WO2009150426A2

WO2009150426A2 - Method

Info

Publication number: WO2009150426A2
Application number: PCT/GB2009/001461
Authority: WO
Inventors: James Hunt; Helen Elizabeth Harries; Samuel Ken En Gan; Andrew John Beavil; Philip John Marsh
Original assignee: King's College London
Priority date: 2008-06-10
Filing date: 2009-06-10
Publication date: 2009-12-17
Also published as: GB0810633D0; WO2009150426A3

Abstract

The present invention relates to a nucleic acid construct encoding an immunoglobulin heavy and/or light chain, wherein said nucleic acid construct comprises one or more non-naturally occuring restriction sites and wherein said non-naturally occuring restriction sites are incorporated at one or more of the following positions: i) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain; and/or ii) between the variable and constant regions of said immunoglobulin heavy and/or light chain; and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain; and/or iii) at or within about 25 nucleotides of the 3' end of the stop codon of the constant region of said immunoglobulin heavy and/or light chain; and/or iv) at or within about 25 nucleotides of the 5' end of the leader sequence of said immunoglobulin heavy and/or light chain.

Description

METHOD

FIELD

The present invention relates to the field of recombinant protein expression. In particular, the present invention relates to the field of recombinant antibody expression and a construct/expression system that can be used to rapidly produce antibodies.

INTRODUCTION

Antibodies are increasingly being used as diagnostic and therapeutic agents for various disorders and diseases (Jain et al 2007; Reichert et al 2005). These applications typically require that functional antibodies, or antibody fragments, are prepared in sufficient quantities (microgram to milligram) for screening of their individual properties. Particularly useful is the application of recombinant methods to this procedure (Jain et al 2007); this allows the use of a variety of expression hosts, and the ability to manipulate DNA sequences to allow tailoring of the final antibody.

Most antibody expression systems require the establishment of cell lines derived from CHO, mouse myeloma, or more recently PER.C6 cells; Bebbington et al 1992, Jain et al

2007, Maynard, Georgiou 2000, Zafir-Laviei et al., 2007, Filpula, 2007 Jones et al., 2003.

Typically, establishing a new cell line requires long selection and screening, often with low success rates, meaning antibody production can become an extended and labour-intensive process. Methods that circumvent selection procedures in these cell-lines can result in very low antibody yields. As such there is a requirement for the development of fast antibody cloning and expression technologies, especially if the properties of a panel of antibodies are to be investigated within a reasonable timeframe. In light of this a body of literature has accumulated on rapid antibody production, commonly applied in human embryonic kidney cells and focussed on IgG (Baldi et al., 2005, Wright et al., 2003,Li et al., 2007, Berntzen et al., 2005). In these systems the potential importance of other antibody isotypes has been neglected, despite the fact that changing antibody isotype markedly alters its ability to mediate a number of biological processes (Gould, HJ. et al 1999, Bruggemann 1987 Nimmerjahn and Ravetch 2007). To fully study the protein it is important to have the ability to swap the elements that define the isotype, without introducing undesired changes to the amino acid sequence. In addition, the ability to swap the antibody regions must be compatible with the vast range of variable-region antibody sequences that define antibodyrtarget specificity.

The present invention seeks to address the problems of the prior art.

SUMMARY ASPECTS AND EMBODIMENTS OF THE PRESENT INVENTION

There is described herein a nucleic acid construct that facilitates easy swapping and rapid expression of antibody regions. In particular, the nucleic acid construct allows for the swapping of elements of an antibody (eg. an antibody variable region in the context of a whole molecule) easily allowing for the production of different antibody combinations. The nucleic acid construct can be used in the transient or stable production of immunoglobulin within a relatively short period of time.

Advantageously, the nucleic acid construct also provides for the rapid exchange of the leader sequence, the plasmid and the variable region and/or constant regions of both the heavy and light chains of an antibody. Isotype swapping is potentially therapeutically important. In addition, many v-region sequences from diverse sources (eg. hybridomas and patients) need to be characterised by recreating functional whole antibodies.

In a first aspect, there is provided a nucleic acid construct encoding an immunoglobulin heavy and/or light chain, wherein said nucleic acid construct comprises one or more non- naturally occuring restriction sites and wherein said non-naturally occuring restriction sites are incorporated at one or more of the following positions: i) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain; and/or ii) between the variable and constant regions of said immunoglobulin heavy and/or light chain; and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain; and/or iii) at or within about 25 nucleotides of the 3' end of the stop codon of the constant region of said immunoglobulin heavy and/or light chain; and/or iv) at or within about 25 nucleotides of the 5' end of the leader sequence of said immunoglobulin heavy and/or light chain.

Suitably, the non-naturally occuring restriction sites incorporated: i) at or within about 25 nucleotides of the 3' end of the leader sequence of the heavy and/or light chain of said immunoglobulin; and ii) between the variable and constant regions of the heavy and/or light chain of said immunoglobulin or at or within about 25 nucleotides of the 3' end of the variable region of the heavy and/or light chain of said immunoglobulin; are silently mutated non-naturally occuring restriction sites.

Suitably, said restriction sites at or within about 25 nucleotides of the 5' end of the leader sequence and at or within about 25 nucleotides of the 3' end of the stop codon of the constant region are compatible with the multiple cloning site of a plasmid or a vector.

Suitably, said leader sequence encodes an Immunoglobulin leader sequence.

Suitably, the Immunoglobulin leader sequence may be derived from a V_H, VK or Vλ leader sequence. For example the leader sequence may be derived from (e.g. be a modified variant of) a sequence set forth in SEQ ID No. 1, 3, 14, 21 and 23.

Suitably, the non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain are selected from Xhol, ApaLI, BspEI, Sail, BssHII, Narl, Nhel, SacII, Agel and/or Notl.

Suitably, more than one (e.g. two, three or four) non-naturally occurring restriction sites may be present at one or more of the above locations. For instance, in one embodiment at least two non-naturally occurring restriction sites are present at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain. In another embodiment, at least two non-naturally occurring restriction sites are present between the variable and constant regions of said immunoglobulin heavy and/or light chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain. In another embodiment, at least two non- naturally occurring restriction sites are present at or within about 25 nucleotides of the 3' end of the stop codon of the constant region of said immunoglobulin heavy and/or light chain. In another embodiment, at least two non-naturally occurring restriction sites are present at or within about 25 nucleotides of the 5' end of the leader sequence of said immunoglobulin heavy and/or light chain. In further embodiments, two or more non- naturally occurring restriction sites are present at each of two or more of the locations defined above. For instance, two or more non-naturally occurring restriction sites are present at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain; and two or more non-naturally occurring restriction sites are present between the variable and constant regions of said immunoglobulin heavy and/or light chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain.

Suitably, the immunoglobulin chain is a heavy chain and the non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy chain are selected from Xhol, ApaLI, BssHII, Narl and/or Notl. More than one non-naturally occuring restriction site(s) may be present at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy chain, for instance in some embodiments two, three or four non-naturally occuring restriction sites may be present at this location. In specific embodiments a Xhol site and a ApaLI site may be present within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy chain, for examples as present in SEQ ID NO:2. Alternatively a Notl site and a BssHII or Narl site may be present at the same location, for instance as present in SEQ ID NO:6.

Suitably, the immunoglobulin chain is a kappa light chain and the non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin kappa light chain are selected from Xhol, ApaLI, Nhel and/or SacII. More than one non-naturally occuring restriction site(s) may be present at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin kappa light chain, for instance in some embodiments two, three or four non-naturally occuring restriction sites may be present at this location. In specific embodiments Xhol and ApaLI sites (for example as present in SEQ ED NO: 2) or Nhel and SacII sites (for example as present in SEQ ID NO: 16) may be present at this location.

Suitably, the immunoglobulin chain is a lambda light chain and the non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin lambda light chain are selected from BspEI, Sail, Xhol, ApaLI and/or

Agel. More than one non-naturally occuring restriction site(s) may be present at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin lambda light chain, for instance in some embodiments two, three or four non-naturally occuring restriction sites may be present at this location. In specific embodiments Xhol and ApaLI sites (for example as present in SEQ ED NO:2) or BspEI and Sail sites (for example as present in SEQ ED NO: 24) may be present at this location.

Suitably, the non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 5' end of the leader sequence of said immunoglobulin heavy and/or light chain are selected from EcoRI, HindIH and/or Notl.

Suitably, said non-naturally occuring restriction site(s) between the variable and constant regions of said immunoglobulin heavy and/or light chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain are selected from Narl, Nhel, Xhol, Kpnl, BsiWI and/or AvrEt. More than one non- naturally occuring restriction site(s) may be present between the variable and constant regions of said immunoglobulin heavy and/or light chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain, for instance in some embodiments two, three or four non-naturally occuring restriction sites may be present at this location. In specific embodiments a Xhol site and a Nhel site may be present at this location; or a BsiWI site and a Kpnl site and a Narl site; or a Avrll site and a Narl site.

Suitably, (a) the immunoglobulin chain is a heavy chain and said non-naturally occuring restriction site(s) between the variable and constant regions of said immunoglobulin heavy chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy chain are selected from Nhel and/or Xhol (for example Nhel and Xhol sites are present, as found in SEQ ID NO: 13); or

(b) the immunoglobulin chain is a kappa light chain and said non-naturally occuring restriction site(s) between the variable and constant regions of said immunoglobulin kappa light chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin kappa light chain are selected from Kpnl and/or BsiWI and/or Narl, (for example Kpnl and BsiWI and Narl sites are present, as found in SEQ ED NO:20); or

(c) the immunoglobulin chain is a lambda light chain and said non-naturally occuring restriction site(s) between the variable and constant regions of said immunoglobulin lambda light chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin lambda light chain are selected from Kpnl and/or Avrll and/or Narl (for example Avrll and Narl are present, as found in SEQ ED NO:28).

Suitably, the non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 3' end of the stop codon of the constant region of said immunoglobulin heavy and/or light chain are selected from Xbal, Hindlll, Notl, and/or Sfil.

Suitably, a naturally-occurring restriction site in said immunoglobulin heavy and/or light chain has been removed by a silent mutation.

Suitably, the leader sequence and/or the variable region and/or the constant region has been swapped for a different leader sequence and/or variable region and/or constant region.

Suitably, the variable region and/or the constant region has been swapped for a different variable region and/or constant region. Suitably, said constant region is replaced with or comprises a tag for protein purification.

In a second aspect, there is provided a method for preparing a nucleic acid construct encoding a light and/or a heavy chain of an immunoglobulin molecule comprising the steps of: a) providing a nucleic acid sequence encoding a heavy and/or light chain of an immunoglobulin molecule; and b) introducing into said nucleic acid sequence one or more non-naturally occuring restrictions sites; wherein said non-naturally occuring restriction sites are incorporated at one or more of the following positions: i) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain; and/or ii) between the variable and constant regions of said immunoglobulin heavy and/or light chain or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain; and/or iii) at or within about 25 nucleotides of the 3' end of the stop codon of the constant region of the heavy and/or light chain of said immunoglobulin; and/or iv) at or within about 25 nucleotides of the 5' end of the leader sequence of the heavy and/or light chain of said immunoglobulin.

Suitably, said method comprises the additional step of: swapping the variable region and/or the leader sequence and/or the constant region of said immunoglobulin heavy and/or light chain with a different variable region and/or leader sequence and/or constant region of the heavy and/or light chain.

Suitably, said method comprises the steps of: i) excising the leader sequence and/or the variable region and/or the constant region of said immunoglobulin heavy and/or light chain using one of more restriction enzymes that cut the one or more restriction sites incorporated therein; ii) amplifying a different leader sequence and/or a different variable region and/or a different constant region of said immunoglobulin heavy and/or light chain to incorporate one or more of the same restriction sites that are incorporated into said immunoglobulin heavy and/or light chain; iii) amplifying the sequence(s) to incorporate one or more of the same restriction sites therein; e) digesting the amplified sequence(s) with restriction enzyme(s) that cut the incorporated restriction sites; and f) inserting said digested sequence(s) into the construct, wherein steps (i) and (ii) are performed in either order or at the same time.

Suitably, one or more of the non-naturally occuring restriction sites incorporated: i) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain; and/or ii) between the variable and constant regions of said immunoglobulin heavy and/or light chain or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain; are incorporated using silent mutations.

Suitably, said restriction sites are restriction sites that do not cut more than once in the construct (cassette) or the vector.

Suitably, the nucleic acid construct encodes a chimeric or a humanised immunoglobulin heavy and/or light chain.

Suitably, said variable and/or constant region is swapped for a humanised or a chimeric variable and/or constant region.

In a third aspect, there is provided a nucleic acid construct obtained or obtainable by the method described herein.

In a fourth aspect, there is provided an isolated nucleic acid sequence comprising the sequence set forth in any of SEQ ID Nos. 1 to 59. In particular, the sequence may be a sequence set forth in any of SEQ ID Nos. 2, 4, 5, 6, 12, 13, 15, 16, 18, 19, 20, 22, 24, 25, 26, 28, 29 or 30 to 59.

In a fifth aspect, there is provided a vector or a plasmid comprising the nucleic acid construct or the nucleic acid sequence. In a sixth aspect, there is provided a host cell comprising the nucleic acid construct or the vector or plasmid.

In a seventh aspect, there is provided a method for expressing an immunoglobulin molecule in a cell comprising the steps of: a) providing the nucleic acid construct or the vector or plasmid; b) transforming or transfecting said nucleic acid construct or said vector into a cell; c) providing for the expression of said nucleic acid construct or said vector in said cell; and d) optionally purifying the immunoglobulin molecule.

Suitably, said nucleic acid construct is inserted into at least two vectors, wherein the first nucleic acid construct or vector comprises the light chain of said immunoglobulin and wherein the second nucleic acid construct or vector comprises the heavy chain of said immunoglobulin.

Suitably, said nucleic acid construct is inserted into one vector comprising the light chain of said immunoglobulin and the heavy chain of said immunoglobulin.

Suitably, the first nucleic acid construct or vector and the second nucleic acid construct or vector are transformed or transfected into the cell with an excess of light chain in concentration..

Suitably, said cells are transfected using the PEI method.

Suitably, said vector is selected from the group consisting of pcDNA3, pcDNA3.1 (+) Hygro, pCEP4, Lonza vectors and pTT3.

Suitably, said cells are bacterial, mammalian or plant cells.

Suitably, said human cells are HEK293 cells and their derivatives - such as HEK293E and HEK293T cells. In a eighth aspect, there is provided a nucleic acid primer comprising the sequence set forth in any of SEQ ID Nos. 30-59.

In a ninth aspect, there is provided a method, a nucleic acid construct, a nucleic acid sequence, a vector, a plasmid, a host cell or a nucleic acid primer substantially as described herein with reference to the accompanying description and drawings.

ADVANTAGES

The present invention is capable of producing immunoglobulins and fragments thereof without the long labour-intensive screening and selection. In particular, the present invention is capable of producing whole immunoglobulins, including chimeric and humanised antibodies. Already, the inventors have been able to rapidly clone, express and purify over 20 immunoglobulins making this a rapid, efficient system without long cell maintenance.

Unlike other antibody expression systems, the system described herein is able to: i) produce large quantities of immunoglobulins and fragments thereof in a human expression system within a month without selection; b) easily conduct heavy chain isotype swapping; c) easily conduct light chain isotype swapping; d) swap v-regions easily on both heavy and light chains; e) able to express chimeric antibodies not just across species, but also cross isotype together thus providing a rapid solution to the bottlenecked antibody expression that may hinder antibody studies and possible immunotherapy; and f) also has the ability to readily swap to other plasmids. In particular, the swapping may be done without the introduction of foreign sequences that would change the amino acid sequence.

The nucleic acid construct described herein is designed for easy swapping or exchange of variable region(s) and/or constant region(s) and/or leader sequence(s) and also for easy swapping or exchange of the vector in which the construct is contained. The design of the nucleic acid construct is such that silent mutations were introduced and so the antibody that is expressed does not have any mutations or extra residues introduced therein. Therefore, the amino acid sequence of the recombinant antibody is the same as the antibody sequence from which it is derived. In the context of the present invention, the use of silent mutations is therefore particularly preferred for at least some of the mutations that are introduced into the nucleic acid construct. Not only does the introduction of restriction sites enable straightforward swapping and exchange of individual components of the immunoglobulin but because the restriction sites have been introduced using silent or synonymous mutations the amino acid sequence of the encoded protein will be the same as the amino acid sequence before the restrictions sites were introduced therein. This is particularly desirable because non-silent restriction sites may affect the biological functionality and/or structure of the antibody which may be problematic for antibody functional and structural studies.

The expression construct/cassette can be used in both eukaryotic and prokaryotic systems.

The use of the described Immunoglobulin leader sequences provides for the rapid production of, for example, IgE. In particular, the leaders used allow silent mutations to be introduced thereby incorporating one or more restriction sites for swapping of leader and variable sequences. Moreover these restriction sites can be used in combination to create a multiple cloning site, for example by the incorporation of two or more restriction sites at or near to the leader/variable region boundary, and/or two or more restriction sites at or near to the variable region/constant region boundary. This allows wider flexibility in planning cloning reactions and provides a fallback if restriction sites are introduced into germline V- regions by somatic hypermutation.

To date, the cassette described herein has been successfully tested in pcDNA3, pcDNA3.1 (+) Hygro, pCEP4, pRY, pEE (GS) and pTT3 vectors. Using the transient HEK293E system, 22 IgEs have been produced and purified within a short period of time with mix- matching of lambda (λ) and kappa (K) light chains and different VH and Vκ/Vλ families. DESCRIPTION OF THE FIGURES

Figure 1 shows the structure of an IgE molecule. The various structural elements of the IgE antibody as shown: Variable Heavy chain (VH); Variable Light chain (VL); Constant region - heavy chain (Cε); Constant region - light chain (CL).

Figure 2 shows the heavy chain cassette SGH. Outlined are the leader/signal peptide, VH, CH regions and the incorporated restriction sites flanking these elements.

Figure 3 shows restriction sites silently mutated into the Humighae 1 leader. Alignment of the wild-type leader from Humighae 1 (Genbank accession J00227) (Top) with the modified leader sequence incorporating 5' restriction sites and silent mutations (shaded, bottom). Underlined sequences show the restriction sites introduced.

Figure 4 shows silent mutations incorporated to the 5' and 3' ends of the IgE constant region. Alignment of 5' and 3' ends of the wild-type human IgE constant sequence (top) 5' and 3' ends of the human IgE constant with incorporated silent mutations (bottom). Underlined sequence denotes restriction recognition sites whereas the shaded region shows a silent mutation.

Figure 5 shows the construction of the IgE heavy chain vector pSGH. IgE constant chain cDNA was PCR amplified from pJJ71 (previously described in (Kenten et al. 1982)) with primers incorporating EcoRI, Nhel and Xbal restriction sites. The PCR product was then digested with EcoRI and Xbal and ligated into EcoRI-Xbal cut pcDNA3 to form pSGCep. Finally, pSGCep was cut with HindIH and Nhel for insertion of PCR amplified VH cDNA incorporating the Humighael leader with HindIII at the 5' site. The final sequence verified plasmid is pSGH (SGH in the pcDNA3 backbone).

Figure 6 shows the full sequence of the SGH heavy chain cassette containing an adapted leader based on Humighael, a human VH4 region and the human IgE constant region, plus the restriction sites used for construction / inserted to enable mobility of V-regions and the whole cassette.

Figure 7 shows the 3' end of the Jk sequence. Alignment of the 3' end of the Jk (top) with the 3' end of the JK where silent mutation have been incorporated (bottom). The Kpnl restriction recognition site added is underlined.

Figure 8 shows the SGLk cassette, with the leader/signal peptide, VL and CL regions and the incorporated restriction sites.

Figure 9 shows the construction of the light chain vector pSGLK. Human kappa constant cDNA was amplified from human nasal turbinate cDNA library using primers incorporating Kpnl and Xbal restriction sites at the 5' and 3' ends. This was cut and ligated into pcDNA 3.1 to form pSGkC. Light chain variable-region cDNA was then PCR amplified with primers incorporating the Humighael leader and HindIII and Kpnl at the 5' and 3' ends respectively for insertion into pSGkC to form pSGLk (SGLk in pcDNA 3.1 (+) Hygro backbone).

Figure 10 shows the whole SGL cassette with kappa constant and in-house Vk (SGLk). The sequence shown comprises the Humighael leader with silent mutations, a V_LK sequence and the human kappa constant sequence. Restriction sites incorporated during assembly/used for subsequent cloning reactions are indicated.

Figure 11 shows restriction sites incorporated into the SGLλ-1 cassette, (top) 5' and 3' ends of Rat SPE7 Jλ with silently mutated Humighael leader (as described in SGH and SGLk) and silently incorporated Kpnl site at the 3' end. (bottom) 5' and 3' ends of the human lambda-constant- 1 with Xbal attached to the 3' end.

Figure 12 shows the SGLλ cassette, with the leader/signal peptide, VL and CL regions and the incorporated restriction sites. Figure 13 shows the full SGLλ-1 cassette, comprising the Humighael leader with silent mutations, a rat Vu sequence and the human lambda constant- 1 sequence. Restriction sites incorporated during synthesis for subsequent use in cloning reactions are indicated.

Figure 14 shows the construction of pOriPs (pSGH-OriP & pSGLk-OriP). The pcDNA based vectors pSGH and pSGLk were adapted for compatibility with EBNA-I expression systems by insertion of the OriP element in the vector backbone. OriP was amplified from the vector pCEP with Nrul sites added with overhanging PCR primers. The PCR product was then ligated as a Nrul fragment into Nrul cut pSGH and pSGLk to create pSGH-OriP & pSGLk-OriP.

Figure 15 shows the construction of pCEP4-SGH and pCEP4-SGLk. The SG cloning cassettes (SGH & SGLk) were first PCR amplified from pSGH and pSGLk using primers incorporating HindHI at both ends. The SG cassettes were then ligated into HindHI cut pCEP4 (Invitrogen) and sequence verified to confirm correct orientation of the cloning cassette.

Figure 16 shows the formation of pTT based vectors pSGH-E and pSGLk-E pTT3 (Durocher et al, 2002) was modified to form pSG-E by cutting the MCS with BamHI and Nhel, followed by incubation with DNA Pol I Klenow fragment to abolish part of the MCS. SG cassettes were PCR amplified using primers incorporating a 5' EcoRI site and 3' Xbal site, then restriction cut and ligated into pSG-E, to create pSGH-E & pSGLk-E.

Figure 17 shows production rates of IgE by HEK293E cells with varying ratios of heavy chain plasmid to light chain plasmid. 1 x 10⁵ HEK293E cells were transfected according to a previously published method (Durocher, Perret & Kamen 2002) with 1 μg/ml DNA with PELDNA ratio at 2:1. Heavy chain plasmid (pSGH-E):light chain plasmid (pSGLk-E) were varied as shown above. Inserts contained the HHM- VH4 heavy and light chain variable sequences. Supernatant was collected 14 days post-transfection and subjected to IgE ELISA. Samples for ELISA were performed in triplicate. Table is representative of 2 independent transfections.

Figure 18 shows SDS-PAGE non-reduced and reduced of purified IgE. (Left panel) Coomasie blue stained SDS-PAGE (8 %) gel of purified Herceptin IgE (1 μg) in non- reducing conditions purified from culture supernatants of transfected HEK293E cells. (Center panel) Coomasie blue stained SDS-PAGE (8 %) gel of purified human SPE7 IgE (1 μg) in non- reducing conditions purified from culture supernatants of transfected HEK293E cells (lane 1) compared to rat SPE7 IgE from Sigma-Aldrich (lane 2) and purified myeloma U266B1 IgE (lane 3). (Right panel) Coomasie blue stained SDS-PAGE 12 % gel of purified HHM- VH4 IgE (Reconstructed human IgE) in reducing condition to show the heavy and light chain constituents (1 μg).

Figure 19 shows surface plasmon resonance analysis of purified IgE in comparison with their commercial partners using a Biacore 3000 (GE Biosciences). A) Binding kinetics of chimeric-human SPE7 and rat SPE7 to immobilised DNP and human FcεRI. Purified IgE was analyzed for its binding to surface immobilized FcεRIα according to a previously published method (Hunt et al, 2005). Briefly, IgE diluted in HBS Biacore run buffer was flowed over a CM5 chip amine coupled with DNP-BSA (-200 RU) (Sigma Aldrich) or human sFcεRIα-IgG4-Fc fusion protein(~200 RU) at 20μL / min with an exposure time to analyte of 180 seconds. B) Binding kinetics of Herceptin IgE and Herceptin IgGl to the extracellular domain of Her2 and human sFcεRIα-IgG4-Fc fusion protein, purified IgE diluted in HBS Biacore run buffer was flowed at a rate of 20μL / min over a CM5 chip surface amine coupled with the extracellular domain of Her2 (Kind gift of Manuel Penichet, UCLA) or human sFcεRIα-IgG4-Fc fusion protein, (200 RU ) with exposure time to analyte of 180 seconds IgE concentrations shown in the range 125 - 8 nM.

Figure 20 shows gel filtration chromatography traces of purified IgE. Samples (left to right) of myeloma IgE (AFlO), Rat SPE7, human VH3, VH4, VH5, VH6 and VH7 IgE,

Herceptin IgE and Human SPE7. Each trace is representative of 2 purifications from different transfections of the same IgE combination. Inset IgE schematics indicate the domains swapped for each antibody analysed. Gel filtration analysis of purified IgE was carried out as described in (Hunt et al. 2005) using a Sephadex S200 column at 0.75 ml/min equilibrated with Tris buffer (0.5M Tris, 0.25M NaCl, pH 7.2).

Figure 21 shows pTT3 -based p SG-Es with the Xhol site in the plasmid removed, allowing efficient V-region swapping.

Figure 22 shows adapted pCEP4-SG vectors with SGH and SGL inserts suitable for the swapping of VH, VL, utilizing Xhol-Nhel, Xhol-Kpnl, respectively.

Figure 23 shows a map of the SGH2 heavy chain cassette. The figure shows a pSGH2 vector in IgE expression mode illustrating positions of restriction sites required for Ig heavy chain domain swapping. The Nar I site at the 3' end of the V_H 1-02 leader sequence and the Nhe I site at the 5' end of Cε allow for exchange of any murine or human V_H gene. The Nhe I site can also be utilised in conjunction with the Sfi I site 3' of the Cε to replace the existing Cε with human Cγl-4, human Cα2 or murine Cε. In addition the whole cassette can be transferred to alternative vectors using Sfi I with the Eco RI or Not I sites that exist 5' of the leader sequence. Alignment shows the introduction of a Nar I restriction site by silent mutation 12 bases 5' of the start of the V_H gene, thus removing the requirement for PCR incorporation of a leader sequence for each V_H gene used, as described in Example 1.

Figure 24 shows an alignment of coding DNA and amino acid translation of bases 1-171 of IgG isotypes 1-4 (derived from Genbank deposists J00228, J00230, X03604 and K01316), highlighting Nar I restriction sites. IgG encoding DNA features one or two Nar I restriction motifs (boxed), at bases 32-37 / 33-38 of IgG2, 3 and 4, and at bases 129-134 / 130-135 of IgGl, 3 and 4. At each of these positions, the IgG isotype that does not feature a Nar I restriction motif exhibits no difference in amino acid sequence, demonstrating the potential to remove these restriction sites by silent mutation. Figure 25 shows primers for modifying a V_RI family gene segment and Cε gene segment for insertion into the SGH2 cassette. The primers comprise a region that anneals to the gene of interest (normal text), which may vary depending on the target, and a region encoding the required restriction sites (bold text), which is fixed for each primer type. Non-specific filler sequence is shown in italic text and restriction enzyme recognition sequences are underlined.

Figure 26 shows the silent mutation of the VK leader A26 allowing incorporation of aNhe I cut site 11 bases upstream of the start of the VK gene segment.

Figure 27 shows the introduction of a restriction site for Bsi WI by silent mutation into the most 5' region of the CK gene.

Figure 28 shows a map of the SGK2 kappa chain cassette. The figure shows the pSGK2 vector illustrating positions of restriction sites required for Ig kappa chain domain swapping. The Nhe I site at the 3' end of the VκA26 leader sequence and the Bsi WI site at the 5' end of CK allow for exchange of any murine or human VK gene. The whole cassette can be transferred to alternative vectors using Sfi I with the Eco RI or Not I sites that exist 5' of the leader sequence.

Figure 29 shows primers used for modifying a VKI family gene in preparation for insertion into the SGK2 cassette. The primers comprise a region that anneals to the gene of interest (normal text), which may vary depending on the target, and a region encoding the required restriction sites (bold text), which is fixed for each primer type. Non-specific filler sequence is shown in italic text and restriction enzyme recognition sequences are underlined.

Figure 30 shows primers for amplification of a CK gene segment.

Figure 31 shows a map of the SGL2 lambda chain cassette. The figure shows a pSGL2 vector illustrating positions of restriction sites required for Ig lambda chain domain swapping. The Age I site at the 3' end of the Vλl-e leader sequence and the Avr II site at the Jλ-Cλ junction allow for exchange of any murine or human Vλ gene, with one exception. The whole cassette can be transferred to alternative vectors using Sfi I with the Eco RI or Not I sites that exist 5' of the leader sequence.

Figure 32 shows the introduction of a restriction motif for Age I by silent mutation of Vλ leader sequence 1-e at a position 14 bases 5' of the start of the Vλ gene segment.

Figure 33 shows the position of a native Avr II restriction site at the junction between the Jλ portion of the Vλ domain (bold) and the Cλ domain (normal text).

Figure 34 shows examples of primers for use with SGL2. The primers may be used for modification of a Vλl family gene and Cλl isotype gene for introduction into the SGL2 cassette. They comprise a region that anneals to the gene of interest (normal text), which may vary depending on the target, and a region encoding the required restriction sites (bold text), which is fixed for each primer type. Non-specific filler sequence is shown in italic text and restriction enzyme recognition sequences are underlined.

Figure 35 shows a map of the pSGH3 heavy chain cassette. The figure shows the pSGH3 vector in IgE expression mode illustrating positions of restriction sites required for Ig heavy chain domain swapping. The BssHH site at the 3' end of the V_H 1-02 leader sequence and the Nhel site at the 5' end of Cε allow for exchange of any murine or human

V_H gene. The Nhel site can also be utilised in conjunction with the Sfil site 3' of the Cε to replace the existing Cε with human Cγl-4, human Cα2 or murine Cε. In addition the whole cassette can be transferred to alternative vectors using Sfil with the EcoRI or Notl sites that exist 5' of the leader sequence.

Figure 36 shows the introduction of a BsstHI restriction site into a V_H leader sequence using the V_H 1-02 leader sequence listed on the Vbase database, by silent mutation 11 bases 5' of the start of the V_H gene. Figure 37 shows a V_H forward primer which can be used to add a BssHII restriction site onto a V_H1 family gene. The region applicable to all V_H forward primers is shown in bold text, whereas the target annealing portion, which may vary depending on target, is shown in normal text. Filler sequence is italicised and the restriction enzyme recognition sequence is underlined.

Figure 38 shows a map of the SGK3 kappa chain cassette. The figure illustrates the pSGK3 vector illustrating additional SacII restriction site alongside pre-existing cut sites required for Ig kappa chain domain swapping. The Nhel and SacII sites at the 3' end of the VκA26 leader sequence plus the B si WT site at the 5' end of CK allow for exchange of any murine or human VK gene. The whole cassette can be transferred to alternative vectors using Sfil with the EcoRI or Notl sites that exist 5' of the leader sequence.

Figure 39 shows the introduction of Nhel and SacII restriction sites into VK leader sequence A26. VK leader A26 can tolerate the introduction of a Nhel restriction site by silent mutation. Webcutter analysis of the A26 leader sequence highlighted that a further silent mutation enables a SacII restriction motif to be incorporated immediately 3' of the

Nhel site.

Figure 40 shows examples of primer sequences for use with pSGK3. The primers enable addition of SacII alone, or both Nhel and SacII to a VKI family gene. The region applicable to all V_H forward primers is shown in bold text, whereas the target annealing portion, which may vary depending on target, is shown in normal text. Filler sequence is italicised and the restriction enzyme recognition sequence is underlined.

Figure 41 shows a SGH cassette with optimal choices of restriction sites at the boundaries and the 5' & 3' ends. XhoI/ApaLI are for the VHl/Humighael leader (see example 1). Narl (example 2) or BssHII (example 3) are used with leader V_HI-02. Figure 42 shows a SGL cassette with optimal choices of restriction sites at the boundaries and the 5' & 3' ends. XhoI/ApaLI are used with the Humighael leader (Example 1), Nhe I/SacII are used with the VK A26 leader (Example 3) and Agel is used with the Vλ 1-e leader. At the V_L-C_L boundary, use of Kpnl is described in Example 1. Alternatively BsiWI can be used to cut the VK-CK boundary (Example 3) and AvrEI can be used at the Vλ-Cλ boundary (Example 2).

Figure 43 shows a map of a pSGL3 lambda chain cassette, illustrating positions of restriction sites required for Ig lambda chain domain swapping. The BspEI or Sail sites at the 3' end of the Vλ 8a leader sequence and the Avrll or Narl sites at the 5' end of the Cλ sequence allow for exchange of any human Vλ gene. The whole cassette can be transferred to alternative vectors using the 3' Sfil site in conjunction with the 5' EcoRI or Notl sites.

Figure 44 shows the introduction of two restriction sites, BspEI and Sail, into the 3' region of the Vλ leader 8a by silent mutation.

Figure 45 shows the introduction of an additional Narl restriction site by silent mutation 13 nucleotides into the 5' region of the Cλ gene segment.

Figure 46 shows primers which may be used for modification of a Vλl family gene and Cλl isotype gene for introduction into the SGL3 cassette.

DETAILED DESCRIPTION OF THE INVENTION

NUCLEIC ACID CONSTRUCT

The nucleotide sequences encoding an immunoglobulin molecule may be present in a nucleic acid (eg. a DNA) construct. The term "construct" as used herein is synonymous with terms such as "conjugate" or "cassette". The nucleic acid construct described herein therefore comprises one or more non-naturally occuring, artificial or synthetic restriction sites. The restriction sites are engineered into the construct such that each of the variable region(s) and/or the constant region(s) and/or the leader sequence(s) of the heavy and/or light chains of said immunoglobulin can be replaced, exchanged or swapped. Suitably, restriction sites are also engineered into the 5' and 3' ends of the nucleic acid construct to allow for the swapping of the vector in which the construct is contained.

Suitably, the restriction sites define approximately each end of the variable region(s) and/or the constant region(s) and/or the leader sequence(s) of the light and/or heavy chains such that each of the complete variable region(s) and/or the complete constant region(s) and/or the complete leader sequence(s) can be excised or removed from the construct and replaced or exchanged with a different sequence.

Suitably, the restriction sites cut the least possible number of known human germline v- regions whether heavy or light chain. This may be determined using, for example, V-Base.

The leader sequence(s) and/or the variable region(s) and/or the constant region(s) of the heavy and/or light chain that is to be inserted (in-frame) into the construct in place of the excised sequence(s) is amplified such that one or more restriction sites are incorporated into the amplified sequence(s) that are compatible with the restriction sites used to excise the sequence from the construct. The amplified sequences are then digested using the appropriate restriction enzymes in order to create the compatible restriction sites. The amplified/digested sequences are then inserted into the construct, thereby effectively exchanging or swapping the leader sequence and/or the variable region and/or the constant region of the heavy and/or light chain. Typically, this will involve making a pair of primers containing suitable restriction enzyme recognition sites flanking a region of the sequence which it is desired to amplify, bringing the primers into contact with the sequence, performing an amplification reaction - such as PCR - under conditions which bring about amplification of the sequence, isolating the amplified fragment (eg. by purifying the reaction mixture on an agarose gel) and recovering/digesting the amplified DNA. The region(s) may be excised or PCR amplified from a holding vector.

The nucleotide sequence encoding the variable region can be excised from the construct and exchanged or swapped for a different nucleotide sequence encoding a variable region; and/or the nucleotide sequence encoding the constant region can be excised from the construct and exchanged or swapped for a different nucleotide sequence encoding a constant region; and/or the nucleotide sequence encoding the leader sequence can be excised from the construct and exchanged or swapped for a different nucleotide sequence encoding a leader sequence; and/or the vector in which the nucleotide sequence encoding the immunoglobulin is contained can be exchanged or swapped. This facilitates the preparation of a construct that can easily express different (whole) antibodies - such as chimeric antibodies or humanised antibodies.

As will be appreciated by the skilled person, the variable region of the light chain may be swapped for a different variable region of the light chain; the constant region of the light chain may be swapped for a different constant region of the light chain; the variable region of the heavy chain may be swapped for a different variable region of the heavy chain; and the constant region of the heavy chain may be swapped for a different constant region of the heavy chain.

The whole cloning cassette comprising the leader, the variable region and the constant region may be transferred into another plasmid with compatible cloning sites or have modified cloning sites for compatibility. This can be done for both heavy and light chain cassettes. Both cassettes may be swapped onto a single vector to create a one-vector expression system.

As used herein, the term "different" in the context of the leader sequence and the constant/variable regions is used in its broadest sense and means that at least one nucleic acid is different. Suitably, the restriction sites are created using one or more silent mutations - such as one or more in-frame silent mutations.

The term "silent mutation" has its ordinary meaning in the art and means that the one or more DNA mutations that are introduced into the nucleic acid construct do not result in a change to the amino acid sequence of the encoded protein.

The silent mutations may occur in a non-coding region (ie. outside of a gene or within an intron). Typically, the silent mutation(s) will occur within an exon in a manner that does not alter the final amino acid sequence of the antibody.

The term "silent mutation" is used interchangeably with the term "synonymous mutation", however, synonymous mutations are a subcategory of a silent mutation and refer to silent mutations occurring only within exons.

In the context of the present invention, the use of silent in-frame mutations is particular preferred. Not only does the introduction of restrictions sites enable the rapid swapping and exchange of individual components of the immunoglobulin but because the restriction sites have been introduced using silent or synonymous mutations the amino acid sequence of the encoded protein will be the same as the amino acid sequence before the restrictions sites were introduced therein. This is particularly desirable because non-silent restriction sites may affect the biological functionality and/or structure of the antibody which may be problematic for antibody functional and/or structural studies.

When using silent mutations, it is desirable to leave no gaps between codon 1 of the variable region and the leader sequence and so the protein that is expressed does not include extra or superfluous amino acids that would not normally be present in the expressed immunoglobulin. Suitably, the leader sequence is cleaved leaving the rest of the immunoglobulin sequence intact. In choosing the restriction sites, it is also desirable to choose restriction sites that are unique in the construct such that the restriction enzymes only cut in a single position within the construct. Suitably, only a single point in the vector is cut.

In choosing the restriction sites, it is also desirable to choose restriction sites that are unique in the vector into which the construct may be inserted such that the restriction enzymes only cut in a single position within the construct (cassette) or the vector. In some instances, it may be necessary to modify the sequence of the vector to remove restriction sites that would otherwise prevent the restriction site from being unique.

Suitably, the restriction enzyme sites are therefore unique restriction enzyme sites.

The construct itself may be directly or indirectly attached to a promoter. Alternatively, the vector into which the construct is inserted can comprise a promoter.

Suitably, the nucleic acid construct is present in a vector- such as an expression vector.

For some embodiments, the nucleic acid construct may comprise the nucleotide sequences encoding the heavy and the light chains of the antibody.

For some embodiments, the nucleic acid construct may comprise the nucleotide sequence encoding only the heavy chain of the antibody.

For some embodiments, the nucleic acid construct may comprise the nucleotide sequence encoding only the light chain of the antibody.

For some embodiments of the present invention, at least two nucleic acid constructs may be provided, one comprising the heavy chain of the antibody and the other comprising the light chain of the antibody. As described herein, non-naturally occuring restriction sites are incorporated within about 25 nucleotides of the 3' or 5' end of a given sequence - such as 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides.

In one embodiment, the non-naturally occuring restriction sites described herein may be incorporated at:

i) the 3' end of the leader sequence of the heavy and/or light chain of said immunoglobulin; and/or

ii) between the variable and constant regions of the light chain of said immunoglobulin or at the 3' end of the variable region of the heavy chain of said immunoglobulin; and/or

iii) downstream of the stop codon of the constant region of the heavy and/or light chain of said immunoglobulin; and

iv) optionally, at the 5' end of the leader sequence of the heavy and/or light chain of said immunoglobulin.

The non-naturally occuring restriction site incorporated at the 3' end of the leader sequence is located, in one embodiment, before the 5'-gtccactcc-3' sequence at the 3' end of the leader sequence - such as the humighael leader sequence shown in SEQ ED No. 1. In one embodiment, the non-naturally occuring restriction site, Xhol is incorporated between nucleotides 40 and 45 of the humighael leader sequence shown in SEQ ID No. 2. In one embodiment, the non-naturally occuring restriction site is incorporated by introducing mutations at nucleotide 41 of the leader sequence - such as the humighael leader sequence shown in SEQ ID No. 2. In one embodiment, the restriction site is a Xhol restriction site. Suitably, the mutation(s) is a silent mutation.

The non-naturally occuring restriction site incorporated at the 3' end of the leader sequence is located, in one embodiment, before the 5'-tcc-3' sequence at the 3' end of the leader sequence - such as the humighael sequence shown in SEQ ID No. 1. In one embodiment, the non-naturally occuring restriction site ApaLI is incorporated between nucleotides 45 and 50 of the leader sequence - such as the humighael leader sequence shown in SEQ BD No. 2. In one embodiment, the non-naturally occuring restriction site is incorporated by introducing mutations at nucleotide 47 of the leader sequence — such as the humighael leader sequence shown in SEQ TD No. 2. In one embodiment, the restriction site is a ApalΛ restriction site. Suitably, the mutation(s) is a silent mutation. In further embodiments, the restriction site may be a silently mutated Narl site (as found in SEQ ID NO:4) or a silently mutated BssHII site (as found in SEQ ED NO:5). Further suitable restriction sites which can be incorporated by silent mutation are described in the examples below.

The non-naturally occuring restriction site incorporated between the variable and constant regions of the light chain of said immunoglobulin is located, in one embodiment, in the sequence 5'- ggtaccaaactg -3', e.g. the restriction site is a Kpnl site within the light chain V-C boundary, as found in SEQ DD NO:s 18 and 26. In another embodiment, a naturally occuring restriction site may be present between the variable and constant regions of the light chain of said immunoglobulin. The naturally occurring restriction site may be, for example, Avrll. Suitably, the mutation(s) is a silent mutation. In other embodiments, the restriction site may be a silently mutated Narl restriction site.

The non-naturally occuring restriction site incorporated between the variable and constant regions of the kappa constant region for the light chain of said immunoglobulin may comprise silently mutated Kpnl, BsiWI and Narl restriction sites (for example as found in SEQ DD NO: 20).

The non-naturally occuring restriction site incorporated at the 3' end of the variable region of the heavy chain of said immunoglobulin is incorporated, in one embodiment, at the junction between the J_H segment of the variable region and the constant region (for example as shown in SEQ DDs 7-11). The non-naturally occuring restriction site incorporated at the 3' end of the variable region of the heavy chain of said immunoglobulin is incorporated, in one embodiment, in the sequence set forth in SEQ ID 12. Two amino acids of the protein encoded by this sequence are conserved in all human Ig isotypes and murine IgE. A Nhe\ site can be introduced at this conserved region (as found in SEQ ID 12). In one embodiment, the non-naturally occuring restriction site is incorporated at nucleotides 10 to 15 of this sequence. In one embodiment, the non-naturally occuring restriction site is incorporated by introducing mutations at nucleotides 12, 13 and 14 of this sequence. In one embodiment, the non-naturally occuring restriction site that is incorporated is Nhel. Suitably, the mutation(s) is a silent mutation.

The non-naturally occuring restriction site incorporated downstream of the stop codon of the constant region of the heavy and/or light chain of said immunoglobulin can be any restriction site since this region is not translated into protein and will thereof not effect the amino acid sequence of the immunoglobulin. In one embodiment, the non-naturally occuring restriction site may be incorporated immediately downstream of the stop codon or further away - such as 10, 100 or 1000 base pairs away, for example. Suitably, the restriction site does not appear in the heavy or light chain leader, variable or constant regions.

The restriction site added to the 5' end of the leader sequence of the heavy and/or light chain of said immunoglobulin can be any restriction site sequence since this sequence does not interfere with the leader sequence. Suitably, the restriction site does not appear in the heavy or light chain leader, variable or constant regions. For some embodiments, any sequences before the Ncol site or Kozak sequence (5'-accatgg-3') can be be deleted.

As used herein, the term "restriction site" has its conventional meaning as used in the art and refers to the site in a nucleotide sequence that is recognised and cleaved by a restriction enzyme/endonuclease.

As used herein, the term "non-naturally occuring restriction site" means in its broadest sense that the restriction site does not occur in the immunoglobulin sequence into which the site is to be incorporated. The restriction site is typically incorporated in to the immunoglobulin sequence by methods known in the art - such as mutagenesis, gene synthesis or nucleotide synthesis. The immunoglobulin sequence into which the site is to be incorporated may be a naturally occuring (eg. wild type) sequence or it may be a mutated or an engineered sequence - such as an immunoglobulin encoding a chimeric or a humanised antibody. The non-naturally occuring restriction site may be an artificial, a synthetic or an engineered restriction site.

Suitably, restriction sites and restriction enzymes are chosen that cut a minimal number of the variable and/or constant region(s) and/or the leader sequences(s) of the heavy and/or light chain.

In some instances, it may be necessary to modify the multiple cloning site of a vector to allow the exchange or swapping.

In some instances, it may be necessary to modify the restriction sites that are engineered into the 5' and 3' ends of the nucleic acid construct and so they are compatible with the multiple cloning site of a vector.

AMPLIFICATION

As described herein, the leader sequence and/or the variable region and/or the constant region of the heavy and/or light chain that is to be exchanged or swapped into the construct is amplified such that one or more restriction sites are incorporated into the amplified sequence that are compatible with the restriction sites used to excise the sequence from the construct.

Suitably, the amplification method uses primers that incorporate the one or more desired restriction sites.

In some instances, it may be necessary to incorporated many or all of the silent restriction sites by the use of a long primer. Suitably, the amplification is an exponential amplification, as exhibited by, for example, the polymerase chain reaction.

Many target and signal amplification methods have been described in the literature. General reviews of these methods can be found in Landegren, U., et al., Science 242:229- 237 (1988) and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990). These amplification methods can be used in the methods described herein, and include polymerase chain reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase hybridisation, Q-beta bacteriophage replicase, transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based amplification (NASBA) and in situ hybridisation. Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.

PCR is a nucleic acid amplification method described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR consists of repeated cycles of DNA polymerase generated primer extension reactions. The target DNA is heat denatured and two oligonucleotides, which bracket the target sequence on opposite strands of the DNA to be amplified, are hybridised. These oligonucleotides become primers for use with DNA polymerase. The DNA is copied by primer extension to make a second copy of both strands. By repeating the cycle of heat denaturation, primer hybridisation and extension, the target DNA can be amplified a million fold or more in about two to four hours. PCR is a molecular biology tool, which must be used in conjunction with a detection technique to determine the results of amplification. An advantage of PCR is that it increases sensitivity by amplifying the amount of target DNA by 1 million to 1 billion fold in approximately 4 hours. PCR can be used to amplify any known nucleic acid in a diagnostic context (Mok et al., (1994), Gynaecologic Oncology, 52: 247-252).

LEADER SEQUENCE A leader sequence is a short sequence that directs the newly synthesized antibody through the cellular membrane of a cell.

The leader sequences may be endogenous or exogenous to the host cell.

The leader sequence may be native or it may be different to the antibody that is expressed.

The leader sequence is located at the 5' position of the DNA sequence or the N-terminal portion of the antibody and is typically removed enzymatically between biosynthesis and secretion of the antibody. Thus, the secretion signal sequence is usually not present in the final antibody product.

Advantageously, the leader sequence is selected such that the antibody that is expressed is secreted into the cell culture medium. This assists in the purification of the expressed antibody since it is not necessary to extract the expressed antibody from the cells in which it is expressed.

Whilst the leader sequence may be a component of the vector, it is preferred that the leader sequence forms part of the nucleic acid construct that is inserted into the vector. The leader sequence will be one that is recognized and processed (eg. cleaved by a signal peptidase) by the host cell.

Suitably, the DNA encoding the leader sequence is ligated in reading frame to the 5'-end of the DNA encoding the light or heavy chain, resulting in a fusion polypeptide.

Suitably, there are no intervening nucleotides between the end of the leader sequence and the 5' end of the V-region.

In particular embodiments, the leader sequence may be a modified Humighael, V_H 1-02, VK A26, Vλ 1 -e or Vλ 8a leader. In one embodiment, the leader sequence comprises a modified Humighae I leader including mutations that incorporate an Xhol and a ApάLl restriction site near the 3' end of the leader sequence. In one embodiment, this leader sequence comprises the sequence set forth in SEQ ID No.2.

In one embodiment, the leader sequence comprises a modified V_H 1-02 leader including mutations that incorporate a Narl site near the 3' end of the leader sequence. In one embodiment, this leader sequence comprises the sequence set forth in SEQ ID No.4.

In one embodiment, the leader sequence comprises a modified V_H 1-02 leader including mutations that incorporate a BssHII site near the 3' end of the leader sequence. In one embodiment, this leader sequence comprises the sequence set forth in SEQ ID No.5.

In one embodiment, the leader sequence comprises an optimised V_H leader including mutations that incorporate Notl and BssHU sites near the 3' end of the leader sequence. In one embodiment, this leader sequence comprises the sequence set forth in SEQ ID No.6.

In further embodiments, the leader sequence comprises a modified VK A26 leader incorporating a Nhel site (e.g. as defined in SEQ ID NO. 15), a modified VK A26 leader incorporating a Nhel site and a SacII site (e.g. as defined in SEQ ID NO. 16), a Vλ 1-e leader incorporating an Agel site (e.g. as defined in SEQ ID NO. 22) or a Vλ 8a leader incorporating a BspEI site and a Sail site (e.g. as defined in SEQ ID NO. 24).

In some embodiments, further restriction sites may be added at the 5' end of the leader sequence to facilitate cloning and swapping of the cassette between vectors. For instance a sequence comprising EcoRI, HindIII and/or Notl sites may be added to the 5' end of the leader. In one embodiment the sequence added to the 5' end of the leader comprises Notl and EcoRI sites, for example this sequence is as defined in SEQ ID NO: 29. Suitably, the Xhol, ApaLI, Notl, BssHII, Nhel, SacII, Agel, BspEI and Sail restriction sites are incorporated using silent mutations.

VARIABLE REGION

Suitably, restriction sites are engineered into the construct in order to join the variable region to the constant region in the heavy and light chains.

hi one embodiment, a restriction site is incorporated between the 3' end of the variable region and the 5' end of the constant region of the heavy chain in order to facilitate the swapping of variable and/or constant regions. The amino acid sequence "Ala-Ser" is conserved at the 5' end of human IgE, IgG 1-4, Ig A2 and murine IgE. Mutations (suitably, a silent mutation) can be introduced into this region in order to incorporate an in-frame

Nhel restriction site comprising the nucleotide sequence 5'-gctagc-3' (for example as present in SEQ ID 12).

In one embodiment, a restriction site is incorporated within the 3' end of VJ in the light chain in order to facilitate the swapping of variable and/or constant regions. In one embodiment, a Kpn\ site is engineered into this sequence, for example as present in SEQ ID NO: 18.

CONSTANT REGION

The 3' end of the constant region comprises a stop codon. As will be appreciated by the skilled person, it is not necessary for the restriction sites downstream of this codon to be silent mutations since this portion of the nucleotide sequence will not form part of the expressed protein. The same is true for restriction sites 5' of the leader sequence, i.e. these sites do not need to be introduced by silent mutation.

hi one embodiment, one or more restriction sites that are added downstream of this stop codon are used in order to facilitate swapping/exchange. In one embodiment, one or more terminal restriction sites are incorporated downstream (eg. immediately downstream) of this stop codon that facilitate swapping/exchange.

Suitably, the restriction site may comprise more than one restriction site to facilitate incorporation into a vector.

In one embodiment, the restriction site is εaxXbal restriction site (5'-tctaga-3).

In one embodiment, the restriction site is HindΩl (5'-aagctt-3').

In one embodiment, the restriction sites are Xba\ and HindΩl restriction sites (5'- tctagaagctt-3). In another embodiment, the restriction site is Sfil (5'- GGCCNNNNNGGCC-3 ').

EXPRESSION

The present invention contemplates the production of an antibody/ies, for instance in eukaryotic cells. The antibody may be stably transferred into a host cell.

A stable transfer means that the polynucleotide of interest is continuously maintained in the host. For long-term, high-yield production of recombinant antibodies, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Host cells may be transformed with DNA controlled by appropriate expression control elements (e.g. promoter, enhancer, sequences, transcription terminators and/or polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then switched to a selective media. The selectable marker in the recombinant vector confers resistance to the selection and allows cells to stably integrate the vector into their chromosomes and grow and which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

The antibodies may be transiently expressed n a host cell. Transiently transfected host cells typically lose the exogenous DNA during cell replication and growth.

For some embodiments, the antibodies are expressed transiently.

Eukaryotic expression

The expression of the polypeptide in eukaryotic cells may be controlled by any promoter or enhancer element known in the art. Promoters which may be used to control expression include, but are not limited to, the SV40 early promoter region, the promoter contained in the 3¹ long terminal repeat of Rous sarcoma virus, the herpes thymidine kinase promoter, the regulatory sequences of the metallothionein gene or the tetracycline promoter.

Suitable vectors for eukaryotic expression will typically include eukaryotic-specific replication origins and promoter regions, which include specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. Additionally, promoter regions may include sequences that modulate the recognition, binding and transcription initiation activity of RNA polymerase. Such sequences may be cis acting or may be responsive to trans acting factors. Depending upon the nature of the regulation, the promoter that is used may be constitutive or regulated.

HOST CELL

The host cell may be a naturally occurring host cell in which the nucleotide sequence encoding the antibody is naturally present. Thus, for example, if the nucleotide sequence encoding the antibody is a human sequence then it may be present in a human host cell. Suitably, the host cell is a host in which the nucleotide sequence encoding the antibody is not naturally present. Thus, for example, if the nucleotide sequence is a human sequence then the host cell may be a eukaryotic cell from a different species. According to this embodiment, the term "host cell" (or "recombinant host cell") is intended to refer to a cell that has been genetically altered, or is capable of being genetically altered by introduction of an exogenous polynucleotide, such as a recombinant vector.

It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

Thus, a further embodiment of the present invention provides host cells transformed or transfected with one or more of the nucleotide sequences {eg. nucleic acid constructs or a vector comprising the same) encoding the antibody. Typically, said nucleotide sequence is carried in a vector for the replication and expression of the nucleotide sequence. The cells will be chosen to be compatible with the said vector and may, for example, be eukaryotic cells (for example, fungal, yeast or plant cells).

Suitable eukaryotic host cells are also known in the art. For example, host cells may include yeast, VERO, HeLa, CHO, W138, BHK, COS-7, MDCK, HEK, HEK293 and HEK293E cells.

In one embodiment, the host cells are HEK293E cells.

The host cell may be transfected or transformed with a vector encoding the heavy chain derived polypeptide and a vector encoding the light chain derived polypeptide or alternatively, be transfected with a single vector encoding both the heavy and light chain cassette. The host cell may be transfected or transformed with a vector encoding the heavy and light chain derived polypeptide. The host cell may be transfected or transformed with a vector encoding the heavy chain derived polypeptide and a vector encoding the light chain derived polypeptide. In other words, the host cell may be co-transfected with dual expression vectors, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.

Where more than one vector is used, the vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, the vectors may contain different selectable markers.

In some situations, the light chain may be placed before the heavy chain to avoid an excess of toxic free heavy chain.

The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

TRANSFORMATION

Host cells are transformed or transfected (the terms "transformed" and "transfected" are used interchangeably herein) with the above-described construct or vector to generate stable or transient cell lines that express the antibodies.

The host cells may be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Such transformation techniques are well known in the art. A preferred transformation method is the PEI method as described in at least Godbey, et al. 1999a. Following transformation, cells (e.g. eukaryotic cells) used to produce the antibodies are grown in media known in the art and suitable for culture of the selected host cells. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the skilled person.

Once the host cells are grown to a certain density, the culturing conditions may be modified to promote the synthesis of the antibody/ies.

The light and heavy chain expression may be induced at different times during the synthesis phase.

The light and heavy chain expression may be induced at the same time during the synthesis phase.

VECTOR

The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.

Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.

Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "recombinant vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector.

Typically, the vector will comprise an origin of replication site. The origin of replication site is a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria.

Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that: (i) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (ii) complement auxotrophic deficiencies, or (iii) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

For the purpose of the present invention, either constitutive or inducible promoters can be used. A large number of promoters recognized by a variety of potential host cells are well known. Alternatively the selected promoter sequences can be synthesized. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of a target gene. However, heterologous promoters are preferred, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the α- lactamase and lactose promoter systems, a tryptophan (tip) promoter system and hybrid ) promoters such as the tac or the trc promoter. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are suitable as well.

Promoters that are functional in eukaryotic host cells are also well known in the art, for example as described in US 6,331,415. Examples of such promoters may include those derived from polyoma, Adenovirus 2 or Simian Virus 40 (SV40).

Each translational unit of the recombinant vector of the invention may contain additional untranslated sequences necessary for sufficient expression of the inserted genes. Such sequences are known in the art and may include the Shine-Dalgarno region located 5'-to the start codon and transcription terminator (e.g., [lambda]) located at the 3'-end of the translational unit.

The vector sequences may either be designed to exist in the host cells as episomes, or may be designed to facilitate integration into the host genomic DNA to create stable cell lines, eg., by designing vector to be linearized. For long-term, high-yield production of recombinant antibody, stable expression is preferred. For example, cell lines which stably express the antibody may be engineered.

For some embodiments, the two- vector system as described by Fouser, et al. (1992); Page and M. A. Sydenham (1991), Tada et al. (1994) and Wood et al (1990) is used. To optimize the production of immunoglobulin in the system, the transfection ratio of light and heavy chains can be optimised when using a dual expression vector system. In particular, the ratio of transfection of a first vector comprising the light chain and the second vector comprising the heavy chain can be altered in order to maximise expression.

For some embodiments, the optimum light chainrheavy chain ratio is about 4:1. Total DNA may amount to about 1 μg/ml/2-4xlO⁵ cells.

ANTIBODY As used herein the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multivalent antibodies, multispecific antibodies (eg. bispecific antibodies), and antibody fragments that exhibit the desired biological activity.

Suitably, the antibody is a whole antibody. In the context of the present invention, a "whole antibody" comprises or consists of at least the variable and constant regions of the light chain and the heavy chain of the antibody. In the context of the present invention, the sequence encoding a "whole antibody" comprises or consists of at least the variable and constant regions of the light chain and the heavy chain of the antibody and may also include the leader sequences for the light chain and the heavy chain of the antibody.

A typical naturally occurring antibody comprises four polypeptide chains, two identical heavy (H) chains and two identical light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region, which in its native form is comprised of three domains, CHl, CH2 and CH3. CH4 would be present for IgE. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.

The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda and their subclasses, based on the amino acid sequences of their constant domains.

Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-I, IgG-2, IgA-I, IgA-2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. (2000) Cellular and MoI. Immunology, 4th ed.

In one particular embodiment, the antibody is IgE.

In another particular embodiment, the antibody is IgG

In one embodiment, the antibody is or is derived from a monoclonal antibody.

The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

The antibodies (eg. monoclonal antibodies) described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or host or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Typically, in the context of the present invention, a chimeric antibody will be an antibody in which the leader and/or the variable and/or the constant region of the heavy and/or light chain has been swapped and replaced for a leader and/or a variable and/or a constant region from a different species or host or belonging to a different antibody class or subclass, while the remainder of the other regions is identical with or homologous to corresponding sequences in antibodies derived from another species or host or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

A "functional" "active" or "biologically active" antibody is one that is capable of exerting one or more of its natural activities in structural, regulatory, biochemical or biophysical events. The capability of an antibody to exert one or more of its natural activities depends on several factors, including proper folding and assembly of the polypeptide chains.

The antibodies may be human, chimeric, humanized or affinity-matured antibodies.

ANTBODY FRAGMENTS

The present invention also encompasses fragments - such as fragments of the antibodies described herein.

Examples of antibody fragments include, but are not limited to: (i) the Fab fragment, having VL, CL, VH and CHl domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHl domain; (iii) the Fd fragment having VH and CHl domains; (iv) the Fd' fragment having VH and CHl domains and one or more cysteine residues at the C-terminus of the CHl domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment including two Fab¹ fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g. single chain Fv; scFv) (Bird et al, Science 242:423-426 (1988); and Huston et al, PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies" with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. ScL USA, 90:6444-6448 (1993)); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CHl-VH-CHl) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata etal. Protein Eng. 8(10): 1057-1062 (1995); and US 5,641,870).

Moreover, the present invention contemplates antibody fragments that are modified to improve their stability and or to create antibody complexes with multivalency. For many medical applications, antibody fragments must be sufficiently stable against denaturation or proteolysis conditions, and the antibody fragments should ideally bind the target antigens with high affinity. A variety of techniques and materials have been developed to provide stabilized and or multivalent antibody fragments. An antibody fragment may be fused to a dimerization domain.

ANTIBODY VARIANTS

Amino acid sequence modification(s) of antibodies or fragments thereof are also contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics and provided that the final construct has the necessary restriction sites as described herein.

The amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made. One method for the identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081- 1085. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. To analyze the performance of a mutation at a given site, ala scanning or random mutagenesis may be conducted at the target codon or region and the expressed antibodies are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated.

Nucleic acid molecules encoding the amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non- variant version of the antibody.

As will be appreciated by the person skilled in the art it is important not to introduce mutations into one or more of the restrictions sites of the nucleic acid construct described herein which will render the restriction site non-susceptible to restriction digestion.

HUMAN ANTIBODY

In one embodiment, the antibody may be may be derived from a human antibody - such as a human monoclonal antibody. A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies.

HUMANISED ANTIBODY

In another embodiment, the antibody may be or may be derived from a humanised antibody — such as a humanised monoclonal antibody.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Typically, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.

The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

Various methods for humanizing non-human antibodies are well known in the art. For example, humanization can be essentially as described in Jones et al. (1986) Nature

321:522-525 by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies may be chimeric antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is important to reduce antigenicity. According to the so-called "best- fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies.

Antibodies are typically humanized with retention of high affinity for the antigen and other favourable biological properties. In this respect, humanized antibodies may be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding. AFFINITY MATURED ANTIBODY

In a further embodiment, the antibody may be or may be derived from an affinity matured antibody - such as an affinity matured monoclonal antibody.

An "affinity matured" antibody is one with one or more alterations in one or more CDRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s).

Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art (eg. see Bio/Technology 10:779-783 (1992)). Random mutagenesis of CDR and/or framework residues can also be used as is described in Jackson et ah, J. Immunol. 154(7):3310-9 (1995).

The antibody may be a bi-specific antibody.

NUCLEOTIDE SEQUENCES

The present invention involves the use of nucleotide sequences which may be available in databases.

The nucleotide sequence may be DNA or RNA of genomic, synthetic or recombinant origin e.g. cDNA. For example, recombinant nucleotide sequences may be prepared using a PCR cloning techniques.

The nucleotide sequence may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.

The nucleotide sequence may comprise exons and/or introns. In one embodiment, the nucleotide sequence is free of introns.

The nucleotide sequences may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5¹ ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in to enhance the in vivo activity or life span of nucleotide sequences useful in the present invention.

The use of nucleotide sequences that are complementary to the sequences described herein, or any homologue, fragment or derivative thereof is also contemplated. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.

PURIFICATION

The expressed light and/or heavy chains may be secreted into, and recovered from, the host cells. Protein recovery typically involves disrupting the cell/microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography.

Alternatively, the proteins may be transported into the culture medium and isolated therein. Cells may be removed from the culture and the culture supernatant filtered and concentrated for further purification of the proteins produced.

The expressed antibodies may be subjected to at least one purification step. Examples of suitable purification steps include hydroxy lapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Other techniques for protein purification - such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE(TM), chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

For affinity chromatography, the suitability of a particular protein as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.

The purified antibody may be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.

The antibody may be analyzed for its biological activity - such as its antigen binding activity. Many different antigen binding assays are known in the art and can be used herein and include without limitation any direct or competitive binding assays using techniques T such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), immunoprecipitation assays and fluorescent immunoassays.

FUSION PROTEIN

An antibody may be part of a larger fusion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.

A further aspect relates to the creation of a fusion protein with a C-terminal antibody constant region - such as an IgE constant region which can be used for easy purification by seamless attachment to heavy and/or light chain constant regions. Between the leader sequence and the constant region any nucleic acid sequence can be exchanged or swapped and is not limited to only antibody variable regions. Accordingly, the constant region may be used as a tag with a cleavage site inserted at the front (ie. the 5 'end) of the constant domain in order to assist in the cleavage of the constant region from the expressed protein. Non-limiting examples of such tags include the His and GST tags.

USES

The antibody may be used, for example, to purify, detect, and target a specific polypeptide it recognizes, including both in vitro and in vivo diagnostic, prophylactic or therapeutic methods for a variety of disorders or diseases.

The antibodies that are created may be used for anti-cancer immunotherapy.

The antibodies that are created may be used to determine their specificity (especially in diagnostics).

The antibodies that are created may be used to determine the importance of structural elements of the antibodies.

Partially humanized antibodies of known specificity from animals may also be created.

VARIANTS/HOMOLOGUES/DERΓVATΓVES

The present invention encompasses the use of variants and homologues of nucleic acid sequences.

Here, the term "homologue" means an entity having a certain homology with the subject nucleotide sequences described herein. Here, the term "homology" can be equated with "identity".

In the present context, a homologous sequence is taken to include a nucleotide sequence, which may be at least 70, 75, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to the subject sequence. Although homology can also be considered in terms of similarity (ie. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.

Various computer programs for carrying out alignments are available - such as the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et ah, 1999 ibid - Chapter 18), FASTA (Atschul et al, 1990, J. MoI. Biol, 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al, 1999 ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8).

Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

RECOMBINANT DNA TECHNIQUES

DNA sequences encoding the light and heavy chains of the antibody molecule can be obtained using standard recombinant DNA techniques. Desired DNA sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, the DNA can be synthesized using a nucleotide synthesizer or PCR techniques.

The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IrI Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

The invention will now be further described by way of Example, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES Example 1

Background Immunoglobulin E (IgE) plays a central role in mediating the allergic response (Gould et al. 2003) and has recently been proposed as an alternative to IgG based immunotherapy of cancer (Gould et al. 1999, Karagiannis et al. 2003). However, it is difficult to rapidly produce panels of monoclonal IgE in sufficient quantities for research analysis. Current systems are either unsuitable for the IgE isotype due to low yields (Rodin et al. 2004), or require laborious selection procedures and as such are appropriate for only a restricted antibody repertoire (Bebbington et al. 1992, Trill 1995, Bruggemann et al. 1987). To overcome this, we have designed cloning cassettes that allow swapping of isolated human V- and C- regions within the context of the human IgE framework. We have combined these cassettes with vectors for transient protein production, to create a method that allows the rapid expression of a large number of human IgE antibodies. This example describes the construction of this system, its application to the production of two antigen-specific and fourteen unknown specificity IgE antibodies, and their subsequent biochemical characterization.

The basic structure of an immunoglobulin is a tetramer of two identical light chains and two identical heavy chains (see Fig 1 for IgE). These chains can be split into two further functional elements; the variable (V) region and the constant (C) region. It is the variable region that defines antigen specificity. The constant region acts as a scaffold for presentation of these variable regions and, in the case of the heavy chain, a route to activation of the immune system's effector mechanisms. The particular effector mechanisms recruited can be altered by switching the gene that defines the heavy chain constant region. This is possible because Immunoglobulins are made up of discrete genetic elements that can be rearranged, such that specific V regions can be paired with different constant regions. Furthermore, the antibody light chain, which can be broadly classified into either λ- or K- families, can be paired with different heavy chains. For a comprehensive review of Immunoglobulin gene structure and rearrangement see (Gellert 2002).

In this example we wanted to exploit the modular gene structure of Immunoglobulins, to create a system that allows the swapping of variable regions within an IgE framework; alongside complementary light chain vectors that allow the creation of a fully functional antibody. One further element is required for the production of an Immunoglobulin in mammalian cells; a leader/signal peptide that encodes a sequence that directs the expressed protein to be secreted from the cell. This sequence is cleaved from the protein during the transport process. Therefore, the basic structure of the expression cassette required is, 5' — 3': leader, variable region, constant region. We have generated cassettes for the cloning and expression of such a gene. At the boundaries of these defined regions we have incorporated restriction sites by silent mutation, thus allowing the swapping of gene elements within these boundaries without altering the wild-type amino acid sequence.

(A) Cloning cassette SGH <heaw chain> and SGL <light chain > construction We have designed cloning cassettes for the IgE heavy chain (SGH) and for the light chain (SGL). The benefit of this is that it enables: 1) Independent expression of light and heavy chains in different plasmids, allowing optimisation of protein expression; 2) If the light- and heavy-constant components are considered separately, a wider range of restriction sites can be used when designing the vector for swapping gene segments. For the selection of restriction sites for the SGH and SGL cassettes, we have aimed to use those that have been previously characterized to not cut, or have minimal cuts, on germline variable regions according to the V-Base database (see Table 1). Number of functional segments which are cut

Restriction Sequence (total number of functional segments) enzyme cleaved VH(51) VK(40) VL(31) D(25) JH(6) JK(5) JK(4) TOTAL(162)

ApaLI GTGCAC

Ascl GGCGCGCC 0 0 0 0 0 0 0 0

BamHI GGATCC 14 0 15 0 0 0 0 29

BgIII AGATCT 12 0 0 0 0 0 0 12

BssHII GCGCGC 0 0 0 0 0 0 0 0

BStEII GGTNACC 4 0 8 0 5 0 1 18

CIaI ATCGAT 0 0 0 0 0 0 0 0

Eagl CGGCCG 23 0 0 0 0 0 0 23

EcoRI GAATTC 0 4 0 0 0 0 0 4

EcoRV GATATC 1 0 0 0 1 1 0 3 HaeIII (AG) GCGC (CT) 3 4 3 0 0 0 0 10

HindIII AAGCTT 1 0 2 0 0 0 0 3

Kpnl GGTACC 0 18 23 0 0 0 0 41

Ncol CCATGG 0 0 1 0 0 0 0 1 Ndel CATATG 4 0 3 0 0 0 0 7 Nhel GCTAGC 0 0 0 0 0 0 0 0

Notl GCGGCCGC 0 0 0 0 0 0 0 0

Pstl CTGCAG 14 33 4 0 0 0 0 51

Pvul CGATCG 0 0 1 0 0 0 0 1

Sad GAGCTC 11 1 1 0 0 0 0 13

SacII CCGCGG 8 0 0 0 0 0 0 8

Sail GTCGAC 0 0 0 0 0 0 0 0

Smal CCCGGG 2 1 4 0 0 0 0 7

Spel ACTAGT 2 0 1 0 0 0 0 3

Sphl GCATGC 3 9 1 0 0 0 0 13

Sfil GGCCNNNNNGGCC 0 0 0 0 0 0 0 0

Xbal TCTAGA 0 0 0 0 0 0 0 0

Xhol CTCGAG

Table 1: V-Base list of restriction enzymes and their cuts on germ-line V-regions.

Underlined enzymes are those incorporated for the SGH and SGL cloning cassettes. Table taken from http://vbase.mrc-cpe.cam.ac.uk/restriction2.php.

i) Heavy chain cassette (SGH) The heavy chain cassette, SGH, with details of restriction sites required for swapping antibody elements is shown in Figure 2.

The VHl leader encoded in the IgE sequence, Humighael (Genbank accession J00227), was chosen for the SGH cloning cassette, as it can be silently mutated close to the 3' end to allow future swapping of VH gene elements (Xhol and ApaLI -Figure 3). To facilitate movement of the cassette to other plasmids, the low frequency restriction sites HindIII / EcoRI and Notl were incorporated at the 5' end of the leader (Figure 3).

The silently encoded restriction sites, Xhol and ApaLI, do not cut the human epsilon gene and are either absent (Xhol), or show low frequency (ApaLI) within the germline genes listed on V-Base (Table 1). Furthermore, both sites can be incorporated within the leader sequence as they are not mutually exclusive. Other restriction sites can also be incorporated by silent mutation within the last 5 codons of the leader sequence (see Examples 2-4). However, for practical cloning purposes Xhol and ApaLI were considered most suitable, as they are commonly used and commercially available enzymes. Furthermore, the sites are close to the 3' end of the leader, reducing the length of the primer required when PCR amplifying V-region cDNA for incorporation into the cassette.

To enable exchange of VH-regions at the 3' end, a second restriction site, Nhel, was incorporated by silent mutation at the 5' start of the epsilon gene (Figure 4). Nhel is absent from human epsilon and VH germline gene sequences (Table 1).

To facilitate movement of the cassette between protein expression plasmids, Xbal and HindIII restriction sites were incorporated at the end of the constant region. Xbal is absent, and Hindm is present at very low frequency (<10%), in VH and VL sequences; both are absent in human C_L and Cε sequences (Table 1). Mobility of the cloning cassette between protein expression vectors is discussed in section B.

Specifically, the sequence of steps leading to the generation of the heavy chain cassettes (SGH) was as follows:

The heavy chain cassette was reconstructed by PCR, restriction endonuclease digestion and ligation according to standard protocols (Maniatis, Fritsch & Sambrook 1982) using the plasmid pcDNA3 (Invitrogen) as a destination. Primers (see Table 2) HCFl and HCRl specific for the human IgE constant (Cε) incorporating the desired silent mutations EcoRI- Nhel and Xbal, were used to PCR amplify the constant region from the plasmid pJJ71 (Kenten et al. 1982) which contains the wild-type Cε gene sequence. The PCR product was restriction cut and ligated as an EcoRl - Xbal fragment into pcDNA3 to form pSGHCep; EcoRI was placed before Nhel for abolishment of the multiple cloning site of pcDNA3 to allow for subsequent insertion of VH regions (Figure 5). A previously cloned VH4-region was PCR amplified from VH holding plasmids with primers VHFl and VHRl (Table 2) containing the Humighael leader and desired restriction sites. The PCR product was then restriction cut and ligated into pSGHCep as a Hindlll-Nhel fragment to finalise the assembly of the heavy chain cassette-SGH (Figure 5). This vector is termed pSGH. Sequences of maps and inserts describing the cloning procedure are shown in Figures 5 and 6 respectively.

Table 2: Primer sequences used to generate IgE expression cassettes.

Annealing temperature as calculated by MWG-Biotech (Germany) according to data sheets supplied with primers.

H) Light chain cassette

Cassettes for expression of both K and λ light chain constant regions were assembled for pairing with the heavy chain vector pSGH. As for the heavy chain cassette, the leader / signal peptide encoded in the IgE protein sequence Humighael (Genbank accession J00227) was chosen for the light chain cloning cassettes. This is because it is compatible with the silent mutations close to the 3' end that will allow swapping of VL gene elements. Specifically, Xhol cuts only 7.5% of germline human VL genes (see Table 1), and neither the human kappa, or lambda constant genes. A second restriction site, Kpnl, was incorporated by silent mutagenesis at the 3' end of the light chain variable region (the JK gene sequence) to facilitate swapping of the V_LK genes (Figure 7).

The elements assembled into a light chain cassette encoding the κ-chain, referred to as SGLK, are shown in Figure 8, along with details of restriction sites flanking constant and variable elements of the gene.

To allow mobility of the cassette between protein expression vectors, the light chain cloning cassette was completed by the addition of unique restrictions sites at the 5' of the Humighael leader (Hindiπ/EcoRI, Not!) and the 3' of the constant regions (Xbal, HindET); as described for the heavy chain SGH. Mobility of the cassette between protein expression vectors is discussed in section B.

Specifically the kappa light chain cloning cassette (SGLK) was generated as follows:

The kappa light chain cassette (SGLk) was assembled in pcDNA 3.1 (Invitrogen). Firstly, human κ-chain constant region (IGKC accession: BCl 10394) was PCR amplified from a human cDNA library with primers KCFl and KCRl (Table 2) comprising the restriction sites Kpnl and Xbal respectively, restriction digested and ligated as a Kpnl-Xbal fragment into pcDNA 3.1 (+) Hygro to form 'pSGkC. Vk regions were then amplified with primers HLVkFl and KVJRl (Table 2) encoding for the Humighael leader and the desired restriction sites (EcoRI, Hindlll, Notl, Kpnl) and transferred by restriction digest and ligation as a HindlJJ-Kpnl fragment into' pSGkC to form pSGLk. See Figures 9 and 10 for the maps and full sequence of the insert describing the cloning procedure.

In addition to the kappa light chain cassette, pSGLK, a lambda light chain cassette, SGLλl, was also created. In this case a human λ-constant-1 region sequence (accession: BC073786; obtained from the IMGT database: www.ebi.ac.uk/imgt/) was paired with a rat Vλ-7 sequence (James, Tawfik 2003). The leader previously described in SGH and SGLK was added to the 5' end of the rat Vλ, with restriction sites HindlH/EcoRI-Notl added at 5' end of the leader to facilitate mobility of the cassette between expression vectors. A Kpnl site was added by silent mutation to the 3 'end of Vλ and Xbal-Hindlϋ to the 3' end of the λ-constant-1 sequence (see Figure 11).

Further Kpnl restriction sites appearing within the genes were removed by silent mutation. The full gene, elements of which are outlined in Figure 12, 13 was not cloned from cDNA as previously described for SGH and SGLK, but synthesized by GeneArt, Germany.

(B) Expression, purification and characterisation of IgE antibodies produced using the SG cloning cassettes (SGH and SGL)

Section A describes the creation of three cloning cassettes: SGH, a human epsilon constant region cassette containing a human VH4 variable region; SGLK, a human kappa light chain cassette, incorporating a kappa variable sequence obtained from the same cell from which the VH4 gene was cloned, and SGLλ-1, a human λ-1 cassette incorporating a synthesised rat hapten-specific variable region, paired with a human λ-1 constant region. In this section we describe the testing of these cassettes for protein expression, and subsequent inserts formed by swapping variable regions using the restriction sites silently mutated into these cassettes. We also characterise the IgE produced in terms of purity, IgE high-affinity receptor (FcεRI) binding and antigen binding.

i) Transfer of SGH and SGLK expression cassettes to EBNA-I compatible vectors / transfer of EBNA-I compatibility topcDNA vectors

The first two cassettes generated, pSGH and pSGLk, were constructed in the protein expression vectors pcDNA 3.1 and pcDNA 3.0, respectively (Invitrogen). These two cassettes were used as the basis for initial mammalian protein expression trials. As rapid production of antibody panels was one of the prerequisites of the system, the SG cassettes were also transferred to expression vectors compatible with the EBNA-I transient expression system vectors pTT3 (Durocher, Perret & Kamen 2002) and pCEP4 (Invitrogen). Furthermore, an origin of replication (OriP) was transferred into the pcDNA vectors to confer compatibility with EBNA-I expressing cells, as previously described by Berntzen et al (Berntzen et al. 2005).

Transfer of OriP element intopcDNA vectors

Insertion of an OriP element into pcDNA vectors was previously found to increase protein production from EBNA-I expressing HEK-293E cells (Berntzen et al. 2005). Therefore an OriP element was amplified by PCR from pCEP4 (Invitrogen) using the primers OriP- Nrul-For and OriP-NruI-Rev (Table 2) that incorporated Nrul sites on the 5' and 3' ends. The PCR fragment was then inserted into Nrul-cut pcDNA3 and pcDNA3.1(+) Hygro (Figure 14) by restriction digest and ligation. The final sequencing verified heavy chain vectors were termed pSGH-OriP, and the light chain vector pSGLk-OriP (see Figure 14).

Transfer ofpSGHandpSGLk into pCEP4

The SGH and SGLK cassettes were also transferred to the EBNA-I system compatible plasmid pCEP4 (Invitrogen). Heavy and light chain cassettes were amplified from pSGH and pSGLic using primers HLFl and HCRl (Table 2). These primers incorporated HindIII sites on both the 5' and 3' ends of the PCR product for ligation into pCEP4 (see Figure 15).

Transfer of SGH and SGLλ-1 into pTT3 vectors

For rapid transient antibody production in the EBNA-I expression system, the SG cloning cassettes were also inserted into pTT3 (Durocher, Perret & Kamen 2002). In order to confer the ability to swap variable elements from these vectors, the pTT3 polylinker sites were adapted prior to the transfer of the SGH and SGLK cassettes. Firstly, pTT3 was cut with Nhel and BamHI restriction enzymes and blunt-ends created by incubation with DNA Pol I-Klenow fragment enzyme. The plasmid was circularised by blunt-end ligation to form pSG-E. Secondly, the SG cassettes were PCR amplified with primers EcoRIHLF and HCRl (Table 2) which incorporated EcoRI and Xbal restriction sites at the 5' and 3' end of the cassettes. Lastly, EcoRI-Xbal digested SG cassettes were inserted into EcoRI-Xbal digested pSG-E to form pSGH-E (heavy chain plasmid) and pSGLλ-1 or pSGLk-E (light chain plasmid) as shown in Figure 16.

H) Comparison of expressed levels of IgE from HEK-293E transfected cells using the cassettes SGH andSGL-k in combination withpcDNA, pcDNA OriP, pCEP4 and pTT

Protein production achieved using the different protein expression vectors described in section (i) was compared in HEK 293E cells (ATCC number CRL-1573). These cells express the EBNA-I gene constitutively. The light and heavy chain vectors were mixed such that they were not only paired with those of the same vector backbone; this was in order to assess which vector was limiting production. The pCEP4 plasmid also carries antibiotic selection, this was also applied to assess whether selection increased IgE production. Supernatants were assayed for IgE production rate by enzyme linked immunosorbant assay (ELISA), as previously described in (McCloskey et al. 2007)

Table 3: Production rates of HEK293E cells transfected with the different combinations of heavy and light chain plasmids encoding the SGH and SGLk cassettes. Hygromycin selection was also applied when using pCEP4 based vectors. The following method, based on that of (Durocher, Perret & Kamen 2002), was applied: 2 x 10⁵ HEK293E cells were transfected with 1 μg/ml DNA with PEIrDNA ratio at 2:1. Heavy chain plasmid:light chain plasmid were 1:1 (w/w) concentration. Supernatant was collected 14 days post-transfection and subjected to IgE ELISA. Samples for ELISA were performed in triplicate. Table is representative of 3 independent transfections.

Overall we have found that the EBNA-based vectors pCEP4 and pTT3 consistently provided the highest levels of IgE production. The addition of OriP to pcDNA-based plasmids (pSGs) also improved protein expression levels (Table 3). When used as a pair the pCEP4 based vectors gave the overall highest level of production, 4.3 mg/L compared to the pTT3-based pSG-Es at 1.1 mg/L. Intriguingly, the mixing of pCEP and pTT vectors gave even higher expression (~10 mg/L). This may be because using weight-to-weight vector ratios led to further optimisation by altering the light- to heavy-chain ratio in terms of plasmid copy number; this is discussed in the following sub-section. The size of the pCEP4 plasmids (>10kb) means it is practically more difficult to manage for cloning purposes (poorer ligation and transformation efficiency). Unfortunately, the pTT3 backbone replicates the Xhol site used for the swapping of V-regions, but the pTT3-based pSG-Es do permit the ligation of EcoRI-Nhel/EcoRI-Kpnl fragments for heavy and light chain leader- V-region respectively. This means that larger primers are needed when preparing inserts for ligation into the pTT vectors, as the whole leader sequence must be incorporated to avoid using Xhol for insertion. This is however preferable to using pCEP4-SGs, where any exchange of antibody elements is not possible at all without adaptation to remove repeating restriction sites in the polylinker. Therefore, the smaller pTT3-based pSG-Es were selected for further optimization for antibody production. Alterations to improve the ability to exchange V-region elements for both pCEP and pTT are discussed at the end of this section.

Optimization of Heavy:light chain ratio

To maximize production, the transfection ratios of antibody heavy and light chain plasmids were optimized. Ratios of 1:2 and 2:1 heavy:light were initially used, as previously suggested by studies on IgG (Baldi et al. 2005, Schlatter et al. 2005). However, we have found that increasing the ratio in favour of light chain (Figure 17) improved IgE production further. A 1:4 - heavy:light chain transfection ratio gave the highest level of protein production, close to 3 times that of a 1:1 ratio. \

Following the selection of the preferred vector system and the transfection conditions for IgE production, the cassettes described were used to create a panel of IgE molecules incorporating different V-regions.

Hi) Creation of a panel of IgE molecules using pTT3 as the expression vector for the SGH and SGLK / λ-1 cloning cassettes

To demonstrate the versatility of the SGH and SGL cloning cassettes we set out to produce an IgE panel from a previously assembled repertoire of VH and VL sequences. In addition, we also created two antigen-specific IgEs, using previously published V- region sequences.

In table 3, we detail 16 antibodies produced from 7 heavy chains and 3 light chains: VH3, 4, 5, 6, 7 heavy chains and a Vk4 light chain are V-region sequences of unknown specificity isolated from human B-cells; Herceptin VH3 chain and VkI light chain are the V-regions of Traztuzamab®, obtained from the PDB database (PDB ID: IN8Z) and reverse translated and synthesized by Gene Art (Germany); SPE7 VHl (accession: AY331040) and Vλ7 (accession : A Y331039) are rat V-regions synthesized by Gene Art (Germany) following sequences described in (James, Tawfik 2003). Both the Traztuzamab® and Spe7 VH and VL sequences were adapted; at the 5' end by addition of the leader sequence, and at the 3' end by inclusion of a Nhel site for the heavy chain and a Kpnl site for the light chain, as described in section A.

The VH3, 4, 5, 6, 7 were obtained by PCR amplification of V-regions isolated previously as described in (Coker, Durham & Gould 2003) utilizing forward primers comprising the VHl/Humighael leader HLVHxF (Table 2 - where x denotes the VH class) and VHRl reverse primers (Table 2). These primers introduced the EcoRI and Nhel sites at the 5' and 3' ends respectively for cloning procedures. For the gene-synthesized Herceptin, the synthesized VH and VL regions were cut out from the GeneArt holding vectors with EcoRl-Nhel for the heavy chain and EcoRI-Kpnl for the light chain and cloned into pTT3- based pSGH-E as EcoRI-Nhel fragments. The SPE7 heavy and light chain sequences were added in an identical fashion.

In the reconstruction of whole IgE using the sequences detailed above, VH regions were paired with the various Vk and Vλ chains. The various pairings of the heavy and light chains and the production of this IgE panel is summarized in Table 4. Certain VH-VL pairing combinations were found to not be produced in detectable amounts (VH3-HerVkl, VH3-ratVλ7, VH6-ratVλ7 in Table 4). Whilst some of the IgEs had unmatched VH-VL, they were able to be produced beyond the rate of their matched chained counterparts e.g. VH3-Vk4, VH7-Vk4 and Herceptin- Vk4). Based on the results obtained with this panel it was concluded that the production system is capable of expressing unmatched rat-human chimeras, as well as fully matched antibodies, in a wide range of production rates from 0.2 to 7 mg/L. See Table 4.

Table 4: Average production levels for pTT based expression of an IgE panel determined by IgE ELISA achieved in a 250-30Om culture volume.

- = Non-applicable, denoting that the particular IgE could not be produced by the SGH- L/HEK293E system. Most values are the average of n=2 with the exception of the 3 matched chains that are in bold, where n= 5. The yields represent the final amount of IgE recovered after purification by affinity chromatography using an IgE receptor column (Shi et al. 1997) and are expressed as total IgE recovered divided by the volume of the starting culture. All transfections were carried out at a PELDNA ratio 2:1, and a heavy: light chain ratio of 1:4; with total DNA lμg/ml. Cells were seeded at 2 x 10⁵cells/ml with lOOmls per triple-layered flask prior to transfection. The transfected cells were allowed to grow for up to 3 weeks before the supernatant was harvested and filtered as done in (Durocher, Perret & Kamen 2002). Each individual IgE in the panel above was cloned, mammalian expression carried out and purification completed within 4 weeks. iv) Biochemical and biophysical characterization of produced IgEs

Following expression and purification of the IgE panel described in Table 4 the protein product was characterised for purity by gel filtration chromatography and SDS-PAGE, and for antigen binding in the case of the two synthesised IgE antibodies. AU IgEs were purified using affinity chromatography based on an IgE receptor fusion protein (Shi et al. 1997) and can therefore be considered active for receptor binding. The two antigen specific IgE molecules were characterised for their FcεRI affinity constant.

SDS-PAGE gel analysis of purified proteins

Human SPE7 (chimeric rat- V, human-C IgE) , Herceptin (anti-Her2 humanized antibody, also known as Trastuzamab®) and HHM- VH4 (in-house matched V-regions from a single B cell) IgE antibodies were purified using a human FcεRIα-IgG4 fusion protein affinity column (for the full method see Shi et al. 1997). The purified antibodies were subject to SDS-PAGE analysis. Under non-reducing conditions human SPE7 appeared similar to well-characterized human IgE (AFlO) from cell line U266B1 (first described in (Nilsson et al. 1970), which was purified in an identical manner. AFlO and human Spe7 IgE migrated as a single band of apparent molecular weight ~ 20OkDa however, the commercial rat SPE7 (Sigma) is larger than both the Human SPE7 and myeloma IgE (Figure 18 centre panel). The right hand panel of Figure 18 shows HHM- VH4 IgE analyzed under reducing SDS- PAGE. Under these conditions two bands were apparent, a larger band (between 62 and 49 kDa of the marker) corresponding to the antibody heavy chain (-60 kDa) and a smaller band (between 28 kDa and 17 kDa of the marker) corresponding to the light chain of about 25 kDa (top right panel of Figure 18). Finally, the left hand panel of Figure 18 shows purified Herceptin IgE under non-reducing conditions. All the purified antibodies showed no significant contamination with other proteins, as judged by the absence of bands of molecular weight inconsistent with those expected for an IgE antibody polypeptide chain.

Surface Plasmon analysis of antizen-specific IsE In order to characterize the antigen binding capabilities of those antibodies produced with known specificity, human SPE7 and Herceptin IgE, the purified IgE was compared with its commercial counterpart: rat SPE7 and Herceptin IgGl (Traztuzamab ®). In the analysis of rat and human SPE7 binding to the antigen DNP (Dinitrophenyl), and Herceptin IgE and IgG binding to Her2, there was no observable difference in kinetic profile between the in- house produced antibodies and the commercial product. In addition to antigen recognition, binding to the IgE receptor human FcεRIα, was also examined. The binding of human SPE7 and Herceptin IgE to FcεRI were indistinguishable. Anti-her2 IgGl did not bind to human FcεRI, as expected; rat IgE bound human FcεRI with similar kinetics to the human IgE (Figure 19). When analyzed using the BIAevaluation package, which is provided with the Biacore 3000 instrument, the affinity constant derived for the IgE:FcεRI interaction yielded an affinity constant (K_A) of - 10^"10 M^"1; a figure consistent with previous affinity measurements (Cook et al. 1997). This analysis demonstrates that the system produces biologically functional antibodies in terms of antigen and receptor binding capabilities. Size-exclusion chromatography purified IgE

In addition to SDS-PAGE purified IgE was also subjected to gel filtration analysis, as previously described (Hunt et al. 2005). This was to determine whether there was any non- covalent aggregate apparent in the preparations. We have found that after affinity column purification antibody purity comparable to commercially available preparations was achieved (rat SPE7 from Sigma). As expected, myeloma IgE AFlO and the commercial rat SPE7 eluted out in a single peak without presence of other peaks (Figure 20). In comparison, the SG-HEK293E system produced IgE antibodies (VH3-Vk4, VH4-Vk4, VH5-Vk4, VH6-Vk4, VH7-Vk4, Herceptin IgE, Human SPE7 in Figure 20) were found to also elute predominately as a single peak with very small satellite peaks indicative of aggregation or impurities.

v) Modification ofpCEP andpTTto allow full compatibility with the SGH and SGL variable region swap sites In transferring the SGH and SGL cassettes to pTT the exchange of Variable regions within the vector became problematic, due to the presence of an Xhol site in the vector backbone. This was addressed by using larger primers, incorporating the whole leader sequence, to amplify the Variable regions used to create the panel described in section B, part iii. This allowed the V-regions to be inserted as EcoRI-Nhel/EcoRI-Kpnl fragments. The use of such large primers is expensive and can make PCR conditions more difficult to optimise. Therefore, we have modified the pTT3-based pSG-Es to abolish the Xhol site present. This was achieved by restriction digest of pSGe (pTT3 with modified polylinker as shown in Example IB) with Xhol followed by digestion with DNA Poll-Klenow fragment to form blunt ends. The blunt end pTT3 was then ligated to abolish the Xhol site before inserting the SG cassettes, using EcoRI-Xbal directional cloning procedures as described in Example IB. The extra step of abolishing Xhol in pTT3 thus created modified versions of pSG-Es (pSGH-E and pSGLλ-1/pSGLk-E) that allow more efficient exchange of VH and VL elements using Xhol - Nhel and Xhol - Kpnl fragments. See Figure 21 for full maps of the final pTT based vectors.

The EBNA-I vector pCEP4 was also evaluated for the production of IgE, with the achieved expression level equivalent, or higher than pTT in some cases. However, using pCEP4 alongside the SG cassettes for the swapping of V region was in this case impossible. This was the result of the presence of Kpnl, Xbal, Nhel in the plasmid polylinker. However, it is also possible to adapt the vector such that these sites are removed. We have achieved this by cutting the plasmid with Nhel and BamHI, followed by Klenow fragment incubation to blunt-end the cut pCEP4. The SG cassettes, PCR amplified with VHLF 1/HLVkLFl and HCRl/kCRl (Table 2) for SGH and SGL respectively, were blunt-end ligated into the blunt-end cut pCEP4. To complete the modification, the plasmid was cut with Kpnl and Notl, followed by blunt-ending with Klenow fragment. The final ligation to close up the plasmid abolished the interfering restriction sites in the polylinker. This created the version of pCEP4-SGs that allow the full swapping ability of the various VH-CH and VL-CL by use of Xhol - Nhel - HindIII (heavy chain) and Xhol - Kpnl - HindIII (light chain) (Figure 22). EXAMPLE 2

A vector system designed to express human IgE has been described in Example 1. Incorporated into this system is the potential to exchange, by restriction digest and ligation, the variable domain encoding portion of the heavy and light chain genes, and thus alter IgE specificity.

In this second example, we outline an optimised version of the vector system described in Example 1, in, which the capacity for V domain swapping is enhanced to include any human or murine V_H, VK or Vλ gene segment, expressed as human or chimeric humanised IgE₅ IgGl-4, or IgA2.

(A) Heavy Chain Cassette

The optimised heavy chain cassette, SGH2, with details of restriction sites required for domain swapping within the heavy chain cassette, is illustrated in Figure 23. Each component of the heavy chain cassette is flanked by restriction sites, which facilitate domain swapping and transfer of the whole cassette between vectors.

(i) V_H Leader Sequence and Nar I restriction site

Using the V_HI -02 leader sequence listed on the Vbase database (http://vbase.mrc- cpe.cam.ac.uk/) enables a Nar I restriction site to be introduced by silent mutation 12 bases 5' of the start of the V_H gene, thus removing the requirement for PCR incorporation of a leader sequence for each V_H gene used, as described in Example 1 (see Figure 23).

Nar I may also be introduced by silent mutations at the same positions of V_H leader sequences 1-03, 1-18, 1-46, 1-58 and 7-04.1. Gene synthesis may be used to incorporate the leader into the cassette, retaining the Eco RI and Not I restriction sites detailed in Example I in the final heavy chain vector (see Figure 23).

(H) C_H Gene Segment and Nhe I restriction site

The SGH-E cassette (Example 1) introduces a Nhe I restriction site into the 5' region of the Cε gene by silent mutation of the first two codons, Alanine and Serine. As these are also the first two residues of human IgG 1-4 and IgA, as well as murine IgE, the Nhe I restriction site can be retained when using any of these additional five human isotypes and murine IgE:

1 2 3

A S T/P

IgE: GCC TCC ACA

IgA2: GCA TCC CCG

IgGl: GCC TCC ACC

IgG2: GCC TCC ACC

IgG3: GCT TCC ACC

IgG4: GCT TCC ACC

A S I

Murine IgE: GCC TCT ATC

Nhe I will not introduce additional cuts into murine Cε, or human Cε, Cγl-4 or Cα2. Nar I restriction sites are absent from murine and human Cε and human Cα2, but are present in Cγl-4. However the sites can be removed by silent mutation of the Cγl-4 genes (Figure 24) and the genes may be synthesised taking this into account. An SfI I restriction site may be added 3' to the C_H stop codon (replacing Xba I from Example I) to facilitate C_H and IgH cassette exchange.

(Ui) V_H Gene Segment V_H gene segments can be swapped into and out of the heavy chain cassette utilising the Nar I and Nhe I restriction sites that have been incorporated at the V_H-C_H boundary (Figure 23). The 51 functional human germline V_H genes listed on the Vbase database and the 203 functional murine germline V_H genes and alleles listed on the EMGT database (http ://imgt.cines. fr) were screened for the presence of Nar I and Nhe I cut sites. Motifs were absent from all human germline genes. Of the murine genes, the Nhe I site was identified in only one V_H allele, IGHV1S137 (Accession number AF304558), while the Nar I motif was located in three alleles, IGHV2S1, IGHV2S3 and IGHV2S4 (Accession numbers V00767, M27021 and U53526). Subsequent analysis of the four gene sequences demonstrated that it is possible to remove the restriction sites by silent site directed mutation, should their use in this vector system be required.

Therefore a heavy chain cassette has been designed that enables the linkage of any human or murine V_H gene segment (provided Nar I or Nhe I restriction sites are not introduced by somatic hypermutation in source gene) to human Cε, Cγl-4 or Cα2.

(iv) Examples of primers for use with SGH2

The primers shown in Figure 25 may be used for modifying a V_H1 family gene segment and Cε gene segment for insertion into the SGH2 cassette. They comprise a region that anneals to the gene of interest (normal text), which may vary depending on the target, and a region encoding the required restriction sites (bold text), which is fixed for each primer type. Non-specific filler sequence is shown in italic text and restriction enzyme recognition sequences are underlined.

(B) Kappa Chain Cassette

The SGK-E cassette described in Example 1 has been optimised to enhance VK exchange tolerance by replacing the leader sequence and utilising restriction sites that are compatible with all human and murine VK germline genes (Figure 28). (i) VK Leader Sequence andNhe I restriction site

The VK leader sequences listed on the Vbase database were screened for the potential to introduce a restriction site at the 3' end that could be tolerated by the VK and CK gene segments. Silent mutation of the VK leader A26 allows incorporation of a Nhe I cut site 11 bases upstream of the start of the VK gene segment, as shown in Figure 26.

The Nhe I restriction site can also be introduced by silent mutation at the same position of VK leaders AlO and A14.

Eco RI and Not I restriction sites may be included 5' of the leader sequence (Figure 28), which may be constructed by gene synthesis.

(H) CK Gene Segment and Bsi WI restriction site

The restriction site for Bsi WI can be introduced by silent mutation into the most 5' region of the CK gene, as shown in Figure 27.

The Bsi WI restriction site does not repeat in the remainder of the single CK isotype. A Sfϊ

I restriction site may be added 3' of the CK stop codon (Figure 28).

(Ui) VK Gene Segment

VK genes can be swapped into and out of the kappa chain cassette utilising the Nhe I and Bsi WI restriction sites positioned in the leader sequence and CK gene respectively. The 40 functional human germline VK genes listed on the Vbase database and the 125 functional murine germline VK genes and alleles listed on the IMGT database were screened for the occurrence of Nhe I and Bsi WI restriction sites: the motifs were absent from all sequences analysed.

Therefore a cassette has been designed that facilitates expression of human CK in conjunction with any human or murine VK gene segment (provided required restriction sites are not introduced by somatic hypermuation). The Igκ cassette can be transferred to any compatible expression vector using the flanking restriction sites Not I or Eco RI with Sfi l.

(iv) Examples of primer sequences for use with SGK2

The primers shown in Figure 29 may be used for modifying a VKI family gene in preparation for insertion into the SGK2 cassette. They comprise a region that anneals to the gene of interest (normal text), which may vary depending on the target, and a region encoding the required restriction sites (bold text), which is fixed for each primer type. Non-specific filler sequence is shown in italic text and restriction enzyme recognition sequences are underlined.

The CK gene segment may be constructed by gene synthesis, but may also be introduced by restriction digest / ligation after using the primers shown in Figure 30.

(C) Lambda Chain Cassette

A cassette for expressing human or humanised chimeric lambda light chains is illustrated in Figure 31. The cassette has been designed with restriction sites that enable exchange of the Vλ domain.

(i) Vλ leader sequence and Age I restriction site The restriction motif for Age I can be introduced by silent mutation of Vλ leader sequence 1-e at a position 14 bases 5' of the start of the Vλ gene segment, as shown in Figure 32.

The Age I motif can be similarly introduced by silent mutation at the same position of Vλ leaders Ib, 2c, 2e, 2a2, 2d, 2b2, 3r, 3j, 3a, 3h, 3e, 3m, 4c, 4a, 4b, 5e, 5c, 5b, 6a and 9a.

Eco RI and Not I restriction sites may be included 5' of the Vλ leader sequence.

(H) Cλ gene segment andAvr II restriction site

A native Avr II restriction site exists at the junction between the Jλ portion of the Vλ domain (bold text) and the Cλ domain (normal text), as shown in Figure 33.

This can be exploited to facilitate Igλ domain swapping as both Age I and Avr II motifs are absent from the functional Cλ isotypes Cλl, 2, 3 and 7. A Sfi I restriction site may be included 3' of Cλ stop codon to enable Cλ isotype and Igλ cassette exchange.

(Ui) Vλ gene segment

Vλ genes can be swapped into and out of the lambda chain cassette utilising the Age I and Avr II restriction sites positioned in the leader sequence and Jλ-Cλ junction respectively. The 31 functional human germline Vλ genes listed on the Vbase database and the 14 functional murine germline Vλ genes and alleles listed on the IMGT database were screened for the occurrence of Age I and Avr II restriction sites. Age I motifs are absent from all murine and human sequences. Avr II is absent from the murine sequences but occurs in one of the 31 human sequences, Igλ3a (Accession number X97471). It is possible to remove this native Avr II site by silent site directed mutation in order to make this Vλ gene compatible with the SGL2 cassette. Therefore this cassette can be used to express Igλ comprising any human Cλ isotype with any human or murine Vλ gene segment (provided required restriction sites are not introduced by somatic hypermutation). The Igλ cassette may be transferred to alternative vectors using the flanking sites Not I or Eco RI with that for Sfi I.

(iv) Examples of primers for use with SGL2

The primers described in Figure 34 may be used for modification of a Vλl family gene and Cλl isotype gene for introduction into the SGL2 cassette. They comprise a region that anneals to the gene of interest (normal text), which may vary depending on the target, and a region encoding the required restriction sites (bold text), which is fixed for each primer type. Non-specific filler sequence is shown in italic text and restriction enzyme recognition sequences are underlined.

EXAMPLE 3

Following the Webcutter analysis of possible restriction sites as described in Example 5 below, it was noted that additional modifications could be made to the leader sequences of pSGH and pSGK that would further enhance the flexibility of these vectors. , The development of pSGH3 and pSGK3 is outlined below.

(A) PSGH3

Version 3 of pSGH, which utilises BssHII rather than Narl to cut the 3' end of the leader sequence, is described in Figure 35. The ability to swap each of the heavy chain domains is retained without the need to remove Narl restriction sites from human Cγl-4 and murine IGHV2S1,3 and 4 gene sequences by silent mutagenesis.

(i) V_H Leader sequence and BssHII restriction site Using the V_H 1-02 leader sequence listed on the Vbase database (http ://vbase.mrc- cpe.cam.ac.uk/) enables a BssHII restriction site to be introduced by silent mutation 11 bases 5' of the start of the V_H gene, as shown in Figure 36.

BssHII may also be introduced by silent mutation into the same position of V_H leaders 1-03, 1-08, 1-18, 1-46, 1-58, and 7-04.1.

The 5' Notl and EcoRI restriction sites may be retained and this region can be constructed by gene synthesis.

(H) C_H Gene Segment and Nhel restriction site

Introduction of Nhel to the 5' region of murine Cε, or human Cε, Cγl-4 or Cα2 is described in Example 2. However, the replacement of a Narl restriction site with a motif for BssHU in the leader sequence makes possible the use of native Cγl-4 gene sequences, which do not contain BssHII motifs, thus removing the need to mutate Cγl-4 (Figure 24).

(Hi) V_H Gene Segment

V_H gene segments can be swapped into and out of pSGH3 utilising the new BssHU and previously existing Nhel restriction sites present in the heavy chain cassette. BssHII is absent from the 51 functional human germline genes listed on the Vbase database. It is also absent from the three murine germline genes that contain a Nar I restriction site, as well as representatives from each of the 15 murine V_H gene families.

Therefore an enhanced version of pSGH has been designed that can be used to express all IgH domain combinations outlined in Example 2, without the need for restriction site removal by silent mutagenesis.

(iv) Example of primers for use with pSGH3 Primers for modifying C_H and the reverse primer for adding Nhel onto V_H are unchanged from the description in Example 2. The V_H forward primer shown in Figure 37 may be used to add the BssHII restriction site onto a V_HI family gene. As in Example 2, the region applicable to all V_H forward primers is shown in bold text, whereas the target annealing portion, which may vary depending on target, is shown in normal text. Filler sequence is italicised and the restriction enzyme recognition sequence is underlined.

(B) PSGK

Version 3 of pSGK incorporates an additional restriction site in the leader sequence at a region closer to the leader- VK boundary (Figure 38), thus providing more flexibility to the user when swapping VK gene segments.

(i) VK Leader Sequence and Nhel / SacII restriction sites

The VK leader A26 can tolerate the introduction of a Nhel restriction site by silent mutation, as described in Example 2. Webcutter analysis of the A26 leader sequence highlighted that further silent mutation enables a SacII restriction motif to be incorporated immediately 3' of the Nhel site, as shown in Figure 39.

The SacII restriction site can also be incorporated by silent mutation into the same position of VK leaders AlO and A14.

(U) CK Gene Segment and BsiWI restriction site

Construction of the CK portion of the pSGK3 vector, including the incorporation of the BsiWI restriction site is unchanged from the description in Example 2. The CK gene segment does not contain SacII restriction motifs so is compatible with the modified leader. (Hi) VK Gene Segments

Human and murine VK gene segments can be swapped into and out of pSGK3 utilising either of the 5' Nhel or SacII sites in conjunction with the 3' BsiWI site. The new SacII site is absent from all human VK germline genes listed on the Vbase database and was not identified in a selection of murine VK germline genes representative of each murine VK gene family.

Therefore a vector for Igκ expression offering increased restriction site options for domain swapping has been designed. This may be of particular use if Nhel or SacII is introduced by somatic hypermutation into a VK gene required for expression, or if the sites are present in a required vector.

(iv) Examples of primer sequences for use withpSGK3

Primers for modification of CK and the reverse primer that facilitates BsiWI addition to VK are unchanged from example below. There are three forward primer options for VK modification; either Nhel, SacII or both may be added to the target VK gene. The forward primer that adds only Nhel is described in Example 2. The primers shown in Figure 40 enable addition of SacII alone, or both Nhel and SacII to a VKI family gene. As in Example 2, the region applicable to all V_H forward primers is shown in bold text, whereas the target annealing portion, which may vary depending on target, is shown in normal text. Filler sequence is italicised and the restriction enzyme recognition sequence is underlined.

EXAMPLE 4

The SGL2 cassette described in Example 2 uses the Vλ leader 1-e, into which an Agel restriction site was introduced at the 3' end. Subsequent screening failed to identify a suitable secondary restriction site that could be incorporated into Vλ leader 1-e (Webcutter analysis, Example 5), so the other Vλ leaders stored on the Vbase database were analysed to investigate the possibility of introducing to restriction sites into the Vλ leader - Vλ boundary of the SGL cassette. An additional restriction site was also introduced into the 5' end of the Cλ gene, thus generating a vector, pSGL3 with a multiple cloning site for immunoglobulin lambda chains (Figure 43).

(i) Vλ Leader sequence and BspEI / Sail restriction sites

After analysing several Vλ leaders with Webcutter 2.0, the potential to introduce two restriction sites, by silent mutation, into the 3' region of the Vλ leader 8a was identified. The sites, BspEI and Sail, reside at adjacent positions within 15 nucleotides of the Vλ leader - Vλ boundary, as shown in Figure 44.

Vλ leader 8a is the only Vλ leader into which these restriction sites can be introduced by silent mutation.

(H) Cλgene segmentand Avrll / Narl restriction sites

hi addition to the native Avrll restriction site located at the Jλ-Cλ boundary, an additional Narl restriction site can be introduced by silent mutation 13 nucleotides into the 5' region of the Cλ gene segment, as shown in Figure 45.

Narl can be incorporated into each of the functional Cλ isotypes, Cλl, 2, 3 and 7 without altering amino acid sequence and can be exploited as an alternative to the Avrll restriction site. None of the proposed cloning restriction sites cut in additional regions of the Cλ sequences.

(Hi) Vλ gene segment Vλ gene segments can be swapped into and out of the lambda chain vector using BspEI or Sail at the leader- Vλ junction with Avrll or Narl at the Vλ-Cλ junction. The human germline Vλ genes and alleles listed on the Vbase database were screened for the presence of the restriction sites and all were shown to be devoid of at least one of the leader - Vλ boundary cut sites and at least one of the Vλ-Cλ boundary cut sites, meaning all human Vλ genes are compatible with pSGL3.

(fv) Examples of primers for use withpSGL3

The primers shown in Figure 46 may be used for modification of a Vλl family gene and Cλl isotype gene for introduction into the SGL3 cassette. They comprise a region that anneals to the gene of interest (normal text), which may vary depending on the target, and a region encoding the required restriction sites (bold text), which is fixed for each primer type. Non-specific filler sequence is shown in italic text and restriction enzyme recognition sequences are underlined.

Example 5 WEBCUTTER ANALYSIS The Webcutter 2.0 online tool (http ://rna.lundberg. gu. se/cutter2Λ was used to check for restriction sites that are either naturally occurring or can be introduced by silent mutation in the sequences flanking the V domain gene i.e. the leader sequence and the J-C boundary. Webcutter search parameters were restricted such that only enzymes that cut once in the sequence by recognition of sequence motifs of at least 6 bases were displayed in the results.

(1) Humighael Leader used in originalpSGH-E:

MetAspTrpThrTrpIleLeuPheLeuValAlaAlaAlaThrArgValHisSer atggactggacctggatcctcttcttggtggcagcagccacgcgagtccactcc basepairs atggaytggacntggathctnttyctngtngcngcngcnacncgngtncaytcn 1 to 54

ENZYME CUT POSITION RECOGNITION

SEQUENCE

AfIIII 40 a/crygt AAllww2211II 5500 gwgcw/c Alw44I 46 g/tgcac

Ama87I 41 c/ycgrg

ApaLI 46 g/tgcac

AspHI 50 gwgcw/c

Aval 41 c/ycgrg

BamHI *14* g/gatcc

BbrPI 43 cac/gtg

Bbvl2I 50 gwgcw/c

Bcol 41 c/ycgrg

Bmyl 50 gdgch/c

BsaAI 43 yac/gtr

BsaMI 53 gaatgc

BsaOI 35 cgry/cg

Bshl285I 35 cgry/cg

BsiEI 35 cgry/cg

BsiHKAI 50 gwgcw/c

Bsml 53 gaatgc

BsoBI 41 c/ycgrg

Bspl286I 50 gdgch/c

Bspl407I 45 t/gtaca

BsrGI 45 t/gtaca

BstDSI 32 c/crygg

Bstl *14* g/gatcc

BstMCI 35 cgry/cg

BstSFI 32 c/tryag

BstX2I *14* r/gatcy

BstYI *14* r/gatcy

BstZI 32 c/ggccg

Cac8I 46 gcn/ngc

CciNI 32 gc/ggccgc

Cfr42I 35 ccgc/gg

Cfr9I 41 c/ccggg

Dsal 32 c/crygg

Eagl 32 c/ggccg

EamllO4I *24* ctcttc

Earl *24* ctcttc

EcIXI 32 c/ggccg

Eco52I 32 c/ggccg

Eco72I 43 cac/gtg

Eco88I 41 c/ycgrg

Ksp632I *24* ctcttc

Kspl 35 ccgc/gg

MfII *14* r/gatcy

MIuI 40 a/cgcgt

MsII 43 caynn/nnrtg

MspAlI 34 cmg/ckg

Mval269I 53 gaatgc

Notl 32 gc/ggccgc

NspBII 34 cmg/ckg

PaeR7I 41 c/tcgag

PmaCI 43 cac/gtg

PmII 43 cac/gtg

PshAI 43 gacnn/nngtc

PspAI 41 c/ccggg

PspALI 43 ccc/ggg

Pstl 36 ctgca/g PvuII 34 cag/ctg

SacII 35 ccgc/gg

Sdul 50 gdgch/c

Sfcl 32 c/tryag

Sfr274I 41 c/tcgag

Sfr303I 35 ccgc/gg

Smal 43 ccc/ggg

SspBI 45 t/gtaca

SstII 35 ccgc/gg

Vnel 46 g/tgcac

XhoI 41 c/tcgag

XhoII *14* r/gatcy

Xmal 41 c/ccggg

XmaIII 32 c/ggccg

In addition to Xhol (underlined), it is also possible to use PaeR71 (bold) to cut at the same position of the Humighael leader sequence (recognises the same 6 base motif).

All other possible restriction sites were discarded on the basis of one or more of the following points:

• Motif occurs in one or more V_H and / or VK germline genes listed on the Vbase database

• Motif occurs in Cε and / or CK gene • Motif occurs more than 20 bases 5' of V gene start

• Motif is used for transfer of whole cassette

• Motif contains redundancies

• Enzyme produces a blunt cut >

• Enzyme cuts at region outside recognition motif • Enzyme is unavailable from standard commercial suppliers

The ApaLl motif (italic) was introduced to the Humighael leader but it is not compatible for use with all germline V genes. It is also possible to incorporate a Notl site into the Humighael leader and use an alternative enzyme such as EcoRI to transfer the cassette between vectors.

(2) VH Leader used in optimised pSG: MetAspTrpThrTrpArglleLeuPheLeuValAlaAlaAlaThrGlyAlaHisSer atggactggacctggaggatcctcttcttggtggcagcagccacaggagcccactcc base pairs atggaytggacntggcgnathctnttyctngtngcngcngcnacnggngcncaytcn 1 to 57

ENZYME CUT POSITION RECOGNITION

SEQUENCE

Acclβl 50 tgc/gca

AccBlI 46 g/gyrcc

Accl 18 gt/mkac

Acsl 17 r/aatty

Acyl 47 gr/cgyc

AgeI 43 a/ccggt

Alw21I 51 gwgcw/c

Alw44I 47 g/tgcac

AIwNI 43 cagnnn/ctg

Apal 51 gggcc/c

ApaLI 47 g/tgcac

Apol 17 r/aatty

AspHI 51 gwgcw/c

Avill 50 tgc/gca

BamHI *17* g/gatcc

Banl 46 g/gyrcc

BanII *51* grgcy/c

Bbel 50 ggcgc/c

BbiII 47 gr/cgyc

Bbvl2I 51 gwgcw/c

BgII 46 gccnnnn/nggc

Bmyl *51* gdgch/c

Bpml 49 ctggag

BsaHI 47 gr/cgyc

BsaWI 43 w/ccggw

Bsellθl 43 r/ccggy

BsePI 47 g/cgcgc

BshNI 46 g/gyrcc

BsiHKAI 51 gwgcw/c

Bsil 33 ctcgtg

Bspl20I 47 g/ggccc

Bspl286I *51* gdgch/c

Bspl43II 50 rgcgc/y

BsrFI 43 r/ccggy

BssAI 43 r/ccggy

BssHII 47 g/cgcgc

BssSI 33 ctcgtg

BstllO7I 19 gta/tac

BstH2I 50 rgcgc/y

Bstl *17* g/gatcc

BstX2I *17* r/gatcy

BstYI *17* r/gatcy

BstZI 35 c/ggccg

Cac8I 49 gcn/ngc

CciNI 35 gc/ggccgc

CfrlOI 43 r/ccggy

Cfr42I 38 ccgc/gg

DraII 47 rg/gnccy

Eagl 35 c/ggccg

EamllO4I *27* ctcttc Earl *27* ctcttc

Ecll36II 49 gag/etc

EcIXI 35 c/ggccg

Eco24I *51* grgcy/c

Eco52I 35 c/ggccg

Eco64I 46 g/gyrcc

EcoICRI 49 gag/etc

Eco0109I 47 rg/gnccy

EcoRI 17 g/aattc

Ehel 48 ggc/gcc

FriOI *51* grgcy/c

Fspl 50 tgc/gca

Gsul 49 ctggag

HaeII 50 rgcgc/y

Hinll 47 gr/cgyc

Hsp92I 47 gr/cgyc

Kasl 46 g/gcgcc

Ksp632I *27* ctcttc

Kspl 38 ccgc/gg

MfII *17* r/gatcy

Mspl7I 47 gr/cgyc

MspAlI 37 cmg/ckg

Mwol *46* gcnnnnn/nngc

Narl 47 gg/cgcc

Notl 35 gc/ggccgc

NspBII 37 cmg/ckg

PinAI 43 a/ccggt

Pspl24BI 51 gagct/c

PspOMI 47 g/ggccc

Pstl 39 ctgca/g

PvuII 37 cag/ctg

Sad 51 gagct/c

SacII 38 ccgc/gg

Sdul *51* gdgch/c

Sfil 46 ggccnnnn/nggcc

Sfr303I 38 ccgc/gg

SgrAI 43 cr/ccggyg

Sstl 51 gagct/c

SstII 38 ccgc/gg

Vnel 47 g/tgcac

Xho11 *17* r/gatcy

XmaIII 35 c/ggccg

In addition to Narl (underlined), it is possible to use Kasl (bold) to cut in the same place (recognises same 6 base motif) or to change the Narl / Kasl restriction site to that for BssHII (italic). BssHII is preferable to Narl as Narl sites need to be silently removed from Cγl-4. It is also possible to incorporate Notl in addition to BssHII, thus providing two exchange site options. This optimal leader sequence is detailed in SEQ ID 6. AU other possible restriction sites were discarded on the basis of one or more of the following points:

• Motif occurs in one or more V_H germline genes • Motif occurs in one or more C_H gene and cannot be removed by silent mutation.

• Motif occurs more than 20 bases 5 ' of V_H gene start

• Motif is utilised for transfer of whole cassette

• Motif contains redundancies

• Enzyme produces a blunt cut • Enzyme cuts at region outside recognition motif

• Enzyme is unavailable from standard commercial suppliers

(3) JH-CH Boundary:

The last 16 bases of the J_H gene and first 5 bases of the C_H gene are universal to all human J_H gene families and C_H isotypes, respectively. These 21 bases were analysed by Webcutter using the parameters described above:

ValThrValSerSerAlaSer gtcaccgtctcctcagcctcc base pairs gtnacngtntcntcngcntcn 1 to 21

ENZYME CUT POSITION RECOGNITION SEQUENCE

Acyl 16 gr/cgyc

Afel 15 agc/gct

Alw21I 13 gwgcw/c

Ama87I 9 c/ycgrg

Aor51HI 15 agc/gct

AspHI 13 gwgcw/c

Aspl 6 gacn/nngtc

Atsl 6 gacn/nngtc

Aval 9 c/ycgrg

BanII 13 grgcy/c

BbiII 16 gr/cgyc

Bbsl 14 gaagac

Bbvl2I 13 gwgcw/c

Bbvl6II 14 gaagac

Bcol 9 c/ycgrg

BIpI 12 gc/tnagc

Bmyl 13 gdgch/c Bpil 14 gaagac

Bpml 14 ctggag

BpullO2I 12 gc/tnagc

BpuAI 14 gaagac

BsaHI 16 gr/cgyc

Bsal 11 ggtctc

BsaJI 11 c/cnngg

BseDI 11 c/cnngg

BseRI *14* gaggag

BsiHKAI 13 gwgcw/c

BsitiBI *11* cgtctc

BsoBI 9 c/ycgrg

Bspl286I 13 gdgch/c

Bspl43II 17 rgcgc/y

Bspl720I 12 gc/tnagc

BstH2I 17 rgcgc/y

Cacθl 18 gcn/ngc

CeIII 12 gc/tnagc

Cfrl 14 y/ggccr

Drdl 9 gacnnnn/nngtc

Eael 14 y/ggccr

EamllO4I 14 ctcttc

Earl 14 ctcttc

Ecll36II 11 gag/etc

Eco24I 13 grgcy/c

Eco31I 11 ggtctc

Eco47III 15 agc/gct

Eco57I 16 ctgaag

Eco88I 9 c/ycgrg

EcoICRI 11 gag/etc

Esp3I *11* cgtctc

FriOI 13 grgcy/c

Gsul 14 ctggag

HaeII 17 rgcgc/y

Hinll 16 gr/cgyc

Hsp92I 16 gr/cgyc

Ksp632I 14 ctcttc

Mspl7I 16 gr/cgyc

Mwol 17 gcnnnnn/nngc

Nhel 16 g/ctagc

PaeR7I 9 c/tcgag

Pspl24BI 13 gagct/c

PstNHI 16 g/ctagc

Sad 13 gagct/c

Sdul 13 gdgch/c

Sfr274I c/tcgag

Sstl 13 gagct/c

Tthllll 6 gacn/nngtc

XhoI 9 c/tcgag

Nhel (underlined) is the optimal enzyme available for cutting in the J_H-C_H boundary. Xhol may also be considered when not being exploited in the Humighael leader, but its motif needs to be removed from Cα2. The J_H-C_H boundary incorporating both Xhol and Nhel as exchange site options is detailed in SEQ ID 13.

All other restriction sites were discarded for one or more of the following reasons:

Motif occurs in one or more V_H germline genes

Motif occurs in one or more C_H gene and cannot be removed by silent mutation

Motif contains redundancies

Enzyme produces a blunt cut

Enzyme cuts at region outside recognition motif

Enzyme is unavailable from standard commercial suppliers

(4) VKLeader:

MetLeuProSerGlnLeuIleGlyPheLeuLeuLeuTrpValProAlaSerArgGly atgttgccatcacaactcattgggtttctgctgctctgggttccagcctccaggggt base pairs atgctnccntcncarctnathggnttyctnctnctntgggtnccngcntcncgnggn 1 to 57

ENZYME CUT POSITION RECOGNITION

SEQUENCE

Acc65I 39 g/gtacc

AccBlI 39 g/gyrcc

Acyl 46 gr/cgyc

Ama87I 50 c/ycgrg

Asp718I 39 g/gtacc

Aval 50 c/ycgrg

Banl 39 g/gyrcc

BbiII 46 gr/cgyc

BbrPI 52 cac/gtg

Bcol 50 c/ycgrg

Bmyl 44 gdgch/c

BsaAI 52 yac/gtr

BsaHI 46 gr/cgyc

BsaJI *50* c/cnngg

Bsell8I 42 r/ccggy

BseDI *50* c/cnngg

BseRI 33 gaggag

BshNI 39 g/gyrcc

Bsil 55 ctcgtg

BsoBI 50 c/ycgrg

Bspl286I 44 gdgch/c

Bsp68I 51 tcg/cga

BspMI 47 acctgc

BsrFI 42 r/ccggy BssAI 42 r/ccggy

BssSI 55 ctcgtg

BstDSI 51 c/crygg

BstXI 19 ccannnnn/ntgg

Cac8I 44 gcn/ngc

CfrlOI 42 r/ccggy

Cfr42I 54 ccgc/gg

Cfr9I 50 c/ccggg

DraII 39 rg/gnccy

Dsal 51 c/crygg

Eco64I 39 g/gyrcc

Eco72I 52 cac/gtg

Eco88I 50 c/ycgrg

EcoO109I 39 rg/gnccy

Hinll 46 gr/cgyc

Hsp92I 46 gr/cgyc

Kpnl 43 ggtac/c

Kspl 54 ccgc/gg

MroNI 42 g/ccggc

Mspl7I 46 gr/cgyc

Nael 44 gcc/ggc

NgoAIV 42 g/ccggc

NgoMI 42 g/ccggc

Nhel 46 g/ctagc

Nrul 51 tcg/cga

PaeR7I 50 c/tcgag

PmaCI 52 cac/gtg

PmII 52 cac/gtg

PpuMI 39 rg/gwccy

Psp5II 39 rg/gwccy

PspAI 50 c/ccggg

PspALI 52 ccc/ggg

PvuII 15 cag/ctg

SacII 54 ccgc/gg

Sdul 44 gdgch/c

Sfr274I 50 c/tcgag

Sfr303I 54 ccgc/gg

Smal 52 ccc/ggg

SstII 54 ccgc/gg

XhoI 50 c/tcgag

Xmal 50 c/ccggg

In addition to Nhel (underlined) a restriction site for SacII (bold) can also be introduced into the A26 leader sequence by silent mutation. It is possible for the Nhel and SacII sites to exist simultaneously without affecting amino acid sequence (SEQ DD 16).

Other restriction sites were excluded on the basis of one or more of the following reasons:

Motif occurs in one or more VK germline genes • Motif occurs in the CK gene

• Motif occurs more than 20 bases 5 ' of VK gene start

• Motif contains redundancies

• Enzyme is unavailable from standard commercial suppliers

(5) JK-CK boundary:

The last 7 bases of the JK, which are universal to all human JK gene families were analysed in conjunction with the first 20 bases of the CK gene using the Webcutter parameters described above:

IleLysArgThrValAlaAlaProSer atcaaacgaactgtggctgcaccatct base pairs athaarcgnacngtngcngcnccntcn 1 to 27

ENZYME CUT POSITION RECOGNITION SEQUENCE

AccBlI 18 g/gyrcc

AccBSI 22 gagcgg

Acyl 19 gr/cgyc

AIwNI 16 cagnnn/ctg

Ama87I 21 c/ycgrg

Aval 21 c/ycgrg

Banl 18 g/gyrcc

Bbel 22 ggcgc/c

BbiII 19 gr/cgyc

Bcol 21 c/ycgrg

BsaHI 19 gr/cgyc

BsaJI 11 c/cnngg

BsaOI 14 cgry/cg

BseDI 11 c/cnngg

Bsgl *22* gtgcag

Bshl285I 14 cgry/cg

BshNI 18 g/gyrcc

BsiEI 14 cgry/cg

BsiWI 7 c/gtacg

BsoBI 21 c/ycgrg

Bspl43II 22 rgcgc/y

BsrBI 22 . gagcgg

BstD102I 22 gagcgg

BstDSI 11 c/crygg

BstH2I 22 rgcgc/y

BstMCI 14 cgry/cg BStSFI 11 c/tryag

Cfrl 14 y/ggccr

Cpol 8 cg/gwccg

Cspl 8 cg/gwccg

Dsal 11 c/crygg

Eael 14 y/ggccr

Eco64I 18 g/gyrec

Eco88I 21 c/ycgrg

Ehel 20 ggc/gcc

HHaaeeIIII 2222 rgcgc/y

Hinll 19 gr/cgyc

Hsp92I 19 gr/cgyc

Kasl 18 g/gcgcc

Mspl7I 19 gr/cgyc

NNaarrll 1199 gg/cgcc

NIaIV 20 ggn/ncc

Pfl23II 7 c/gtacg

PspLI 7 c/gtacg

PspN4I 20 ggn/ncc

RRssrrIIII 88 cg/gwccg

Sfcl 11 c/tryag

SpII 7 c/gtacg

Sunl 7 c/gtacg BsiWI (underlined) is the only restriction enzyme suitable for cutting at the JK-CK boundary. All other possible restriction sites were discarded on the basis of one or more of the following reasons:

• Motif occurs in one or more VK germline genes • Motif occurs in the CK gene

• Motif contains redundancies

• Enzyme produces a blunt cut

• Enzyme cuts at region outside recognition motif

• Enzyme is unavailable from standard commercial suppliers

The Kpnl site introduced into the original pSGK-E vector is 5' of the region analysed here. Both Kpnl and Narl are less preferred as they cut in some VK germline genes. However, they may both be included in the JK-CK boundary in addition to BsiWI as alternative exchange sites, as detailed in SEQ ED 20.

(6) Vλ leader: MetAlaTrpSerProLeuLeuLeuThrValLeuThrHisCysThrGlySerTrpAla atggcctggtctcctctcctcctcactgtcctcactcactgcacagggtcctgggcc base pairs atggcntggtcnccnctnctnctnacngtnctnacncaytgyacnggntcntgggcn 1 to 57

ENZYME CUT POSITION RECOGNITION SEQUENCE

Accll3I 29 agt/act

AccBSI 18 gagcgg

AgeI 43 a/ccggt

Alw21I 32 gwgcw/c

AspHI 32 gwgcw/c

BamHI 46 g/gatcc

BanII 49 grgcy/c

Bbvl2I 32 gwgcw/c

Bsal *13* ggtctc

BsaJI *50* c/cnngg

BsaWI 43 w/ccggw

Bsellβl 43 r/ccggy

BseDI *50* c/cnngg

Bsgl *₄₄* gtgcag

BsiHKAI 32 gwgcw/c

Bsil 53 ctcgtg

Bspl407I 40 t/gtaca

BsrBI 18 gagcgg

BsrDI 42 gcaatg

BsrFI 43 r/ccggy

BsrGI 40 t/gtaca

BssAI 43 r/ccggy

BssSI 53 ctcgtg

BstD102I 18 gagcgg

BstEII 8 g/gtnacc

Bstl 46 g/gatcc

BstPI 8 g/gtnacc

BstX2I 46 r/gatcy

BstXI 43 ccannnnn/ntgg

BstYI 46 r/gatcy

CfrlOI 43 r/ccggy

DraII *47* rg/gnccy

EamllO4I 19 ctcttc

Earl 19 ctcttc

Eco24I 49 grgcy/c

Eco255I 29 agt/act

Eco31I *13* ggtctc

Eco91I 8 g/gtnacc

EcoO109I *47* rg/gnccy

Eco065I 8 g/gtnacc

FriOI 49 grgcy/c

Ksp632I 19 ctcttc

MfII 46 r/gatcy

MspAlI 15 cmg/ckg

Mwol 18 gcnnnnn/nngc

NspBII 15 cmg/ckg

PinAI 43 a/ccggt

PpuMI *47* rg/gwccy

Psp5II *47* rg/gwccy PspEI 8 g/gtnacc

Seal 29 agt/act

SspBI 40 t/gtaca

XhoII 46 r/gatcy

Agel is the only restriction enzyme suitable for cutting at the 3' end of the Vλl-e leader. AU other restriction sites were discarded due to one or more of the following reasons:

• Motif occurs in one or more Vλ germline genes

• Motif occurs in one or more Cλ isotype gene

• Motif occurs more than 20 bases 5 ' of Vλ gene start

• Motif contains redundancies

• Enzyme is unavailable from standard commercial suppliers (?) Jλ-Cλ Boundary:

The boundary formed by the final 10 bases of the Jλ gene segment and the first 14 bases of the Cλ gene, which are universal to all Jλ gene families and Cλ isotypes, respectively, were analysed by Webcutter using the parameters outline above:

ThrValLeuGlyGlnProLysAla accgtcctaggtcagcccaaggcc base pairs acngtnttrggncarccnaargcn 1 to 24

ENZYME CUT POSITION RECOGNITION SEQUENCE

Accll3I 5 agt/act

Alw2LI 8 gwgcw/c

Ama87I 7 c/ycgrg

Aocl 17 cc/tnagg

AspHI 8 gwgcw/c

Aval 7 c/ycgrg

Avrll *6* c/ctagg

Ball 11 tgg/cca

Bbvl2I 8 gwgcw/c

Bcol 7 c/ycgrg

BInI *6* c/ctagg

Bmyl 8 gdgch/c

BsaJI 17 c/cnngg

Bsc4I 7 ccnnnn/nnngg

Bse21I 17 cc/tnagg BseDI 17 c/cnngg

BsiHKAI 8 gwgcw/c

BsiYI 8 8 ccnnnnn/nngg

BsII 8 8 ccnnnnn/nngg

BsoBI 7 c/ycgrg

Bspl286I 8 gdgch/c

BssTII 17 c/cwwgg

Bsu36I 17 cc/tnagg

Cacδl 13 gcn/ngc

Cfrl 9 y/ggccr

Cvnl 17 cc/tnagg

Eael 9 y/ggccr

Ecol30I 17 c/cwwgg

Eco255I 5 agt/act

Ecoδll 17 cc/tnagg

Eco88I 7 c/ycgrg

EcoT14I 17 c/cwwgg

Erhl 17 c/cwwgg

Hindi 13 gty/rac

HindII 13 gty/rac

MIuNI 11 tgg/cca

Mscl 11 tgg/cca

Mwol 12 gcnnnnn/nngc

Seal 5 agt/act

Sdul 8 gdgch/c

Styl ' 17 c/cwwgg

Avrll (underlined) is the only restriction enzyme suitable for cutting at the Jλ-Cλ boundary. All other restriction sites were discarded due to one or more of the following reasons:

• Motif occurs in one or more Vλ germline genes

• Motif occurs in one or more Cλ isotype gene • Motif contains redundancies

• Enzyme produces a blunt cut

• Enzyme is unavailable from standard commercial suppliers

(8) Vλ leader 8a:

MetAlaTrpMetMetLeuLeuLeuGlyLeuLeuAlaTyrGlySerGlyValAspSer atggcctggatgatgcttctcctcggactccttgcttatggatcaggagtggattct base pairs atggcntggatgatgctnctnctnggnctnctngcntayggntcnggngtngaytcn 1 to 57 EHZ¹ZMB COT POSITION RECOGNITION SEQUENCE

Aatl 26 agg/cct

AccIII 43 t/ccgga

Acyl 47 gr/cgyc

Avrll 21 c/ctagg

BamHI 40 g/gatcc

BanII 29 grgcy/c

BbiII 47 gr/cgyc

BbsI 31 gaagac

Bbvl6II 31 gaagac

Bcgl 45 cgannnnnntgc

BInI 21 c/ctagg

Bmyl 29 gdgch/c

Bpil 31 gaagac bpml 49 ctggag

BpuAI 31 gaagac

BsaHI 47 gr/cgyc

Bsal 30 ggtctc

BsaWI 43 w/ccggw

BseAI 43 t/ccgga

BsiMI 43 t/ccgga

BsiWI 35 c/gtacg

Bspl286I 29 gdgch/c

Bspl3I 43 t/ccgga

BspEI 43 t/ccgga

BssTII 21 c/cwwgg

Bstl 40 g/gatcc

BstX2I 40 r/gatcy

BstYI 40 r/gatcy

Cacβl 28 gcn/ngc

Ecol30I 21 c/cwwgg

Ecol47I 26 agg/cct

Eco24I 29 grgcy/c

Eco31I 30 ggtctc

EcoT14I 21 c/cwwgg

Erhl 21 c/cwwgg

FauNDI 36 ca/tatg

FriOI 29 grgcy/c

Gsul 49 ctggag

Hinll 47 gr/cgyc

Hindi 51 gty/rac

HindII 51 gty/rac

Hsp92I 47 gr/cgyc

Kpn2I 43 t/ccgga

MfII 40 r/gatcy

Mrol 43 t/ccgga

Mspl7I 47 gr/cgyc

Mwol 24 gcnnnnn/nngc

Ndel 36 ca/tatg

Nhel 30 g/ctagc

Pfl23II 35 c/gtacg

Pme55I 26 agg/cct

PspLI 35 c/gtacg

PstNHI 30 g/ctagc

Sail 49 g/tcgac

SdUl 29 gdgch/c

SpII 35 c/gtacg

SseBI 26 agg/cct

Stul 26 agg/cct

Styl 21 c/cwwgg

Sunl 35 c/gtacg

XhoII 40 r/gatcy The restriction sites BspEI and Sail (underlined) can be introduced by silent mutation at adjacent positions within 15 nucleotides of the leader - Vλ boundary. Each cuts a single Vλ allele listed on the Vbase database, but they do not occur simultaneously in one Vλ gene. BsiWI may also be incorporated into the Vλ leader 8a, but at a more distant position from the leader - Vλ boundary and the BsiWI site occurs in more than one Vλ allele. Other restriction sites were disregarded on the basis of one or more of the following:

• Motif occurs greater than two Vλ genes

• Motif occurs in one or more Cλ isotype gene

• Motif is used for cutting another region of the cassette (Avrll)

• Motif is greater than 25 bases 5' of the leader - Vλ boundary • Motif contains redundancies

• Enzyme produces a blunt cut

• Enzyme cuts at position separate to recognition sequence

• Enzyme is unavailable from standard commercial suppliers

(9) 5' Region of Cλ:

GlnProLysAlaAlaProSerValThrLeuPheProProSerSerGlu cagcccaaggctgccccctcggtcactctgttcccgccctcctctgag base pairs carccnaargcngcnccntcngtnacnctnttyccnccntcntcngar 1 to 48

ENZSME CDT POSITION RECOGNITION SEQUENCE

AccBlI 12 g/gyrcc

AccBSI 16 gagcgg

Acyl 13 gr/cgyc

Aocl 5 cc/tnagg

Banl 12 g/gyrcc

Bbel 16 ggcgc/c

BbiII 13 gr/cgyc

BbsI 44 gaagac

Bbvl6II 44 gaagac

Bcgl 21 cgannnnnntgc

Bpil 44 gaagac

BpuAI 44 gaagac

BsaHI 13 gr/cgyc

BSC4I 41 ccnnnn/nnngg

Bse21I 5 cc/tnagg

BseRI *44* gaggag

Bsgl 16 gtgcag

BshNI 12 g/gyrcc

BsiYI 42 ccnnnnn/nngg

BsII 42 ccnnnnn/nngg

Bspl43II 16 rgcgc/y

BsrBI 16 gagcgg

BssTII *5* c/cwwgg

BstD102I 16 gagcgg

BstEII 21 g/gtnacc BstH2I 16 rgcgc/y

BstPI 21 g/gtnacc

Bsu36I 5 cc/tnagg

Cvnl 5 cc/tnagg

Ecol30I *5* c/cwwgg

Eco64I 12 g/gyrcc

Ecoβll 5 cc/tnagg

Eco91I 21 g/gtnacc

Eco065I 21 g/gtnacc

ECOT14I *5* c/cwwgg

Ehel 14 ggc/gcc

Erhl *5* c/cwwgg

HaeII 16 rgcgc/y

Hinll 13 gr/cgyc

Hsp92I 13 gr/cgyc

Kasl 12 g/gcgcc

Mspl7I 13 gr/cgyc

Mwol 33 gcnnnnn/nngc

Narl 13 gg/cgcc

NIaIV 14 ggn/ncc

PspEI 21 g/gtnacc

PspN4I 14 ggn/ncc

Sapl 33 gctcttc

Styl *5* c/cwwgg

Narl (or Kasl or Bbel, which recognize the same sequence) can be introduced by silent mutation into the 5' region of the Cλ gene sequence thus providing an alternative to Avrll for cutting at the Vλ-Cλ boundary in the SGL cassette. Other restriction sites were disregarded for one or more of the following reasons:

• Motif occurs in Vλ germline gene that also contains Avrll

• Motif occurs in one or more Cλ isotype gene

• Motif contains redundancies

• Enzyme produces a blunt cut

• Enzyme cuts at position separate to recognition sequence

• Enzyme is unavailable from standard commercial suppliers

The complete set of enzymes available for cutting each of the SG cassettes are summarised in Table 5 and Figures 41 and 42.

Table 5. Enzymes selected for cutting at boundaries of immunoglobulin heavy and light chain domains. Following analysis with Webcutter 2.0 (http://rna.lundberg.gu.se/cutter2), optimal restriction motifs that were naturally occurring or could be introduced by silent mutation were selected for digestion at the domain boundaries of the SG cassettes. The use of these restriction sites is described in full in Examples 1-3. The Webcutter analysis also highlighted a number of additional enzymes, listed here in brackets, which are less preferred for the full range of V or C regions, but which can co-exist with other sites to generate multiple cloning options.

Primer Name Sequence (5'-3')

VHXF / VHXL GAA TTC GCG GCC GCG TTC CTC ACC ATG GAC TGG ACC TGG

EcoRI Notl

ATC CTC TTC TTG GTG GCA GCA GCC ACT CGA GTG CAC TCC

Xhol ApaLI

XXX XXX ....

VHRl CAG TGC TAG CTC AGG AGA CGG TGA C

Nhel

HCFl GAA TTC GCT AGC ACA CAG AGC CCA TCC GTC TT

E∞RI Nhel

Table 6. Primers used to introduce restriction sites at domain boundaries of SG Cassettes. Primer sequences that were used to construct the SG cassettes incorporating the optimal restriction sites illustrated in Figures 41 and 42 are listed. Restriction sites are underlined; where there are two adjacent restriction sites one of the motifs is italicised. XXX denotes the requirement for V or C region specific primer sequences.

NUCLEICACID SEQUENCES (5' to 3")

Sequences are given in 5 '-3' convention. Restriction sites are identified by italic type. Where two adjacent restriction sites are present, the more 3' site is underlined.

SEQ ID 1 (Humighael Leader, used in Example 1):

ATGGACTGGACCTGGATCCTCTTCTΓGGTGGCAGCAGCCACGCGAGTCCACTCC

SEQ ID 2 (Humighael Leader incorporating Xhol and ApaLI restriction sites):

ATGGACTGGACCTGGATCCTCTTCTrGGTGGCAGCAGCCAC7TCG^GTGCACTCC

SEQ ID 3 (V_H 1-02 Leader, used in Examples 2 and 3):

ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCC

SEQ ID 4 (V_H 1-02 Leader incorporating Narl restriction site): ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGCGCCCACTCC

SEQ ID 5 (V_H 1-02 Leader incorporating BssHII restriction site): ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGCσCGCACTCC

SEQ ID 6 (Optimised V_H Leader incorporating Not! and BssHII restriction sites): ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCGGCCGCCACAGGCGCGCACTCC

SEQ ID 7 (Va-Cε Boundary; 3' 10 bases of J_H and 5' 8 bases of Cε): GTCTCCTCAGCCTCCACA

SEQ ID 8 (V_H-CO2 Boundary; 3' 10 bases of J_H and 5' 8 bases of Cα2): GTCTCCTCAGCATCCCCG SEQ ID 9 (Vπ-Cγl/2 Boundary; 3' 10 bases of J_H and 5' 8 bases of Cγl/2): GTCTCCTCAGCCTCCACC

SEQ ID 10 (VirCγ3/4 Boundary; 3' 10 bases of J_H and 5' 8 bases of Cγ3/4): GTCTCCTCAGCTTCCACC

SEQ ID 11 (Murine Vπ-Cε Boundary; 3' 10 bases of J_H and 5' 8 bases of Cε): GTCTCCTCAGCCTCTATC

SEQ ID 12 (VH-CH Boundary; 3' 10 bases of J_H and 5' 8 bases of C_H, incorporating Nhel restriction site):

GTCTCCTCAGCΓΛGCXXX

SEQ ID 13 (Optimised VH-C_H Boundary, incorporating Xhol and Nhel restriction sites): GTCTCGAGCGCTAGCXCX

SEQ ID 14 (VK A26 Leader, used in Examples 2 and 3): ATGTTGCCATCACAACTCATTGGGTTTCTGCTGCTCTGGGTTCCAGCCTCCAGGGGT

SEQ ID 15 (VK A26 Leader incorporating Nhel restriction site):

ATGTTGCCATCACAACTCATTGGGTTTCTGCTGCTCTGGGTTCCAGCT^σCAGGGGT

SEQ ID 16 (VK A26 Leader incorporating Nhel and SacII restriction sites): ATGTTGCCATCACAACTCATTGGGTTTCTGCTGCTCTGGGTTCCAGCT^GCCGCGGT

SEQ ID 17 (JK-CK Boundary; 3' 22 bases of Jκl and 5' 17 bases of CK): GGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCA

SEQ ID 18 (JK-CK Boundary incorporating Kpnl, as used in Example 1): GGΓΛCCAAGGTGGAAATCAAACGAACTGTGGCTGCACCA

SEQ ID 19 (JK-CK Boundary incorporating BsiWI, as used in Examples 2 and 3):

GGGACCAAGGTGGAAATCAAACGΓΛCGGTGGCTGCACCA

SEQ ID 20 (Optimised JK-CK Boundary incorporating Kpn I, BsiWI and Narl restriction sites):

GGΓΛCCAAGGTGGAAATCAAACGΓΛCGGTGGCGGCGCCA SEQ ID 21 (Vλ 1-e Leader, used in Example 2): ATGGCCTGGTCTCCTCTCCTCCTCACTGTCCTCACTCACTGCACAGGGTCCTGGGCC

SEQ ID 22 (Vλ 1-e Leader incorporating Agel restriction site):

ATGGCCTGGTCTCCTCTCCTCCTCACTGTCCTCACTCACTGC^CCGGTTCCTGGGCC

SEQ ID 23 (Vλ 8a Leader, used in Example 4):

ATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTGGATTCT

SEQ ID 24 (Vλ 8a Leader incorporating BspEI and Sail restriction sites): ATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATUCGG^GTCGACTCT

SEQ ID 25 (Rat Vλ-Human Cλ Boundary; 3' 21 bases of Jλ2 and 5' 6 bases of Cλ, as used in Example 1): GGGACCAAACTGACTGTCCTCAGCCC

SEQ ID 26 (Rat Vλ-Human Cλ Boundary incorporating Kpnl restriction site):

GGΓΛCCAAACTGACTGTCCTCAGCCC

SEQ ID 27 (Human Vλ - Cλ Boundary; 3' 10 bases of Jλ and 5' 29 bases of Cλ, as used in Examples 2 and 4): ACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACT

SEQ ED 28 (Human Vλ - Cλ Boundary, incorporating Avrll and Narl restriction sites): ACCGTCCTMGGTCAGCCCAAGGCGGCGCCCTCGGTCACT

SEQ EO 29 (Sequence added 5' of leader sequences including Notl and EcoRI cloning restriction sites): GA4TTCGCGGCCGCGTTCCTCACC

The following sequences are primers utilized in Example 1. Restriction sites are again identified by italic and underlined text:

SEQ EO 30 (VHXF/VHXL; humighael leader encoding primer incorporating Xhol and ApaLI swap sites plus EcoRI and Notl cloning sites. X denotes V gene specific sequence): G^TTOJCGGCCGCGTTCCTCACCATGGACTGGACCTGGATCCTCTTCTTGGTGGCAGCAGCCAC 7UGΛCTOCACTCCXXXXXX...

SEQ ID 31 (VHRl; reverse primer for V_H gene amplification, incorporating Nhel restriction site): CAGTGCΓΛGCTCAGGAGACGGTGAC

SEQ ID 32 (HCFl ; forward primer for Cε amplification incorporating EcoRI cloning site and Nhel swap site):

GA47TCGCTAGCACACAGAGCCCATCCGTCTT

SEQ ED 33 (HCRl ; reverse primer for Cε incorporating HindIH and Xbal cloning sites): A4GC77TCTAGATCATTTACCGGATTTACAG

SEQ ED 34 (EcoRIHLF; humighael leader annealing primer incorporating EcoRI and Notl): CACACGAJTTUGCGGCCGCGTTCCTCACCATGGACTGGACCTGGATCC

SEQ ED 35 (kVFl; forward primer for VK amplification): CACACACTCGAGTGCACTCCGACATCCAGTTGACCCAG

SEQ ED 36 (kVJRl; reverse primer for VK amplification, incorporating Kpnl):

TTTGATCTCCASYTTGGΓΛCC

SEQ ED 37 (kCFl; forward primer for CK amplification, incorporating Xhol cloning site and Kpnl swap site): CΓCGΛGGGTACCAAGCTGGAGATCAAACGAACTGTGGCTGCACC

SEQ ED 38 (kCRl; reverse primer for CK amplification, incorporating Hindiπ and Xbal cloning sites): ^GC77UΓAGACTAACACTCTCCCCTGTTGA

SEQ ED 39 (NruI5'CMV; for priming in CMV promoter region of expression vector): TTAGGGTTAGGCGTTTTGCGCTGCT

SEQ ED 40 (OriP-NruI For; forward primer for OriP amplification, incorporating Nrul restriction site): CACACrcσCGΛA GGAAAAGGACAAGCAGCGAA

SEQ ED 41 (OriP-NruI Rev; reverse primer for OriP amplification, incorporating Nrul restriction site): CACACΓCGCGΛATGCTGGGGGACATGTACCTC

The following sequences are primers utilized in Example 2. Restriction sites are identified by italic or underlined text:

SEQ ID 42 (NarIVHX; forward primer for V_H gene amplification, incorporating Narl restriction site. X denotes VH gene specific sequence): CACACGGCGCCCACTCCXXXXXX

SEQ ID 43 (JHNhel; reverse primer for V_H gene amplification, incorporating Nhel restriction site):

CACACGCΓΛGCTGAGGAGACGGTGACCAGGGT

SEQ ED 44 (NheICXF; forward primer for C_H gene amplification, incorporating Nhel restriction site. X denotes C_H isotype specific sequence): CACACGC7MGCXXXXXX....

SEQ ED 45 (CXRSfil; reverse primer for C_H gene amplification, incorporating Sfil cloning site. X denotes C_H isotype specific sequence):

CACACσGCCGΛGrCGGCCXXXXXX

SEQ ED 46 (NhelVkX; forward primer for VK gene amplification, incorporating Nhel restriction site. X denotes VK gene specific sequence):

CACACGCΓΛGCAGGGGTXXXXXX...

SEQ ED 47 (JkBsiWI; reverse primer for VK amplification, incorporating BsiWI restriction site): CACACCGZ4CGTTTGATCTCCAGCTTGGTCCC

SEQ ED 48 (BsiWICkF; forward primer for CK amplification, incorporating BsiWI restriction site):

CACACCGΓΛCGGTGGCTGCACCATCTGTC

SEQ ED 49 (CkRSfil; reverse primer for CK amplification, incorporating Sfil cloning site):

CACACGGCCGΛGΓCGGCCCTAACACTCTCCCCTGTTGAAGCT

SEQ ED SO (AgelVLX; forward primer for Vλ amplification, incorporating Agel restriction site. X denotes Vλ gene specific sequence):

CACAC^CCGGTTCCTGGGCCXXXXXX... SEQ ID Sl (JLAvrll; reverse primer for Vλ amplification, highlighting Avrll restriction site):

CACACACCΓΛGGACGGTGACCTTGGTCCC

SEQ ID 52 (AvrllCLlF; forward primer for Cλl amplification, highlighting AvrH restriction site):

CACACGTCCΓΛGGTCAGCCCAAGGCCAACCCC

SEQ ID S3 (CLlRSfII; reverse primer for Cλl amplification, incorporating Sfil cloning site):

CACACGGCCGΛGΓCGGCCCTATGAACAΓΓCTGTAGGGGCCACGTG

The following sequences are primers utilised in Example 3. Restriction sites are identified by italic or underlined text.

SEQ ID 54 (BssHπVHX; forward primer for V_H gene amplification, incorporating BssHII restriction site. X denotes V_H gene specific sequence):

CACACGGCGCGCACTCCXXXXXX...

SEQ ID 55 (SacϋVkX; forward primer for VK gene amplification, incorporating SacII restriction site. X denotes VK gene specific sequence): CACACAGCCGCGGTXXXXXX....

SEQ ID 56 (SacϋNhelVkX; forward primer for VK gene amplification, incorporating SacII and Nhel restriction sites. X denotes VK gene specific sequence):

CACACGCΓΛGCCGCGGTXXXXXX....

The following sequences are primers utilised in Example 4. Restriction sites are identified by italic or underlined text.

SEQ ID 57 (BspSalVLX; forward primer for Vλ gene amplification, incorporating BspEI and Sail restriction sites. X denotes Vλ gene specific sequence):

CACACΓCCGGΛGTCGACTCTXXXXXX...

SEQ ID 58 (JLAvrNar; reverse primer for Vλ gene amplification, highlighting Avrll and incorporating Narl restriction sites): CACACGGGCGCCGC AGCCTTGGGCTGACCΓΛGGACGGTGACCTTGGTCCC SEQ ID 59 (AvrNarCLIF; forward primer for Cλl gene amplification, highlighting Avrll and incorporating Narl restriction sites):

CACACGTCCTAGGTCAGCCCAAGGCGGCGCCC

Example 6

EXPRESSION of IgE USING pSGH3 and pSGK3

pSGH3 and pSGK3, described in Example 3, were constructed using V_H and VK gene sequences from a scFv. Correct construction of the new cassettes was confirmed by sequencing and maxi-preparations of the vectors were performed.

pSGH3 and pSGK3 were transfected into HEK293E cells using the optimised PEI protocol described in Example 1. After 8 days in 6-well plate culture, supernatants were analysed by IgE ELISA and a maximum yield of 13.5ng IgE / ml supernatant was observed.

The transfection was repeated in triple layer flasks and transfected cells cultured for 2 weeks. On day 8 of the culture transfected cells were treated with OptiMab (Invitrogen) according to the manufacturer's guidelines. IgE ELISA was performed on the supernatants after 2 weeks and a maximum yield of 48.3ng IgE / ml supernatant was observed.

The optimised vectors pSGH3 and pSGK3 successfully give rise to IgE expression and secretion when transfected into HEK293E cells.

List of Abbreviations

Abbreviation Full name

CH Constant heavy chain region

DNA Deoxyribonucleic acid

DNP-BSA Bovine serum albumin coated with hapten, Dinitrophenyl (DNP) FcεRI High affinity Fc receptor for IgE HEK293E Human Embryonic Kidney cell line with EBNA-I bearing plasmid transfected PCR Polymerase Chain Reaction pCEP4-SGs Refers to both pCEP4-SGH and pCEP4-SGLk pOriPs Refers to both pSGH-OriP and pSGLk-OriP pSGs Refers to pSGH (pcDNA3 based) and pSGLk (pcDNA 3.1 + Hygro based) pSGe pTT3 plasmid with its polylinker modified for SG cassettes pSG-Es Refers to pSGH-E , pSGLk-E and pSGLλ-1 all of which are pTT3 based pSGH pcDNA3 plasmid with the SGH cassette pSGH-E pTT3 plasmid with the SGH cassette pSGH-OriP pSGH with OriP cloned in with Nrul sites pSGLk pcDNA 3.1 (+) Hygro plasmid with the SGLk cassette pSGL-E pTT3-plasmid with the pSGL ( pSGLk-E and pSGLλ-1) pSGLk-E pTT3 plasmid with the SGLk cassette pSGLk-OriP pSGLk with OriP cloned in with Nrul site pSGLλ-1 pTT3 plasmid with the SGL λ-1 casstte SGH Heavy chain cloning cassette (refer to both HindIH or EcoRI at the 5' site)

SGLk Kappa chain cloning cassette (refers to both HindHI or EcoRI at the 5' site)

SGLλ-1 Lambda- 1 chain cloning cassette SPR Surface plasmon resonance PEI Polyethylenimine VDJ Variable, diversity, joining region of immunoglobulin

VH Variable heavy chain region VL Variable light chain region

VK Variable light chain kappa Vλ Variable light chain lambda

Note: Other plasmids with the SG-cloning cassettes will be followed by -SGs -SGH or - SGLk/SGLλ-1.

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Trill, JJ., Schatzman A.R., Ganguly S. 1995, "Production of monoclonal antibodies in COS and CHO cells. Curr. Opin. Biotechnol. Vol.6, pp. 553-560 All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in immunology, molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. A nucleic acid construct encoding an immunoglobulin heavy and/or light chain, wherein said nucleic acid construct comprises one or more non-naturally occuring restriction sites and wherein said non-naturally occuring restriction sites are incorporated at one or more of the following positions:

i) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain; and/or

ii) between the variable and constant regions of said immunoglobulin heavy and/or light chain; and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain; and/or

iii) at or within about 25 nucleotides of the 3' end of the stop codon of the constant region of said immunoglobulin heavy and/or light chain; and/or

iv) at or within about 25 nucleotides of the 5' end of the leader sequence of of said immunoglobulin heavy and/or light chain.

2. The nucleic acid construct according to claim 1, wherein one or more of the non- naturally occuring restriction sites incorporated:

ii) between the variable and constant regions of said immunoglobulin heavy and/or light chain; and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain;

are silently mutated non-naturally occuring restriction sites.

3. The nucleic acid construct according to claim 1 or claim 2, wherein said restriction sites at or within about 25 nucleotides of the 5' end of the leader sequence and at or within about 25 nucleotides of the 3' end of the stop codon of the constant region are compatible with the multiple cloning site of a plasmid or a vector.

4. The nucleic acid construct according to any of the preceding claims, wherein said leader sequence encodes a modified V_H, VK or Vλ leader sequence, preferably a modified Humighael, V_H 1-02, VK A26, Vλ 1-e or Vλ 8a leader..

5. The nucleic acid construct according to claim 4, wherein the leader sequence comprises the sequence set forth in any of SEQ ID Nos. 2, 4, 5, 6, 15, 16, 22 or 24.

6. The nucleic acid construct according to any of the preceding claims, wherein the non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain are selected from Xhol, ApaLI, BssHII, Narl, Nhel, SacII, Agel and/or Notl.

7. The nucleic acid construct according to claim 6, wherein the immunoglobulin chain is a heavy chain and the non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy chain are selected from Xhol, ApaLI, BssHII, Narl and/or Notl.

8. The nucleic acid construct according to claim 6, wherein the immunoglobulin chain is a kappa light chain and the non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin kappa light chain are selected from Xhol, ApaLI, Nhel and/or SacII.

9. The nucleic acid construct according to claim 6, wherein the immunoglobulin chain is a lambda light chain and the non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin lambda light chain are selected from Xhol, BspEI, Sail, ApaLI and/or Agel.

10. The nucleic acid construct according to any of the preceding claims, wherein the non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 5' end of the leader sequence of said immunoglobulin heavy and/or light chain are selected from EcoRI, Hindm and/or Notl.

11. The nucleic acid construct according to any of the preceding claims, wherein said non-naturally occuring restriction site(s) between the variable and constant regions of said immunoglobulin heavy and/or light chain, arid/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain are selected from Nhel, Narl, Xhol, Kpnl, BsiWI and/or Avrll.

12. The nucleic acid construct according to claim 11 , wherein:

(a) the immunoglobulin chain is a heavy chain and said non-naturally occuring restriction site(s) between the variable and constant regions of said immunoglobulin heavy chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy chain are selected from Nhel and/or Xhol; or (b) the immunoglobulin chain is a kappa light chain and said non-naturally occuring restriction site(s) between the variable and constant regions of said immunoglobulin kappa light chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin kappa light chain are selected from Kpnl, Narl and/or BsiWI; or (c) the immunoglobulin chain is a lambda light chain and said non-naturally occuring restriction site(s) between the variable and constant regions of said immunoglobulin lambda light chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin lambda light chain are selected from Kpnl, Narl and/or Avrll.

13. The nucleic acid construct according to any of the preceding claims, wherein said non-naturally occuring restriction site(s) at or within about 25 nucleotides of the 3' end of the stop codon of the constant region of said immunoglobulin heavy and/or light chain are selected from Xbal, Hindϋl, Notl, and/or Sfil.

14. The nucleic acid construct according to any of the preceding claims, wherein a naturally-occurring restriction site in said immunoglobulin heavy and/or light chain has been removed by a silent mutation.

15. The nucleic acid construct according to any of the preceding claims wherein the leader sequence and/or the variable region and/or the constant region has been swapped for a different leader sequence and/or variable region and/or constant region.

16. The nucleic acid construct according to any of the preceding claims, wherein the variable region and/or the constant region has been swapped for a different variable region and/or constant region.

17. The nucleic acid construct according to any of the preceding claims, wherein said constant region is replaced with or comprises a tag for protein purification.

18. A method for preparing a nucleic acid construct encoding a light and/or a heavy chain of an immunoglobulin molecule comprising the steps of:

a) providing a nucleic acid sequence encoding a heavy and/or light chain of an immunoglobulin molecule; and

b) introducing into said nucleic acid sequence one or more non-naturally occuring restrictions sites;

wherein said non-naturally occuring restriction sites are incorporated at one or more of the following positions: i) at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain; and/or

ii) between the variable and constant regions of said immunoglobulin heavy and/or light chain or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain; and/or

iii) at or within about 25 nucleotides of the 3' end of the stop codon of the constant region of the heavy and/or light chain of said immunoglobulin; and/or

iv) at or within about 25 nucleotides of the 5' end of the leader sequence of the heavy and/or light chain of said immunoglobulin.

19. The method according to claim 18, comprising the additional step of:

(c) swapping the variable region and/or the leader sequence and/or the constant region of said immunoglobulin heavy and/or light chain with a different variable region and/or leader sequence and/or constant region of the heavy and/or light chain.

20. The method according to claim 19, comprising the steps of:

i) excising the leader sequence and/or the variable region and/or the constant region of said immunoglobulin heavy and/or light chain using one of more restriction enzymes that cut the one or more restriction sites incorporated therein;

ii) amplifying a different leader sequence and/or a different variable region and/or a different constant region of said immunoglobulin heavy and/or light chain to incorporate one or more of the same restriction sites that are incorporated into said immunoglobulin heavy and/or light chain; iii) amplifying the sequence(s) to incorporate one or more of the same restriction sites therein;

e) digesting the amplified sequence(s) with restriction enzyme(s) that cut the incorporated restriction sites; and

f) inserting said digested sequence(s) into the construct,

wherein steps (i) and (ii) are performed in either order or at the same time.

21. The method according to any of claims 18 to 20, wherein one or more of the non- naturally occuring restriction sites incorporated:

ii) between the variable and constant regions of said immunoglobulin heavy and/or light chain or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain;

are incorporated using silent mutations.

22. The method according to any of claims 18 to 21, wherein said restriction sites are restriction sites that do not cut more than once in the construct (cassette) or the vector.

23. The method according to any of claims 18 to 22, wherein the nucleic acid construct encodes a chimeric or a humanised immunoglobulin heavy and/or light chain.

24. The method according to any of claims 18 to 23, wherein said variable and/or constant region is swapped for a humanised or a chimeric variable and/or constant region.

25. A nucleic acid construct obtained or obtainable by the method according to any of claims 18 to 24.

26. An isolated nucleic acid sequence comprising the sequence set forth in any of SEQ ID Nos. 2, 4, 5, 6, 12, 13, 15, 16, 18, 19, 20, 22, 24, 25, 26, 28, 29 or 30 to 59.

27. A vector or a plasmid comprising the nucleic acid construct according to any of claims 1 to 17 or 25 or the nucleic acid sequence according to claim 26.

28. A host cell comprising the nucleic acid construct according to any of claims 1 to 17 or 25 or the vector or plasmid according to claim 27.

29. A method for expressing an immunoglobulin molecule in a cell comprising the steps of:

a) providing the nucleic acid construct according to any of claims 1 to 17 or 25 or the vector or plasmid according to claim 27;

b) transforming or transfecting said nucleic acid construct or said vector into a cell;

c) providing for the expression of said nucleic acid construct or said vector in said cell; and

d) optionally purifying the immunoglobulin molecule.

30. The method according to claim 29, wherein said nucleic acid construct is inserted into at least two vectors, wherein the first nucleic acid construct or vector comprises the light chain of said immunoglobulin and wherein the second nucleic acid construct or vector comprises the heavy chain of said immunoglobulin.

31. The method according to claim 29, wherein said nucleic acid construct is inserted into one vector comprising the light chain of said immunoglobulin and the heavy chain of said immunoglobulin.

32. The method according to any of claims 29 to 31, wherein the first nucleic acid construct or vector and the second nucleic acid construct or vector are transformed or transfected into the cell with an excess of light chain in concentration.

33. The method according to claim 32, wherein the transfection ratio of the heavy chain vector to light chain vector is approximately 1 :4.

34. The method according to any of claims 29 to 33, wherein said cells are transfected using the PEI method.

35. The method according to any of claims 29 to 34 wherein said vector is selected from the group consisting of pcDNA3, pcDNA3.1, (+) Hygro, pCEP4, a glutamine synthase vector and pTT3.

36. The method according to claim 35, wherein the vector is a pcDNA vector comprising an origin of replication.

37. The method according to any of claims 29 to 36, wherein said cells are bacterial, mammalian or plant cells.

38. The method according to claim 37, wherein said human cells are HEK293 cells or their derivatives, such as HEK293E and HEK293T cells.

39. A nucleic acid primer comprising the sequence set forth in SEQ ID Nos. 22 to 35.

40. A nucleic acid construct according to any of claims 1 to 17, comprising a non- naturally occuring restriction site at each of positions (i) and (ii) as defined in claim 1.

41. A nucleic acid construct according to any of claims 1 to 17, comprising a non- naturally occuring restriction site at each of positions (i), (ii) and (iii) as defined in claim 1.

42. A nucleic acid construct according to any of claims 1 to 17, comprising a non- naturally occuring restriction site at each of positions (i), (ii), (iii) and (iv) as defined in claim 1.

43. A nucleic acid construct according to any of claims 1 to 17 or 40 to 42, wherein two or more non-naturally occurring restriction sites are present at one or more of positions (i),

(ii), (iii) and (iv) as defined in claim 1.

44. A nucleic acid construct according to any of claims 1 to 17 or 40 to 43, wherein two or more non-naturally occurring restriction sites are present at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain.

45. A nucleic acid construct according to any of claims 1 to 17 or 40 to 44, wherein at least two non-naturally occurring restriction sites are present between the variable and constant regions of said immunoglobulin heavy and/or light chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain.

46. A nucleic acid construct according to any of claims 1 to 17 or 40 to 45, wherein two or more non-naturally occurring restriction sites are present at two or more of positions (i),

(ii), (iii) and (iv) as defined in claim 1.

47. A nucleic acid construct according to any of claims 1 to 17 or 40 to 46, wherein:

(a) two or more non-naturally occurring restriction sites are present at or within about 25 nucleotides of the 3' end of the leader sequence of said immunoglobulin heavy and/or light chain; and (b) two or more non-naturally occurring restriction sites are present between the variable and constant regions of said immunoglobulin heavy and/or light chain, and/or at or within about 25 nucleotides of the 3' end of the variable region of said immunoglobulin heavy and/or light chain.

48. A method according to any of claims 18 to 24, further comprising removing a naturally-occurring restriction site in said immunoglobulin heavy and/or light chain by a silent mutation.