision of repertoires of such polypeptides and encoding nucleic acid therefor.
• RNA splice sites such as self- splicing introns
• site-specific recombination system such as lox P (Hoess et al Proc. Natl. Acad. Sci. USA 79 3398-3402, 1982; Sternberg et al J. Mol. Biol . 150 467-486, 1981) .
• the site-specific recombination allows two sequences of nucleic acid to be cloned separately as libraries and be brought together subsequently by a recombination event (Waterhouse et al Nucleic Acids Res.
• One library of sequence is cloned into a first replicon and a second library of sequences into a second replicon. Recombination between the sites brings together libraries of both sequences on the same replicon. This recombination can be performed in vivo e.g. by Pl infection or by using a recombinase encoded by a plasmid in E. coli or in vi tro using soluble recombinase. For lox P, the recombinase is Cre.
• the size of the library is significant for ability to select antibodies or other binding proteins of appropriate affinity and specificity.
• WO 93/19172 describes recombining two libraries of nucleic acid using a site-specific e.g. lox P, system mainly to code for heterodimeric proteins in which two chains encoded by distinct (separate) nucleic acid sequences associate to form a functional binding site. Also described is the bringing together of two polypeptides for continuous open reading frames. However, this imposes the use of an amino acid sequence encoded in the site-specific recombination sequence at the junction between the two parts of the sequence, for instance the linker in single chain Fv molecules. A problem with this is that there is only one open reading frame in the lox P sequence and the amino acids encoded by this may be incompatible with the expression of many proteins in functional form. If alternative lox P sites to the wild-type are used (eg see Figure 4) , further different amino acid sequences may be generated, but the possibilities are still restricted.
• lox P sites to the wild-type are used (eg see Figure 4) , further different amino acid sequences may be generated, but the
• functional single chain Fv molecules can be constructed with 15 amino acid linkers encoded in part by the loxP recombination site.
• the length of the loxP site 34bp
• a minimum of 11 heterologous (“foreign") amino acids must be incorporated into the final expressed protein. This makes the incorporation of a loxP site into a continuous reading frame unsuitable for the construction of a diabody repertoire and also leaves little scope for the modification of scFv linkers to enhance expression.
• the present invention involves RNA splicing, particularly the use of self-splicing introns. This allows the recombination site to be inserted within the intron so that amino acids encoded by nucleotides which are spliced out are not incorporated into the final expressed protein. In such circumstances, the only “foreign" amino-acids which need be incorporated are those derived from the sequences at either end of the self-splicing intron. (Note: the amino acid composition and sequence of the product can be engineered with precision and amino acids inserted, substituted or deleted according to choice and using techniques known in the art. )
• a “diabody” is a multivalent or multispecific multimer (e.g. bivalent or bispecific dimer) of polypeptides wherein each polypeptide in the multimers comprises a first domain comprising a binding portion of an immunoglobulin heavy chain variable region linked to a second domain which comprises a binding protein of an immunoglobulin light chain variable region such that the domain of a given polypeptide cannot associate with each other to form an antigen binding site.
• Antigen binding sites are formed from an antigen binding site.
• Antigen binding sites are formed by multimerisation (e.g. dimerisation) of the polypeptides.
• the use of systems such as lox P which promote recombination allows one polypeptide sequence to be replaced by another one with a similar or different function, originally encoded on another replicon. This is particularly useful with polypeptide chains such as single chain Fvs which have two or more domains which contribute to function.
• the invention allows the use of two repertoires of nucleic acid, with a splice site between the two repertoires and proteins or peptides thus encoded selected. In one embodiment, termed "chain shuffling", one nucleic acid sequence is kept constant and the library of other chains recombined at the lox P site in the intron. Self-splicing introns have been shown to be functional in E .
• the present invention provides a DNA construct comprising a first sequence of nucleotides encoding a first peptide or polypeptide, a second sequence of nucleotides encoding a second peptide or polypeptide and a third sequence of nucleotides between the first and second sequences encoding a heterologous intron between RNA splice sites and a site-specific recombination sequence within the intron.
• RNA splice sites render the intron operable for splicing out of nucleotides from between the first and second sequences upon transcription of the DNA construct into RNA, which may result in splicing together of the first and second sequences .
• one or more nucleotides may remain between the first and second sequences in transcribed RNA following splicing, resulting in one or more amino acids between the first and second peptides or polypeptides in the product of translation of the RNA.
• first and second sequences may be termed "exon" sequences.
• heterologous indicates that the intron is one not found naturally between the first and second sequences in a position operable for removal of nucleotides from between the first and second sequences upon transcription.
• DNA constructs according to the present invention are "artificial” in the sense that they do not occur naturally, ie without human intervention by means of recombinant DNA technology.
• the first and second peptides or polypeptides may be any sequence of amino acids.
• the first and second polypeptides together form a member of a specific binding pair (sbp) , such as the antigen binding site of an immunoglobulin (antibody or antibody fragment) .
• sbp specific binding pair
• the combination of first and second polypeptides may form a polypeptide sbp member which is a scFv antibody fragment consisting of a VH domain linked to a NL domain by a peptide linker which allows the NH and VL domains of the sbp member to associate with one another to form an antigen binding site.
• the DNA construct comprises a first sequence of nucleotides encoding a VH or VL domain, a second sequence of nucleotides encoding a counterpart VL or VH domain and a third sequence of nucleotides, between the first and second sequences, comprising a heterologous intron.
• nucleotides of the third sequence Upon transcription of the DNA construct into RNA and splicing out of nucleotides of the third sequence, nucleotides of the third sequence remaining in the RNA encode, and are translatable into, the peptide linker of the scFv antibody fragment.
• This principle with nucleotides of the third sequence encoding and being translatable into amino acids of a linker joining the first and second peptides or polypeptide chains, may be used for any peptides or polypeptides, for example in the creation of peptide libraries.
• the first and second sequences encode peptides or polypeptides which are not linked in any naturally occuring polypeptide.
• the peptides or polypeptides may be derived from the same naturally occuring molecule but not linked directly by a peptide bond, ie they may be two parts of a polypeptide naturally separated by one or more intervening amino acids .
• One or both of the first and second peptides or polypeptides may be an antibody fragment, for example VH, VL, CH, CL, VH-CH or VL-CL.
• the peptide or polypeptide need not be a complete domain.
• first and second peptides or polypeptides may be encoded by a synthetic nucleotide sequence, eg one created randomly.
• a random sequence peptide or polypeptide library may be created for example by expression from a repertoire or population of DNA constructs, as disclosed, wherein the first and second exon sequences comprise randomly-generated nucleotide sequences.
• the DNA construct may be transcribable into RNA which, following splicing, encodes a "diabody" polypeptide, ie a polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the domains being linked (eg by a peptide bond or peptide linker) but incapable of associating with each other to form an antigen binding site.
• the domains are linked by a peptide linker, the linker may, for instance, be 10 amino acids or fewer in length. See Holliger et al, PNAS USA 90: 6444-6448 (1993) and PCT/US93/02492.
• Polypeptides of this kind are able to associate with one another to form multivalent or multispecific binding proteins.
• DNA constructs which can be transcribed into RNA which, following splicing, encodes such a "diabody” polypeptide may, however, be excluded from the present invention.
• first and second peptides or polypeptides include any polypeptide comprising binding regions of immunoglobulin heavy and light chain variable domains; Va/V ⁇ domains of T cell receptors; T cell receptor/antibody (fragment) fusions; peptides, for example for epitope mapping of an antibody, receptor binding peptides, enzyme, eg protease, inhibitors; mutagenesis libraries of any multiple domain protein, for example nucleotide dehydrogenases which have nucleotide binding domains and substrate binding domains, adhesion molecules such as ICAM-1, receptors such as PDGF-receptor which have a ligand binding domain and a kinase domain, transcription factors which have a DNA binding domain and a second domain which interacts with a ligand - such as the glucocorticoid receptor.
• nucleotide dehydrogenases which have nucleotide binding domains and substrate binding domains
• adhesion molecules such as ICAM-1
• the intron may be a self-splicing group I intron such as ICE10 from Tetrahymena (T.R. Cech Ann. Rev.
• self- splicing group I introns including: Tetrahymena thermophila rRNA intron, Neurospora crassa cytochrome b gene intron 1, Neurospora crassa mitochondrial rRNA, Neurospora crassa cytochrome oxidase subunit 1 gene oxi3 intron, phage T4 thymidylate synthase intron, Clamydomonas reinhardtii 23S rRNA Cr.LSU intron, phage T4 nrdB intron, Anabaena pre tRNA(Leu) intron.
• Group II self-splicing introns include yeast mitochondrial oxi3 gene intron5 ⁇ and Podospora anserina cytochrome c oxidase I gene.
• Self-splicing introns may be used in combination with recombination, for example, at a lox P site, in the construction of molecules.
• a lox P site may be included in a self-splicing intron between the two domains (eg VH and VL) of a polypeptide chain. This may, for example, be recombined at the DNA level through a lox P site on another replicon carrying another variable domain gene and the appropriate region of a self-splicing intron.
• Self-splicing at the RNA level following transcription will now lead to a product polypeptide chain with a new combination of first and second polypeptides.
• the third sequence of nucleotides in the DNA construct, the intron comprises a sequence for site-specific recombination.
• the sequence may be suitable for site- specific recombination in vivo and/or in vi tro . It may be the lox P site, a 34bp site at which recombination is catalysed by the protein Cre (Hoess et al. , PNAS USA 79: 3398-3402, 1982, and Sternberg et al . , J " . Miol . Biol . ; 150 . : 467-486, 1981) .
• the 34bp of the lox P site consists of two 13bp inverted repeats separated by an 8bp non-symmetrical core (see Figure 4) .
• each vector may include two site-specific recombination sequences each of which is different from the other.
• the sequences should then be such that recombination will take place between like sequences on different vectors but not between the different sequences on the same vector.
• site-specific recombination allows first and second nucleic acid sequences originally on different (first and second) vectors/replicons to be brought together onto a single recombinant vector/replicon.
• Each of the first vectors and each of the second vectors may include a first site-specific recombination sequence and a second site-specific recombination sequence different from the first, site-specific recombination taking place between first site-specific recombination sequences on different vectors and between second site-specific recombination sequences on different vectors but not between a first site-specific recombination sequence and a second site-specific recombination sequence on the same vector.
• the first site-specific recombination sequence may be lox P obtainable from coliphage Pl and the second site-specific recombination sequence a mutant lox P sequence, or vice versa. Potentially, both the first and second site-specific recombination sequences may be mutants, as long as the first sequence will not recombine with the each other and second sequences will recombine with each other.
• a suitable mutant lox P sequence is lox P 511. See Figure 4.
• the first vectors may be phages or phagemids and the second vectors plasmids, or the first vectors may be plasmids and the second vectors phages or phagemids.
• This system ie employing site-specific recombination but not intron splicing has been used in the preparation of antibodies displayed on phage (P. Waterhouse et al . , Nuc. Acid Research 2 - 2265-2266, 1993; and W093/19172) .
• the recombination is intracellular and takes place in a bacterial host which replicates the recombinant vector preferentially over the first vectors and the second vectors. This may be used to enrich selection of successful recombination events.
• the intracellular recombination may take place in a bacterial host which replicates plasmids preferentially over phages or phagemids, or which replicates phages or phagemids preferentially over plasmids.
• the bacterial host may be a PolA strain of E. coli or of another gram-negative bacterium.
• PolA cells are unable to support replication of plasmids, but can support replication of filamentous phage and phagemids (plasmids containing filamentous phage intergenic regions) . So, for instance, if the first vectors are plasmids containing a first marker gene, and the second vectors are phage or phagemids containing a second marker gene, selection for both markers will yield recombinant vectors which are the product of a successful recombination event, since recombination transferring the first marker from plasmid must take place in order for that marker to be replicated and expressed.
• nucleic acid for two components or subunits of a product polypeptide bringing together of nucleic acid for two components or subunits of a product polypeptide, initially present on two separate replicons enables favourable combinations of subunit genes to be isolated directly without recourse to extensive recloning, e.g. using phage display. This may be achieved by recombination between the replicons once they have been introduced into the same cell .
• recombination events are effected such that the genes for one of the component is recombined onto a recipient replicon which contains the gene for a partner component.
• the recipient replicon is capable of being packaged into a bacteriophage particle.
• the genes encoding one or more of the subunits is fused to a capsid gene such as gill in order that the functional multimer can be displayed on the surface of the rgdp.
• a capsid gene such as gill
• a variety of recombination systems are known, and many of these could be harnessed in such as way as to effect recombination between replicons.
• the integration event is catalysed by a host encoded factor called IHF and a phage encoded enzyme called Int recombinase, which recognises a 15bp region common to the two att sites.
• the integrated DNA is flanked by sequences derived from att B and att P, and these are called att L and att R.
• the integration event is reversible and is catalysed by Int, IHF and a second bacteriophage encoded enzyme, Xis. It is envisaged that this system could be used for sequence transfer between replicons within E. coli.
• the donor gene could be flanked by att L and att R sites such that when Int and Xis proteins are provided in host cell, recombination between att L and att R sites would create a circular DNA segment containing the donor gene and a recreated att B site. This circular segment could then recombine with an att P site engineered into the recipient plasmid.
• the lox P/Cre system was chosen of the possibilities available because the recombination is highly sequence- specific, very efficient and occurs at a short target site that is readily incorporated into cloning vectors.
• site-specific recombination systems may be used, for instance: flp recombinase (A. Landy, Curr. Opinion Genetics Devel . 3 699-707, 1993) .
• lox P 511 has a G->A point mutation in the central 8bp segment, with the result that it will only recombine with other lox P 511 sites, but not the wild-type lox P sequence (Hoess, R.H. Wierzbicki, A. and Abremski, K.
• Placement of wild-type and mutant lox P sequence combinations can direct which recombination events are possible.
• the sites loxPl, loxP2, loxP3 and loxP4 ( Figure 4) can be used in a similar way to loxP511. These sites do not recombine significantly with loxP511. There is in some cases a degree of recombination between the loxPWT site and these mutant sites, derived from it. For instance, in one experiment 5% recombination was observedbetween loxP3 and loxPWT sites.
• a clone specific for an antigen may be isolated where the gene for a VH domain of a scFv fragment is located between loxP511 and loxP wt of a vector containing 3 loxP sites, such as fd31ox.
• a library of VL domains may then be shuffled with the VH domain gene kept constant by recombining the clon in the 3 loxP site vector with a library of VL genes on a donor vector such as pUC19 which are located between the 2oxP4 site and the loxP 511 site.
• the library of VL domain genes is now encoded in the 3 lox site vector and scFv fragments, eg with improved affinity, may be selected from the phage displayed scFv fragment repertoire.
• this 3loxP system gives more flexibility, particularly to the nature of the replicon, phage or plasmid, where the reshuffled repertoire is expressed, since both repertoires are flanked by loxP sites.
• Example 6 and Figure 13 show the use of a loxP system in model experiments for the construction of a diabody or single chain Fv repertoire where the VH and VL genes are separated by a self-splicing intron containing a loxP site.
• the design of the system will faciliate chain shuffling as above.
• (b) It facilitates the transfer of light and heavy chain gene pairs which have been selected on the surface of filamentous bacteriophage for binding to antigen into a soluble expression vector for expression of e.g. soluble scFv fragments, which at present needs to be done by cloning using restriction enzymes.
• the transfer by recombination could be achieved by creating an expression vector containing a new mutant loxP site such as loxP4 and the WT site and by recombination between these two sites and the corresponding sites on the fd3lox vector. Model experiments for this are described in example 6 and Figure 13.
• loxP sites also allows, for example, the recombination of three sequences in order.
• One sequence to be recombined could be flanked by loxP and loxP ⁇ ll , a second sequence by loxP ⁇ ll and loxP3. These sequences may then be recombined into a third replicon containing a third DNA sequence and three loxP sites.
• the location of 2 loxP sites within different self splicing introns allows the three sequences to be expressed continuously as shown in Figures 7 and 8.
• Selection of productive arrangements may be facilitated by use of a polA strain of bacteria, preferably E. coli or other gram negative bacterium.
• the invention also provides a vector comprising a DNA construct as disclosed.
• the vector comprises nucleic acid necessary for expression.
• the vector may comprise nucleic acid for secretion of the product polypeptide upon expression.
• the present invention also provides a method of producing a polypeptide product which comprises a combination of a first peptide or polypeptide component and a second peptide or polypeptide component, the method comprising: providing a DNA construct comprising a first sequence of nucleotides encoding a first peptide or polypeptide, a second sequence of nucleotides encoding a second peptide or polypeptide and a third sequence of nucleotides between the first and second sequences encoding a heterologous intron with a site-specific recombination sequence within the intron; transcribing DNA of the construct into RNA; causing or allowing splicing of nucleotides of the third sequence to produce an RNA molecule encoding the polypeptide product; translating the RNA molecule into the polypeptide product.
• the transcription, splicing and translation steps may take place in in vi tro or in vivo systems.
• these steps are performed in vivo, eg in E. coli .
• Splicing may also be accomplished, less preferably, using in introns which are not self-splicing, by introducing the components of the splicing apparatus of eukaryotic cells, which promote splicing (J.A. Wise Science 262 1978-1979, 1993; A.J. Lamond, BioEssays 15 595-603, 1993) , into eg E. coli .
• the DNA construct provided may be any as discussed above.
• Suitable vectors for expression can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate, as is well known to those skilled in the art. For further details see, for example, Molecular Cloning: a Laboratory Manual : 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. Transformation procedures depend on the host used, but are well known.
• a phage or phagemid vector is used and the vector, with the DNA construct, packaged into a bacteriophage particle.
• the polypeptide product comprises a domain which is a surface component of an organism such as a bacteriophage, for example a filamentous bacteriophage such as fd or M13.
• the surface component is GUI of bacteriophage fd or the equivalent from another filamentous 'phage. Suitable technology is described in WO92/01047, WO92/20791, WO93/06213, W093/11236, W093/19172 and PCT/GB93/02492.
• the provided DNA construct is packaged into a particle which displays on its surface the polypeptide product of expression from the construct, including the splicing step.
• polypeptide product with binding affinity or enzymatic eg catalytic affinity for a target can be extracted from medium or selected from a mixture of different polypeptide products without such binding affinity or enzymatic activity, by contact with target eg using a chromatographic technique.
• selection may be on the basis of binding affinity for complementary sbp member: eg an immunoglobulin binding domain (such as scFv fragment) can be selected on the basis of binding affinity for antigen.
• the step of provision of a DNA construct may actually involve the provision of a plurality, eg a repertoire, of constructs with different nucleic acid sequences.
• the term "repertoire" is used to indicate genetic diversity, ie variety in nucleotide sequence, and generally implies a large number of different sequences, perhaps of the order of millions (eg 10 7 -10 9 - 10 12 -10 14 ) .
• Highly diverse repertoires may be created when a sequence for site-specific recombination, (as discussed, eg lox P wild-type or mutant) , is included within the third sequence in the DNA construct at a site compatible with splicing upon transcription.
• each recombination may introduce a further level of diversity of 10 7 , thus recombination between a first repertoire encoding 10 7 different VH domains with a second repertoire encoding 10 7 different VL domains yields a recombinant repertoire encoding 10 14 different product polypeptides.
• two libraries of 10 3 clones can be recombined to give a library of 10 6 clones.
• a first repertoire of replicons comprising nucleic acid encoding a repertoire of first peptide or polypeptide component may contain part of a self-splicing intron, while a second repertoire of replicons comprising nucleic acid encoding a repertoire of second peptide or polypeptide component contains a complement part of the self-splicing intron.
• the replicons in each of the first and second repertoires of replicons each comprise a sequence for site-specific recombination, suitably positioned such that recombination of a replicon from the first repertoire of replicons with a replicon from the second repertoire of replicons results in formation of the self-splicing intron in the resultant recombinant replicon.
• replicons in either or both of the first and second repertoires may contain a complete self- splicing intron.
• the first and second repertoires of replicons may be recombined ("crossed") , eg at a site-specific recombination sequence, to produce a third repertoire of (recombinant) replicons which includes nucleic acid encoding a plurality of different combinations of first and second peptide or polypeptide component, with a self-splicing intron between the nucleic acid encoding the first and second peptide or polypeptide components on each replicon.
• the recombination may take place in vivo in bacterial host cells following transfection with the first repertoire of replicons and subsequent transfection with the second repertoire of replicons.
• the recombination may be catalysed by Cre recombinase. Transcription of nucleic acid in the third repertoire of replicons into RNA is followed by splicing out of the intron containing the sequence for site-specific recombination, leaving mRNA coding for polypeptide product which can be translated into the polypeptide product.
• the production of a repertoire of polypeptide products comprising different combinations of first and second peptide or polypeptide components may be followed by a step of selection of products of interest, such as those with a particular binding specificity or enzymatic activity.
• Each replicon in the third repertoire of replicons may comprise a sequence enabling packaging of the replicon into a bacteriophage particle, and the polypeptide product may comprise a surface component of a bacteriophage, as discussed. Then, particles may be selected from a repertoire of particles by their display of polypeptide product with a binding specificity or enzymatic activity of interest. Each selected particle then contains DNA encoding that polypeptide product.
• Figure 5 demonstrates the principle for use in production of a scFv repertoire.
• first polypeptide component of the polypeptide product is a VH domain and the "second polypeptide component” of the polypeptide product is a VL domain.
• a lox P site is included within a Class I self-splicing intron.
• the peptide linker of each scFv fragment in the product repertoire is formed, at least in part, by remnants of the splice sites left after splicing out of the intron between the VH and VL domains upon transcription.
• a single first or second peptide or polypeptide component may be "chain shuffled" against a repertoire of corresponding second or first peptide or polypeptide component.
• a VH or a VL domain known to be able with complementary VL or VH domain to bind to the antigen may be combined with a repertoire of complementary VL or VH domain to produce a repertoire for expression followed by selection on the antigen for pairings able to bind.
• a further aspect of the present invention provides nucleic acid comprising a sequence of nucleotides encoding self-splicing intron with a site- specific recombination sequence, such as a loxP site or a mutant or derivative thereof, within the intron.
• a site-specific recombination sequence such as a loxP site or a mutant or derivative thereof
• nucleic acid consists essentially of a sequence of nucleotides encoding self-splicing intron with a site-specific recombination sequence within the intron.
• Such nucleic acid may be isolated and is suitable for use in creation of constructs for use in a method as herein disclosed.
• the nucleic acid comprises restriction sites flanking the intron, for ligation of nucleic acid encoding or peptides.
• the nucleic acid may be incorporated in a vector operably linked, ie under the control of, a promoter for expression.
• a promoter for expression ie under the control of, a promoter for expression.
• Other preferred features are as disclosed herein with reference to the methods and the DNA constructs.
• the site-specific recombination sequence within the intron is preferably heterologous, as discussed.
• a recombination site may be included in a self-splicing intron between the two antibody domains of the polypeptide chain. This may, for example, be recombined at the DNA level through a lox P site on another replicon carrying another variable domain gene and the appropriate region of a self- splicing intron. Self-splicing at the RNA level following transcription will now lead to a diabody polypeptide chain with a new combination of variable domains or a single chain Fv polypeptide, depending on the length of the linker region encoded. In PCT/GB93/02492 the splicing of an intron from RNA encoding a diabody polypeptide is described. This can readily be extended to single chain Fv fragments by introducing the sequence encoding the extra amino acids on either side of the RNA splice sites encoding the appropriate length of linker.
• Chain shuffling can be performed for bivalent or bispecific diabodies or for single chain Fv fragments using the systems described in Figures 3 and 5. As noted above, a further level of control may be established by the use of a system with 3 loxP sites, as shown in Figure 13. The expression of diabody and single chain Fv molecules from clones containing loxP sites within self splicing introns is demonstrated in examples 1, 2 and 4. Example 3 demonstrates the feasibility of making a large library which recombines two exons into a longer continuous sequence. This methodology for making a repertoire can be applied to other molecules such as single chain Fv fragments and diabodies where the VH and VL genes replace the peptide sequences.
• Example 6 describes model experiments which demonstrate that recombination can be performed between loxP sites configured for the construction of diabody or single chain Fv repertoires. It is concluded that this methodology is suitable for the libraries described in example 3 and Griffiths et al (1994, supra) and that libraries of more than 10 12 independent scFv or diabody clones are feasible.
• introns with splice sites such as self-splicing introns, containing an internal lox P site may be applied to any other system where two functional domains come together, for instance T cell receptors or two domain proteins.
• proteins with natural variants such as antibodies
• for any two domain proteins mutagenesis libraries can be made for the two domains and then combined using the lox P system.
• parts of domains may be spliced together, eg using a self-splicing intron.
• a self-splicing intron containing a recombination site such as lox P in framework 3 of V domains allows recombination of fragments containing CDRs 1 and 2 with fragments containing CDR3, eg in CDR3 shuffling.
• Libraries may be made where two sequence encoding peptides are encoded separated by a self-splicing intron containing a recombination, eg lox P, site. For instance, two separate libraries of ten amino acid peptides can be cloned and then recombined via the lox P 511 and lox P sites as is shown in Figure 6. The amino acids encoded by the region of the 5' and 3' splice sites make this into a total 25 amino acid peptide with 5 constant amino acids in the centre.
• the peptide library can then be used for a number of purposes, for instance the epitope mapping of antibody binding sites or to derive new molecules such as receptor binding proteins, protease inhibitors or substrates.
• Example 3 shows that a large phage display library of ca. 5 x 10 10 recombined 25 amino acid peptides may be constructed using recombination between loxP sites contained in a self splicing intron and peptides containing the epitope recognised by an anti- p53 antibody selected.
• Constrained peptide libraries may be made by incorporating a cysteine residue in each of the 10 amino acid peptides to be recombined so that a disulphide bond is formed and the peptides between the cysteine would form a loop.
• the five amino acid linker may be varied in length and amino acid sequence by varying the 5' and 3' splice sites and the reading frame.
• the number of random amino acids may also be varied and need not be the same on either side of the linker. This example demonstrates the feasibility of making a large library which recombines two exons into a longer continuous sequence.
• Two or more splicing introns may be used to link together three or more nucleic acid sequences encoding polypeptides. This may be particularly advantageous in constructing libraries where V-D-J recombination (for the antibody heavy chain) occurs in E. coli .
• site-specific recombination sequences e.g. lox P
• site-specific recombination sequences e.g. lox P
• lox P site-specific recombination sequences within the introns
• VH, DH and JH regions may be natural V, D and J genomic segments regions or derived from synthetic oligonucleotide sequences, perhaps of different lengths, especially for the D region, so that the range of CDR3 lenghts generated by the recombination may reflect the same (or a modified) distribution of natural CDR3 lengths and the presence or absence of N base addition.
• Figure 7 shows the use of lox P to achieve V-D-J recombination to obtain a single chain Fv molecule and Figure 8 shows the expression of this molecule.
• the introns and splice donor and acceptor sites need to be designed to ensure that splicing does not cut out the exon sited between the two introns.
• Example 1 shows two different amino acid sequences incorporated into a diabody due to this residue of the intron, with variation in expression occurring. There are likely to be differences in the expression of a number of proteins depending on the nature of the Pl and P10 sequences. Therefore, there may be a need in certain cases to identify amino acids which are compatible with successful splicing of the intron and expression of protein.
• Identification of suitable amino acids incorporated due to the bases at the 5'and 3' splice sites may be done by mutating bases (eg randomly) in the region of the internal guiding sequence with complementary bases which form the Pl hairpin loop of the intron. If the intron is now inserted, between the nucleic acid encoding the first and second peptides or polypeptides, for instance between the VH and VL domains of antibody fragment, such that efficiently spliced polypeptide product is produced and may be displayed on phage and selected by binding to target, those sequences compatible with efficient splicing can be selected. Similarly, sequences of the 3' splice site can be varied together with those of the internal guiding sequence and those which are efficiently spliced selected by the expression of the polypeptide sequence.
• This directed mutation procedure may be applicable to other sites of the self splicing intron.
• the GLSSG sequence used in Example 1 may be used as the first trial sequence for the sequence linking the two polypeptides following splicing out of the intron. Further sequences identified, eg using a mutation process as described in Example 4, may be used as alternatives .
• sequences of the splice site at the 5' end of the exon which are retained in the mature protein after splicing of the pre-mRNA that are compatible with self-splicing may be examined (F. Michel and E. Westhof J " . Mol . Biol . 216 581 - 606, 1990; F. Lisacek et al J. Mol . Biol . 235 1206-1217, 1994) . Sequences compatible with self-splicing leading to the incorporation of favourable amino acids may then be chosen.
• Streptomycin prevents self-splicing.
• streptomycin in Str-R E. coli will prevent splicing occurring in transcribed RNA.
• the removal of streptomycin will aloow the generation of a spliced RNA product, leading to, on translation, a protein product which is only generated on splicing.
• Figure 1 shows a schematic of a self-splicing intron, including the Pl and P10 helices and the internal guiding sequence. The splice sites are marked by arrows.
• Figure 2 illustrates the expression of a single chain Fv or diabody polypeptide from DNA containing a self splicing intron. The sequences flanking the self splicing intron will determine the length of the peptide linker. Ribosome binding sites are indicated by open circles, Lg3 is the leader sequence for phage fd gene III.
• Figure 3 illustrates chain shuffling of a diabody (or a single chain Fv) molecule. It shows the replicons generated by Cre-mediated recombination between the acceptor phage vector fdDOG-2dialoxsplice (A) and the donor plasmid vector pUC19-2dialoxsplice (B) .
• A is based on fd-tet-DOGl, with the chain VHA-VLB in one cistron under control of the gene III promoter. Between VHA and VLB is inserted the self-splicing intron from Tetrahymena containing the lox P 511 recombination site inserted at a site compatible with self-splicing activity.
• B is based on pUC19 and contains lox P 511, the distal part of the self- splicing intron from Terahymena, VLA, and the lox P wild type sequence in the same arrangement as A.
• a and B can cointegrate by recombination between either mutant or wild-type loxP sites to create chimaeric plasmids C and D respectively. Further recombination can then occur between the two wild-type or the two mutant loxP sites, to generate the original vectors (A and B) or two new vectors (E and F) .
• the light chains of A and B are therefore exchanged, and product E now encodes fd phage displaying a single chain Fv or a diabody depending on the linker length used.
• Product F contains the VL originally in A.
• Figure 4 shows the sequence of wild type and mutant lox P sites.
• Figure 5 illustrates the generation of a single chain Fv repertoire by recombination between repertoires of VH and VL domains.
• Figure 6 illustrates the generation of a peptide library by recombination between two replicons (a) pUC19-PEP and (b) fdDOG-PEP.
• rbs represents ribosome binding sites
• LpelB is the leader peptide sequence
• gill is fd phage gene III
• lOaa is a random oligonucleotide (NNK) 10 encoding ten amino acid residues (K is an equimolar mixture of G and T)
• * is an ochre stop codon.
• the expressed sequence is: aal-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9-aal0-A-L-L-R-Y- aall-aal2-aal3-aal4-aal5-aal6-aal7-aal8-aal9-aa20.
• Figure 7 illustrates the recombination of V, D and J regions using recombination between lox P sites within self splicing introns.
• the VH, DH and JH regions may be natural VH, DH and JH regions or derived from synthetic oligonucleotides sequences, perhaps of different lengths, especially for the D region, so that the range of CDR3 lengths generated by the recombination, reflects the same (or a modified)- distribution of natural CDR3 lengths.
• the scheme is shown for a single chain Fv molecule with the VL domain fused to gene III protein.
• lxl, 1x2 and 1x3 are 3 different lox P sites e.g. wild type lox P, lox P511 and lox P3.
• in2 and in3 are the two introns which contain 1x2 and 1x3 sites such as the Tetrahymena rRNA and the T4 sunY intron.
• Figure 8 shows the transcription, splicing and expression of a single chain Fv molecule constructed as in Figure 7, containing recombined V, D and J regions, fused to gene III protein.
• the nucleic acid regions encoding the amino acids of the final product are shown as Expressed scFv-gene III fusion.
• Figure 9 shows an alternative final product from recombination which mimics VH, DH and JH recombination in vi tro to generate a new VH domain.
• Two separate libraries of sequences of random nucleotides (x and y) which encode 0 to 15 amino acids are made and recombined using the lox/Cre system, lxl and 1x2 are two distinct lox sites such as lox P5 511 and lox P (wild type) . This scheme requires only one self- splicing intron and two different lox P sequences.
• Figure 10 shows the construction of the vector fdDOG-PEP.
• Figure 11 shows the construction of the vector pUC19-PEP.
• Figure 12 shows construct fdDWT/3 and three different linkers formed on expression from constructs described in example 4.
• Sequence A is derived from the unmutated self splicing intron.
• Sequence B is derived from the self splicing intron mutated at the 3' splice site and in the internal guiding sequence.
• Sequence C shows the sequence derived from the single chain Fv fragment. Bases contributing to the Pl and P10 hairpin loops are underlined. Restriction site bases are outlined. The diagonal slashed line shows the bases between which the self-splicing intron is spliced out.
• T7 is the promoter for T7 RNA polymerase.
• Fx is a site for Factor X protease.
• Part D shows the schematic of the self splicing intron highlighting the bases which are mutated (G to C in the P10 hairpin loop and its complementary base in the internal guiding sequence) .
• Figure 13A shows the fd phage acceptor vector, fdDWT/4 containing 3 lox sites is shown. It contains the VH and VL genes of the anti-NIP clone G6 (Griffiths et al, 1994 supra) .
• the sites loxP511 and loxPWT flank the VH gene and the sites loxPWT and loxP4 flank the VL gene.
• the loxPWT site is in the self splicing intron and the loxP4 site sits between the VL gene and gene III.
• the diabody or single chain Fv polypeptide chain encoded is expressed as a fusion with the gene III protein.
• a site for the factor X protease is included between the VL gene and gene III to allow the possibility of the elution by proteolysis of phage from the antigen during selection procedures .
• Alternative versions of fdDWT/4 were also made with the site loxP4 replaced with loxP3 and loxPl respectively.
• the donor vector PDN8 contains the VH-D10 gene flanked by loxP511 and loxPWT sites.
• the donor vector pRWT/4 contains the VL-DlO gene flanked by loxPWT and loxP4 sites.
• the loxP4 site of pRWT/4 is replaced by the loxP3 or loxPl site respectively.
• the expression vector pEX51l/4 contains the S12 gene, which confers streptomycin sensitivity on bacteria, flanked by loxP511 and loxP4 sites.
• Figure 13B summarises the recombination efficiencies obtained in the experiments described in example 6.
• the left hand loxP site is loxP511
• the middle loxP site is the loxP site within the self splicing intron
• the right hand loxP site is the loxP site between the VL gene and gene III.
• EXAMPLE 1 USE OF SELF -SPLICING INTRONS IN THE CONSTRUCTION OF DIABODY MOLECULES
• a self splicing intron was introduced between the VH and VL domain genes of two antibodies cloned in the diabody format, NQ11 and DI.3 directed against 2-phenyloxazol- 5-one and hen egg lysozyme respectively.
• This self splicing intron was shown to be spliced out following expression, as determined by the expression of functional bivalent diabodies.
• Such a self-splicing intron from clone ICE10 (Ian Eperon, University of Leicester) was inserted between the genes encoding the VH and VL domains of the antibodies D1.3 and NQ11 in such a way as to create upon splicing out an open reading frame encoding a diabody with linker VH-GLSSG-VL. Without splicing no functional diabody can be produced as the self splicing intron contains several stop codons in 3 reading frames.
• a restriction site for BstEII was incorporated at the 5' end of the primer TlbaBstEII and a Sad restriction site introduced in the primer TlfoSac.
• TlbaBstEII primes at the 5' end of the self splicing intron and conserves the internal guidance sequence (IGS) required for splicing activity and inserts a extra glycine residue at the 3' end of the VH domain.
• IGS internal guidance sequence
• TlfoSac primes at the 3' end of the self splicing intron and conserves the thymidine base just 3' of the self splicing intron which, though not part of the intron, is present in Tetrahymena DNA.
• TlfoSac inserts a extra Gly and Ser residue at the 5' end of the VL creating a 5 amino acid linker.
• the self splicing intron was amplified with the primers TlbaBstEII and TlfoSacI using standard conditions (see eg example 14 of PCT/GB93/02492) .
• the product of the PCR reaction was digested with restriction enzymes Sad and BstEII and ligated into BstEII/SacI digested pUC119D1.3 or pUC19NQll in a molar ratio 4:1 (SSI :pUC119Dl.3 or pUC19NQll) and the resulting ligation mixes used to transform E. coli TGI cells .
• Soluble diabody was expressed by growth at 37°C.
• Cells in log phase growth in 2 mL 2YT/0.1% glucose/lOO ⁇ g mL' 1 ampicillin were induced by adding IPTG to a final concentration of ImM IPTG and grown 3 hours 22°C.
• the cells were centrifuged (lOOOg 10 minutes) and the cell pellet resuspended in lOO ⁇ l ice cold PBS/lmM EDTA and left on ice, 60 minutes.
• the cell suspension was centrifuged (lOOOg for 10 minutes) and the diabody- containing supernatant used in ELISA on lysozyme and phOx (as described in example 1 of PCT/GB93/02492) .
• the ELISA signal (absorbance at 405nm) was equivalent (greater than 1.0 after 10 min) for the spliced 5 amino acid linker DI .3 diabody to that obtained with the 5 amino acid linker DI.3 diabody *
• the self splicing intron was amplified with Tlba2BstEII and Tlfo2SacI by PCR. This intron was inserted between the VH and VL domain genes of antibody NQll and creates upon splicing out an open reading frame * encoding a diabody with linker VH-GSLKVG-VL. Without splicing no functional diabody can be produced as the self splicing intron contains several stop codons in 3 reading frames.
• a restriction site for BstEII was incorporated at the 5' end of the primer Tlba2BstEII and a Sad restriction site introduced in the primer Tlfo2Sac.
• Tlfo2Sac primes at the 3' end of the self splicing intron and conserves the thymidine base just 3' of the self splicing intron which, though not part of the intron, is present in Tetrahymena DNA and inserts a extra Gly and Ser residue at the N- terminal end of the VL domain.
• the self splicing intron used in this case contained a lox P site inserted between bp 236 and 237. It was amplified with the primers Tlba2BstEII and Tlfo2SacI using standard conditions. The product of the PCR reaction was digested with restriction enzymes Sad and BstEII and ligated into BstEII/SacI digested pUC19NQll in a molar ratio 4:1 (SSI :pUC19NQll) and the resulting ligation mix used to transform E. coli TGI cells.
• Recombinants were screened for inserts of correct size using the primers specific for self splicing intron, Tlfo2Sac and Tlba2BstEII.
• Soluble diabody was expressed as above and assayed by ELISA. In this case an equivalent signal (greater than 1.0 after 10 min) was obtained with the 6 amino acid linker NQll diabody formed by self splicing as for the 5 amino acid linker diabody constructed in example 1 of PCT/GB93/02492.
• this strategy allows more efficient self splicing in the NQll construct.
• This self-splicing intron is shown to be spliced out following transcription, as determined by the expression of a functional single chain Fv molecule with a 15 amino acid linker.
• a restriction site for BstEII is incorporated at the 5' end of the primer TlbascFvBstEII and a Sad restriction site is introduced in the primer TlfoSac. This allows the self-splicing intron fragment to be cloned in a 2-way ligation reaction into the expression vector pUC119D1.3 (encoding the V domains of the DI.3 anti-lysozyme antibody: Holliger et al (1993) supra) each cut with BstEII and Sad.
• TlbascFvBstEII primes at the 5' end of the self- splicing intron and conserves the sequences at the 5' splice site which pair with the internal guidance sequence (IGS) required for splicing activity, and inserts an extra 10 amino acid residues at the 3' end of the VH.
• TlfoSac primes at the 3' end of the self- splicing intron which, though not part of the intron, is present in Tetrahymena DNA and inserts extra serine and glycine residues at the N-terminal end of the VL domai . +
• the self-splicing intron used in this case contained a lox P site inserted between bp 236 and 237. It was amplified with the primers TlbascFvBstEII and TlfoSacI using standard conditions .
• the product of the PCR reaction was digested with restriction enzymes Sad and BstEII and ligated into BstEII/SacI digested pUC119D1.3 in a molar ratio 4:1 (SSI:pUC19NQll) and the resulting ligation mix used to transform E. coli TGI cells. Recombinants were screened for inserts of correct size using the self-splicing intron specific primers TlfoSac and TlbascFvBstEII.
• Soluble single chain Fv is expressed as in example 1 and assayed for ability to bind lysozyme by ELISA. A signal of greateer than 1.0 is obtained after 10 minutes. Hence, self-splicing introns may be used in nucleic acid encoding single chain Fv molecules.
• EXAMPLE 3 CONSTRUCTION OF A DIVERSE REPERTOIRE OF 25 AMINO ACID PEPTIDES (CONTAINING 20 VARIED RESIDUES) DISPLAYED ON PHAGE USING LOX P RECOMBINATION SITES WITHIN SELF SPLICING INTRONS
• VHCH fragment of the antibody NQ10/12.5 was amplified from the vector pUC19 NQ10 k using oligo 3249, which introduces the lox P 511 site upstream of the pelB leader sequence and an ApaLI restriction site (see Table 1 and Figure 10) and oligo LMB2. The resulting fragment was then cloned into fdDOGl . (T. Clackson et al, supra) cut with ApALI and Notl. The group I self-splicing intron from Tetrahymena (T.R.
• the group I self-splicing intron from Tetrahymena containing a wild type lox P site was amplified with oligo 3194 (which introduces a EcoRI restriction site and includes the random nucleotide (NNK) 10 (Table 1, Figure 11) and oligo 3198 (which introduces a Sfil restriction site.
• the resulting fragment was then cloned into pUC19-21ox (P. Waterhouse et al, 1993 supra) cut with Sfil and EcoRI to create the vector pUC19-PEP ( Figure 11) .
• the culture was then filtered as before.
• the recombined phage, in the filtrate, were precipitated using PEG/NaCl and resuspended in a final volume of 26 ml PBS.
• the phage were titred by infecting exponential phase E. coli (30 mins, 37°C) and by plating on TYE-tet.
• the yield obtained was 6.0 x 10 13 t.u. total (the fdDOG- REC library glycerol stock) .
• a PCR screen was performed by amplifying DNA from individual colonies using oligos 4226 and pelBBACK (Table 1) .
• each bacterium should yield at least one phage containing the peptide from the donor vector and that the overall library size is 4.7 x 10 10 clones.
• the peptide library displayed on phage was selected for the ability to bind an anti-p53 antibody (Pab240) which recognize a linear epitope on the surface of the cell with the amino acid sequence RHSV (C.W. Stephen & D.P. Lane J * . Mol . Biol 1992 225 577- 583) .
• phage from single isolated clones was assessed by ELISA on plates coated with antibody p53.
• Phage were prepared as described by McCafferty et al (supra) and ELISA was performed as described by Griffiths et al, (1993 supra) except that the second antibody used was an anti-sheep antibody coupled to alkaline phosphatase.
• RHSV epitope-containing phage sequence
• 4 KHSV and 5 (R or K)HS(L or I) and 3 (R or K)HSX included the sequence RHSV, 4 KHSV and 5 (R or K)HS(L or I) and 3 (R or K)HSX.
• a large phage display library of ca. 5 x 10 10 recombined 25 amino acid peptides may be constructed using recombination between loxP sites contained in a self splicing intron.
• This method should be particularly valuable for selecting, for example, peptides involved in binding to receptors.
• Constrained peptide libraries could be made by incorporating a cysteine residue in each of the 10 amino acid peptides to be recombined so that a disulphide bond is formed and the peptides between the cysteine would form a loop.
• the amino acid linker could be varied in length and amino acids by varying the 5' and 3' splice sites and the reading frame.
• This example demonstrates the feasibility of making a large library which recombines two exons into a longer continuous sequence.
• This methodology for making a repertoire may be applied to other molecules, including, for example, single chain Fv fragments and diabodies.
• EXAMPLE 4 MUTATION OF THE 3 ' SPLICE AND INTERNAL GUIDING SEQUENCE OF A SELF SPLICING INTRON CONTAINING A LOXP SITE TO ENCODE A NEW DIABODY LINKER WHICH IS COMPATIBLE WITH HIGHER EXPRESSION.
• a loxP site can be included between the two antibody domains, VH and VL of a single chain Fv fragment, in a continuous open reading frame, employing the amino acid sequences encoded by those loxP sequences as a linker.
• linker is dictated by the length and sequence of the loxP sites used.
• An alternative strategy is to employ RNA splicing of a group I self splicing intron inserted between the VH and VL.
• a recombination site such as loxP may be inserted within the intron so that the amino acid sequence encoded by the site is spliced out from the RNA after expression and is therefore not incorporated into the final expressed protein.
• a group I intron is deleted by self-splicing process, a residue of the intron, derived from the 5' and 3 ' splice sites (which pair with the internal guiding sequence in the Pl and P10 hairpin loops respectively) , remains within the coding region of the polypeptide.
• Successful splicing is dependent on base pairing in the Pl and P10 hairpin loops involving the internal guiding sequence (IGS) .
• the 3' splice site and the internal guiding sequence may be mutated so that following splicing the amino acids encoded by the RNA are altered. These amino acids contribute to a 7 amino acid (diabody) linker which is compatible with higher level expression. It is further shown that the mutated 3 ' splice site can be used in the construction of a single chain Fv molecule containing a 15 amino acid linker.
• vectors encoding scFv fragments or diabodies directed against the hapten NIP (3-iodo-4-hydroxy-5-nitrophenyl-acetate) are constructed and expressed using self splicing introns which include loxPWT sites to link the VH and VL domains.
• the intron was amplified by PCR from the vector pUS19Tet-intron-loxP (which contains the loxPWT sequence inserted between bp236-237 of the Tetrahymena ICE10 intron sequence) using #3312 intron-lox-back and 3463 intron-for-2 oligos (Table 1) which contain the sequences of the 5' splice site and the internal guiding sequence of the pl hairpin loop flanked by a Xhol and Ncol sites at the 5' end, and 3' splice site of the P10 hairpin loop flanked by an ApaLI site and
• the amplified product was cloned as a Ncol-EcoRI fragment into pUC19-21ox (Waterhouse et al, 1993 supra) .
• the intron is flanked by Xhol and ApaLI sites.
• the VH and VL genes originate from the Fab fragment clone G6 (anti-NIP; A.D. Griffiths et al EMBO J. 13 3245-3250, 1994) .
• the VH gene was cloned into the pUC vector derivative as a Ncol-Xhol fragment. Promoter sequences for T7 RNA polymerase were introduced into the Hindlll site and were flanked by Sail and Hindlll sites.
• the sail-Notl fragment containing the VH-NIP, self-splicing intron, loxP sites and T7 polymerase promoter was now subcloned from the pUC vector derivative into fd-DOGl (Clackson et al Nature 352 624- 628, 1991) which had its ApaLI site converted to a Sail site.
• the VL gene of G6 was cloned in as a ApaLI-Notl fragment.
• An Ascl site was subsequently introduced at the 3 'end of the VL gene with a loxP3 site and a Factor X protease cleavage site between this Ascl site and a Notl site at the 5' end of gene III.
• fdDWT/3 The resulting construct fdDWT/3 is shown in Figure 12.
• TGI cells were transformed with fdDNA encoding the construct and phage were prepared as described in A.D. Griffiths et al (1994, supra) . This diabody was poorly expressed.
• the phage titer was lower than 10 7 TU/ml (at least a hundred fold lower than would be normally expected) .
• the intron was shown to be spliced correctly as shown by sequencing of the cDNA made from the spliced transcript .
• the vector was first amplified by PCR with the primers fd-PCR-Back and
• RNA template containing the T7 promoter sequence (Table 1) .
• RNA was prepared using an in vi tro transcription kit (Promega, Riboprobe II core System T7 RNA Polymerase, cat.#P2590) .
• the original DNA template was first removed by digestion with DNasel, and cDNA was then prepared using the First-Strand cDNA Synthesis Kit (Amersham) .
• the cDNA was amplified by PCR with VH3BackSfi and JK-FOR primers (J.D. Marks et al, J". Mol . Biol .222 581-597, 1991) , and was sequenced using the same primers.
• the sequence obtained demonstrated accurate splicing resulting in an SLKVSAL linker in the expressed diabody product.
• amino acids more compatible as linkers for the expression of diabody may be identified and used to alter the bases of the splice sites.
• a second anti NIP diabody was constructed in which the first G within the 3 ' splicing signal (P10) was mutated to C.
• the corresponding C within the IGS was changed to G ( Figurel2d) .
• the intron of the vector pUC19Tet-intron-loxP was amplified by PCR with a second set of primers, #3877 encoding Pl and the mutated C to G in the IGS, and #3878 encoding P10 having a G to C mutation (Table 1) .
• the intron was cloned as above to give an analogous fdDWT/3 construct, but in this case after splicing of the intron, the resulting RNA encodes the linker VH-SLNVSAL-VL ( Figure 12b) .
• the splicing of the mutated intron was tested by the cDNA sequencing of expressed RNA as above.
• the ELISA signal indicated that some diabody polypeptide chains are cleaved from the fusion and combine with the glll- diabody polypeptide fusion retained on the surface of the phage, to form a functional bivalent diabody which can bind to a NIP.
• Western blots were performed of phage proteins with detection with an antibody directed against gene 3 protein as described by J. McCafferty et al [Protein Engineering 4 955-961, 1991) . This gave the relative proportions of gill protein-diabody polypeptide fusion to cleaved fusion migrating at the position of native gill protein to be 40% and 60%, respectively.
• the self splicing intron was amplified by PCR from the vector pUC19Tet-intron-loxP using the oligonucleotides 4243 and 4244 (Table 1) . These contain bases encoding a stretch of four glycine residues flanking the 5' and 3' splice sites respectively. Oligonucleotide 4243 contains the mutation of the internal guiding sequence and oligonucleotide 4244 and the mutation of the 3' splice site, to effect the K to N mutation as above.
• the intron is spliced out after transcription and there is functional display of anti-NIP scFv fragments on the surface of phage fd as determined by phage ELISA on NIP-BSA with an absorbance of 1.0. Further, the phage titre was in the range of 5 x 10 8 - 1 x 10 9 TU/ml, indicating that these phage fd clones grew well.
• mutations may be made at the 3' splice site and internal guiding sequence of the self splicing intron to allow the encoding of amino acids compatible with higher expression on self-splicing.
• it may be necessary to mutate the bases of the Pl hairpin loop as well as the P10 hairpin loop or the Pl hairpin loop only.
• EXAMPLE 5 CONSTRUCTION OF THE PLASMID pACYCaraCre EXPRESSING CRE RECOMBINASE UNDER THE CONTROL OF AN ARABINOSE PROMOTER
• a plasmid was constructed in which Cre recombinase is expressed under the control of a promoter inducible by arabinose.
• the origin used pl5A makes it suitable for use in combination with plasmids with ColEl origin and with phage or phagemids with phage origins.
• a fragment was amplified by PCR from pUC119 (Vieira, J. and Messing, J. (1987) . Methods in Enzymol . 153, 3-11) using the primers lacfor2 and lacback2. This fragment extended from within the lad gene fragment (inactive) to the polylinker of pUC119 and the primers incorporate a series of restriction sites at both ends of the fragment.
• This fragment was ligated into pUC119lacipoly cut with the same enzymes to generate pUC119ara.
• the Cre recombinase gene was amplified by PCR from bacteriophage PICm cl.100 r ' m " (Yarmolinsky, M.B., Hansen, E.B., Jafri, S. and Chattoraj , D.K. (1989) . J. Bacteriol . , 171, 4785-4791) using the primers crefor and creback. After digestion with Bsal and Kpnl this fragment was ligated into pUC119ara cut with Ncol and Kpnl to generate pUC119araCre.
• This plasmid can co-exist in E. coli with both the heavy chain donor vector (which has a ColEl origin) and with the acceptor vector (which has a filamentous phage origin) and is useful for the generation of a large phage display library in the lox P format.
• EXAMPLE 6 MODEL EXPERIMENTS FOR THE CONSTRUCTION OF A DIABODY REPERTOIRE USING THE FD3LOX SYSTEM, USING A LOXP SITE WITHIN A SELF- SPLICING INTRON
• model experiments are described which demonstrate that the loxP site within the self- splicing intron may be used in the construction of a diabody or single chain Fv repertoire by recombination of VH and VL gene repertoires.
• model experiments are described using a fd phage acceptor vector containing 3 lox sites encoding an anti-NIP diabody molecule, where recombination is performed with donor vectors encoding VH or VL domains. Recombination of the diabody cassette with an expression vector is also demonstrated.
• the methods of this example are equally applicable to the construction of single chain Fv repertoires using 15 amino acid linkers as described in Example 4, and other polypeptides.
• the fd phage acceptor vector, fdDWT/4 containing 3 lox sites is shown in Figure 13. It contains the VH and VL genes of the anti-NIP clone G6 (Griffiths et al, 1994 supra) .
• the sites loxP511 and loxPWT flank the VH gene and the sites loxPWT and loxP4 flank the VL gene.
• the loxPWT site is in the self splicing intron and the loxP4 site sits between the VL gene and gene III.
• the diabody or single chain Fv polypeptide chain encoded is expressed as a fusion with the gene III protein.
• a site for the factor X protease is included between the VL gene and gene III to allow the possibility of the elution by proteolysis of phage from antigen during selection procedures.
• Alternative versions of fdDWT/4 were also made with the site loxP4 replaced with loxP3 and loxPl respectively (see Figure 13) .
• a VH gene repertoire may then be introduced by recombination with a donor vector containing the VH gene repertoire, flanked by loxP511 and loxPWT sites.
• fdDWT/4 was recombined with the donor vector pDN8 containing the VH-DIO gene flanked by loxP511 and loxPWT sites. This was performed by transforming E.
• Example 5 coli TGI pACYCaraCre (Example 5) with pND8 donor vector containing VH-DIO and then infecting with fdDWT/4 phage containing the genes encoding the variable domains, VH-G6 and VL-G6. Recombination was allowed to continue at 30°C overnight. Recombined phage from the bacterial supernatant were used to infect TG- 1. As a result of recombination between the loxP511 sites of donor and acceptor and between the loxPWT sites of the donor and acceptor, the recombined fd phage contains VH-DIO while keeping the original VL-G6.
• a VH gene repertoire may be cloned between the Ncol and Xhol sites of fdWT/4 and a VL repertoire, flanked by loxPWT and loxP4 sites.
• fdDWT/4 was recombined with the donor vector pRWT/4 containing the VL-D10 gene flanked by loxPWT and loxP4 sites . This was performed by transforming TGI pACYCaraCre (Example 5) with pRWT/4 donor vector containing VL-DIO and then infecting with fdDWT/4 phage containing the genes encoding the variable domains, VH- G6 and VL-G6.
• Recombination was allowed to continue at 30°C overnight. Recombined phage from the bacterial supernatant were used to infect TG-1. As a result of recombination between the loxP4 sites of donor and acceptor and between the loxPWT sites of the donor and acceptor, the recombined fd phage contains VL-DIO while keeping the original VH-G6.
• bivalent diabody results from the association of free diabody polypeptide, cleaved from gene III protein, with diabody polypeptide gene III fusion. It is desirable to express the bivalent diabody directly as a soluble molecule.
• the expression vector pEX511/4 was constructed ( Figure 13) .
• E. coli TGI pACYCaraCre (Example 5) were transformed with pEX511/4 and then infected with fdDST/4 containing the genes encoding the variable domains, VH-G6 and VL-G6.
• a preferred approach to construction of a repertoire may be first to clone the VL genes as ApaLI- Ascl fragments into fdDWT/4 and then recombine with a VH repertoire as Ncol-Xhol fragments in pDN8. This would generate a diabody repertoire (or single chain Fv repertoire, if modified slightly) suitable for phage display. Following selection of diabodies, individual or pooled clones could be subcloned into pEX51l/4 for soluble expression.
• Tlfo2Sac 5 ' -GAG CCA TCA ATC TCG GAG CTC GAT GTC ACC TAC CTT ACG AGT ACT CCA AAA
• TlfoSacI 5' -GAG CCA TCA ATC TCG GAG CTC GAT GTC ACC AGA CGA GTA CTC CAA AAC TAA
• TlbaBstEII 5' -CCA TCA ATC GAT CTG GTC ACC GTC TCC TCA GGT CTC TCT AAA TAG CAA TAT
• TlbascFvBstEII 5' -CCA TCA ATC GAT CTG GTC ACC GTC TCC TCA GGT GGA GGC GGT TCA GGC GGA
• oligonucleotides used fd-pcr-back 5'- GCG ATG GTT GTT GTC ATT GTC GGC-.3'
• Oligonucleotides used for the construction of the vectors fdDOG-PEP, pUC19-PEP and the screening of the peptide library are used for the construction of the vectors fdDOG-PEP, pUC19-PEP and the screening of the peptide library.
• N means an equimolar mixture of all 4 bases and K an equimolar mixture of G and T.

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