The present invention relates to a method for the generation of novel nucleic acid molecules and proteins, and to nucleic acids and proteins produced by such a method. The invention allows the generation in vitro of new biological products formed by applying a combinatorial approach to blocks of nucleic acid sequence.
Advances in recombinant DNA technology over the last decade or so have meant that it has become possible to construct synthetic genes and, consequently, synthetic proteins. Molecules may now be rationally designed and produced with the aim of improving their efficacy over that of a sequence that occurs naturally.
Various techniques now exist for the generation of libraries of proteins, most using methods that allow the random combination of a number of peptides to produce a library of variants. Molecules having the desired characteristics can be isolated through selection regimes that select for a desired phenotype, such as a particular biochemical or biological activity.
Phage display provides one example of a technology that has been highly successful in allowing for the selection of a displayed protein (for reviews see Clackson and Wells, 1994, Hoogenboom HR, 1997 and Lowman HB, 1997). Additionally, combinatorial chemistry can be used to generate peptides of random sequence (Lom KS, 1997).
Existing methods for the generation of libraries of peptides or proteins are limited by pre-design requirements, the number of molecules in the library and the small size of the products obtained. The present invention advantageously provides a method that facilitates the generation of new genes and proteins of unlimited size, assembled in either a predetermined and/or random order, and also allows their subsequent analysis. This method utilises compatible restriction enzymes and ligation to build DNA molecules from smaller, naturally occurring and/or synthetic DNA in a desired orientation.
Thus according to one aspect of the present invention there is provided a method of generating a library of DNA molecules of varying length and sequence in a desired orientation comprising the steps of:
Where desired the mixture of double-stranded DNA molecules in step a) may also be ligated to a cut vector. Advantageously this allows for the subsequent analysis and utilisation of the assembled DNA molecules. Thus according to a further aspect of the invention, there is provided a method of generating a library of DNA molecules of varying length and sequence in a desired orientation in a vector comprising the steps of:
For each of description of the invention, each component of the mixture of double-stranded DNA molecules initially present at the start of the method will be referred to hereinafter as a “sequence block” (SB) unless stated otherwise. Each SB will in general be naturally occurring or synthetic DNA. The size of each SB is advantageously not crucial and may be varied widely as desired, for example from a few bases up to and beyond 10 Kb in length. Each end of a SB comprises half a restriction enzyme site and may be blunt-ended or preferably single-stranded thereby forming a cohesive end. Each half-size of each SB is compatible with the other. Particular sites include those equivalent to the half-sites obtainable by cutting with the restriction enzymes described hereinafter. Additionally, the SB may contain a recognition site for a restriction enzyme, which is preferably distinct from any recognition site capable of being formed by the half-sites of the SB.
Where desired the SB may define a specific motif. The motif may be biologically functional at the protein or at the nucleic acid level. In the latter case the motif may represent, for example, a promoter element, a binding motif for a regular or inhibitor protein, a response element, an enhancer element, a nuclease size, a hairpin motif or a spacer or linker domain that is required for the correct assembly of the double-stranded nucleic acid molecule.
In the case of an SB encoding a motif with a biological function as part of a portion, the motif may be a binding domain, especially for example a SH2 or SH3 domain, and a SH2 or SH3 binding domain, a dimerisation domain, a signalling sequence (such as an immunoglobuilin tyrosine based activation motif; ITAM), a recognition site for an enzyme, an immunoglobulin domain or fragment thereof, an epitope, a transmembrane domain, a catalytic domain, a regulatory domain, or α helical motif or other structural or functional domain. Particular examples of such domains will be readily clear to the man of skill in the art.
Each SB in the starting mixture may be generated by any convenient method, for example by PCR cloning from naturally occurring complementary DNA sources and subsequent cutting with appropriate restriction enzymes (see for example those described below). This allows the selection of any naturally-occurring sequence module of interest, of almost any length. More usually, the SBs may be generated by annealing two synthetic strands of nucleic acid (oligonucleotides) so that the 5′ end and the 3′ end form two appropriate overhangs. Combinations of these two approaches may be applied.
Restriction enzymes for use in the methods according to the invention recognise symmetric double-stranded DNA sequences and cleave within the sequences leaving a 3′-hydroxyl on one side of the cut and a 5′ phosphate on the other. Depending on the type of restriction enzyme, a fragment with either a cohesive end (having a 5′ or 3′ single-stranded overhang) or a blunt-ended end (no single-stranded overhand) is produced. Cohesive DNA fragments can be ligated to other DNA fragments if their single-stranded overhangs are compatible. Depending on the sequence of the cleavage product of each fragment that is ligated together, either the original recognition sequence, a new recognition sequence or even no recognition sequence may be formed on ligation. Different restriction enzymes that produce compatible overhangs, which may be ligated together such that the may or may not produce a recleavable ligation product, are termed “compatible restriction enzymes”. When overhangs that have been generated by the same restriction enzyme religate together, they reform a recognition site for that enzyme.
In the method of the invention, compatible restriction enzymes that have a 6 base pair or longer recognition sequence are preferred, since such enzymes cut DNA infrequently. Assuming a 50% G-C content of the DNA, a restriction enzyme with a 6 base pair recognition sequence will cleave, on average, every 46 (4096) base pairs in a given DNA sequence. Restriction enzymes that produce cohesive ends are preferred since they ligate much more efficiently.
One example of two compatible restriction enzymes that form cohesive ends on digestion is the BamHI/BcII pair. These enzymes produce overhangs that may be ligated to each other, but which form a ligation product that is not cleavable by either enzyme. In fact, the ligation product may be cleaved by the unrelated enzymes AlwI and DpnI.
Preferred restriction enzymes that form cohesive ends are: AvaI, BamHI, BclI, BglII, BstEI, BstBI, BstYI, EcoRI, MluI, NarI, NheI, NotI, PstI, PvuI, SacI, SalI, SpeI, StyI, XbaI, XhoI and XmaI. Most preferably, the compatible set BglII, BamHI and BclI is used. Preferred enzymes that form blunt ends are EcoRV, FspI, NaeI, NruI, PvuII, SmaI, SnaBI and StuI.
It is envisaged that more than one pair of restriction endonucleases that are different but produce compatible cleavage products, may be employed in the method of the invention.
The recognition sites of these enzymes, the sites with which they are compatible, and the cleavable products are listed in categories of most restriction enzyme suppliers, for example the New England Biolabs catalogue.
In the present invention, compatible restriction enzymes are selected on the basis of the amino acid sequence that is desired, the necessity to maintain the reading frame of the protein sequence, and the rarity with which the enzymes cut the chosen DNA sequence. Also relevant is whether during subsequent steps of the process, it will be desirable to recleave at the ligation point.
Where in a method according to the invention the SBs are ligated to a cut vector, the latter may be any double-stranded DNA vector which has been cut with a restriction enzyme, for example, selected from those described above. The vector may contain such features necessary to enable the vector to be grown, maintained and selected in a host cell, including prokaryotic, yeast and higher eukaryotic e.g. mammalian cells.
The vector may be an expression vector and, in this event, may comprise an appropriately-positioned promoter, polyadenylation signal and transcription termination sequence, as well as features to allow expression of a protein encoded by a combination of SBs, such as a ribosome binding site and signal sequences. Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if so desired. Expression constructs are normally maintained in a replicon, such as an episomal element (e.g. plasmid) capable of stable maintenance in a host, such as mammalian cells or bacteria. For further details, see Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger) and Molecular Cloning: A Laboratory Manual (Sambrook et al. (1989)).
Suitable vectors for use in the invention are widely available, for example from commercial suppliers such as Clontech Laboratories, Inc., Palo Alto, USA and Stratagene, La Jolla, USA, and may be modified as desired using conventional techniques.
Particularly suitable vectors for use in processes according to the invention include viral vectors such as retroviral and adenoviral vectors in mammalian cells.
In general, a desirable feature of the vector is that, once the SBs have been assembled into the vector and analysed, the SB combination(s) of choice may be transferred from the cloning vector and inserted into alternative vectors that allow the function or structure of the assembled SBs to be assessed. In most instances, this will be achieved by utilising a unique restriction site that is recognised by an enzyme that does not cut at any site in the sequence of the assembled SBs.
A vector of choice is a cloning cassette system derived from pBluescript SK+ (Stratagene). This vector is a modification of the cassette system described in International Patent Specification WO97/23613, the contents of which are incorporated herein in their entirety.
Where desired, the process according to the invention may be adapted to use a solid phase. Thus, according to a further aspect of the invention there is provided a method of generating a library of DNA molecules of varying length and sequence in a desired orientation of a solid phase comprising the steps of:
The solid phase may comprise any solid matrix to which a DNA molecule may be attached, for example a synthetic bead, column, or any other solid surface. Suitable methods of attachment of DNA molecules to a solid phase are known in the art, such as, for example by biotin capture (Sterky F., et al 1998, Journal of Biotechnology 60, 119-125) and by digoxigenin/anti-digoxigenin interaction (Ioannon, P. and Christopoulos, T. 1998, Analytical Chemistry 70, 698-702).
The methods according to the invention employ standard DNA ligatation reactions and restriction enzyme digestion to generate a DNA library. Such techniques are well-known and routinely practised, and are described for example in laboratory manuals such as “Molecular Cloning” [Maniatis et al., Cold Spring Harbour Laboratory, New York, 1998]. Suitable ligases, e.g. T4 DNA ligase, and necessary cofactors such as ATP, are commercially available and may be used according to the manufacturer's instructions. Similarly, restriction enzymes, such as those described above are commercially available and may be used as instructed by the supplier.
The ligation step in each method may be controlled by manipulation of SB concentration, incubation time and/or temperature to determine the degree of ligation. For maximum efficiency the ligation conditions, such as SB concentrations, may need to be determined empirically, for example by titration, for any particular application.
Where desired ligated SBs may be purified from other components of the ligation reaction, such as enzymes and ATP, using standard separation procedures, for example, by gel exclusion and other size fractionation techniques.
Of course, as will be apparent to the skilled artisan, any of the nucleic acids generated using the methods of the present invention may be mutagenised in accordance with standard techniques as describe, for example, in Molecular Cloning: a Laboratory Manual; 2nd edition, (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press) or in Protein Engineering; A practical approach (edited by A. R. Rees et al., IRL Press 1993). A particularly suitable method for introducing mutations within individual SBs is described below. This method allows the introduction of random mutations at a specific site (or sites) within a SB.
This is achieved through the use of substantially complementary oligonucleotides, one or both of which is degenerate. These oligonucleotides are annealed, ligated into a suitable vector and then transformed into a suitable host. Mutations may thus be introduced at the sites of degeneracy; the precise mutation introduced will depend on the degree of degeneracy at a particular nucleotide position and the operation of the mismatch repair system of the host organism. This will repair any nucleotides that are mismatched between the substantially complementary oligonucleotide pairs during DNA replication, as the cells grow and divide.
Thus, the method of the invention may employ at least one mutant double-stranded DNA molecule, which has been generated by annealing substantially complementary oligonucleotides as described above, the sequences of which are based on a parent nucleic acid, to form a plurality of double-stranded DNA molecules, ligating said plurality of double-stranded DNA molecules with vector, transforming the modified vector molecules into a host cell, culturing said transformed cell under conditions suitable for growth and cell division and isolating mutated DNA from the host cell.
The oligonucleotides are preferably based on a parent nucleic acid. Such parent nucleic acid may encode a polypeptide (this aspect of the invention, where an SB encodes a string of amino acids is described below in more detail).
Mutations may be introduced by annealing complementary oligonucleotides, only one of which is degenerate. Alternatively they may be introduced by annealing complementary oligonucleotides, both of which are degenerate, either at corresponding, or at different, nucleotide positions.
More than one mutation may be introduced into each SB. However, it will be appreciated that there will be a maximum number of mutations that can be introduced at any one time. This number will depend on the length of the oligonucleotide and other factors that govern efficient annealing. However, we have found that the method works satisfactorily when an oligonucleotide exhibits degeneracy at a ratio equal to, or less than 1 degenerate nucleotide in every 5 nucleotides. This ratio is calculated as an average over the entire length of the oligonucleotide, and is meant to be used only as guide when designing a degenerate oligonucleotide, as it may be possible to increase the amount of degeneracy in certain circumstances.
In some instances, it may be desirable to cluster degeneracy in, for example, groups of three adjacent nucleotides. Clustering degeneracy in such groups of three is particularly useful if one wishes to effect change in the amino acid sequence that a particular SB encodes. Thus, an individual codon may be altered by making 1 out of 3, 2 out of 3 or all 3 adjacent nucleotides degenerate. It will be clear to the skilled man that whilst the number of nucleotides in a degenerate cluster may exceed three, the mismatch repair system of the host organism, into which the annealed oligonucleotides will be introduced, will only tolerate and repair a maximum number of unpaired nucleotides before invoking other repair mechanisms, e.g. excision of the unpaired region of DNA.
Whilst the method of mutagenesis allows one to target precisely which nucleotide(s) is mutated, the nature of the mutation will be random due to the degeneracy of the oligonucleotide at the site of mutations. It may be desirable to bias this mutation away from (or towards) a particular nucleotide base, for example, to reduce the probability of introducing a stop codon. This can be achieved by limiting the degree of degeneracy created at a given position within the oligonucleotide. One example of this limited degeneracy wold be where only C, G or T (and not A) are present at a particular position. It will be clear to the skilled reader that any desired bias may be introduced in this manner. Alternatively, if the degeneracy of the oligonucleotide at a given position is maximal (i.e. all four nucleotide bases or inosine are present at the desired sit of mutation), the mutation will be random.
The conditions that are required for annealing degenerate oligonucleotides will, in general, be similar to those described herein in the examples. However, it will be appreciated by the skilled reader that these conditions may vary and may be dependable on factors such as length of oligonucleotide, the percentage of nucleotides that are degenerate, and the percentage GC content. Annealed oligonucleotides will then be ligated with any suitable vector and transformed (for example, for calcium chloride transformation, electroporation or any other method that is well known in the art) into a suitable host organism, preferably bacteria or yeast. The host organism will then be cultured under conditions suitable for cell growth and maintenance of the vector, examples of which are readily available in the art, see for example, Sambrook et al., (Molecular cloning: a laboratory manual. 2nd edition. Cold Spring Harbour Press, N.Y.) and Glover (DNA cloning: a practical approach, Volume II: Expression systems. IRL press).
The methods of the invention may be repeated in iterative steps, for example, to incrementally increase in size and complexity the DNA molecules generated. For example, a library of molecules generated by a first round may itself be isolated and the isolated molecules used in a subsequent round. In this fashion, the number and type of motifs in the molecule may gradually be increased. Such iterative steps are of particular value for the generation of a selection of polypeptide species, each comprised of a different combination of protein modules. After each round of combination, the DNA molecules generated may be analysed as described below and promising candidate DNA molecules may then be selected and used in the next of the method.
In general, the method according to the invention allows the production of DNA libraries of any desired size and diversity. Particular libraries include those, for example, in which the DNA has been rationally designed. Thus, SBs of a predetermined length may be incorporated into a cut vector in sequential steps. This will be particularly advantageous when the DNA is intended to encode a multifunctional polypeptide sequence. For instance, in the Examples described herein relating to the construction of chimeric receptor sequences, the individual modules or domains that constitute the protein must be ordered appropriately, for example: binding components, extracellular spacer, transmembrane component, signalling component. Therefore, the individual SBs should be inserted in this order. Insertion in the correct orientation can be checked by restriction analysis or by nucleic acid sequencing.
In another example, the method allows the production of libraries generated from the random combination of SBs. In this method, a ligation reaction is carried out in which a large number of different SB components are employed, any of which may insert in either orientation. Depending upon the ligation reaction conditions, for example as described above, a large number of different SB combinations is possible. Strings of multiple ligated SBs are then restricted with both the SB 5′ restriction enzyme and the SB 3′ restriction enzyme, to destroy combinations that contain one or more SBs in the incorrect orientation, prior to insertion of the assembled blocks into a cloning vector.
In the above examples and in general selective order of ligation of SBs, of either random or pre-determined length may be controlled by the selective use of kinases and phosphatases to add or remote phosphate groups from some or all of the SB components. As will be clear to the skilled artisan, a 5′ terminal phosphate group must be donated by the ligating species in order that ligase-directed ligation is possible. In most instances, the vector should be phosphatased so as to prevent its self-ligation.
Thus according to a further aspect of the invention there is provided a DNA library and each nucleic acid component thereof generated by the method according to any one of the above-described aspects of the invention. The invention also provides a host cell transformed or transfected by such nucleic acid molecules and protein molecules expressed from said nucleic acid. Thus, the method of the invention also provides for the generation of a library of protein or polypeptide molecules.
The method of the invention is ideally suited to the generation of novel peptide or polypeptide compounds that are either wholly or partly derived from a combination of motifs, modules or domains. Such novel peptides, polypeptides, and indeed any protein library, may be expressed in a host cell(s), following transformation of the host with a nucleic acid (or library of nucleic acids) generated by the method of the invention. This may be achieved by culturing host cells under conditions that are well known in the art to be suitable for expression of a polypeptide from a nucleic acid (see for example: Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press; DNA cloning: a practical approach, Volume II: Expression systems, encoded by D. M. Glover IRL Press, 1995; DNA cloning: a practical approach, Volume IV: Mammalian systems, edited by D. M. Glover IRL Press, 1995). Desirable peptide/polypeptide products may then be identified through any suitable screening process, and isolated, if required, by standard protein purification techniques that are well known in the art.
In this embodiment of the invention, the SBs are designed so as to encode an in-frame string of amino acids when the SBs are linked together in the correct orientation. In a preferred embodiment of the invention, the SBs are designed so that a stop codon is formed by the linkage of any two SBs in an incorrect orientation.
In the case of insertion of one or more SBs into a vector to express a polypeptide, the vector can be designed so that a stop codon is present after insertion of the laser SB in the correct orientation. Preferably, the vector will have stop codons in all reading frames.
In the case of peptides, the SBs will encode short lengths of amino acid sequence, such as antigenic epitopes or signal sequences. However, the SBs may be significantly longer. Entire proteins may be assembled from component domains by the combination of SBs in either a random or a rational order. For example, many proteins are composed of domains that are identifiable by the presence of known consensus sequences or a prevalence of certain amino acid residues.
Binding proteins such as antibodies are examples of such proteins, being composed of constant and variable domain. Receptors are also usually composed of known domain structures, such as an extracellular binding component for recognition of ligand, a transmembrane region, a dimerisation or oligomerisation domain, linker domains, signalling domains and other intracellular domains. Combinations of synthetic and natural sequences can also be constructed. In this respect, spacer and linker domains may be used to maintain the steric configuration of the molecule, as and when appropriate for optimisation of function.
Other domains suitable for use in the invention may be derived from sources that will be clear to those of skill in the art and will include enzymatic effector domains such as protease, kinase or phosphatase domains.
Suitable applications for peptides formed by the combination linkage of encoding SBs include many immunological applications. Most linear epitopes are fairly short in length, often comprising no more than 6-10 amino acid residues. It is envisaged that these epitopes may be combined to generate long stretches of sequence containing a number of different epitopes. Such a molecule might find application as a component of a vaccine, for example, if each of the epitopes is capable of eliciting a separate immune response. For example, such a polyepitope peptide might contain known epitopes from bacterial pathogens that cause disease such as tetanus, cholera and diphtheria. Other epitopes that selectively stimulate populations of the immune system might be used, such as carrier peptides comprising epitopes recognised by T helper cells.
Short stretches of polypeptide comprising a number of peptides encoded by SBs may also be inserted into whole proteins or protein domains. For example, it may be desirable to include a plurality of signalling sequences into an existing protein. In this event, the assembled SB component would be inserted in-frame into the appropriate site of an existing nucleic acid sequence or gene. As exemplified below, one example of such an application is the creation of a synthetic signalling component from the assembly of multiple individual signalling motifs that have been derived from different naturally-occurring signalling regions. This ready-assembled component can then be inserted into a specific site in the sequence of a protein so as to confer on that protein the functional properties of each signalling motif.
Many similar applications may be envisaged, as will be clear to those of skill in the art.
As has been mentioned above, in one embodiment, the method of the present invention allows the design of nucleic acid molecules derived from a plurality of SBs that do not necessarily encode proteins, but which constitute regulatory elements that affect the rate of transcriptional or translational events. Each individual SB may encode an element such as a promoter element, a binding motif for a regulator or inhibitor protein, a response element, an enhancer element, a nuclease site, a hairpin motif, or any other element of a nucleic acid which may be transformed from its natural position in a gene and used in a different context without losing its function. For example, it may be desirable to create an improved transcriptional element to increase the transcription of a gene or to make such transcription dependent upon the presence of a particular activator or inhibitor compound.
Analysis of the DNA molecules that have been generated by the assembled SBs which have been produced in accordance with the present invention, may be by any suitable transcription or translation system. For example, when the method of the invention is being used to design an improved or modulated promoter or other regulatory element affecting the levels of transcription, the analysis system may be a reporter plasmid, in which the assembled SBs are inserted upstream of an unrelated reporter gene so as to regulate their expression of the encoded reporter protein. Suitable reporter genes may be luciferase, β-galactosidase, alkaline phosphatase or green fluorescent protein (GFP).
An efficient promoter will give a high level of transcription that will be reflected in the levels of translated protein. Furthermore, the effect of an inhibitor or of an activator compound may be assessed by allowing transcription to take place in the presence of a suitable amount of that compound. Similarly, when the method of the invention is being used to generate DNA encoding a functional protein, the contents of any library may be assessed using an appropriate expression system with a suitable assay to screen for the desired, expressed, functional protein, for example, as described in the Examples herein.
The invention will now be described in further detail with specific reference to chimeric receptor molecules generated by the combination of SBs in a pBLUESCRIPT-based system. It will be appreciated that variation may be made from these specific Examples without departing from the scope of the invention.
All documents cited herein are incorporated by reference.
FIG. 1: Schematic diagram showing system for assembly of SBs.
FIG. 2: Cloning cassette for construction of chimeric receptors with synthetic signalling components.
FIG. 3: Sequence of cloning cassette.
FIG. 4: Oligonucleotide sequences for chimeric receptor construction.
FIG. 5: Predicted amino acid sequence of SBs.
FIG. 6: Level of expression and degree of activation of chimeric receptors containing synthetic signalling components by surface-bound antigen.
FIG. 7: Activation of chimeric receptors containing synthetic signalling components by surface-bound antigen.
FIGS. 8-10: Activation of chimeric receptors containing synthetic signalling components by surface-bound antigen.
In these examples, we aim to construct a synthetic signalling component, from individual blocks of sequence, that is more potent than a naturally occurring signalling region for use in chimeric receptors. The individual sequence blocks are designed to include predicted signalling motifs from different naturally occurring signalling regions.
These sequence blocks (SBs) are constructed by annealing two synthetic strands of DNA (oligos) so that the 5′ end forms a Bcl I overhang and the 3′ end forms a BamH I overhang. Both these restriction site overhangs are compatible with a BamH I site in the cloning vector. Insertion of the SB in the correct orientation destroys the 5′ BamH I site and retains a 3′ BamH I site, allowing subsequent 3′ insertion of SB(s). Insertion of the SB in the wrong orientation retains a 5′ BamHI site and generates a 3′ stop codon (FIG. 1). In this example the SBs have 5′ and 3′ phosphate groups and the BamH I site in the construction vector (
To facilitate construction of chimeric receptors with different binding, extracellular spacer, transmembrane and signalling components, a cloning cassette system was devised in pBluescript SK+ (Stratagene). This is a modification of our cassette system described in International Patent Specification No. WO 97/23613.
This new cassette system is shown in FIG. 2. The binding component has 5′ Not I and Hind III restriction sites and a 3′ Spe I restriction site. The extracellular spacer has a 5′ Spe I site (Tbr, Ser) and a 3′ Nar I site (Gly, Ala). The transmembrane component has a 5′ Nar I site (Gly, Ala) and 3′ Mlu I (Thr, Arg) and BamHI sites (Gly, Ser). The signalling component may be cloned into the BamHI site. Following this BamH I site there is a stop codon for transcription termination and there is an EcoRI site situated 3′ of this for subsequent rescue of whole constructs.
To generate this cassette, a 200 bp fragment was PCR assembled using oligos: S0146, A6081, A6082 and A6083 (FIG. 4). This fragment starts with a SpeI site and consists of the extracellular spacer h.CD28, the human CD28 transmembrane region, a stop codon and finishes with an EcoRI site (see FIG. 3). This PCR fragment was then restricted with SpeI and EcoRI and substituted for the same fragment in our previously described cloning cassette system to join the binding component (International Patent No. WO 97/237613 FIG. 2).
This cloning vector termed pHMF393 contains P67scFv/h.CD28/CD28TM and forms the base vector molecule into which synthetic signalling regions were built.
Each sequence block was generated by annealing two oligos such that they have single-stranded overhangs forming half a Bcl I site at the 5′ end and half a BamH I site at the 3′ end. Oligos were annealed at a concentration of 1 pmole/μl in a buffer consisting of: 25 mM NaCl, 12.5 mM Tris-HCl, 2.5 mM MgCl2, 0.25 mM DTE, pH 7.5 by heating in a boiling water bath for 5 minutes and then allowing the bath to cool slowly to room temperature.
The predicted amino acid sequences of these examples of SBs are shown in
1) SB1
This sequence is based on the first ITAM of human TCR ζ and was constructed by annealing oligos A8816 and A8817 (FIG. 4). Both these oligos have a 5′ phosphate group.
2) SB2
This sequence is based on the second ITAM of human TCR ζ and was constructed by annealing oligos A8814 and A8815 (FIG. 4). Both these oligos have a 5′ phosphate group.
3) SB3
This sequence is based on the third ITAM of human TCR ζ and was constructed by annealing oligos A8812 and A8813 (FIG. 4). Both these oligos have a 5′ phosphate group.
4) SB 4
This sequence is based on the ITAM of the γ chain of human FcεR1 and was constructed by annealing oligos A8810 and A8811 (FIG. 4). Both these oligos have a 5′ phosphate group.
5) SB4*
This sequence was originally generated in error by mis-annealment of the above oligos but was subsequently made by annealing oligos A8810B and A8811B (FIG. 4). Both these oligos have a 5′ phosphate group.
6) SB5
This sequence is based on the ITAM of the β chain of human FcεR1 and was constructed by annealing oligos A9000 and A9001 (FIG. 4). Both these oligos have a 5′ phosphate group.
7) SB6
This sequence is based on the ITAM of the γ chain of human CD3 and was constructed by annealing oligos A9002 and A9003 (FIG. 4). Both these oligos have a 5′ phosphate group.
8) SB7
This sequence is based on the ITAM of the δ chain of human CD3 and was constructed by annealing oligos A9004 and A9005 (FIG. 4). Both these oligos have a 5′ phosphate group.
9) SB8
This sequence is based on the ITAM of the ε chain of human CD3 and was constructed by annealing oligos A9006 and A9007 (FIG. 4). Both these oligos have a 5′ phosphate group.
10) SB9
This sequence is based on the ITAM of human CD5 and was constructed by annealing oligos A9008 and A9009 (FIG. 4). Both these oligos have a 5′ phosphate group.
11) SB10
This sequence is based on the ITAM of human CD22 and was constructed by annealing oligos A9010 and A9011 (FIG. 4). Both these oligos have a 5′ phosphate group.
12) SB11
This sequence is based on the ITAM of human CD79a and was constructed by annealing oligos A9012 and A9013 (FIG. 4). Both these oligos have a 5′ phosphate group.
13) SB12
This sequence is based on the ITAM of human CD79b and was constructed by annealing oligos A9014 and A9015 (FIG. 4). Both these oligos have a 5′ phosphate group.
14) SB13
This sequence is based on the ITAM of human CD66d and was constructed by annealing oligos A9016 and A9017 (FIG. 4). Both these oligos have a 5′ phosphate group.
15) SB28
This sequence is based on the co-stimulation motif of human CD28 and was constructed by annealing oligos A9018 and A9019 (FIG. 4). Both these oligos have a 5′ phosphate group.
16) SB29
This sequence is based on the co-stimulation motif of human CD154 and was constructed by annealing oligos A9020 and A9021 (FIG. 4). Both these oligos have a 5′ phosphate group.
Exemplification of mutation of SB1.
SB1, based on the first ITAM of TCR ζ, has the naturally occurring amino acid sequence of: QNQLYNELNLGRREEYDVLDKRRGRDPEM (SEQ ID NO. 3). Degenerate oligonucleotides (D7001 and D7002) were designed to alter the three amino acid residues following each of the ITAM-defining tyrosine residues (ie, the six residues highlighted in bold, above). The oligonucleotides were designed such that any residue cold be introduced at the first two positions following the tyrosine, but only leucine, or isoleucine, or valine would be introduced at the third position. Thus, mutated SBs with the following sequence would be generated (where X is any amino acid):
Degenerate oligonucleotides D7001 and D7001 were annealed at a concentration of 1 pmole/μl in a buffer consisting of 25 mM NaCl, 12.5 mM Tris.HCl, 2.5 mM MgCl2, 0.25 mM DTE, pH 7.5, by heating in a boiling water bath for 5 minutes and then allowing them to cool slowly to room temperature. Approximately 1 pmole of annealed oligonucleotides were mixed with approximately 1 ng of BamHI-digested pBluescript, T4 DNA ligase and ATP in a 10 μl reaction volume and ligation performed under the manufacturer's recommended conditions. Competent Escherichia coli, strain XL1-Blue, were transformed with 2 μl of ligation reaction, and plated onto LB agar containing ampicillin. Ampicillin resistant colonies were picked and grown up in L-broth containing ampicillin, and plasmid DNA prepared. The presence and sequence of any insert in the pBluescript vector was confirmed by sequencing with oligonucleotides corresponding to regions 5′ and 3′ to the BamHI site in the vector. Representative examples of mutant SBs generated using this method with degenerate oligonucleotides D7001 and D7002 are given as SBW1A, SBW1B, SBW1C and SBW1D in Table 1.
The vector, pHMF393 was digested with the restriction enzyme BamH I under the manufacturer's recommended conditions and then treated with alkaline phosphatase for 10 minutes at 37° C. in the same buffer. The linearised vector fragment was eluted from an agarose gel and purified. Approximately 1 ng of vector fragment was ligated to approximately 1 pmole of SB or mixture of SBs using T4 ligase and ATP under the manufacturer's recommended conditions in a 10 μl reaction. 2 μl of the ligation reaction was transformed into XL-1 blue competent E. coli and plated onto L-broth/Ampicillin plates. Ampicillin resistant colonies were picked and grown up in L-broth containing Ampicillin, and plasmid DNA prepared. Correct orientation of the inserted SB was established by digesting the DNA with BamH I and an enzyme within the Vector (Nar I). Optionally before picking, colonies were screened for the presence of inserted SBs by PCR using oligos corresponding to regions 5′ and 3′ to the BamH I site in the vector. Positive colonies were then grown up in L-broth containing Ampicillin and plasmid DNA prepared. Correct orientation of the inserted SB was established by digesting the DNA with BamH I and an enzyme within the Vector (Nar I).
Correct plasmids were then digested with BamH I, treated with alkaline phosphatase, gel purified and ligated to an SB, or mixture of SBs, as described above to insert a second SB. Colonies were screened again in the same way to find vectors with a second SB in the correct orientation. These vectors were then put through further rounds of digestion, purification, ligation and screening, as desired, to generate the required number of SBs in the correct orientation in a vector.
Specific Example: (see Table 2)
Construction of synthetic signalling regions with random combinations of SBs by adding mixtures of more than one SB at a time was done by the following methods:
a) SBs were ligated to each other in the absence of vector and then digested with both Bcl I and BamH I to cut SBs ligated to each other in incorrect orientations. If a specific number of SBs were required, then the desired size fragments were gel eluted and purified; if not, the ligated SB fragments were purified from the restriction enzymes and then ligated to the digested, phsophatased and purified vector as in Example 3. Ampicillin-resistant colonies were then generated and screened as in Example 3 to select vectors with more than one SB in the correct orientation in a vector.
b) SBs were ligated to the digested, phosphatased and purified vector and then digested with both Bcl I and BamH I to cut Sbs ligated to each other and to the vector in incorrect orientations. Fragments larger than the unligated vector were gel purified and then ligated to recircularise. Ampicillin-resistant colonies were then generated and screened as in Example 3, to select vectors with more than one SB in the correct orientation in a vector.
c) SBs were ligated to the digested, phosphatased and purified vector at a ratio of SB to vector that favoured the insertion of multiple SBs; Ampicillin resistant colonies were then generated and screened as in Example 3 to select vectors with more than one SB in the correct orientation in a vector.
It was found that for maximum efficiency it was desirable to use method c and establish a titration of SB for each vector. A de-stimulation of colony number from vector only controls was a good indication of insertion of multiple SBs. It was also found to be more efficient to perform two rounds of ligation and screening for correct insertion of a low number of SBs rather than one round of ligation and screening for many SBs in the correct orientation. Statistically, adding 1 SB, 50% should be in the correct orientation; adding 2 SBs, 25% should be in the correct orientation but when 4SBs are added at one time only 6% of vectors would have all 4 SBs in the desired orientation.
In the case of multiple rounds of multiple insertion, mixtures of both vector and SBs were ligated to each other to increase diversity of the library produced.
Specific examples: (see Table 2)
a) Construction of expression plasmids
The chimeric receptor constructs were subcloned from pBluescript KS+ into the expression vector pEE6hCMV.ne (C. R. Bebbington (1991), Methods 2, 136-145) on a HindIII to EcoRI restriction fragment. The expression vector with no chimeric receptor genes is used as a negative control in subsequent experiments.
b) Stable transfection into Jurkat E6.1 cells
The expression plasmids were linearised and transfected into Jurkat E6.1 cells (ECACC) by electroporation using a Bio-rad Gene Pulser. 10 μg of DNA per 2.5×106 cells were given two pulses of 1000V, 3 μF in 1 ml of PBS. Cells were left to recover overnight in non-selective medium before being selected and cultured in medium supplemented with the antibiotic G418 (Sigma) at 1.5 mg/ml. After approximately four weeks the cells were ready for analysis.
c) FACS analysis of surface expression
Approximately 5×105 Jurkat cells were stained with 1 μg/ml FITC labelled antigen, CD33 extracellular region. Fluorescence was analysed by a FACScan cytometer (Becton Dickinson).
d) IL-2 production analysis of function
2×105 cells were incubated at 37° C./8% CO2 for 20 hours in 96 well plates with CD33 expressing HL60 target cells at an effector: target ratio of 1:1. Cell supernatants were then harvested and assayed for human IL-2 (R & D Systems Quantikine kit).
The library of synthetic signalling regions produced is listed in Table 2. These were constructed by the methods described in example 3 and example 4. All signalling regions were sequenced prior to analysis and some additional diversity of the library was found to arise from mis-annealment and recombination events and these are listed in the notes to Table 2.
This diversity library of synthetic signalling receptors were demonstrated to function specifically in response to antigen (see
1) SB4* is a single amino acid different from SB4, initially generated by mis-annealment of oligos but subsequently deliberately generated by annealing oligos A8810B and A8811B (see
SB4*: GSYEKSDGVYTGLSTRNQETYDTLKHEKPS (SEQ ID NO. 8)
2) SB1* is a truncated version of SB1 generated by a recombinant event during cloning.
SB4*: GSGQNQLYNELNLGRREEYDVALAK (SEQ ID NO. 4)
3) R to G change at the 5′ end of SB3
4) A to T change at the 3′ end of SB3
5) K to R change at the 5′ end of SB4*
6) SB10* is a truncated version of SB10 with an altered 3′ end.
SB10*: GSPPRTCDDTVTYSALHKRQVGDYENVIPER (SEQ ID NO. 16)
7) S to L change at the 5′ end of SB4 and a S to G change in the middle of SB4.
SB4″: GLYEKSDGVYTGLGTRNQETYETLKHEKPGS (SEQ ID NO. 9)
Number | Date | Country | Kind |
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9908814 | Apr 1999 | GB | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/GB00/01498 | 4/17/2000 | WO | 00 | 10/16/2001 |
Publishing Document | Publishing Date | Country | Kind |
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WO00/63360 | 10/26/2000 | WO | A |
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5093251 | Richards et al. | Mar 1992 | A |
5863730 | Masson et al. | Jan 1999 | A |
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