WO1993012246A1 - Vecteurs d'expression-secretion servant a produire des fragments fv biologiquement actifs - Google Patents

Vecteurs d'expression-secretion servant a produire des fragments fv biologiquement actifs Download PDF

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WO1993012246A1
WO1993012246A1 PCT/US1991/007625 US9107625W WO9312246A1 WO 1993012246 A1 WO1993012246 A1 WO 1993012246A1 US 9107625 W US9107625 W US 9107625W WO 9312246 A1 WO9312246 A1 WO 9312246A1
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expression
dna sequence
sequence encoding
dna
sequences
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PCT/US1991/007625
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Shi Chung Ng
James G. Anthony
Sui-Lam Wong
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The University Of Calgary
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Priority to JP05510854A priority Critical patent/JP2000505641A/ja
Priority to EP92923150A priority patent/EP0618977A4/en
Priority to PCT/US1991/007625 priority patent/WO1993012246A1/fr
Publication of WO1993012246A1 publication Critical patent/WO1993012246A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins

Definitions

  • the Fv fragment is the smallest complete antigen binding site presently known. This fragment is composed of only the variable domains of the immunoglobulin variable heavy (V warrant) and variable light (V.) chains.
  • VERT immunoglobulin variable heavy
  • V. variable light
  • the small size of the Fv fragment has generated a great deal of interest in the antibody and protein engineering fields because of its potential application in imaging, therapeutics, and structural studies.
  • Initial attempts to generate Fv were made using proteolytic cleavage of whole antibody. However, this technique was hindered by difficulties in controlling both quality and yield (Inbar et al., Proc. Natl. Acad. Sci. USA 9, 2659 [1972]). Recombinant DNA techniques were later employed in attempts to express native Fv in bacterial cells.
  • the present invention concerns expression- secretion systems for the production of biologically active Fv fragments and single chain Fv molecules.
  • the present invention concerns an expression-secretion vector capable of producing a biologically active Fv fragment comprising a DNA sequence encoding the T7 promoter, a DNA sequence encoding the variable domain of an immunoglobulin heavy chain (V gris), a DNA sequence encoding the variable domain of an immunoglobulin light chain (V-), and one or more DNA sequences encoding one or more signal peptide sequences.
  • the present invention further concerns a host cell containing an expression-secretion vector capable of producing a biologically active Fv fragment comprising a DNA sequence encoding the T7 promoter, a DNA sequence encoding the variable domain of an immunoglobulin heavy chain, a DNA sequence encoding the variable domain of an immunoglobulin light chain, and one or more DNA sequences encoding one or more signal peptide sequences.
  • the present invention additionally concerns a method for producing a biologically active Fv fragment comprising culturing a host cell containing an expression-secretion vector capable of producing a biologically active Fv fragment which comprises a DNA sequence encoding the T7 promoter, a DNA sequence encoding the variable domain of an immunoglobulin heavy chain, a DNA sequence encoding the variable domain of an immunoglobulin light chain, and one or more DNA sequences encoding one or more signal peptide sequences under conditions permitting expression- secretion of the biologically active Fv fragments.
  • the present invention also concerns an expression-secretion vector capable of producing a biologically active single chain Fv (sFv) molecule comprising a DNA sequence encoding the T7 promoter, a DNA sequence encoding a single chain Fv molecule, and a DNA sequence encoding a signal peptide sequence.
  • sFv biologically active single chain Fv
  • Figure 1 shows the nucleotide and deduced amino acid sequences of the (A) V ⁇ [SEQ. ID NO. 1] and (B) V L [SEQ. ID NO. 2] portions of antidigoxin monoclonal antibody 26-10. Restriction sites are shown.
  • Figure 2 shows the modified DNA sequences encoding and the deduced amino acid sequences of the signal peptide sequences (A) ompA [SEQ. ID NO. 3] and (B) phoA [SEQ. ID NO. 4]. Arrows indicate the change of nucleotides at these particular positions to generate desirable restriction enzyme recognition sites.
  • Figure 3 shows the various plasmids produced in generating plasmid FvpD: (A) Construction of plasmid V H pD; (B) Construction of plasmid L pD; (C) Construction of plasmid V-pD-Xbal; (D) Construction of plasmid FvpD.
  • Figure 4 is a sodium dodecyl sulphate (SDS) polyacrylamide gel demonstrating the production of the 26-10 Fv fragment.
  • Lanes 1-4 Eluted fraction numbers 3 to 6 from ouabain column. Peak of Fv is at fraction 3.
  • Lane 5 Protein size standards 16*9, 14*4, 8*2 kd.
  • Lane 6 Prestained protein size standards 110, 84, 47, 33, 24, 16 kd.
  • Lane 7 Fv periplasmic fraction before column purification.
  • Figure 5 shows the constructs of plasmid pT7PhoA 26-10sFv.
  • Figure 5 shows the construction of plasmid pT7PhoA26-10sFv.
  • Figure 6 shows the DNA sequence encoding and the amino acid sequence of the 26-10 single chain Fv molecule. Restriction sites and some 5 1 and 3' non-coding sequences are shown.
  • the present invention concerns an expression- secretion vector capable of producing a biologically active Fv fragment comprising a DNA sequence encoding the T7 promoter, a DNA sequence encoding the variable domain of an immunoglobulin heavy chain, a DNA sequence encoding the variable domain of a immunoglobulin light chain, and one or more DNA sequences encoding one or more signal peptide sequences.
  • the present invention also concerns an expression-secretion vector capable of producing a biologically active single chain Fv (sFv) molecule comprising of DNA sequence encoding the T7 promoter, a DNA sequence encoding a single chain Fv molecule, and a DNA sequence encoding a signal peptide sequence.
  • sFv single chain Fv
  • the biologically active Fv fragment or sFv molecule has authentic N-termini (i.e., the mature Fv fragment or sFv molecule is generated by cleavage of the peptide bond between the carboxy terminus of the signal peptide sequence and the amino terminus of the variable domain of the immunoglobulin heavy or light chain).
  • expression- secretion vectors wherein the signal peptide sequences are ompA and phoA.
  • expression-secretion vectors wherein the DNA sequences encoding the signal peptide sequences have been modified to generate additional restriction enzyme sites without changing the amino acid sequences of the signal peptide sequences.
  • an expression-secretion vector capable of producing a biologically active Fv fragment comprising a DNA sequence encoding the T7 promoter operatively linked to a DNA sequence encoding the variable domain of an immunoglobulin heavy chain, a DNA sequence encoding the variable domain of an immuno ⁇ globulin light chain, and one or more DNA sequences encoding one or more signal peptide sequences.
  • operatively linked means that the T7 promoter is capable of directing the transcription of the DNA sequences encoding the variable domains of the immunoglobulin heavy and light chains.
  • Fv fragment means the non-covalently associated variable domains of the immunoglobulin heavy and light chains which can bind antigen but which lack the effector functions of the constant regions of the immunoglobulin heavy and light chains.
  • single chain Fv molecule means a molecule in which variable domains of the immunoglobulin heavy and light chains which can bind antigen but which lack effector functions of the constant regions of the immunoglobulin heavy and light chains are joined using an amino acid linker.
  • biologically active Fv fragment or “biologically active sFv molecule” means that the Fv fragment or sFv molecule is capable of specifically binding one or more of the same antigens as the full length antibody from which it is derived.
  • Expression-secretion vectors of utility in the present invention are often in the form of "plasmids", which refer to circular double stranded DNAs which, in their vector form, are not bound to the chromosome.
  • the invention is intended to include such other forms of expression-secretion vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • the expression-secretion vectors of the present invention capable of producing a biologically active Fv fragment at a minimum contain a DNA sequence encoding the T7 promoter, a DNA sequence encoding the variable domain of an immunoglobulin heavy chain, a DNA sequence encoding the variable domain of an immunoglobulin light chain, one or more DNA sequences encoding one or more signal peptides sequences (e.g., ompA, phoA, pelB) and the remaining vector.
  • the expression-secretion vectors of the present invention capable of producing a biologically active sFv molecule at a minimum contain a DNA sequence encoding a T7 promoter, a DNA sequence encoding a single chain Fv molecule, a DNA sequence encoding a signal peptide sequence and the remaining vector.
  • ompA and phoA are signal peptide sequences which are encoded by DNA sequences identical to or derived from the Escherichia coli (E. coli ) ompA and phoA loci.
  • the ompA locus is the structural gene for an E. coli outer membrane protein
  • the phoA locus is the structural gene of E. coli alkaline phosphatase.
  • the remaining vector must, of course, contain an origin of replication, for example, a colEI origin of replication.
  • the expression-secretion vectors may also include other DNA sequences known in the art, for example, stability leader sequences which provide for stability of the plasmid, transcription termination sequences, regulatory sequences which allow expression-secretion of the structural gene to be modulated (e.g., by the presence or absence of nutrients or other inducers in the growth medium), marker sequences (e.g., for ampicillin and kanamycin resistance) which are capable of pro ⁇ viding phenotypic selection in transformed host cells, and sequences which provide sites for cleavage by restriction endonucleases.
  • stability leader sequences which provide for stability of the plasmid
  • transcription termination sequences e.g., regulatory sequences which allow expression-secretion of the structural gene to be modulated (e.g., by the presence or absence of nutrients or other inducers in the growth medium)
  • marker sequences e.g., for ampicillin
  • the characteristics of the actual expression-secretion vector used must be compatible with the host cell which is to be employed.
  • the expression-secretion vector should contain DNA sequence (e.g., the T7 promoter) capable of functioning in that system.
  • An expression-secretion vector as contemplated by the present invention is capable of directing the replication and the expression of DNA sequences encoding the variable domains of the immunoglobulin heavy and light chains or single chain Fv molecules.
  • Suitable expression-secretion vectors containing the desired coding and control sequences may be constructed using standard recombinant DNA techniques known in the art, many of which are described in Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
  • an integral component of the expression-secretion vectors of the present invention are DNA sequences coding for immuno ⁇ globulin V suspend and V- chains or for single chain Fv molecules.
  • DNA sequences can be generated in various ways.
  • the DNA sequences of the present invention coding for immunoglobulin V favor and j . chains or for single chain Fv molecules can be chemically synthesized.
  • chains or for single chain Fv molecules can be synthesized as a series of 100 base oligonucleo- tides that can then be sequentially ligated (via appropriate terminal restriction sites) so as to form the correct linear sequence of nucleotides [on the condition that the nucleotide sequences of the V render 11 and VL ⁇ i chains or single chain Fv molecules are known] .
  • DNA sequences coding for immunoglobulin V suspend and V, chains or for single chain Fv molecules can be generated using polymerase chain reaction (PCR). Briefly, pairs of synthetic DNA oligonucleotides at least 15 bases in length (PCR primers) that hybridize to opposite strands of the target (template) DNA sequence are used to enzymatically amplify the intervening region of DNA on the target sequence. Suitable template DNA sequences may be generated, for example, by isolating mRNA from a hybridoma of interest and reverse transcribing the mRNA.
  • Suitable PCR primers may be chemically synthesized, and may be designed by sequencing mRNA from a hybridoma of interest, by sequencing the antibody molecule itself and producing degenerate primers, or by using generic primers [See, Orlandi et al., Proc. Natl. Acad. Sci. USA 8(5, 3833 (1989); Sastry et al., Proc. Natl. Acad. Sci. USA 86, 5728 (1989)].
  • Suitable 5' primers include, for example, those based on mature termini of the immunoglobulin V favor and V- chains
  • suitable 3' primers include, for example, those based on the heavy and light chain J regions.
  • the present invention further concerns a host cell containing an expression-secretion vector capable of producing a biologically active Fv fragment comprising a DNA sequence encoding the T7 promoter, a DNA sequence encoding the variable domain of an immunoglobulin heavy chain, a DNA sequence encoding the variable domain of an immuno- globulin light chain, and one or more DNA sequences encoding one or more signal peptide sequences.
  • the present invention also concerns a host cell containing an expression vector capable of producing a biologically active single chain Fv molecule comprising a DNA sequence encoding a T7 promoter, a DNA sequence encoding a single chain Fv molecule and a DNA sequence encoding a signal peptide sequence.
  • the biologically active Fv fragment or single chain Fv molecule has authentic N-termini (i.e., the mature Fv fragment or single chain Fv molecule is generated by cleavage of the peptide bond between the carboxy terminus of the signal peptide sequence and the amino terminus of the variable domain of the immunoglobulin heavy or light chain).
  • expression-secretion vectors wherein the signal peptide sequences are ompA and phoA.
  • expression-secretion vectors wherein the DNA sequences encoding the signal peptide sequences have been modified to generate additional restriction enzyme sites without changing the amino acid sequences of the signal peptide sequences.
  • host cells containing an expression-secretion vector capable of producing a biologically active Fv fragment comprising a DNA sequence encoding the T7 promoter operatively linked to a DNA sequence encoding the variable domain of an immunoglobulin heavy chain, a DNA sequence encoding the variable domain of an immunoglobulin light chain, and one or more DNA sequences encoding one or more signal peptide sequences.
  • Suitable host cells include Escherichia coli cells, such as Escherichia coli MC1061 cells.
  • Other suitable E. coli strains include GM-1, SG-935 and 1023.
  • Particularly preferred host cells are those containing an integrated copy of the T7 RNA polymerase gene, such as E. coli strains JM109 DE3 and BL21/DE3/pLysS.
  • the expression-secretion vectors of the present invention may be introduced into host cells by various methods known in the art. For example, transformation of host cells with expression- secretion vectors can be carried out as described in Maniatis et al., supra. However, other methods for introducing expression-secretion vectors into host cells, for example, electroporation, liposomal fusion, or viral or phage infection can also be employed.
  • Host cells producing active Fv fragments or single chain Fv molecules and which contain an expression-secretion vector comprising a DNA sequence encoding the T7 promoter, a DNA sequence encoding the variable domain of an immunoglobulin heavy chain, a DNA sequence encoding the variable domain of an immunoglobulin light chain, and one or more DNA sequences encoding one or more signal peptide sequences, or which contain an expression-secretion vector comprising a DNA sequence encoding a T7 promoter, a DNA sequence encoding a single chain Fv molecule and a DNA sequence encoding a signal peptide sequence can be identified by one or more of the four general approaches: (a) DNA-DNA hybridization; (b) the presence or absence of marker gene functions; (c) assessing the level of transcription as measured by production of immunoglobulin V render or V L chain or single chain Fv molecule mRNA transcripts in the host cells; and (d) detection of the gene product biologically.
  • the presence of DNA sequences coding for immunoglobulin V secret or V L chains or single chain Fv molecules can be detected by DNA-DNA or RNA-DNA hybridization using probes complementary to the DNA sequences.
  • the recombinant expression-secretion vector host system can be identified and selected based upon the presence or absence of certain marker gene functions (e.g. , ampicillin and kanamycin resistance to antibiotics).
  • a marker gene can be placed in the same plasmid as the DNA sequence coding for the immunoglobulin V favor or V_ chains or single chain Fv molecule under the regulation of the same or a different promoter used to regulate the immunoglobulin V H or V L chain or single chain Fv molecule coding sequences. Expression of the marker gene can be used to select for cells harbouring the plasmid containing the DNA sequences coding for the immunoglobulin V favor or V. chain or single chain Fv molecule.
  • the production of immunoglobulin V or V. chain or single chain Fv molecule mRNA transcripts can be assessed by hybridization assays. For example, total RNA can be isolated and analyzed by Northern blotting or nuclease protection assay using a probe complementary to the RNA sequence.
  • the expression of immunoglobulin V render or V- chains or single chain Fv molecules can be assessed biologically, for example, by Western blotting or binding to antigen, or by sequencing of the protein product.
  • Fv fragments or single chain Fv molecules may be used in the same manner as the full length antibody molecules from which they are derived. For example, they may be used for in vivo and in vitro immunological diagnostic procedures, and may be used thera-plastically, either alone or after conjugation to drugs and toxins. They may also be used for structural studies, for example, using nuclear magnetic resonance (NMR) and X-ray crystallography. If desired, the Fv fragments or single chain Fv molecules produced in this manner may be isolated and purified to some degree using various protein purification techniques. For example, chromatographic procedures such as ion exchange chromatography, gel filtration chro atography and im unoaffinity chromatography may be employed.
  • DNA sequences of expression-secretion vectors, plasmids or DNA molecules of the present invention may be determined by various methods known in the art. For example, the dideoxy chain termination method as described in Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977), or the Maxam- Gilbert method as described in Proc. Natl. Acad. Sci. USA 74, 560-564 (1977) may be employed.
  • the methodology described herein can be used to prepare Fv fragments or single chain Fv molecules derived from animal species other than mice, and Fv fragments or single chain Fv molecules for a wide variety of different antigens, for example, digoxin and fibrin. It should also be understood that the methodology described herein can be used in the production of modified Fv fragments or single chain Fv molecules.
  • the DNA sequences coding for the variable domain of the immunoglobulin heavy chain, or the variable domain of the immunoglobulin light chain, or both, or for the single chain Fv molecule can be modified (i.e., mutated) to prepare various mutations that change the amino acid sequence encoded by the mutated codon.
  • modified DNA sequences may be prepared, for example, by mutating the DNA sequences coding for the variable domain of the immunoglobulin heavy chain, or the variable domain of the immunoglobulin light chain, or both, or for the single chain Fv molecule, so that the mutation results in the deletion, substitution, insertion, inversion or addition of one or more amino acids in the encoded polypeptide using various methods known in the art. For example, the methods of site-directed mutagenesis described in Taylor, J. W. et al., Nucl. Acids Res. 13, 8749-8764 (1985) and Kunkel, J. A., Proc. Natl. Acad. Sci. USA 82 ⁇ , 482-492 (1985) may be employed.
  • kits for site-directed mutagenesis may be purchased from commercial vendors.
  • a kit for performing site- directed mutagenesis may be purchased from Amersham Corp. (Arlington Heights, IL).
  • Contemplated modifications include, for example, humanization of Fv fragments derived from mice. See, Jones et al., Nature 321, 522 (1986). All such variations are included within the scope of the present invention.
  • modified when referring to a nucleotide or polypeptide sequence, means a nucleotide or polypeptide sequence which differs from the wild-type sequence found in nature.
  • Antidigoxin monoclonal antibody 26-10 is a high affinity (5 x 10 9 M- 1 ) antibody produced against digoxin conjugated to bovine serum albumin (Mudgett-Hunter et al. Mol. Immunol. 2_2, 477 [1985]).
  • cDNA clones of the genes encoding the Vr_l and V_ portions of the 26-10 antibody were made by PCR amplification of cDNA generated by reverse transcription of mRNA isolated from the 2610 hybridoma and sequenced by the dideoxy chain termination method ( Figures 1-A [SEQ. ID NO. 1] and 1-B [SEQ. ID NO. 2]). The DNA sequences were compared to genomic 2610 sequences [See, Near, R. I. et al., Mol. Immunol., 27, 901-909 (1990)] to verify that the authentic genes encoding 26-10 had been cloned.
  • a T7 promoter based expression-secretion vector was made through modification of the pT7-7 plasmid described in Tabor, S. et al., Proc. Natl. Acad. Sci. USA 82 ⁇ , 1074 (1985).
  • the restriction sites in the polylinker region of pT7-7 were altered in such a way that convenient restriction sites were available for cloning DNA fragments containing both 26-10 V H and V_ and their respective signal sequences ( Figure 2 [SEQ. ID NO. 3 and SEQ. ID NO. 4]) on the same plasmid as an artificial operon.
  • the signal sequences, ompA (Mowa et al., J. Biol. Chem.
  • the initial step in the construction of the expression-secretion vector was the previously mentioned modification of the pT7-7 polylinker region.
  • the pT7-7 plasmid was cut with BamHl and Sail, then filled in with Klenow to effectively destroy the BamHl, Sail and Xbal sites in the pT7-7 polylinker.
  • the resulting vector was then cut with Xbal and filled in with Klenow to destroy the Xbal site upstream of the ribosome binding site.
  • This plasmid was then cut with Smal and ligated using T4 DNA ligase with an Xbal linker to generate the pT7-ll vector.
  • the pT7-ll plasmid was cut with Ndel and EcoRl and ligated using T4 DNA ligase with an Ndel/EcoRl fragment containing the ompA signal sequence, to generate the pT7-ll OmpA vector.
  • the pT7-ll OmpA plasmid was then cut the Nrul and EcoRl and ligated using T4 DNA ligase in frame with a fragment encoding the V favor chain of 2610 generated by PCR using as template mRNA isolated from the 2610 hybridoma and the following oligonucleotide primers: 5' Primer
  • the phoA signal sequence was PCR amplified and cloned as a Ndel/EcoRl fragment into unmodified pT7-7.
  • the 26-10 V- DNA was amplified by PCR using the oligonucleotide primers indicated below to generate a fragment suitable for cloning into the pT7phoA expression-secretion vector: 5' Primer
  • V.pD-Xbal plasmids were then used to construct the FvpD expression-secretion vector.
  • the V.pD-Xbal plasmid was cut with Xbal and Hindlll to release the light chain containing the signal peptide sequence but lacking the T7 promoter sequence. This fragment was then ligated using T4 DNA ligase with Xbal/Hindlll cut V tensionpD to yield FvpD ( Figure 3D).
  • Gpl-2 a second compatible plasmid called Gpl-2 (Tabor et al., supra), which contains the T7 RNA polymerase gene under the control of the temperature sensitive lambda cl repressor and the kanamycin resistance gene, was co-transformed as described in Maniatis et al., supra, with FvpD into MC1061 E. coli cells using selection for both ampicillin and kanamycin resistant transformants.
  • MC1061 cells may be obtained from Clontech (Palo Alto, CA) or the American Type Culture Collection (Rockville, MD).
  • the cells containing the Gpl-2 plasmid are shifted to 42°C for thirty minutes this inactivates the temperature sensitive repressor protein and permits expression of the T7 R ⁇ A polymerase gene.
  • the T7 R ⁇ A polymerase protein is then able to promote transcription of the 26-10 V fi and V L genes by utilizing the T7 promoter present upstream of the two genes.
  • the cells were then shifted to 25°C for 30 minutes to facilitate the proper processing and assembly of the V ⁇ H and V ⁇ Li polypeptides. As shown in Figure 4
  • the 26-10 Fv was purified by affinity chromatography of the periplasmic fraction on a ouabain-Sepharose affinity column (ouabain is a digoxin congener).
  • the periplasmic fraction was harvested by osmotic shock as described in Skerra et al., Science 240, 1038 (1988). All steps were performed on ice or at 4°C. After induction, the cells from a 1 liter culture were harvested by centrifugation at 4000 x g for 10 minutes. The cell pellet was suspended in 10 ml of TES buffer (0.2 M Tris HCl pH 8.0, 0.5 mM EDTA, 0.5 M sucrose).
  • the suspended cells were then subjected to osmotic shock by the addition of 15 mis of diluted TES (TES diluted 1:4 with H 2 0) to release the proteins present in the periplasmic space. After a 30 minute incubation on ice, the cells were removed by successive centrifugations of 5000 x g for 10 minutes and 38,000 g for 15 minutes. The supernatant containing the periplasmic fraction was then subjected to affinity chromatography. Upon elution of the bound material with 20 mM ouabain, fractions 3 and 4 ( Figure 4, lanes 1 and 2) revealed two polypeptides of the correct size that were selectively purified.
  • TES diluted 1:4 with H 2 0
  • protease deficient strains as host, by optimizing fermentation conditions, by using alternative signal sequences, and by co-expressing enzymes and chaperones (e.g., heavy chain binding protein [BIP] ) that are normally employed for immunoglobulin chain assembly in mammalian cells.
  • the 26-10 Fv made by this method is stable for at least a two months, and probably longer, when stored at 4°C at nM range protein concentrations.
  • a second method was also used to express 26-10 Fv from FvpD.
  • the FvpD plasmid was transformed into E. coli strain
  • JM109/DE3 [Promega; See also, Studier, F.W. et al., Methods in Enzymology 185, 60-88 (ed. D.V. Goeddel) Academic Press (1990)].
  • JM109/DE3 contains an integrated copy of the T7 RNA polymerase gene under the control of a lac promoter.
  • JM109/DE3 cells harboring the FvpD plasmid were grown until the A600nm of the cells measured between 1.0 and 2.4 in modified 2 x YT medium (2% bacto tryptone, 1% yeast extract, 0.5% sodium chloride, 0.2% glycerol, 50 M potassium phosphate pH 7.2) with glucose (0.4%), ampicillin (50 mg/liter) at 37°C.
  • IPTG isopropyl beta-D-thiogalactoside
  • osmotic shock supernatants the periplasmic fractions from IPTG-treated JM109/DE3 cells containing the FvpD plasmid
  • MC1061/Gpl-2 cells the proteins that comigrated with proteins found in heat-treated MC1061/Gpl-2 cells. These two polypeptides appeared to be greatly enriched in the osmotic shock supernatant.
  • 26-10 Fv was purified from the osmotic shock supernatant using a ouabain-Sepharose column (see Example 3), two polypeptides were isolated of the approximate sizes expected for the 26-10 V £ society1 and VL r i chains (15 and 12 kD).
  • the yield of affinity purified 26-10 Fv from the JM109/DE3 strain was 14 mg/liter.
  • Example 5 Expression of Biologically Active Single Chain Fv
  • the expression systems of the present invention were also used to express biologically active single chain Fv (sFv) molecules.
  • the sFv form of the 26-10 antibody was constructed by PCR amplification with mutagenic oligonucleotides to create novel restriction sites (and to insert sequences encoding a peptide linker between the two chains.) Briefly, as summarized in Figure 5, the genes encoding the variable regions of the light (V- ) and heavy (V preference) chains were separately PCR amplified under the conditions described in Example 2 using as a template the cDNA clones of the genes encoding the V H and V L portions of the 26-10 antibody (see Example 1) and the following oligonucleotide primers: 3' V L 26-10 Sequence Overlap Extension (SOE)
  • oligonucleotides had complementary sequences, and included sequences encoding the peptide linker engineered between the V. and V preparation chains, so that the V and V makeup sequences could later, after a second round of PCR amplification, form a complete double stranded DNA molecule encoding a single chain Fv molecule containing a 15 amino acid linker with the following sequence:
  • This DNA construct was designated PCR amplified 26-10 sFv.
  • the PCR amplified 26-10 Fv and the plasmid designated pT7PhoA (See Example 2) were both cut with the restriction enzymes BstE2 and Sail and ligated, resulting in the plasmid designated pT7PhoA26-10sFv.
  • This plasmid encodes a single chain protein with the following domains (going from the N-terminus to the C-terminus: PhoA leader - 26-10 variable light chain- linker- 26-10 variable heavy chain) (See Figure 6 [SEQ. ID NO. 18] for the DNA and encoded amino acids sequences of this construct).
  • the pT7PhoA26-10sFv plasmid was transformed by the CaCl 2 method ( See, Maniatis et al., supra) into E. coli strain BL21 DE3/pLysS [ See, Studier, F.W. et al., Methods in Enzymology 185, 60-88 (ed. D.V. Goeddel) Academic Press (1990)].
  • AGC AGA GTG GAG GCT GAA GAT CTG GGA ATT TAT TTC TGC TCT CAA ACT Ser Arg Val Glu Ala Glu Asp Leu Gly He Tyr Phe Cys Ser Gin Thr

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Abstract

Cette invention concerne des vecteurs d'expression-sécrétion capables de produire des fragments Fv ou des molécules Fv monocaténaires biologiquement actifs, des cellules hôtes contenant ces vecteurs d'expression-sécrétion, ainsi que des procédés de production de fragments Fv ou de molécules Fv monocaténaires biologiquement actifs.
PCT/US1991/007625 1991-12-18 1991-12-18 Vecteurs d'expression-secretion servant a produire des fragments fv biologiquement actifs WO1993012246A1 (fr)

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JP05510854A JP2000505641A (ja) 1991-12-18 1991-12-18 生物学的に活性なｆｖフラグメント生産用発現分泌ベクター
EP92923150A EP0618977A4 (en) 1991-12-18 1991-12-18 Expression-secretion vectors for the production of biologically active fv fragments.
PCT/US1991/007625 WO1993012246A1 (fr) 1991-12-18 1991-12-18 Vecteurs d'expression-secretion servant a produire des fragments fv biologiquement actifs

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PCT/US1991/007625 WO1993012246A1 (fr) 1991-12-18 1991-12-18 Vecteurs d'expression-secretion servant a produire des fragments fv biologiquement actifs

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WO1993012246A1 true WO1993012246A1 (fr) 1993-06-24

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EP (1) EP0618977A4 (fr)
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WO1997015601A1 (fr) * 1995-10-20 1997-05-01 Novartis Ag Procede de preparation d'un fragment d'anticorps fv a simple chaine

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DE3744595A1 (de) * 1987-12-31 1989-07-13 Andreas Dr Plueckthun Verfahren zur gentechnischen herstellung von antikoerpern
AU4507589A (en) * 1988-10-11 1990-05-01 United States of America, as represented by the Secretary, U.S. Department of Commerce, The New plasmid constructions for high level production of eukaryotic proteins
JPH03187383A (ja) * 1989-09-04 1991-08-15 Takeda Chem Ind Ltd 発現プラスミドおよびその用途

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Methods in Enzymology, Volume 185, issued June 1990, STUDIER et al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes", pages 60-89, see entire article. *
Proceedings of the National Academy of Sciences, Volume 82, issued February 1985, TABOR et al., "A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes", pages 1074-1078, see entire article. *
Proceedings of the National Academy of Sciences, Volume 85, issued August 1988, HUSTON et al., "Protein Engineering of antibody binding sites: Recovery of specific activity in an anti-dioxin single-chain Fv analogue produced in Escherichia coli", pages 5879-5883, see entire article. *
Science, Volume 240, issued 20 May 1988, BETTER et al., "Escherichia coli Secretion of an Active Chimeric Antibody Fragment", pages 1041-1043, see entire article. *
Science, Volume 240, issued 20 May 1988, SKERRA et al., "Assembly of a Functional Immunoglobulin Fv Fragment in Escherichia coli", pages 1038-1040, see entire article. *
Science, Volume 242, issued 21 October 1988, BIRD et al., "Single-Chain Antigen-Binding Proteins", pages 423-426, see entire article. *
See also references of EP0618977A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015601A1 (fr) * 1995-10-20 1997-05-01 Novartis Ag Procede de preparation d'un fragment d'anticorps fv a simple chaine

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EP0618977A4 (en) 1997-02-12
EP0618977A1 (fr) 1994-10-12
JP2000505641A (ja) 2000-05-16

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