AU628310B2

AU628310B2 - A process for the preparation of antibodies by genetic engineering

Info

Publication number: AU628310B2
Application number: AU27617/88A
Authority: AU
Inventors: Andreas Dr. Pluckthun; Arne Skerra
Original assignee: Individual
Current assignee: Individual
Priority date: 1987-12-31
Filing date: 1988-12-30
Publication date: 1992-09-17
Anticipated expiration: 2008-12-30
Also published as: AU2761788A; DK732988A; IE62257B1; PT89362B; ES2063022T3; EP0324162A1; DE3888337D1; ATE102651T1; PT89362A; DK732988D0; KR890010203A; JPH02799A; IE883893L; DE3744595A1; JP2771204B2; ZA889711B; EP0324162B1; KR970007861B1; HUT49169A; HU212592B

Abstract

Functional antibody fragments can be produced in bacteria by coupling each of the genes for the individual chains to signal sequences which bring about the transport of the prepeptides through the cytoplasmic membrane, expressing the gene structures in a bacterium, and isolating the functional protein from the periplasmic space or the medium. The functional protein is advantageously isolated by affinity chromatography on an adsorbent which is loaded with the hapten or antigen, and elution with a solution of the hapten or antigen.

Description

To The Commissioner of Patents i; 9 'i i :n i i :iB~ 1~ Dr., Andreas PLuckthun.

Dipl.-Ing. Arne Skerri, HOE 87/S 025 Dr. KL/ST 0 0

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Description A process for the preparation of antibodies by genetic engineering The expression of antibodies in yeast has been described Wood, M.A. Boss, J.H. Kenten, J.E. Calvert, N.A.

Roberts and J.S. Emtage, Nature 314, 446 but only a very small proportion of the expressed protein proved to be functional. In E. coli, to date it has been possible to obtain antibody proteins only in denatured form Boss, J.H. Kenten, C.R. Wood and J.S. Emtage, Nucleic Acids Res. 12, 3791 (1984); S. Cabilly, A.D.

Riggs, H. Pande, J.E. Shively, W.E. Holmes, M. Rey, L.J.

Perry, R. Wetzel and H.L. Heyneker, Proc. Natl. Acad.

Sci. U.S.A. 81, 3273 (1984)). The purification of active antibodies or antibody fragments from yeast or other microorganisms has not been disclosed. Attempts at protein folding have to date resulted in only a very small percentage of correctly folded recombinant antibody proteins. In addition, it is difficult to separate the desired functional proteins from undesired and non-functional proteins, which impedes accurate measurement of binding constants, yields of folding, spectral properties 20 and the like, and use in therapy and industry. Optimization of the folding conditions is thus extremely difficult, especially since there are no proven processes and measurement methods for the refolding.

The expression of complete functional antibodies or functional binding domains of antibodies in bacterial expression systems has not been disclosed to date, and the prospects of bringing it about have been assessed pessimistically Morrison, Science 229, 1202 (1985), M.A. Boss and C.R. Wood, Immunol. Today 6, 12 (1985)).

Such a system would be very desirable because the genetic -2 engineering processes have been thoroughly worked out, especially for E. coli, and mass production is facilitated by the rapid growth, which is of considerable economic importance.

Hence the invention relates to the preparation of functional antibodies, functional fragments thereof, or fusion proteins composed of antibody domains and other proteins in bacteria, preferably Gram-negative bacteria, especially in E. coli. The process according to Lhe invention comprises coupling the genes for the individual chains of the antibody molecule or fragment each tc a signal sequence which brings about the transport of the polypeptide chains through the cytoplasmic membrane and which S 15 can be split off, bringing about the expression of the Sgene structures, and isolating the functional protein from the periplasmic space or the medium. Preferred enmbodiments of this invention are explained in detail hereinafter and defined in the patent claims.

The coupling of the genes for the individual chains pro-

S.

vided with signal sequences preferably takes place in the manner of a regulatable operon system which brings about simultaneous expression by a common control region.

25 In this way, the individual protein chains are expressed together in approximately the stoichiometric ratio, and are transported into the periplasmic space, where joining G*of' to form a functional molecule takes place. Proteins are S* transported out of the cytoplasm by methods known per se, as are described, for example, in European Patent Specification 0,006,694.

Suitable as control region is every suitable regulatable gene regulatory region, for example lac, tac, trp or synthetic sequences. Particularly preferred are regulatory regions which can be reliably turned off.

The proteins are preferably isolited Vrom the periplasmic -3space by exerting a mild osmotic shock on the harvested cells, and subjecting the liquid phase obtained by this to concentration by ultrafiltration or precipitation, for example with salts such as ammonium sulfate.

A "mild" osmotic shock brings about the ejection of the periplasm with, however, the cytoplasmic membrane remaining intact.

The protein concentrate is, expediently after a dialysis, applied to an adsorbent, advantageously in the form of an affinity column, which is loaded with the appropriate antigen or hapten. The antibody or the functional antibody fragment is then obtained by suitable elution, ad- 15 vantageously with the antigen or hapten.

In the eukaryotic cell, antibodies are formed in the Lumen of the endoplasmic reticulum probably with the cooperation of dr'sulfide isomerases, proline cis-transisomerases and p'ssibly other enzymes or proteins. It was surprising that the bacterial cell is also able to prepare the two Lhains in approximately the same stoichiometric amount, to transport the two precursor proteins into the periplasmic space or the medium surrounding it, to eliminate the signal sequences correctly, to fold the globular and soluble domains correctly, to form the intramolecular disulfide linkages, and to associate the two chains to give a heterodimer, because it is regarded as improbable that the bacterial cell is equipped with enzymes which are of this nature or have the same effect Thus, it has surprisingly emerged that, in the case of Gram-negative bacteria, the bacterial periplasm is in this respect functionally equivalent to the lumen of the eukaryotic endoplasmic reticulum.

The process according to the invention has a number of advantages: apart from the easy and low-cost large-scale bacterial 4fermentation already mentioned, there is direct formation of functional proteins, which thus means that the cLeavag? of fusion proteins, with subsequent isolation of the desired protein or protein fragment, its oxidation or in vitro refolding, are avoided.

Nor have problems with cellular proteases been found in the process according to the invention. It is, after all, known that, because of these cellular proteases, proteins are normally expressed in bacteria in the form of fusion proteins, especially as insoluble inclusion bodies, which, however, is associated with the stated elaborate further processing steps. In contrast, the separation and puri- 0* fication to homogeneity is rapid and straightforward in 15 the process according to the invention.

*o Thus the invention permits easy access to antibodies, functional fragments thereof and modified antibodies which S differ from the natural antibodies by the insertion, elimination and/or exchange of amino acids. Thus, for example, cysteines can be elir'inated or replaced by other S. amino acids in order to suppress undesired folding. It is possible in just the same way to convert a murine into a huma'n antibody, or to introduce other mutations. Thus, besides the possible pharmacological and industrial applications, access is facilitated for research into antibody structure and function and into the fundamentals of enzymatic catalysis Raso and B.D. Stollar, Biochemistry 14, 584 (1975); V. Raro end B.D. Stollar, Biochemistry 14, 591 (1975); A. Tramontano, K.D. Janda and R.A. Lerner, Science 234, 1566 (1986); S.J. Pollack, J.W. Jacobs and P.G. Schultz, Science 234, 1570 (1986); J. Jacobs, P.G.

Schultz, R. Sugasawara and M. Powell, J. Am. Chem. Soc.

109, 2174 (1987)).

The invention furthermore permits the application of test systems (assays) directly to the bacterial cell in which the functional antibody is formed, and thus rapid 1 5 investigation for possibly mutated antibodies.

Besides the stated variations in the antibody molecule by altering individual, or a few, amino acids, it is also possible to insert into the antibody gene other gene regions, or to exchange parts for non-critical gene regions. It is possible in this way to couple marker enzymes Neuberger, G.T. Williams and R.O. Fox, Nature 312, 604 (1984)), toxins Mller "Antibody carriers of drugs and toxins in tumor therapy", Immunol. Rev.

62, Munksgaard, Copenhagen (1982)) or immunoglobulin regions of another class Neuberger, G.T. Williams, E.B. MitcheLL, S.S. Jouhal, J.G. Flanagan, and T.H.

Rabbitts, Nature 314, 268 (1985)) or of another species Jones, P.H. Dear, J. Foote, M.S. Neuberger, and G.

Winter, Nature 321, 522 (1986)) to the antibody molecule.

a fee The process according to the invention is explained nereinafter taking the example of the variable domains of the phosphorylcholine-binding antibody myeloma protein The three-dimensional structure of this mouse immunoglobulin A is known Segal, E.A. Padlan, G.H. Cchen, S.

Rudikoff, M. Potter and D.R. Davies, Proc. Natl. Acad.

Sci. 71, 4298 (1974)). Synthetic genes for the variable light chain VL and heavy chain VH were used. Such syn- Sthetic genes are proposed in German Offenlegungsschrift 3,715,033 and published European Patent Application 0,290,005 and published Australian Patent Application 15631/88 (Tables 1 and 2 therein). A particularly expedient embodiment of such synthetic genes is shown in the Table, in which the DNA sequence of the complete expression plasmid is depicted, and the genes for the two chains are emphasized by indicating the amino acids.

Codons which are rarely used by E. coli were avoided in the construction of these DNA sequences. Furthermore, unique restriction enzyme cleavage sites were incorporated, and account was taken of tho secondary structure of the RNA.

i b I: i 0* 9 0 0@ 6

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S* 0 0 90 69 0 9* 0* 9*

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9 6 In the functional antibody fragment serving as model, each domain has an intramolecular disulfide bridge (from Cys 23 to Cys 94 in VL and from Cys 22 to Cys 98 in VH).

There is no disuLfide bridge between the chains, nor is there any free cysteine.

The expression vector used, pASK 22, is depicted diagrammatically in the following formula po ac Iri ompA-V

H

phoA-VL pASV 22 3.5 kb Ap r In it the synthetic genes for the VL and VH domains are coupled to gene fragments for the bacterial signal sequence of the outer membrane protein A (ompA) on the one hand, and of alkaline phosphatase (phoA) on the other hand. The genes for the two precursor proteins are located in a synthetic operon-like structure downstream of the lac promoter, which ensures that both genes undergo simultaneous induction, coexpression and cosecretion.

After induction of gene expression, the cells are harvested and exposed to a mild osmotic shock. The liquid

M

-7phase obtained by this, which contains the periplasmic proteins, is concentrated by ultrafiltration, dialyzed and applied directly to an affinity column which contains a phosphorylcholine derivative Chesebro and H. Metzger, Biochemistry 11, 766 (1972)) as affinity ligand. Elution with phosphorylcholine results in a homogeneous FV fragment which is gel electrophoretically homogeneous. It can be deduced from the SDS polyacrylamide gel that the two chains of the purified FV fragment are present in a molar ratio of 1:1, and the expected molecular weights of the mature proteins (VH: 13600 D, VL: 12400 D) are present. To demonstrate correct elimination of the two signal sequences, the six N-terminal amino acids both chains were sequenced. It emerged that both chains have the correct N-termini for the mature proteins. Thus, S both heterologous preproteins have been correctly cleaved by the bacterial signal peptidase, and there is no detectable evidence that imprecise processng or an N-terminal degradation reaction has taken place.

The affinity constant of the recombinant F V fragment of McPC603 was measured by equilibrium dialysis. The conditions used for this were the same as were applied in the determination of the affinity constant of the natural '25 antibody MCPC603 isolated from mouse ascites. The value of 1.21 0.06 x 105 M-1 found for the FV fragment is identical, within the accuracy of the experiment, to the value of 1.6 0.4 x 105 M-1 reported for the natural antibody. The Scatchard binding plot (Ann. N.Y.

Acacd. Sci. 51, 660 (1949)) is linear, and extrapolation indicates that approximately 1 mol of hapten is bound per mol of FV fragment. This confirms that there is only one type of binding site per Fy fragment, and that there are no inactive components present in the isolated protein.

Thus, it has emerged, surprisingly, that it is possible to prepare the F

V

fragment of the antibody McPC603 as 8 completely functional and stable protein in E coli. This demonstrates the functional equivalence of the transport into the periplasm of the bacterial cell with the transport into the lumen of the endoplasmic reticulum of the eukaryotic cell. This equivalence has not been disclosed or even suspected, because the bacterial protein which has hitherto been characterized best and which could be defined as soluble heterodimeric protein in the periplasm E. coli penicillin acylase is produced by proteolytic processing from a single-chain precursor in the periplasm Schumacher, D. Sizmann, H. Hang, P. Buckel and A.

Bock, Nucleic Acids Res. 14, 5713 k1986)). In addition, it has already been pointed out that, according to present knowledge, bacteria have no enzymes or proteins which 15 might in eukaryotes be involved in the folding.

It was also surprising that the FV fragment of McPC603 has essentially the same affinity constant for phosphorylcholine as has the intact antibody McPC603 itself. This finding is unexpected because the functionality of FV fragments is controversial in the literature Sen and S. Beychok, Proteins 1, 256 (1986)). It emerges from this 0 that the functionality can be maintained on modification or even complete deletion of the constant domains.

In contrast to the only method used hitherto for the preparation of FV fragments, namely by proteolysis o' an antibody, there are no problems in the process according to the invention with non-functional, incorrectly folded, incorrectly reassociated or chemically modified proteins.

Moreover, the preferred isolation process using an antigenor hapten-loaded adsorbent is also suitable for purifying those FV fragments obtained by known processes, because such impurities would either not be bound or not be eluted.

Example 1: Preparation of the plasmid pASK22: -9 The expression plasmid pASK22 is constructed from the large EcoRI-HindIII fragment of pUC12 Yanisch-Perron, J. Vieira, and J. Messing, Gene 33, 103 (1985)), from fragments of the vectors pIN III-OmpAl Masui, J.

Coleman and M. Inouye in "Experimental manipulation of gene expression", M. Inouye, ed., Academic Press 1983) and pHI61 Inouye, W. Barnes and J. Beckwith, J. Bacteriol.

149, 434 (1982)) as well as various synthetic DNA fragments, in several stages using methods known per se (T.

Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning, Cold Spring Harbor 1982). The complete DNA sequence of the resulting vector pASK22 is depicted in the Table.

The ligation mixture is used to transform competent E.

15 coli cells, and the latter are selected for ampicillin resistance. The plasmids with the desired gene structure are characterized by restriction analysis and sequencing of the critical junctions.

Example 2: Preparation of the FV fragment A culture of the E. coli strain W3110 transformed with S" pASK22 is cultivated in LB medium containing 100 mg/L ampicillin to an 00D 5 5 0 of 0.5. Expression is induced by addition of IPTG to a final concentration of ImM.

After 45 minutes, the cells are harvested by centrifugation at 4000 x g (10 minutes at 4 0 Cell fractiona- S tion is carried out by resuspension of the cell peLlet Sin TES buffer (0.2 M tris.HCL, pH 8.0; 0.5 mM EDTA; 0.5 M sucrose) in 10 ml/l of the original culture. The cells are exposed to a mild osmotic shock by addition of 15 ml/l of the original culture of TES buffer which is diluted 1:4 with water and contains 2 mM phosphorylcholine. After the suspension has been incubated on ice for 30 minutes it is centrifuged at 5000 x g for 10 minutes, and the supernatant is subjected to renewed centrifugation at 48000 x g for 15 minutes. The resulting supernatant, which contains all the soluble periplasmic proteins, is 10 concentrated by ultrafiltration ((R)AMICON YM5 membrane) to a volume of about 2.5 ml/l of the original culture, and is dialyzed against BBS buffer (0.2 M borate/NaOH, pH 8.0; 0.16 M NaCI). This concentrated solution is applied to an affinity column loaded with a phosphorylcholine derivative Chesebro and H. Metzger, loc.cit.) (1-4 ml of solution per ml of bed volume), which is washed with BBS buffer, and the pure FV fragment is eluted with a solution of 1mM phosphorylcholine in BBS buffer.

Example 3: Equilibrium dialysis of* a 0 0 s ee

S.

S. S 0@ S CS

OS

S

In a dialysis chamber with a volume of 100 pl on each side of the membrane, 50 ul of purified F V fragment in BBS buffer were placed on one side, and a solution of pl of phosphoryl(methyl- C)choline (50 mCi/mmol) in BBS buffer was placed on the other side. The concentration of the FV fragment determined from the OD 2 0 5 of 6.85 was 0.22 mg/ml Scopes, Protein Purification Principles and Practice, Springer-Verlag, New York, 1982, p. 241). After 22 hours at room temperature, equilibrium had been reached, and 20 pl samples of each solution were measured in a scintillation counter (Beckman LS 1801), and the data were subjected to Scatchard plotting (Loc. cit.).

The affinity constant derived from the gradient of the resulting line is Ka 1.21 0.06 x 105 M 1

TABLE

DNA sequence of pASK22 with the amino acid sequences of ornpA-VH and phoA-VL GCGCCCA ATACGCAACCGCCWTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCA

CGCGGGTTATGCGTTTGGCGGACAGGGGCGCGCAACCGGCTAAGTAATTACGTCGACCGT

CGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCT

61 120 GCTGTCCAAAGGCTr2ACCTTTCGCCCGTCACTCGCGTTGCGTTAATTACACTCAATCGA

CACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAAT

121 GTGAGTAkTCCGTGGGGTCCGAAATGTGAAATACGAAGGCCGAGCA'OACAACACACCTTA TGTGAGCGGATAALC.; TTTCACACAGGAAACAGCTATGACCATGATTACGAATTTCTAGA

SACACTCGCCTATTGTTAAGTTGTCCTTTGTCGA.ACTGGTACTAATGCTTAAAGATCT

TAACGAGGGCAAAAAATGAAAAAGACAGCTATCGCGATTGCAGTGGCATGGCTGGTTTC

9 024 300 0 0: AVrTCTCCCG ,'TTTTTACTTTTTC~fTCGATAGCGCTMCGTCACCGTGACCGACCAAAG )fetLysLysThrklallaIAkaVaAlaLeuAlaGlyPhe 0*0301 360 10696 CGATGGCATCGCGTCCGGCTTCAATTTGACCATCTCAGACCACCACCAGACCATGTCGGC of AlaThrValAlaGlnAlaGluValLyuLeuValGluSerGlyGlyGlyLeuValGlnPro

GGTGGATCCCTGCGTCTGTCTTGCGCTACCTCAGGTTTCACCTTCTCTGACTTCTACATG

420

CCACCTAGGGACGCAGACAGAACGCGATGGAGTCCAAAGTGGAAGAGACTGAAGATGTAC

'eoc GlyGlySerLeuArgLeuSerCyuAlaThrSerGlyPheThrPheSerApPheTyrMet *S GAGTGGGTACGTCAGCCCCCGGGTAAACGTCTCGAGTGGATCGCAGCTAGCCGTAACAAA 421 480

CTCACCCATGCAGTCGGGGGCCCATTTGCAGAGCTCACCTAGCGTCGATCGGCATTGTTT

GluTrpVa lArgGlnProProGlyLysArgLeuGluTrpIl1.AlAlaSerArgAu nLys GGTAACAGTATACCACCGAATACAGCGCTTCTGAAGTCGTTTATCGTrTCTCGT 540 CCATGTTCATATGGTC-GCTTATGTCGCGAAGACMhT'CCAGCAAAGTAGCAAGAGCA GlyAsnLyuTyrThrT7,1rGLuTyrSrAlaSerVaLyGyArgPhll.ValSerArg

GACACTAGTCMATCGATCCTGTACCTGCAGATGAATGCATTGCGTGCTGAAGACACCGCT

51eeeeeeeeeeeeeeee 600

CTGTGATCAGTTAGCTAGGACATGGACGTCTACTTACGTACGCACGATTCTGTGGCGA

AapThrSerGlnSerl eLeuTyrLeu~l1nI4tAsnA1aLou~rgAlaGluAspThrAla. 12 ATCTACTACTrGCGCGCGTAACTACTATGG7CAGCACTTGGTACTTCGACC.i 1 GGTGCA 601

TAGATGATGACGCGCGCATTGATGATACCGTCGTGAACCATGAAGCTGCACCC'-CACGT

I leTyrTyrCysAl aArgAatnTyrTyrGlySerThrTrpTyrPhekspva lTrpGlyAl a

GGTACCACCGTTACCGTTTCTTCTTGATAACATGGAGAAMATAAAGTGAAACAAAGCACT

661

CCATGGTGGCAATGGCAAA.GAAGAACTATTGTACCTCTTTTATTTCACTTTGTTTCGTGA

GlyThrThrValThrValSerSerEnd MetLysGlnSerThr

ATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCCGATATCGTTATG

780

TAACGTGACCGTGAGAATGGCAATGACAAATGGGGACACTGTTTTCGGCTATAGCAATAC

IleAlaLeuAlaLeuLeuPnroLeuLeuPheThrProValThrLysAlaAspI leValMet

ACCCAGTCTCCGAGCTCTCTGTCGTATCTGCGTGAACGTGTTACTTCTTGCA

781 TGGGTCAGAGGCTCGAGAGACAGACATAGACGTCCACTT,3CACAATGGTACAGAACGTTT

S.

006

S

0***ee

S

00 0 0 @600 841 0 S. S S 9 06 ThrGlnSerProSerSerLeuSerValSerAaGlyGluArgValThrMetSerCys~ys

TCTTCTCAGTCCTGCTGAACTCTGGTAACCAGAM~CTTCCTGTATCA

900

AGAAGAGTCAGAGACGACTTGAGACCATTGGTCTTTTTAACGACCGACCATAGTCGTT

SerSerGlnSerLeuLeuAsnSerGlyAsnGlnLyesnPheLeu~laTrpTyrGlnGln

AAGCCTGGCCAACCGCCGAACTGCTGATCTACGGTGCGTCGACCCGTGATCTGTGTT

*S.~901 960

TTCGGA.CCGGTTGGCGGCTTTGACGACTAGAGCCACGCGCGGCACTTAGACCC

LysProGiyGlnProProLysLeuLeulleTyrGlyAlaSerThrArgGluSerGlyval CCGCGTT CGT*CGAGGTCGATCCCTACTTCTTT 961 1020

GGCCTGGCAAATGGCCATCGCCATCGCCATGGCTGAGTGAACTGGTAAGAGACAT

S...ProAspArgPheThrGlySerGlySerGlyThrAapPhsThrLeuThrl leSerSerVa 1 goes CAGGCTGAAGATCTGGCTGTTTACTACTGTCAACGACCACTC!IUrACCCGCTGACCTTT o 1021 1080

GTCATCAACAAAGTAAGTTCGTAATGCATGA

GlnAlaGluApLuAlaVa lTyrTyrCyuGlnAsnAspHisaSerTyrProLeuThrPhe

GGGCGACACGACGACCCTGTACTGATGCTGT

1081 1140

CCCGCT,'TTACTATCCCACATGACTACGACA

GlyAlaGlyThrLyuLeuG 1uL~uLy- Arg~laEnd TACAACGTCGTGACTGGGAAACCCTGGCGT',"ACC Ck-TTAATCGCCTTGCAGC.ACATC 1200 ATTS. "'CGCC7'TGACWAGGTATACOCTGGA

CCCTCCACGCTAACAGAGCGACACCCTCACG

1260 GGGGAAAGCGGTCGACCGCA ?ITATCGCTTCTCCGGGC,3TGGCTAGCGGGAAGGGTTGTCA proteins composed 01 antibody domains and other proteins; and isolating mne tunctionai antibodies, fragments or fusion proteins from periplasmic space of the bacterium or a medium in which the bacterium is cultured.

13

TGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCG

1261 1320

ACGCGTCGGACTTACCGCTTACCGCGGACTACGCCATAAAAGAGGAATGCGTAGACACGC

GTATTTCACACCGCATATGGTGCACTCTCAG-TACAATCTGCTCTGATGCCGCATAGTTAA

1321 1380 CATAAAGTGTGGCGTATACCACGTiGAGAGTCATGTTAGACGAGACTACGGC(GTATCAATT GCCAGCCCCGACAC-CCGCCAACACCCG%-cTl CGCGCCCTGACGGGCTTGTCTGCTCCCGG 1440 CGGTrCGGGGCTGTGGGCGGTTGTGGGCGACTGCGCGGGACTGCCCGAACAGACGAGGGCC

CATCCGCTTACAGACAAGCTGTG:ACCGTCTCCGGGAGCTGCATGTGTCAGAGG!'TTTCAC

1441 1500

GTAGGCGAATGTCTGTTCGACACTGGCAGAGGCCCTCGACGTACACAGTCTCCAAAAGTG

0 000 CGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTA 1~501 +1560 S. *GCAGTAGT2GGCTGCGCGCTCTGCTTTCCCGGAGCACTATGCGGATAAAAATATCCAAT

ATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTLCGGGGAAATGTGCGCG

7561 1620

TACAGTACTATTATTACCAAAGAATCTGCAGTCCACCGTGAAAAGCCCCTTTACACGCGC

GAACCCCTATTTGTTTATTTTCTASATACATTCAAATATGTATCCGCTCATGAGACAAT

.***1621 1680

AACCCTGATAAATGCTTCAATAATATTGAAAALGGAAGAGTATGAGTATTCAACATTTCC

1740

TTGGGACTATTTACGAAGTTATTATAACTTTCCTTCTCATACTCATAAGTTGTAAAGG

GTGTCGCCCTTATrtCCCTTTTTGCGGCATTTGrCCTTCCTGTTTT-GCTCC,CAGAAA 1800 cAcAG(GGGAATAAGGGAAAAAAcGcCGTAAAACGGAAGGACAAAAALCGAGTGGGTCTTT 181CGCTGGTGAAGTAAAGAGCTGAAGATCAGTTGGGTGCACGATGGGTTACATCGAAC 16

GCGACCACTTTCATTTCTACGACT~CTAGTCICCCAGTGCTCACCCAATGTAGCT'G

TGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTCGCc GAAGAACGTTTTCCAATGA9 1861 1920 ACCTAGAGTTGTCGCCATTCTAG3GAACTCTCAAAGCGGGGC TCTGCAAAAGGTTACT

TGAGC"LCTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGVATTGACGCCGGGCAAG

1921 1980

ACTCGTGAAAA',TTTCAAGACGATACACCGCGCCAPTAATAGGGCATAACTGCGGCCCGTTC

AGCACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCA

2 04 0 TCGTGAGCCACGGCGTA~tGTrakTAAGAGTCTTACTGACCAACTCATGAGTGGTCAGT

ENGINEERING

The following statemen~t.; a iull description of this invention, including the best method of performing it known to' -14 CAGAAAAGCATCTTA CGGATGGCATGACAGTAAGAG)ATTATGCAGTGCTGCCATAACCA 2041 2100 GTCTTTCGTAGAATGCCTACCGTACTGTCATTwCTCTTAATACGTCACGACGGTATTGGT TGAGTGATA.1,CACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAA 2101 2160 ACTCACTATTGTGACGCCGGTTGATGA.k,AC.-GTTGCTAGCCTCC"TGGCTTC%'-TCGAFTr-- CCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGvGAACCGGAGC 2161 2220 GGCGAAAAAACGTGTT~wGTACCCCCTAGTACATTGAGCGGAACTAGCAACCCTTGGCCTCG TGAATGAAGCCAT%4*ACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAA 2221 2280 ACTTACTTCGGTA'GGTTTGCTGCTCGCACTGTGGTGCTACG7GACATCGTTACCGTTGTT

S.

i281 0 a.

C

4S*6 2341 e0 a B S aO 2401

'C

a 0O

S.

C

6e 2A61 S a.

C

a ~5 21

*OSS

.5 S C a

CGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAG

2340

GCACGGTTTGATAATTGACCGCTTGATGATGAGAI~CGAAGGGCCCTTGTTAJ'TATC

ACTGGATGGAGGCGGATAGTTGCGGACCACTCTGCGCTCGCCCTTCCGGCTGGCT

2400 TSl-AC CTACC TCCGC CTATTTCAACGTCC TGGTGAAGAC GCGAGC CGGGAAGGCCGCC GA GG TTTATTG"1"MTGATAAAT CTGGAGC CGGTGAGC GTGGG T CT CGC GGTATCATTGCAG CAC 2460 CCAAATAACGACTATTTAGACCTCGG CCACTCGCACC'-CAGAGCGCCATAGTAACGTCGTG

TGGGGCCAGATGGTAAGC-CCTCCCGTATCGTAGTATCT.CACGACGGGGAGTCAGGCAA

2520 %C CC CGGTCTACCZATTCGGG GGCATAGCATCAATAGATGTGCTGCC CC TCAGTCCGTT CTATGGATGA~GAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCAT-rGGT 2580 GvATACCTACTTGCTTTATCTGTCTAGCGACTCTATC.ACGGAGTGACTAATTCGTAACCA AACTTCAGACCAAGTTTACTCAT.*LTATACTTTAGATTGAII MCTTCAT aAAT 2581 2600

TTGACAGTCTGGTTCAATGAGTATATATGAAATCTAACTAAATTTGAAGTAAAPATTA

TTAAAGGATCTAGGTGAAGATCCTTTTGATAATC'rCATGKCCAAAATCCCTTAMCGTG 2700

AATTTTCCTAGATCCACTTCTAGGAAAACTATTAGAGTACTGGTTTAGGGAATTGCAC

AGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAGATCAAGATCTTCTTGAGATC

2701 fa m 276 0

TCAACAGGCCCGCGGCTTTCATTCAAIGCCA

CTTTT CGGCGTAATCTGCTG-TCAi ki'hhi i 1

CACGCCTACGT

2820 GAAAhGGATAuAGhGTTTTTTGCCGTvTGCC spects of bringing it about have been assessed pessimistic- ~1 ally Morrison, Science 229, 1202 (1985), M.A. Boss and C.R. Wood, ImmunoL. Today 6, 12 (1985)).

Such a system would be very desirable because the genetic 15 TTTGTGCCGGATCAGAGCTACCACTCTTTTTCCGAAGTAACTGGCTTCAGCArGAG 2821 AACAACGGCCTAGTTCTCGATGGTTGAGAAA &GGCTTCCAiTTGACCGAAGTCGTCTC 281CGCAGATACCAAATACTG. Z:CTTCTAGTGTAGCCGTAGTTAGGCcAccACTTCAAGAACT 2 94 GCGTCTATi'GTTTATGACAGGAAGATCACATCGGCATCA'.TCCGG2TGGTGAAGTTCTTGA CTGTAGCALCCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGT3- 2941 GACATCG TGGCGGATGTATGGAGCGAGACGATTAGGACMATGGTCACCGACGACGGTCAC G7CGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGC CGCTATCAGCAAGAAT C-CAACCTGAGTTCTGCTATCAATGGCCTATTCCGCGTCG CGTCGGGC'CGAACGGGGG;GTTCGTGCACACAGCCCAGC?PGGACG CG CCTAcACC, a3061 GA G C 34*CCAGCCCGACTTGCCCCCCAAGCACGTGTGTCGG;GTCGAACCTCGCTTGCTGGATGTGGC 32 APACTGPAGATACCTACAGCGTGAGCTATGAGAANGCGCCACGCTTCCCGMGGGAGjAAGG :.3121 3180

TTGACTCTATGGATGTCGCACTCGATACTCTTTCGCGGTGCGAA.GGGCTTCCCTCTTTCC

CGGACAGGTATCCGGTAAGCGGCAGGGTCGGALACAGGAGAGCGCACGAGGGACTCA

3240A 0 GCCTGTCCATAGGCCATTCGCCGTCCCAGCCTTGTCCTCTCGCGTGCTCcCTCGAAGGTC GGGGAAACGCCTGGTATCTTAATCGCGTTGCCTT.cTAcT 3.24 3300 3 CCCTTTCGGACATAGAAATATCAGGACAGCCCAAAGCGGTGGAGACTGAACTCGCAG

GATTGTGATGCTCGTCGGGGGGAGCCTATGGAAACGCCAGCAACGCG

3360 3,.4108 CTAAAAACACTACGA'GC"'TCCCCCCGCCTCGGATACCTTTTTGCGGTCGTTGCGCCGGA TTTTACGGTTCCTGGCCII'rTTGCTGGCCTTTTGcTcAcATGTTcTrCCGACC 3420 AAAATGCCAAACCGGAACGACCGGAAAACGAGTGTACAA GACGCATAGGG 3421 3480 GACTAAGACACCATTGCATAAGCGG-%pACTCACTCACTAGJ

CGCGTCGG

GAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGMkMCGM 3523

CTTGCTGGCTCGCGTCGCTCAGTCACTCGCTCCTTCGCCTTCT

Claims

1. A gene construct for the preparation of a functional antibody, a functional fragment of an antibody or a fusion protein composed of an antibody domain and other proteins, said gene construct consisting of two or more sagments each said segment comprising a gene encoding an individual antibody chain and linked thereto a signal sequence, said two or more segments being coupled to each other so as to form a regulatable operon and so as to enable said genes to undergo simultaneous induction, co- expression and co-secretion in a bacterial host.

2. A process for the in vivo preparation of functional antibodies, functional fragments of the antibodies or fusion proteins composed of antibody domains and other proteins, which comprises coupling genes encoding individual antibody chains each linked to a signal sequence, such that the signal sequence brings about the transport of the antibody chains through the cytoplasmic membrane of a bacterial cell and is then eliminated; bringing about the expression of the genes encoding individual antibody chains to produce functional antibodies, functional fragments of antibodies or fusion proteins composed of antibody domains and other proteins; and isolating the functional antibodies, fragments or fusion proteins from periplasmic space of the bacterium or a medium In which the bacterium is cultured.

3. A process for the in vivo preparation of a functional antibody, a functional fragment of the antibody or a fusion protein comprising an antibody domain, comprising placing on a single plasmid a first DNA sequence consisting of: a first nucleotide sequence that encodes an antibody heavy chain or a '.functional fragment thereof, and a first signal sequence derived from a prokaryote, and a second DNA sequence consisting of: a second nucleotide sequence that encodes an antibody light chain or a functional fragment thereof, and R a second signal sequence derived from the prokaryote; hh.T 109, 2174 (1987)). The invention furthermore permits the application of test systems (assays) directly to the bacterial cell in which the functional antibody is formed, and thus rapid 1' 17 bringing about expression of the first and second nucleotide sequences in a bacterium to produce a functional antibody, a functional fragment of the antibody or a fusion protein comprising an antibody domain, and isolating the functional antibody, functional fragment or the fusion protein from periplasmic space of the bacterium or from a medium in which the bacterium is cultured.

4. The process as claimed in claims 2 or 3, wherein the bacterium is a Gram- negative bacterium.

The process as claimed in claim 4, wherein the bacterium is E. coli.

6. The process as claimed in claims 2 5, wherein the genes are coupled in the form of a regulatable operon system.

7. The process as claimed in one or more of the preceding claims, wherein, in order to isolate the functional protein from the periplasmic space, the bacteria which have been separated off are exposed to an osmotic shock such that the periplasm is ejected but the cytoplasm remains in tact, the liquid phase obtained by this is enriched by centrifugation, and the desired proteins are obtained from this concentrate.

8. The process as claimed in one or more of the preceding claims, wherein the solution containing the functional protein is applied to an absorbent loaded with the appropriate antigen or hapten, and the desired proteins are isolated by elution with a S solution containing this antigen or hapten.

9. A functional antibody, a functional fragment of an antibody or a fusion protein composed of an antibody domain and other proteins prepared from a gene constr:ct as claimed in claim 1. Codons which are rarely used by E. coli were avoided in the construction of these DNA sequences. Furthermore, unique restriction enzyme cleavage sites were incorpor- ated, and account was taken of the secondary structure of the RNA. c II: I a 11 -7 1 g m cJ48e~ i- 1 k r 1-i I 18 A functional antibody, a functional fragment of an antibody or a fusion protein composed of an antibody domain and other proteins prepared according to the process of any one of claims 2 8. DATED this 11th day of March 1992. DR ANDREAS PLUCKTHUN and ARNE SKERRA o r WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRAUA DBM/KJS/CH (DOC.10) AU2761788.WPC ~I