WO2009150426A2 - Procédé - Google Patents

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Publication number
WO2009150426A2
WO2009150426A2 PCT/GB2009/001461 GB2009001461W WO2009150426A2 WO 2009150426 A2 WO2009150426 A2 WO 2009150426A2 GB 2009001461 W GB2009001461 W GB 2009001461W WO 2009150426 A2 WO2009150426 A2 WO 2009150426A2
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WIPO (PCT)
Prior art keywords
light chain
nucleic acid
immunoglobulin
acid construct
sequence
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PCT/GB2009/001461
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English (en)
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WO2009150426A3 (fr
Inventor
James Hunt
Helen Elizabeth Harries
Samuel Ken En Gan
Andrew John Beavil
Philip John Marsh
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King's College London
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Publication of WO2009150426A2 publication Critical patent/WO2009150426A2/fr
Publication of WO2009150426A3 publication Critical patent/WO2009150426A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]

Definitions

  • the present invention relates to the field of recombinant protein expression.
  • the present invention relates to the field of recombinant antibody expression and a construct/expression system that can be used to rapidly produce antibodies.
  • 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.
  • the present invention seeks to address the problems of the prior art.
  • nucleic acid construct that facilitates easy swapping and rapid expression of antibody regions.
  • 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.
  • 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.
  • nucleic acid construct encoding an immunoglobulin heavy and/or light chain
  • 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.
  • 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.
  • 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.
  • said leader sequence encodes an Immunoglobulin leader sequence.
  • the Immunoglobulin leader sequence may be derived from a V H , VK or V ⁇ leader sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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-natural occurring restriction sites are present at each of two or more of the locations defined above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Xhol and ApaLI sites for example as present in SEQ ED NO:2
  • BspEI and Sail sites for example as present in SEQ ED NO: 24
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • 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).
  • 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.
  • a naturally-occurring restriction site in said immunoglobulin heavy and/or light chain has been removed by a silent mutation.
  • 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.
  • variable region and/or the constant region has been swapped for a different variable region and/or constant region.
  • said constant region is replaced with or comprises a tag for protein purification.
  • 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
  • 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.
  • 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.
  • said restriction sites are restriction sites that do not cut more than once in the construct (cassette) or the vector.
  • the nucleic acid construct encodes a chimeric or a humanised immunoglobulin heavy and/or light chain.
  • variable and/or constant region is swapped for a humanised or a chimeric variable and/or constant region.
  • nucleic acid construct obtained or obtainable by the method described herein.
  • an isolated nucleic acid sequence comprising the sequence set forth in any of SEQ ID Nos. 1 to 59.
  • 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.
  • a vector or a plasmid comprising the nucleic acid construct or the nucleic acid sequence.
  • a host cell comprising the nucleic acid construct or the vector or plasmid.
  • 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.
  • 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.
  • said nucleic acid construct is inserted into one vector comprising the light chain of said immunoglobulin and the heavy chain of said immunoglobulin.
  • 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.
  • said cells are transfected using the PEI method.
  • said vector is selected from the group consisting of pcDNA3, pcDNA3.1 (+) Hygro, pCEP4, Lonza vectors and pTT3.
  • said cells are bacterial, mammalian or plant cells.
  • said human cells are HEK293 cells and their derivatives - such as HEK293E and HEK293T cells.
  • a nucleic acid primer comprising the sequence set forth in any of SEQ ID Nos. 30-59.
  • 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.
  • the present invention is capable of producing immunoglobulins and fragments thereof without the long labour-intensive screening and selection.
  • the present invention is capable of producing whole immunoglobulins, including chimeric and humanised antibodies.
  • the inventors have been able to rapidly clone, express and purify over 20 immunoglobulins making this a rapid, efficient system without long cell maintenance.
  • 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.
  • 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.
  • 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.
  • Immunoglobulin leader sequences provides for the rapid production of, for example, IgE.
  • the leaders used allow silent mutations to be introduced thereby incorporating one or more restriction sites for swapping of leader and variable sequences.
  • 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.
  • FIG. 1 shows the structure of an IgE molecule.
  • VH Variable Heavy chain
  • VL Variable Light chain
  • C ⁇ Constant region - heavy chain
  • CL Constant region - light chain
  • 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.
  • FIG. 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 SGL ⁇ -1 cassette, (top) 5' and 3' ends of Rat SPE7 J ⁇ 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 SGL ⁇ cassette, with the leader/signal peptide, VL and CL regions and the incorporated restriction sites.
  • Figure 13 shows the full SGL ⁇ -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 10 5 HEK293E cells were transfected according to a previously published method (Durocher, Perret & Kamen 2002) with 1 ⁇ g/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.
  • Figure 19 shows surface plasmon resonance analysis of purified IgE in comparison with their commercial partners using a Biacore 3000 (GE Biosciences).
  • IgE diluted in HBS Biacore run buffer was flowed over a CM5 chip amine coupled with DNP-BSA (-200 RU) (Sigma Aldrich) or human sFc ⁇ RI ⁇ -IgG4-Fc fusion protein( ⁇ 200 RU) at 20 ⁇ L / min with an exposure time to analyte of 180 seconds.
  • Figure 20 shows gel filtration chromatography traces of purified 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 C ⁇ 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 C ⁇ to replace the existing C ⁇ with human C ⁇ l-4, human C ⁇ 2 or murine C ⁇ .
  • 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 R I family gene segment and C ⁇ 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.
  • Normal text normal text
  • bold text region encoding the required restriction sites
  • 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 V ⁇ A26 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.
  • Normal text normal text
  • bold text region encoding the required restriction sites
  • 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 V ⁇ l-e leader sequence and the Avr II site at the J ⁇ -C ⁇ junction allow for exchange of any murine or human V ⁇ 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 V ⁇ leader sequence 1-e at a position 14 bases 5' of the start of the V ⁇ gene segment.
  • Figure 33 shows the position of a native Avr II restriction site at the junction between the J ⁇ portion of the V ⁇ domain (bold) and the C ⁇ domain (normal text).
  • Figure 34 shows examples of primers for use with SGL2.
  • the primers may be used for modification of a V ⁇ l family gene and C ⁇ l 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 C ⁇ allow for exchange of any murine or human
  • the Nhel site can also be utilised in conjunction with the Sfil site 3' of the C ⁇ to replace the existing C ⁇ with human C ⁇ l-4, human C ⁇ 2 or murine C ⁇ .
  • 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 H 1 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 V ⁇ A26 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.
  • FIG 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
  • 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 H I-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)
  • Agel is used with the V ⁇ 1-e leader.
  • Example 2 use of Kpnl is described in Example 1.
  • BsiWI can be used to cut the VK-CK boundary (Example 3) and AvrEI can be used at the V ⁇ -C ⁇ 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 V ⁇ 8a leader sequence and the Avrll or Narl sites at the 5' end of the C ⁇ sequence allow for exchange of any human V ⁇ 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 V ⁇ 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 C ⁇ gene segment.
  • Figure 46 shows primers which may be used for modification of a V ⁇ l family gene and C ⁇ l isotype gene for introduction into the SGL3 cassette.
  • the nucleotide sequences encoding an immunoglobulin molecule may be present in a nucleic acid (eg. a DNA) construct.
  • a nucleic acid eg. a DNA
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the restriction sites are created using one or more silent mutations - such as one or more in-frame silent mutations.
  • 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.
  • 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.
  • silent in-frame mutations are 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.
  • the leader sequence is cleaved leaving the rest of the immunoglobulin sequence intact.
  • 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.
  • 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.
  • the restriction enzyme sites are therefore unique restriction enzyme sites.
  • the construct itself may be directly or indirectly attached to a promoter.
  • the vector into which the construct is inserted can comprise a promoter.
  • the nucleic acid construct is present in a vector- such as an expression vector.
  • the nucleic acid construct may comprise the nucleotide sequences encoding the heavy and the light chains of the antibody.
  • the nucleic acid construct may comprise the nucleotide sequence encoding only the heavy chain of the antibody.
  • the nucleic acid construct may comprise the nucleotide sequence encoding only the light chain of the antibody.
  • nucleic acid constructs may be provided, one comprising the heavy chain of the antibody and the other comprising the light chain of the antibody.
  • 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.
  • non-naturally occuring restriction sites described herein may be incorporated at:
  • 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.
  • 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.
  • 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.
  • the restriction site is a Xhol restriction site.
  • 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.
  • 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.
  • 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.
  • the restriction site is a Apal ⁇ restriction site.
  • the mutation(s) is a silent mutation.
  • 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.
  • 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.
  • the mutation(s) is a silent mutation.
  • 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).
  • 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.
  • 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.
  • 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.
  • the restriction site does not appear in the heavy or light chain leader, variable or constant regions.
  • any sequences before the Ncol site or Kozak sequence (5'-accatgg-3') can be be deleted.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the amplification method uses primers that incorporate the one or more desired restriction sites.
  • the amplification is an exponential amplification, as exhibited by, for example, the polymerase chain reaction.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the leader sequence may be a modified Humighael, V H 1-02, VK A26, V ⁇ 1 -e or V ⁇ 8a leader.
  • the leader sequence comprises a modified Humighae I leader including mutations that incorporate an Xhol and a Ap ⁇ Ll restriction site near the 3' end of the leader sequence.
  • this leader sequence comprises the sequence set forth in SEQ ID No.2.
  • 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.
  • 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.
  • 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.
  • this leader sequence comprises the sequence set forth in SEQ ID No.6.
  • 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 V ⁇ 1-e leader incorporating an Agel site (e.g. as defined in SEQ ID NO. 22) or a V ⁇ 8a leader incorporating a BspEI site and a Sail site (e.g. as defined in SEQ ID NO. 24).
  • further restriction sites may be added at the 5' end of the leader sequence to facilitate cloning and swapping of the cassette between vectors.
  • a sequence comprising EcoRI, HindIII and/or Notl sites may be added to the 5' end of the leader.
  • 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.
  • the Xhol, ApaLI, Notl, BssHII, Nhel, SacII, Agel, BspEI and Sail restriction sites are incorporated using silent mutations.
  • restriction sites are engineered into the construct in order to join the variable region to the constant region in the heavy and light chains.
  • 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).
  • 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.
  • a Kpn ⁇ site is engineered into this sequence, for example as present in SEQ ID NO: 18.
  • the 3' end of the constant region comprises a stop codon.
  • restriction sites downstream of this codon 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.
  • restriction sites 5' of the leader sequence i.e. these sites do not need to be introduced by silent mutation.
  • one or more restriction sites that are added downstream of this stop codon are used in order to facilitate swapping/exchange.
  • one or more terminal restriction sites are incorporated downstream (eg. immediately downstream) of this stop codon that facilitate swapping/exchange.
  • the restriction site may comprise more than one restriction site to facilitate incorporation into a vector.
  • the restriction site is ⁇ axXbal restriction site (5'-tctaga-3).
  • the restriction site is Hind ⁇ l (5'-aagctt-3').
  • the restriction sites are Xba ⁇ and Hind ⁇ l restriction sites (5'- tctagaagctt-3). In another embodiment, the restriction site is Sfil (5'- GGCCNNNNNGGCC-3 ').
  • 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.
  • stable expression is preferred.
  • 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.
  • 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.
  • the antibodies are expressed transiently.
  • 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 3 1 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.
  • the host cell may be a naturally occurring host cell in which the nucleotide sequence encoding the antibody is naturally present.
  • the nucleotide sequence encoding the antibody is a human sequence then it may be present in a human host cell.
  • the host cell is a host in which the nucleotide sequence encoding the antibody is not naturally present.
  • the host cell may be a eukaryotic cell from a different species.
  • 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.
  • 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.
  • 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.
  • host cells may include yeast, VERO, HeLa, CHO, W138, BHK, COS-7, MDCK, HEK, HEK293 and HEK293E cells.
  • 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.
  • 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.
  • the vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • the vectors may contain different selectable markers.
  • 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.
  • 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.
  • 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.
  • cells e.g. eukaryotic 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.
  • 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 is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Plasmid 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.
  • 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
  • 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”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • 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.
  • 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.
  • origins of replication or autonomously replicating 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.
  • Selection genes 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.
  • either constitutive or inducible promoters can be used.
  • a large number of promoters recognized by a variety of potential host cells are well known.
  • 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.
  • 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 ⁇ - lactamase and lactose promoter systems, a tryptophan (tip) promoter system and hybrid ) promoters such as the tac or the trc promoter.
  • PhoA promoter the ⁇ - lactamase and lactose promoter systems
  • a tryptophan (tip) promoter system and hybrid ) promoters
  • other promoters that are functional in bacteria 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.
  • 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.
  • the transfection ratio of light and heavy chains can be optimised when using a dual expression vector system.
  • 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.
  • the optimum light chainrheavy chain ratio is about 4:1.
  • Total DNA may amount to about 1 ⁇ g/ml/2-4xlO 5 cells.
  • 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.
  • the antibody is a whole antibody.
  • 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.
  • 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).
  • CDR complementarity determining regions
  • FR framework regions
  • 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,
  • 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.
  • antibodies can be assigned to different classes.
  • immunoglobulins 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.
  • the antibody is IgE.
  • the antibody is IgG
  • the antibody is or is derived from a monoclonal antibody.
  • 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.
  • 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.
  • the present invention also encompasses fragments - such as fragments of the antibodies described herein.
  • 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 Fab 1 fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g.
  • the present invention contemplates antibody fragments that are modified to improve their stability and or to create antibody complexes with multivalency.
  • 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.
  • 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.
  • 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.
  • 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.
  • variants are 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.
  • 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.
  • 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.
  • 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.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues.
  • 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.
  • 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.
  • Fc immunoglobulin constant region
  • humanization can be essentially as described in Jones et al. (1986) Nature
  • 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.
  • 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.
  • variable domains both light and heavy
  • 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.
  • 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.
  • 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.
  • the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
  • 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.
  • 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.
  • 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 5 1 ends of the molecule.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention encompasses the use of variants and homologues of nucleic acid sequences.
  • homologue means an entity having a certain homology with the subject nucleotide sequences described herein.
  • homology can be equated with “identity”.
  • 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.
  • 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.
  • 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).
  • % homology can be measured in terms of identity
  • the alignment process itself is typically not based on an all-or-nothing pair comparison.
  • 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.
  • 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.
  • Immunoglobulin E 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).
  • IgE Immunoglobulin E
  • 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).
  • 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.
  • Immunoglobulins are made up of discrete genetic elements that can be rearranged, such that specific V regions can be paired with different constant regions.
  • the antibody light chain which can be broadly classified into either ⁇ - or K- families, can be paired with different heavy chains.
  • 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.
  • 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).
  • 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.
  • Nhel a second restriction site, was incorporated by silent mutation at the 5' start of the epsilon gene (Table 4). Nhel is absent from human epsilon and VH germline gene sequences (Table 1).
  • 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 C ⁇ sequences (Table 1). Mobility of the cloning cassette between protein expression vectors is discussed in section B.
  • 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 (C ⁇ ) 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 C ⁇ 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.
  • the light chain cloning cassette was completed by the addition of unique restrictions sites at the 5' of the Humighael leader (Hindi ⁇ /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.
  • SGLK kappa light chain cloning cassette
  • the kappa light chain cassette (SGLk) was assembled in pcDNA 3.1 (Invitrogen).
  • human ⁇ -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.
  • pSGLK a lambda light chain cassette, SGL ⁇ l
  • a human ⁇ -constant-1 region sequence accession: BC073786; obtained from the IMGT database: www.ebi.ac.uk/imgt/
  • a rat V ⁇ -7 sequence James, Tawfik 2003.
  • the leader previously described in SGH and SGLK was added to the 5' end of the rat V ⁇ , 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 V ⁇ and Xbal-Hindl ⁇ to the 3' end of the ⁇ -constant-1 sequence (see Figure 11).
  • 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 SGL ⁇ -1, a human ⁇ -1 cassette incorporating a synthesised rat hapten-specific variable region, paired with a human ⁇ -1 constant region.
  • 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
  • SGL ⁇ -1 a human ⁇ -1 cassette incorporating a synthesised rat hapten-specific variable region, paired with a human ⁇ -1 constant region.
  • 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).
  • 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).
  • the SG cloning cassettes were also inserted into pTT3 (Durocher, Perret & Kamen 2002).
  • 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.
  • 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.
  • EcoRI-Xbal digested SG cassettes were inserted into EcoRI-Xbal digested pSG-E to form pSGH-E (heavy chain plasmid) and pSGL ⁇ -1 or pSGLk-E (light chain plasmid) as shown in Figure 16.
  • 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 10 5 HEK293E cells were transfected with 1 ⁇ g/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.
  • cassettes described were used to create a panel of IgE molecules incorporating different V-regions.
  • 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 V ⁇ 7 (accession : A Y331039) are rat V-regions synthesized by Gene Art (Germany) following sequences described in (James, Tawfik 2003).
  • 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.
  • 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.
  • VH regions were paired with the various Vk and V ⁇ 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-ratV ⁇ 7, VH6-ratV ⁇ 7 in Table 4).
  • VH3-HerVkl, VH3-ratV ⁇ 7, VH6-ratV ⁇ 7 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).
  • 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.
  • 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 Fc ⁇ RI affinity constant.
  • Human SPE7 chimeric rat- V, human-C IgE
  • Herceptin anti-Her2 humanized antibody, also known as Trastuzamab®
  • HHM- VH4 in-house matched V-regions from a single B cell
  • IgE antibodies were purified using a human Fc ⁇ RI ⁇ -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 well-characterized human IgE
  • 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.
  • IgE antibodies VH3-Vk4, VH4-Vk4, VH5-Vk4, VH6-Vk4, VH7-Vk4, Herceptin IgE, Human SPE7 in Figure 20
  • 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.
  • 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.
  • 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.
  • 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.
  • Example 1 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.
  • 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.
  • V H I -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).
  • the SGH-E cassette introduces a Nhe I restriction site into the 5' region of the C ⁇ 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:
  • IgE GCC TCC ACA
  • IgA2 GCA TCC CCG
  • IgGl GCC TCC ACC
  • IgG2 GCC TCC ACC
  • IgG3 GCT TCC ACC
  • IgG4 GCT TCC ACC
  • Murine IgE GCC TCT ATC
  • Nhe I will not introduce additional cuts into murine C ⁇ , or human C ⁇ , C ⁇ l-4 or C ⁇ 2.
  • Nar I restriction sites are absent from murine and human C ⁇ and human C ⁇ 2, but are present in C ⁇ l-4. However the sites can be removed by silent mutation of the C ⁇ l-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.
  • 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.
  • 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.
  • 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 C ⁇ , C ⁇ l-4 or C ⁇ 2.
  • the primers shown in Figure 25 may be used for modifying a V H 1 family gene segment and C ⁇ 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.
  • 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).
  • 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.
  • 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.
  • I restriction site may be added 3' of the CK stop codon ( Figure 28).
  • 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.
  • the Ig ⁇ cassette can be transferred to any compatible expression vector using the flanking restriction sites Not I or Eco RI with Sfi l.
  • 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.
  • FIG. 31 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 V ⁇ domain.
  • V ⁇ leader sequence and Age I restriction site The restriction motif for Age I can be introduced by silent mutation of V ⁇ leader sequence 1-e at a position 14 bases 5' of the start of the V ⁇ gene segment, as shown in Figure 32.
  • the Age I motif can be similarly introduced by silent mutation at the same position of V ⁇ 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 V ⁇ leader sequence.
  • a native Avr II restriction site exists at the junction between the J ⁇ portion of the V ⁇ domain (bold text) and the C ⁇ domain (normal text), as shown in Figure 33.
  • a Sfi I restriction site may be included 3' of C ⁇ stop codon to enable C ⁇ isotype and Ig ⁇ cassette exchange.
  • V ⁇ 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 J ⁇ -C ⁇ junction respectively.
  • the 31 functional human germline V ⁇ genes listed on the Vbase database and the 14 functional murine germline V ⁇ 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, Ig ⁇ 3a (Accession number X97471). It is possible to remove this native Avr II site by silent site directed mutation in order to make this V ⁇ gene compatible with the SGL2 cassette.
  • this cassette can be used to express Ig ⁇ comprising any human C ⁇ isotype with any human or murine V ⁇ gene segment (provided required restriction sites are not introduced by somatic hypermutation).
  • the Ig ⁇ cassette may be transferred to alternative vectors using the flanking sites Not I or Eco RI with that for Sfi I.
  • the primers described in Figure 34 may be used for modification of a V ⁇ l family gene and C ⁇ l 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.
  • 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.
  • 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.
  • 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 H I 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.
  • 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.
  • 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.
  • SacII restriction site can also be incorporated by silent mutation into the same position of VK leaders AlO and A14.
  • 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.
  • Primers for modification of CK and the reverse primer that facilitates BsiWI addition to VK are unchanged from example below.
  • 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.
  • 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.
  • the SGL2 cassette described in Example 2 uses the V ⁇ 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 V ⁇ leader 1-e (Webcutter analysis, Example 5), so the other V ⁇ leaders stored on the Vbase database were analysed to investigate the possibility of introducing to restriction sites into the V ⁇ leader - V ⁇ boundary of the SGL cassette. An additional restriction site was also introduced into the 5' end of the C ⁇ gene, thus generating a vector, pSGL3 with a multiple cloning site for immunoglobulin lambda chains ( Figure 43).
  • V ⁇ leader 8a is the only V ⁇ leader into which these restriction sites can be introduced by silent mutation.
  • an additional Narl restriction site can be introduced by silent mutation 13 nucleotides into the 5' region of the C ⁇ gene segment, as shown in Figure 45.
  • Narl can be incorporated into each of the functional C ⁇ isotypes, C ⁇ l, 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 C ⁇ sequences.
  • V ⁇ gene segment V ⁇ gene segments can be swapped into and out of the lambda chain vector using BspEI or Sail at the leader- V ⁇ junction with Avrll or Narl at the V ⁇ -C ⁇ junction.
  • the human germline V ⁇ 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 - V ⁇ boundary cut sites and at least one of the V ⁇ -C ⁇ boundary cut sites, meaning all human V ⁇ genes are compatible with pSGL3.
  • the primers shown in Figure 46 may be used for modification of a V ⁇ l family gene and C ⁇ l 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/cutter2 ⁇ 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.
  • 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.
  • VH Leader used in optimised pSG: MetAspTrpThrTrpArglleLeuPheLeuValAlaAlaAlaThrGlyAlaHisSer atggactggacctggaggatcctcttttggtggcagcagccacaggagcccactcc base pairs atggaytggacntggcgnathctnttyctngtngcngcngcnacnggngcncaytcn 1 to 57
  • 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:
  • 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 C ⁇ 2.
  • the J H -C H boundary incorporating both Xhol and Nhel as exchange site options is detailed in SEQ ID 13.
  • Enzyme produces a blunt cut
  • 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).
  • Motif occurs in one or more VK germline genes • Motif occurs in the CK gene
  • 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:
  • Kpnl site introduced into the original pSGK-E vector is 5' of the region analysed here.
  • 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.
  • V ⁇ leader MetAlaTrpSerProLeuLeuLeuThrValLeuThrHisCysThrGlySerTrpAla atggcctggtctcctctcctcactgtcctcactcactgcacagggtcctgggcc base pairs atggcntggtcnccnctnctnacngtnctnacncaytgyacnggntcntgggcn 1 to 57
  • Agel is the only restriction enzyme suitable for cutting at the 3' end of the V ⁇ l-e leader. AU other restriction sites were discarded due to one or more of the following reasons:
  • Avrll (underlined) is the only restriction enzyme suitable for cutting at the J ⁇ -C ⁇ boundary. All other restriction sites were discarded due to one or more of the following reasons:
  • V ⁇ leader 8a V ⁇ leader 8a

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Abstract

La présente invention concerne une construction d'acide nucléique codant pour une chaîne lourde et/ou légère d'immunoglobuline, ladite construction d'acide nucléique comprenant un ou plusieurs sites de restriction n'existant pas naturellement et lesdits sites de restriction n'existant pas naturellement étant incorporés à l'une ou plusieurs des positions suivantes : i) au niveau ou à l’intérieur de 25 nucléotides de l'extrémité 3' de la séquence de tête de ladite chaîne lourde et/ou légère d'immunoglobuline; et/ou ii) entre les régions variables et constantes de ladite chaîne lourde et/ou légère d'immunoglobuline; et/ou au niveau ou à l’intérieur d’environ 25 nucléotides de l'extrémité 3' de la région variable de ladite chaîne lourde et/ou légère d'immunoglobuline; et/ou iii) au niveau ou à l’intérieur d’environ 25 nucléotides de l'extrémité 3' du codon stop de la région constante de ladite chaîne lourde et/ou légère d'immunoglobuline; et/ou iv) au niveau ou à l’intérieur d’environ 25 nucléotides de l'extrémité 5' de la séquence de tête de ladite chaîne lourde et/ou légère d'immunoglobuline.
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US20040033561A1 (en) * 2001-10-19 2004-02-19 Millennium Pharmaceuticals, Inc. Immunoglobulin DNA cassette molecules, monobody constructs, methods of production, and methods of use therefor

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US20040033561A1 (en) * 2001-10-19 2004-02-19 Millennium Pharmaceuticals, Inc. Immunoglobulin DNA cassette molecules, monobody constructs, methods of production, and methods of use therefor

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