EP0618977A1

EP0618977A1 - Expression-secretion vectors for the production of biologically active fv fragments

Info

Publication number: EP0618977A1
Application number: EP92923150A
Authority: EP
Inventors: Shi Chung Ng; James G. Anthony; Sui-Lam Wong
Original assignee: University of Calgary
Current assignee: University of Calgary
Priority date: 1991-12-18
Filing date: 1991-12-18
Publication date: 1994-10-12
Also published as: JP2000505641A; EP0618977A4; WO1993012246A1

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.The present invention relates to expression-secretion vectors capable of producing biologically active single-stranded Fv fragments or Fv molecules, host cells containing these expression-secretion vectors, and methods of producing single-stranded Fv fragments or Fv molecules. biologically active.

Description

EXPRESSION-SECRETION VECTORS FOR THE PRODUCTION OF BIOLOGICALLY ACTIVE FV FRAGMENTS

BACKGROUND OF THE INVENTION 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„) and variable light (V.) chains. 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. Some groups have tried to express the individual V„ and V- chains and reassociate the chains in vi tro . The production of these proteins intra- cellularly resulted in insoluble proteins which had to be denatured and renatured to generate functional antibody. It is not clear what the exposure to denaturants will do to native antibody structure and hence these systems are less than ideal. More recently, the production of soluble native Fv (Skerra and Pluckthun, Science 24 , 1038 [1988]) and other related fragments (Better et al., Science 240, 1041 [1988]) has been reported. However, the yields of native Fv reported in these systems are quite low (0.2 mg/liter of cells) for many practical applications (e.g., isotope labeling for 3-D NMR analysis). It would be useful to have improved expression-secretion systems for the production of biologically active Fv fragments and single chain Fv molecules.

SUMMARY OF THE INVENTION The present invention concerns expression- secretion systems for the production of biologically active Fv fragments and single chain Fv molecules.

In particular, 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„), 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. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the nucleotide and deduced amino acid sequences of the (A) V_β [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_HpD; (B) Construction of plasmid _LpD; (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 5¹ and 3' non-coding sequences are shown.

DETAILED DESCRIPTION OF THE INVENTION 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. Preferably, 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). Further preferred are expression- secretion vectors wherein the signal peptide sequences are ompA and phoA. Additionally preferred are 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. Also preferred is 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. As used in this context, the term "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.

As used in the present application, the term "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.

As used in the present specification, the term "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.

As used in the present specification, the terms "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. However, 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, and 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. The characteristics of the actual expression-secretion vector used must be compatible with the host cell which is to be employed. For example, when cloning in a bacterial system, 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.

Particularly preferred are the expression- secretion vectors designated FvpD or pT7PhoA

26-10sFv, described herein below, or expression- secretion vectors with the identifying characteristics of FvpD or pT7PhoA26-10sFv. 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).

Of course, an integral component of the expression-secretion vectors of the present invention are DNA sequences coding for immuno¬ globulin V„ and V- chains or for single chain Fv molecules. Such DNA sequences can be generated in various ways. In one approach, the DNA sequences of the present invention coding for immunoglobulin V„ and _j. chains or for single chain Fv molecules can be chemically synthesized. For example, DNA sequences coding for immunoglobulin V„ and V. 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„ 11 and VL_τi chains or single chain Fv molecules are known] .

In a second approach, DNA sequences coding for immunoglobulin V„ 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„ and V- chains, and suitable 3' primers include, for example, those based on the heavy and light chain J regions. Repeated cycles of heat denaturation of the template, annealing of the primers and extension of the 3'-termini of the annealed primers with a DNA polymerase results in amplification of the segment defined by the 5¹ ends of the PCR primers. See, U.S. Patent Nos. 4,683,195 and 4,683,202.

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. Preferably, 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). Further preferred are expression-secretion vectors wherein the signal peptide sequences are ompA and phoA. Additionally preferred are 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. Also preferred are 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„ or V_L chain or single chain Fv molecule mRNA transcripts in the host cells; and (d) detection of the gene product biologically. In the first approach, the presence of DNA sequences coding for immunoglobulin V„ 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. In the second approach, 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„ 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„ or V. chain or single chain Fv molecule.

In the third approach, 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.

In the fourth approach, the expression of immunoglobulin V„ 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.

Once an expression-secretion vector has been introduced into an appropriate host cell, the host cell may be cultured under conditions permitting expression of large amounts of Fv fragments or single chain Fv molecules. Such 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- peutically, 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.

The 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.

It should be understood that 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. In this case, 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. These 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. In addition, kits for site-directed mutagenesis may be purchased from commercial vendors. For example, 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.

As used above and elsewhere in the present application, the term "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.

The following examples are further illustrative of the present invention. These examples are not intended to limit the scope of the present invention, and provide further understanding of the invention. Exa ple 1

Cloning of Genes Encoding Antibody Fragments

Antidigoxin monoclonal antibody 26-10 is a high affinity (5 x 10⁹ 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.

Example 2

Construction of T7 Promoter Based Expression-Secretion Vec

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. 255, 27 [1980]) and phoA (Inouye et al., J. Bacteriol. 149, 434 [1982]) were engineered such that novel restriction sites were generated without changing the amino acid sequence of the signal peptides. Hence, correct processing would be expected to generate the authentic N-terminal sequences of both chains, and for the proteins to be secreted into the periplasmic space. These modified signal sequences were made by PCR using E. coli chromosomal DNA as template. For ompA, the following oligonucleotides were used as PCR primers: 5' Primer

5^»-AACATATGAAAAAGACAGCTATCGCCATT-3^■ [SEQ. ID NO. 5] 3^f Primer 5^f-GAATTCGGCCTGCGCAACGGTCGCGAAACCAGCTAGCGCCACTGC-3 ' [SEQ. ID NO. 6]. For phoA, the following oligonucleotides were used as PCR primers: 5' Primer 5^•-AACATATGAAACAAAGCACTATTGCACTGGCA-3^» [SEQ. ID NO. 7] 3' Primer

5ⁱ-GAATTCGGCCTTGGTCACCGGGGTAAACAGTAA-3' [SEQ. ID NO. 8] The modified signal sequences are shown in Figure 2 [SEQ. ID NO. 3 AND SEQ. ID NO. 4]. In both cases, and for all PCR procedures, PCR was performed using a GeneAmp Kit (Perkin-Elmer Cetus, Norwalk, CT) as recommended by the manufacturer.

The initial step in the construction of the expression-secretion vector was the previously mentioned modification of the pT7-7 polylinker region.

In order to generate a plasmid containing the 26-10 V„ region (see Figure 3A), 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„ chain of 2610 generated by PCR using as template mRNA isolated from the 2610 hybridoma and the following oligonucleotide primers: 5' Primer

5'- AACATATGTTCGCGACCGTAGCGCAGGCCGAGGTCCAGCTGCAACAGTCCGGA [SEQ. ID NO. 9] 3' Primer

5'- TTGAATTCTTATTATGAGGAGACGGTGACTGAGGCTCC-3' [SEQ. ID NO. 10] Oligonucleotides used for the amplification generated Nrul and EcoRl sites at the 5* and 3' ends, respectively, of the amplified V„ fragment. The resulting plasmid was designated V_Hp .

In order to generate a plasmid containing the 26-10 V_ region (see Figure 3B), 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

5'-AACATATGACCAAGGCCGATGTTGTGATGACCCAAACTCCA-3 [SEQ. ID NO. 11]

3¹ Primer

5^•-TTCTGCAGTTATTACCGTTTGATTTCCAGCTTGGTGCC-3' [SEQ. ID NO. 12] The amplified 26-10 V- fragment was cut with Styl and ligated using T4 DNA ligase with the pT7phoA plasmid which had been cut with Styl and Smal, resulting in the ligation of the 26-10 v_τ as a Styl/blunt ended fragment. The resulting plasmid was designated V-pD. The V.pD plasmid was then cut with BamHl and Sail, filled in with Klenow and religated with T4 DNA ligase to generate plasmid V.pD-Xbal (Figure 3C).

The V„pD and 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„pD to yield FvpD (Figure 3D).

The final construct (FvpD) as well as the intermediate constructs were sequenced by dideoxy DNA sequencing to assure no alterations affecting amino acid sequences had occurred during their construction/amplification. Example 3 Expression of 26-10 Fv from Plasmid FvPD

To express 26-10 Fv from FvpD, 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 transformants were inoculated into 2 x YT medium containing 20 μg/ml kanamycin and 50 μg/ml ampicillin, and grown at 25°C to OD₆oo = 2.0 prior to induction of 26-10 Fv. When 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 RΝA polymerase gene. The T7 RΝA 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_π H and V_τLi polypeptides. As shown in Figure 4

(lane 7), temperature induced cells containing both the Gpl-2 and FvpD plasmids expressed two polypeptides that migrated with apparent molecular weights of 12 kd and 15 kd. The size of the 12 kd polypeptide was essentially identical to the size of the 26-10 V L_τi chain (12.2 kd) predicted from the polypeptide encoded by the 26-10 V. DNA sequence. The 15 kd polypeptide appeared to migrate slower than the predicted size (13.2 kd) of the polypeptide encoded by the 26-10 V_β DNA sequence. These two polypeptides also appeared to be properly localized as they were found to be greatly enriched in the periplasmic fraction. 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 H₂0) 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. The following evidence indicated that this is 26-10 Fv: (1) It competed with ¹²⁵I-26-10 whole antibody in com¬ petitive RIA assays; (2) It bound ¹²⁵I-digoxin with =1.3 x 10⁹ M~^x compared to 5 x 10⁹ M^"1 for 26-10 whole antibody; (3) N-terminal sequencing of V L_ri and Vn„. variable domains indicated correct processing and expected N-terminal sequences. The yield of purified Fv was demonstrated to be 1 mg/L. It should be possible to further improve the yield by using 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.

Example 4 Alternate Expression of 26-10 Fv from FvpD

A second method was also used to express 26-10 Fv from FvpD. In this method, 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. The cells were then cooled to 24°C. Subsequently, isopropyl beta-D-thiogalactoside (IPTG) was added to a final concentration of 0.05 mM to induce transcription of the T7 RNA polymerase gene. After the addition of IPTG, the cells were allowed to incubate at 24°C for 16 hours, and screened for periplasimic proteins.

It was found that osmotic shock supernatants (the periplasmic fractions from IPTG-treated JM109/DE3 cells containing the FvpD plasmid) contained two 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. When 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 £„1 and VL_ri chains (15 and 12 kD). The yield of affinity purified 26-10 Fv from the JM109/DE3 strain was 14 mg/liter. In the

JM109/DE3 strain, the maximum level of 26-10 Fv accumulation was observed approximately 16 hours after induction, while in MC1061/GP1-2 (Example 3), the maximum level of 26-10 Fv accumulation occurred one hour after the start of induction.

Coomassie staining of protein fractions separated by SDS-PAGE indicated that in both JM109/DE3 and MC1061/Gpl-2 (Example 3), most of the 26-10 Fv protein was found in the periplasm. N-terminal protein sequence analysis of the JM109/DE3 produced protein revealed that both the V„ (approximately 15 kD) and V_L (12 kD) chains had been correctly processed by the bacterial export system. In both the JM109/DE3 and MC1061/Gpl-2 (Example 3) strains, it was necessary to cool the cells to 25 C after the protein inductions. Incubation at temperatures exceeding 27 C resulted in the accumulation of proteins of approximately 17 kD and 12 kD that did not bind to the ouabain-Sepharose column.

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„) 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)

5^«-AGAGCCGGATCCACCGGAACCGGAGCCGCCAGAACCAGAACCACCCCGTTTGATT CAGCTTGGT-3' [SEQ. ID NO. 13] 5^! VL_τi 26-10 BstE2 (^sfor PhoA *pT7) 5^«-CCATCGGTGACCAAAGCCGATGTTGTGATGACCCAAACT-3' [SEQ. ID NO. 14] 5¹ V_p 26-10 SOE

5'-GGTGGTTCTGGTTCTGGCGGCTCCGGTTCCGGTGGATCCGGCTCTGAGGTCCAGCT CAACAGTCC-3' [SEQ. ID NO. 15] V_H 26-10 Sail

5'-CCCGTCGACCTGCAGGCATGCGGATCCTTATGAGGAGACGGTGACTGAGGCTCC- [SEQ. ID NO. 16]

These oligonucleotides had complementary sequences, and included sequences encoding the peptide linker engineered between the V. and V„ chains, so that the V and V„ 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:

-Gly-Gly-Ser-Gly-Ser-Gly-Gly-Ser-Gly-Ser-Gly-Gly-Ser- Gly-Ser

[SEQ. ID NO. 17] 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 CaCl₂ 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)]. Cells harboring the pT7PhoA26-10sFv plasmid were grown overnight in Minimal Medium (7.6 mM NH.S0₄, 11.0 mM sodium acetate, 12.7 mM succinic acid, 60.3 mM K 3PO., 68 μM CaCl₂.2H₂0, 35 μM ZnS0₄.7H₂0, 59 μM MnS0₄.H₂0, 741 μM thia in, 2032 μM niacin, 12 μM biotin, 40 μM FeCl₃.6H₂0, 3 μmM Na₂MθO₄.2H₂0, 3 μM CuS0₄.5H₂0, 3 μM H₃B0₃, 3 μM vitamin B-12, 4 mM

MgS0₄, 22 mM glucose, 50 μg/ml ampicillin, 20 μg/ml chloramphenicol) at 37°C, then diluted 1:20 into 2 x YT medium (as in Example 4) at 37°C and grown until the A_6Q0 of the cells measured between 0.5 and 1.0. The cells were then cooled to 24°C. Subsequently IPTG was added to a final concen¬ tration of 0.2 mM to induce transcription of the T7 RNA polymerase gene. After the addition of IPTG the cells were allowed to incubate at 24 C for 16 hours and screened for periplasmic proteins and proteins in the culture supernatant. Under these conditions, a yield of 3 to 10 mg of affinity purified 26-10 sFv protein per liter cell culture was obtained. The affinity purified material had a molecular weight of about 29 kd, as shown by SDS-PAGE, which is in good agreement with the theoretically predicted molecular weight of about 26 kD. The fact the 26-10 sFv protein is biologically active was shown by its ability to bind to and be purified using a ouabain-Sepharose affinity column (see Example 3).

All publications and patents referred to in the present application are incorporated herein by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: Ng, Shi Chung; Anthony, James G. ; Wong, Sui-Lam

(ii) TITLE OF THE INVENTION: Expression- Secretion Vectors for the Production of Biologically Active Fv Fragments

(iii) NUMBER OF SEQUENCES: 12

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Burton Rodney - Squibb

Corporation

(B) STREET: P.O. Box 4000

(C) CITY: Princeton

(D) STATE: New Jersey

(E) COUNTRY: USA

(F) ZIP: 08543-4000

(2) INFORMATION FOR SEQ. ID NO.: 1 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 357 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO.:l

GAG GTC CAG CTG CAA CAG TCC GGA CCT GAG CTG GTG AAG CCT GGG GCT Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15

TCA GTG AGG ATG TCC TGC AAG TCT TCT GGA TAC ATA TTC ACT GAC TTC Ser Val Arg Met Ser Cys Lys Ser Ser Gly Tyr lie Phe Thr Asp Phe 20 25 30

TAC ATG AAC TGG GTG AGG CAG AGC CAT GGA AAG AGC CTT GAT TAC ATT Tyr Met Asn Trp Val Arg Gin Ser His Gly Lys Ser Leu Asp Tyr lie 35 40 45

GGA TAT ATT TCT CCT TAC AGT GGT GTT ACT GGC TAC AAC CAG AAG TTC Gly Tyr lie Ser Pro Tyr Ser Gly Val Thr Gly Tyr Asn Gin Lys Phe 50 55 60

AAA GGC AAG GCC ACA TTG ACT GTA GAC AAG TCC TCC AGC ACA GCC TAC 2 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80

ATG GAG CTC CGC AGC CTG ACA TCG GAG GAT TCT GCA GTC TAT TAC TGT 2 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

GCA GGA TCG TCG GGG AAT AAG TGG GCT ATG GAC TAC TGG GGT CAC GGA 3 Ala Gly Ser Ser Gly Asn Lys Trp Ala Met Asp Tyr Trp Gly His Gly 100 105 110

GCC TCA GTC ACC GTC TCC TCA 3 Ala Ser Val Thr Val Ser Ser 115

(2) INFORMATION FOR SEQ. ID NO.: (i) SE UENCE CHARACTERISTICS:

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO,

GAT GTT GTG ATG ACC CAA ACT CCA CTC TCC CTG CCT GTC AGT CTT GGA Asp Val Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15

GAT CAA GCC TCC ATC TCT TGC AGA TCT AGT CAG AGC CTT GTA CAC AGT Asp Gin Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser 20 25 30

AAT GGA AAT ACC TAT TTA AAT TGG TAC CTG CAG AAG GCA GGC CAG TCT Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gin Lys Ala Gly Gin Ser 35 40 45

CCA AAG CTC CTG ATC TAC AAA GTT TCC AAC CGA TTT TCT GGG GTC CCA Pro Lys Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60

GAC AGG TTC AGT GGC AGT GGA TCA GGG ACA GAT TTC ACA CTC AAG ATC Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80

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

85 90 95

ACA CAT GTT CCT CCG ACG TTC GGT GGA GGC ACC AAG CTG GAA ATC AAA Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys 100 105 110

CGG Arg (2) INFORMATION FOR SEQ. ID NO. : 3 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 68 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double stranded

(D) TOPOLOGY: circular

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO. : 3

AACAT ATG AAA AAG ACA GCT ATC GCC ATT GCA GTG GCG CTA GCT GGT Met Lys Lys Thr Ala He Ala He Ala Val Ala Leu Ala Gly 1 5 10

TTC GCG ACC GTT GCG CAG GCC

Phe Ala Thr Val Ala Gin Ala 15 20

(2) INFORMATION FOR SEQ. ID NO. : 4 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 68 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double stranded

(D) TOPOLOGY: circular

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO. : 4

AACAT ATG AAA CAA AGC ACT ATT GCA CTG GCA CTC TTA CCG TTA CTG Met Lys Gin Ser Thr He Ala Leu Ala Leu Leu Pro Leu Leu 1 5 10

TTT ACC CCG GTG ACC AAG GCC

Phe Thr Pro Val Thr Lys Ala 15 20

(2) INFORMATION FOR SEQ. ID NO. : 5 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO.: 5 AACATATGAA AAAGACAGCT ATCGCCATT (2) INFORMATION FOR SEQ. ID NO.: 6 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO.: 6

GAATTCGGCC TGCGCAACGG TCGCGAAACC AGCTAGCGCC ACTGC

(2) INFORMATION FOR SEQ. ID NO. : 7 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO.: 7

AACATATGAA ACAAAGCACT ATTGCACTGG CA

(2) INFORMATION FOR SEQ. ID NO. : 8 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO. : 8

GAATTCGGCC TTGGTCACCG GGGTAAACAG TAA

(2) INFORMATION FOR SEQ. ID NO. : 9 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 53 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO.: 9 AACATATGTT CGCGACCGTA GCGCAGGCCG AGGTCCAGCT GCAACAGTCC GGA 5 (2) INFORMATION FOR SEQ. ID NO.: 10 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO. : 10

TTGAATTCTT ATTATGAGGA GACGGTGACT GAGGCTCC 3

(2) INFORMATION FOR SEQ. ID NO.: 11 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO. : 11 AACATATGAC CAAGGCCGAT GTTGTGATGA CCCAAACTCC A 4

(2) INFORMATION FOR SEQ. ID NO.: 12 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single stranded

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ. ID NO. : 12 TTCTGCAGTT ATTACCGTTT GATTTCCAGC TTGGTGCC 38

Claims

CLAIMSWhat is claimed is:

1. 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, and 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.

2. An expression-secretion vector capable of producing a biologically active single chain Fv 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.

3. The expression-secretion vector according to Claim 1 wherein the Fv fragment has authentic N-termini.

4. The expression-secretion vector according to Claim 1 wherein the DNA sequences encoding the signal peptide sequences have been modified to generate addtional restriction enzyme sites without changing the amino acid sequences of the signal peptide sequences.

5. The expression-secretion vector according to Claims 1 wherein the signal peptide sequences are ompA and phoA.

6. The expression-secretion vector according to Claim 2 wherein the signal peptide sequence is phoA.

7. The expression-secretion vector according to Claim 1 wherein the Fv fragment has been modified by modifying either the DNA sequence encoding the variable domain of the immunoglobulin heavy chain or the DNA sequence encoding the variable domain of the immunoglobulin light chain or both.

8. 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.

9. A host cell comprising an expression- secretion vector according to Claims 1, 2, 3, 4, 5, 6, 7 or 8.

10. The host cell according to Claim 9 wherein the host cell contains a stably integrated copy of the T7 RNA polymerase gene.

11. The host cell according to Claim 10 wherein the host cell is E. coli strain JM109/DE3 or E. coli strain BL21/DE3/pLysS.

12. A method for producing a biologically active Fv fragment or single chain Fv molecule comprising culturing a host cell according to Claim 9 under conditions permitting expression of the biologically active Fv fragment or single chain Fv molecule.

13. A polypeptide molecule comprising a biologically active Fv fragment which specifically binds digoxin.