Recombinant DNA-molecule for the expression of the FV-fragment of an antibody

Abstract: The invention provides a recombinant DNA-molecule for the expression of a functional Fv-fragment of a desired antibody, directed towards interferon, comprising a homologous promoter sequence, a homologous terminator sequence and dicistronic heterologous DNA-sequence, the cistrons of which encoding the variabel part of a heavy chain (V H ) of the desired anti-interferon antibody and the variabel part of a light chain (V K ) of the desired anti-interferon antibody, respectively. The recombinant Fv-fragment produced by expressing the recombinant DNA-molecule of the invention in a suitable host can be used to neutralize the biological activity of interferon-Γ, which may be harmful for the host. The use of a Fv-fragment reduces the anti-immunoglobulin respons provoked in a host to which foreign immunolgobulins are administered.

Description: The present invention relates to a recombinant DNA-molecule for the expression of a functional Fv-fragment of a desired antibody, directed toward interferon. The invention further relates to a recombinant E.coli-strain , comprising the recombinant DNA-molecule, a method for preparing the recombinant DNA-molecule, and a method of preparing a recombinant Fv-fragment of a desired anti-interferon antibody. The invention relates in particular to a recombinant DNA-molecule for the expression of a functional Fv-fragment of a anti-human-interferon-Γ (anti-HuIFN-Γ) antibody. The invention also relates to biological active preparations comprising said Fv-fragments and the use of said biological active preparations. Immunoglobulins (antibodies) are glycoproteins present in the blood and bodyfluids of higher animal species. They recognize and bind certain categories of substances that are foreign to the body (antigens) and induce effector functions that eliminate or neutralize these antigens and their carriers (viruses, bacteria, cancer cells). An immunoglobulin comprises two identical light and two also identical heavy polypeptide chains (called L- and H-chains respectively), that are linked by means of disulphide bridges. The sequences of the N-terminal ends of the four chains are very variable and together responsible for the binding of the antigen. The variable regions therefore determine the specificity of the antibody. The C-terminal part of the chains has however a relatively constant structure and determines the nature of the effector functions, such as complement activation, antibody dependent cellular cytotoxicity and others. Interferon-Γ (IFN-Γ) is a glycoprotein that is produced by T-cells in response to antigenic or mitogenic stimuli. In addition to its antiviral activity, interferon-Γ can also show strong immunoregulatory effects making it an important factor in the defense mechanism against bacteria, viruses and other antigens, but also in auto-immune diseases and cancer. The contribution of IFN-Γ in these pathological reactions can however also be harmful for the host. Animal model studies have clarified that monoclonal antibodies against interferon-Γ have a positive effect on endotoxic shock, local inflammation, cerebral malaria and auto-immune arthritis. The monoclonal antibodies against interferon-Γ could therefore possibly also be used for the treatment of related human diseases. Because of the ongoing development of hybridoma techniques a great number of monoclonal antibodies has become available that can be applied for the treatment of diseases. From numerous studies it is however found that the administration of xenogenic antibodies generates an immunoglobulin response in the recipient. The administration of human monoclonal antibodies could reduce or preclude such anti-immunoglobulin response. The production of those human monoclonal antibodies encounters ethical as well as practical objections. To avoid the above problems it has been tried recently to strongly reduce the unwanted anti-immunoglobulin response by means of in vitro manipulation of the immunoglobulin genes, such as chimerization or humanization. Important results have already been achieved herewith. It has now been found that the Fv-fragment of a antibody directed towards human interferon-Γ has a affinity for interferon-Γ and is capable of neutralizing the biological activity thereof. It is expected that with the use of such a Fv-fragment the anti-immunoglobin response can also be reduced. This is caused by the fact that the immunogenic C-regions are removed form the antibody, while the antigen binding properties of the V-regions are maintained. The present invention provides a recombinant DNA-molecule for expressing a functional Fv-fragment of a desired antibody directed towards human interferon-Γ. The recombinant DNA-molecule therefore comprises a homologous promoter sequence, a homologous terminator sequence, and a dicistronic heterlogous DNA-sequence, the cistrons of which encode the variable part of a heavy chain (V H ) of the desired antibody and the variable part of a light chain (V K ) of a desired antibody, respectively. Such a recombinant DNa-molecule provides the necessary simultaneous production of the genes of the variable regions of the light and heavy chain. Both variabel genes are preferably controled by the lac-promoter. This promoter can be inhibited of activated according to need. Thus it can be avoided that the Fv-fragment, which might be toxic to E-coli is produced at a undesirable moment. It is of course also possible to use other inducible promoters. Both variable genes are preferably preceded by a signal peptide sequence of pel B, trough which both V-regions are secreted into the peri plasm. It is also possible to connect both variable regions by means of a linker. Said linker preferably has the sequence according to formule Ic. In this case the signal peptide sequence is only comprised upstream from the cistron for the heavy chain. Downstream of the cistron for the light chain a DNA-sequence encoding a small polypeptide, e.g. the TAG1-polypeptide, is preferably comprised. This polypeptide is used for detecting the Fv-fragment by means of a antibody directed towards the TAG1-polypeptide. The present invention will be further illustrated by the following examples, which are not intended to be limiting to the scope of the invention. EXAMPLE 1 Isolation and amplification of the V H - and V K -fragment of anti-HuIFN-Γ. The nucleotidesequence of both the 5'- and the 3'-ends of the V-regions of immunoglobulins is strongly conserved. Because of that it is possible to define oligonucleotides capable of amplifying the V H - and V K -regions of practically every mouse antibody (see Orlandi et al. (1989). Proc. Natl. Acad. SCI. USA 86: 3833-3837). The primers for the amplification of the V H -regions are: wherein : S = C or G M = A or C R = A or G W = A or T The primers for the amplification of V K -regions are: By the introduction of restriction sites in the amplified DNA-fragment the cloning of the fragments is considerably simplified. For the amplification of 1 µg mRNA of D9D10 the first cDNA strand was synthesized in a way well known in the art. For this 100 pmol random hexanucleotide was used as a primer because after PCR a more specific DNA-fragment was obtained therewith than when a PCR-oligonucleotide was used for the first strand synthesis. The amplification of the desired fragment was performed on the reaction product of the first strand cDNA synthesis with the two specific primers in a 50 µl reaction mixture comprising 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 0.1 mg/ml gelatine, 0.25 mM dNTP, 25 pmol per oligonucleotide primer and 2.5 U Taq-polymerase . The reaction mixture was covered with 2 drops paraffine oil and subjected to 30 to 35 cycles of 30 sec. 95°C (denaturation), 35 sec. 61°C (annealing) and 25 sec. 72°C (filling-in reaction) with a last filling-in of 5 min. at 72°C. For performing the PCR a programmable thermic block was used (PCH-2; Techne, Cambridge, GB). Electroforesis of 5 µl of the reaction mixture on a 2% agarose gel showed the degree of purity and the yield of the amplified DNA-fragment. The other 45 µl were subjected to chloroform extraction (400 µl) after which the DNA was precipitated with NaA-c/ethanol. On this the desired reactions with the restriction enzymes were subsequently performed. EXAMPLE 2 Construction of an expression vector for Fv-fragments of anti-HuIFN-Γ antibody. For the construction of an expression vector for the Fv-fragments of anti-HuIFN-Γ the vector pSW1-VHD1.3-VKD1.3-TAG1 was used as described by Ward et al. (1989) Nature 341: 544-546. The V H -domain of the antibody D1.3 was eliminated out of the vector by digestion with PstI and BstEII and replaced by the V H -fragment of an anti-human-interferon-Γ of the hybridoma cell line D9D10, according to formula Ia. The fragment was obtained in sufficient amounts as described in example 1. The V K -domain of the antibody D1.3 was then replaced by a SacI-XhoI-fragment according to formula Ib comprising the V K -region of anti-HuIFN-Γ. The expression vector thus obtained was named pSW1-Fv-anti-HuIFN-Γ. The nucleotide sequences of the V H - and V K -regions respectively (formula Ia and Ib respectively) of the D9D10 antibody are represented in the accompanying formula form. The parts in bold represent the primers used in the PCR. The underlined parts represent the restriction sites. The strategy of construction is schematically represented in figure 7. EXAMPLE 3 Transformation of E. coli (DH5α) Plasmid DNA was brought into E. coli cells according to the CaCl 2 method of Mandel et al. ((1970) J. Mol. Biol. 53:159-162) except for the difference that only one heat shock was administered (instead of five) namely 20 sec. at 37°C for a 50 µl reaction, 45 sec at 37°C for a 100 µl reaction. E. coli DH5α cells (supE44, hsdR17(r k -,m k +), recA1, endA1, gyrA96, thi-1, relA1, del(argF-laczya)U169, 80d-lacZdelM15) were spread out after transformation in LB-medium (1% Bacto-trypton; 0.5% yeast extract and 0.8% softagar) on a solid LB-plate (1.5% agar) containing 100µg/ml amplicillin, 0.02% X-gal en 0.25 mM IPTG. They were incubated overnight at 37°C. In this manner clone P330 was obtained. EXAMPLE 4 Expression of the Fv-fragment of anti-HuIFN-Γ. The bacterial clone comprising the expression vector for the production of the Fv of the anti-HuIFN-Γ-antibody was grown for 16 hours at 37°C in LB-medium containing 100 µM ampicillin and 1% glucose. Glucose suppresses the lacpromotor which prevents the production of antibody fragments. Then the cells were washed twice in 50 mM NaCl and resuspended in LB-medium containing 100 µM ampicilline and 0.1-1 mM IPTG for induction of the promotor. This suspension was incubated for 2 to 20 hours in a shaking incubator at 37°C. The cells were then isolated by centrifugation (7000 rpm, 4°C) and the supernatant tested in an ELISA (see example 8) are stored at 4°C. The periplasma was isolated as described by Skerra et al. (1988) Science 240: 1038-1041. After induction with IPTG the cells were precipitated by centrifugation, resuspended in TES-buffer (0.2 M Tris-HCl pH 8.0; 0.5 mM EDTA; 0.5M sucrose; 10 ml/l culture) and subjected to an osmotic shock by addition of a 1/4 dilution of TES-buffer in water (15 ml/l culture). After 30 minutes on ice the suspension was centrifuged by 8000 rpm for 10 minutes at 4°C. The supernatant was centrifuged again at 20,000 rpm for 30 minutes to remove the remaining celdebris. The supernatant was directly tested, stored at 4°C or frozen at -20°C. EXAMPLE 5 Detection of the Fv-fragment of anti-HuIFN-Γ The binding of the Fv-fragment to the human IFN-Γ molecule was tested in an ELISA. The wells of a 96-well "MaxiSorp Nunc-Immuno Plate" were incubated overnight at 4°C with 2 µg/ml recombinant HuIFN-Γ (Bioferon, Laupheim, Germany) in 100 µl of a 50 mH Tris-HCl pH 8.5 and 10 mM NaCl buffer. The stock solution of recombinant HuIFN-Γ was dissolved in 50 mM natrium phosphate and 100 mM ammonium acetate buffer with 12.5 mg/ml sucrose, to a concentration of 0.7 mg/ml pure proteine. The culture plate was decanted and to every well 250 µl blocker (PBS with 0.5% caseine and 0.01% merthiolate pH 7.5) was added. After incubation for 1 hour at 37°C the cells were washed with 0.05% Tween-20 in PBS containing 0.01% merthiolate (washing buffer), after which the Fv-fragment samples were added (any dilutions in blocker). After shaking for 2 hours at 37°C the material that was not bound was washed away and 100 µl of a 1 µg/ml anti-TAG1 antibody in blocker was added to every well. Subsequently the plate was again shaken for two hours and washed three times. After that a biotinylated sheep anti-mouse-Ig-antibody was added (Amersham, Buckinghamshire, GB, 100 µl of a 1/1000 dilution). After one hour shaking at 37°C and three times washing the reaction was finished with the biotinylated peroxidase-streptavidin complex, H 2 O 2 and ABTS. Because no anti-idiotype antibody was available the detection was performed by means of the TAG1-polypeptide that was linked to the V K -region. The highest expression of Fv in the supernatant of a 18 hours incubated cell culture amounted to 30 binding units. This means that a 1/30 dilution of the fluid was still positive (green colour) in the ELISA for HuIFN-Γ binding. Almost no Fv was demonstrated in the periplasma for the same induction. This is caused by the fact that the Fv-concentration in the periplasma reaches its maximal value after an induction of two hours after which the Fv leaks away into the culture liquid and after 18 hours is found substantially therein. This "leakage" out of the cells could be due to a stress situation in the bacteria caused by a high production of a strange protein. In the fluid of cells that were not induced (absence of IPTG) a basal amount of Fv was always present while the fluid of cells with a suppressed promotor, because of the presence of glucose, contained no detectable Fv-material. The periplasma preparation method provided already a substantial enrichment of the produced Fv because the volume was much smaller in comparison to that of the supernatant. The periplasmic fraction of one liter culture amounted to 12 ml (83 times more concentrated) and of a 1 to 3 ml culture this amounted 75 µl (13 to 40 times more concentrated). After a two hours induction with 0.1 mM IPTG (the combination giving the highest induction in the periplasma) the fluid as well as the periplasmic fraction were tested in the ELISA. The periplasma had a titer of 200 binding units; in the supernatant no Fv was detected. From these experiments it is clear that E. coli, wherein the Fv-gen was introduced produced indeed Fv and that the Fv was capable of binding HuIFN-Γ. This means that neither the aminoacid alterations in the 5'- and 3'-ends of teh V-regions nor the linking of TAG1 to V K have an important negative influence on the binding of the Fv of D9D10 to the HuIFN-Γ-molecule. EXAMPLE 6 Inhibition of the antiviral activity of HuIFN-Γ To verify whether the Fv-fragment produced by E. coli can inhibit or neutralize the activity of HuIFN-Γ in vitro neutralization tests were performed. For these A549-cells were used, a human carcinoma cell line. This cell line was cultured in MEM with 10% "Newborn Calf Serum" (NCS) and is sensitive to the antiviral activity of HuIFN-Γ. A human-IFN-Γ being just antivirally active (167 ng HuIFN-Γ with a specific activity of 2.8 x 10 4 U/µg on Wish cell; Biogen, Gent) were mixed with serial 1/2 logarithmic dilutions of the samples to be tested. These were incubated for 4 hours at 37°C after which the mixtures were desinfected for 6 minutes under UV light. To all the wells 50,000 A549 cells were added and the whole was incubated for 20 hours in a CO 2 -incubator until the cells had formed a mono-layer. The infection was performed with a 1/300 dilution of the Encephalo Myocarditis Virus (EMCV). After 48 hours, when the virus control was totally affected, the living cells were coloured with 100 µl of a neutral red solution (1/30 dilution neutral red in PBS with Ca 2 + and Mg 2 +) and stored for 2 to 4 hours in the dark. Subsequently they were washed with PBS (with Ca 2 + and Mg 2 +) and 100 µl acidified alcohol was added to every well. After 20 minutes the plates were read in a Multiscan Titertek at 542 nm. As controls the samples in the absence of HuIFN-Γ and IFN-Γ in the absence of the sample were used. Cell- and virus controls were also tested. A 1/3 dilution of this fraction appeared to be capable to block the antiviral activity completely. When a combination of the 1/3 dilution together with a low IFN-Γ dose was brought on the cell the EMC-virus could infect and kill the cells, contrary to the sample with the low IFN-Γ concentration alone, with which protection occurred. This inhibiting effect was not due to a possible toxicity of a component from the periplasma because in the absence of virus the cells were not affected, neither to a stimulation of the virus growth, because the cells were not faster affected in the presence of the periplasmic fraction than in the absence thereof. In a 1/6 dilution of the periplasmic fraction no inhibition of the antiviral activity was observed. The Fv-fragment that was purified on a affinity column showed the same neutralizing effects. This indicates that it is no other component from the periplasma that blocks the antiviral activity of HuIFN-Γ. Moreover periplasma of non-induced cells was not capable of neutralizing. This means that the Fv of the anti-HuIFN-Γ antibody of D9D10 is also capable of neutralizing the antiviral activity of HuIFN-Γ, despite of minor changes in the 5' and 3'-ends of the V-regions and the linking of TAG1 to V K . The above examples show that the introduction of the V H -V L PCR-fragments behind the LacZ-promoter provides for a dicistronic mRNA being formed which produces the F H -F L , proteins separately but probably in equal amounts. Upstream from every V-gene a ribosome binding site is present. Since the the pel B signal peptide provides the transport of the pectate lyase enzyme from the cytoplasma to periplasm both V-proteins will be transported the periplasm. This means that the Fv-fragment can easily be isolated and obtained in a concentrated form. It is then also not subject to the degradation by cytoplasmic proteases. As was shown by ELISA and neutralization tests the functional HuIFN-Γ binding Fv-fragment was secreted into the periplasm. The Fv-fragment, binding the HuIFN-Γ molecule and neutralising its anti-viral activity, and only consisting of both variabel regions and a small polypeptide (TAG1) will probaly have a reduced HAMA response as compared to the parental antibody. The absence of the C-regions has no further influence on a good immunoresponse because no effect or functions are necessary for this antibody. The stability of the Fv-fragment can be increased by chemical cross-linking of the V-regions, by introduction of an intermoleculair disulphidebridge or by coupling of the V-regions with a protein linker. Coupling of a toxine, cytotoxic substance or radio-isotope to the Fv-fragment can be performed in case it is used as a magic bullet. this coupling also increases the half life in the serum.

Claims: A recombinant DNA-molecule for the expression of a functional Fv-fragment of a desired antibody, directed towards interferon, comprising a homologous promoter sequence, a homologous terminator sequence and dicistronic heterologous DNA-sequence, the cistrons of which encoding the variable part of a heavy chain (V H ) of the desired anti-interferon antibody and the variable part of a light chain (V K ) of the desired anti-interferon antibody, respectively. A recombinant DNA-molecule as claimed in claim 1, characterized in that the cistrons of the heterologous DNA-sequence encode the variable part of the heavy chain, and light chain of an antibody directed towards human interferon-γ, respectively. A recombinant DNA-molecule as claimed in claim 2, characterized in that the cistrons are comprised in the following nucleotide sequences, or mutated versions thereof: or Recombinant DNA-molecule as claimed in claim 3, characterized in that the cistron encoding the variable part of the light chain is represented by the formula Ib and the cistron encoding the heavy chain is represented by formula Ia, or by mutated versions thereof that do not adversely affect the functionality of the Fv-fragment. Recombinant DNA-molecule as claimed in any one of the claims 1-4, characterized in that the homologous promoter sequence is the lac-promoter. Recombinant DNA-molecule as claimed in any one of the preceding claims, characterized in that the recombinant DNA-molecule comprises a signal peptide sequence upstream from each of the cistrons and a sequence encoding the TAG1-polypeptide downstream of the second cistron. A recombinant DNA-molecule as claimed in any one of the claims 1-5, characterized in that the cistrons of the heavy and light chain, respectively are connected by means of a linker sequence as represented by formula Ic, that upstream of the cistron encoding the heavy chain a signal peptide sequence is comprised and that downstream of the cistron encoding the light chain the sequence encoding the TAG1-polypeptide is comprised. A recombinant DNA-molecule as claimed in claim 6 or 7, characterized in that the signal peptide sequence is derived from pel B. E.coli strain comprising a recombinant DNA-molecule as claimed in any one of the preceding claims. E.coli strain as claimed in claim 9, characterized in that the strain expresses the Fv-fragment of an antibody directed towards human-interferon-γ. E.coli strain as claimed in claim 10, characterized in that the strain expresses the Fv-fragment of the monoclonal anti-human-interferon-γ antibody characterized by V H - and V K -nucleotide sequences comprised respectively in formula Ia and Ib as defined in claim 3, or mutated versions thereof. A method for preparing a recombinant DNA-molecule as claimed in any one of the preceding claims, comprising the steps of: a) linearizing a DNA-molecule comprising the homologous promoter and terminator regions to obtain a linear DNA-molecule; b) isolating and multiplying a variable part of a heavy chain (V H -fragment) of a desired antibody; c) ligating the linear DNA-molecule and the V H -fragment to obtain a first recombinant DNA-molecule; d) linearizing the first recombinant DNA-molecule to obtain a first linear DNA-molecule; e) isolating and multiplying a variable part of a light chain (V K -fragment) of a desired antibody; and f) ligating the linearized first DNA-molecule and the V K -fragment to obtain a second recombinant DNA-molecule. A method as claimed in claim 12, characterized in that the desired DNA-fragments are multiplied by means of Polymerase Chain Reaction. A method as claimed in claim 12 or 13, characterized in that the V K -fragment has a DNA-sequence comprised in the sequence as represented in formula Ib as defined in claim 3, or mutated versions thereof. A method according to any one of the claims 12 to 14, characterized in that the V H -fragment has a DNA-sequence comprised in the sequence as represented in formula Ia as defined in claim 3, or mutated versions thereof. A method according to any one of the claims 12 to 15, characterized in that between the sequences for the heavy and light chain, respectively, a linker sequence comprised in the sequence as represented in formula Ic: or mutated versions thereof, is cloned. A method for preparing a recombinant Fv-fragment of a desired antibody, comprising: a) producing a recombinant DNA-molecule as claimed in any one of the claims 1 to 8; b) transforming a host cell culture with the recombinant DNA-molecule to obtain a recombinant host cell culture; c) culturing the host cell culture under conditions allowing the expression of the DNA-sequence encoding the recombinant Fv-fragment; and d) isolating the recombinant Fv-fragment. A method as claimed in claim 17, characterized in that the recombinant Fv-fragment is the Fv-fragment of the monoclonal anti-human-interferon-γ antibody characterized by V H - and V K -nucleotide sequences comprised respectively in formula Ia and Ib as defined in claim 3, or mutated versions thereof. A method as claimed one of the claims 17 or 18, characterized in that the host cell is E.coli . A recombinant Fv-fragment of an antibody directed towards human interferon-γ, prepared according to the method as claimed in any one of the claims 17-19. A biological active preparation comprising an antihuman interferon-γ Fv-fragment as claimed in claim 20. A biological active preparation as claimed in claim 21 having a neutralizing effect on the antiviral activity of human interferon-γ. Biological active preparation as claimed in claim 21 or 22 for use in the treatment of pathological reactions caused by interferon-γ. Biological active preparation as claimed in claim 21 or 22 for use in the treatment of endotoxic shock, local inflammation, cerebral malaria, allergic encephalomyelitis, skin allograft rejection, auto-immune arthritis, tumor development and lupus-like disease.

Inventors: BILLIAU ALFONS JOZEF DENIS ALI, FROYEN GUIDO FRANS VALENTIUS, BILLIAU, ALFONS JOZEF DENIS ALIDA, FROYEN, GUIDO FRANS VALENTIUS
Applicants: STICHTING REGA V Z W, STICHTING REGA V.Z.W.
Application year: 1992
Main classification: C12N15
IPC codes: C12N1, A61K38, C07K16, C12N15