EXPRESSION OF FUSION PROTEINS

Abstract: This invention provides a method of producing a fusion protein comprising an antibody portion and a further biologically active portion, the method comprising forming a virus-based construct including a DNA sequence encoding the fusion protein and infecting cells or an organism with the thus constructed virus-based construct whereby the fusion protein is expressed in in vitro in the cells, or in vivo in the organism.

Description: Expression of Fusion Proteins This invention relates to the delivery of fusion proteins comprising antibodies or recombinant fragments thereof to organs, tissues, cells and the like. More particularly, the invention relates to use of virus based, constructs to deliver fusion proteins comprising antibody fragments thereof to target tumour cells where the fusion proteins are exposed. It is known in the art that antibodies have enormous potential for the therapy of malignant and other diseases. For the therapy of malignant disease, the use of small fragments of antibodies, and fusion proteins, derived from them has potential advantages (Chester K.A. and Hawkins R.E. Advanced Drug Delivery Reviews. 1996; 22: 303-314 and Chester K.A. and Hawkins R.E. TIBTECH 1995;13: 294-300). A particular form of antibody fragments which has been extensively studied in recent years is the single chain Fv ("scFv") ( Huston J.S. et al, Proc. Natl. Acad.Sci. USA 1988; 85:5879-5883). These fragments have great potential for therapy as they are small and penetrate tumours and solid tissues relatively well. They can be isolated directly using phage technology (Hawkins R.E. et al J. Mol. Biol. 1992; 226:889-896) and can be subsequently engineered to enhance their affinity (Chester K.A., et al Lancet 1994; 343: 455-456). Indeed such fragments made from phage libraries (Verhaar M.J., et al Int J Cancer 1995; 61: 497-501) appear to be superior to those derived from pre-existing monoclonal antibodies in tumour targeting models (Begent R.H.J., et al Nature Medicine 1996; 2: 979-984). Recently their activity in patient imaging studies has been established and this work also confirms the short half-life of such antibody fragments in the human circulation.(Savage P., et al Br. J. Cancer 1993; 67: 304-310.) Fusion proteins with such scFv fragments are potentially attractive for therapeutic use but the short half life of the antibody fragments means that continuous infusion for a period of time may be required to obtain sufficient target tissue concentrations and that large quantities will need to be administered (see Hawkins et al supra). Difficulties in the production and purification of bulk quantities of fusion protein remain. Hitherto, the fusion proteins have been made in vitro using hybridoma technology. It is therefore an object of the present invention to provide an attractive and alternative method for the production of antibody fragments to overcome disadvantages of known techniques. According to a first aspect of the invention, there is provided a method of producing a fusion protein comprising an antibody portion and a further biologically active portion, the method comprising forming a virus-based construct including a DNA sequence encoding the fusion protein and infecting cells or an organism with the thus constructed virus-based construct, whereby the fusion protein is expressed in in vivo in the cells, or in vitro in the organism. Preferably the antibody portion or fragment, or construct, is fused to GM-CSF so that it is able to function as a cytokine in order to induce a systemic immune response. The use of GM-CSF is particularly attractive since, as well as having a direct effect on targeted cell killing, it is a particularly effective cytokine for the induction of systemic immune responses to tumour cells. There are a number of different fusion proteins which could be used (see Chester K.A and Hawkins R.E. supra) but antibody-cytokine fusion molecules are preferably used because of their potential to recruit specific effector cells. Such fusion proteins, produced using hybridoma technology, have previously been described with regard to use with interleukin-2 (Hornick J.L., et al. Blood 1997; 89: 4437-4447) and with GM-CSF (Dranoff G., et al. Proc. Natl. Acad. Sci. U S A 1993; 90: 3539-3543.and Lee C.T., et al. Hum. Gene Ther. 1997; 8: 187-193). This is achieved by altering the local immunological environment to increase the immunogenicity of the tumour cells (Lee C.T., et al. Hum Gene Ther 1997; 8: 187-193). Practically, however, it is preferable to treat tumours directly in vivo. Systemic toxicity means that it is necessary to devise methods that increase the concentration of cytokine in the immediate vicinity of the tumour. One approach has been to use gene transfer to introduce cytokine genes into tumour cells in vivo. Recombinant adenovirus has been used to introduce the GM-CSF gene into a murine lung tumour model system and to suppress growth of the tumour cells (Stevenson F. K., et al. Immunol Rev 1995; 145: 211-228). However, tumours are often not readily accessible for the direct introduction of adenoviruses and in the adjuvant setting (where immunotherapy is most likely to be effective) all known tumour has been removed so it is generally preferable to target the cytokine itself if appropriate methods can be devised. It will be appreciated that the recombinant adenoviruses encoding scFv fusion proteins as a model for the production of antibodies and their fragments in vivo overcome practical problems associated with the production and clinical use of antibodies and scFvs for immunotherapy of cancer. The recombinant adenoviruses,Y.B1.8.mGM-CSF and Y.MFE-23.mGM-CSF,(Ψ.B1.8.mGM-CSF and Ψ.MFE-23.mGM-CSF) have been generated by a method that utilises Cre recombinase (Hardy S et al J Virol 1997; 71: 1842-1849) rather than relying on the more inefficient process of overlap recombination (Bett AJ et al Proc Natl Acad Sci U S A 1994; 91: 8802-8806). They contain a gene fusion encoding murine GM-CSF fused to the scFvs B1.8 or MFE-23, which bind to the antigens NIP or CEA, respectively. These adenoviruses are able to infect a variety of human and murine cell lines in vitro and promote expression and secretion of the scFv.mGM-CSF proteins. In the case of MFE-23.mGM-CSF, the presence of CEA on the cell surface in a number of these lines does not appear to have a major effect on secretion of the fusion protein. This suggests that secretion of the fusion protein is not prevented by aggregation with or binding to the CEA intracellularly. Importantly, both parts of the scFv.mGM-CSF fusion proteins are bioactive. The scFvs are able to bind specifically to their respective antigens in vitro and the mGM-CSF domain promotes in vitro proliferation of the murine myeloid cell line FDC-P1, which is dependent on either GM-CSF or IL-3 for growth. Quantitive assessment of the mGM-CSF activity suggests full biological activity is retained and importantly no significant proteolysis can be detected. The Y.MFE-23. mGM-CSF virus when injected intravenously into a mouse infects murine cells efficiently enough to produce substantial amounts of the fusion protein. The MFE-23. mGM-CSF protein can be detected in mouse serum by ELISA for at least 13 days post infection, which is in accordance with previous data for the expression of proteins from adenoviral vectors in vivo (Kozarsky KF et al J Biol Chem 1994; 269: 13695-13702). One of the potential advantages of using an antibody-GM-CSF fusion protein for therapy in comparison to systemic GM-CSF administered alone or in conjunction with a monoclonal antibody, is that it should be possible to achieve high concentrations of GM-CSF in the immediate vicinity of the targeted cells but maintain low levels of systemic GM-CSF hence reducing toxicity. However, these proteins have a short half life in the body. ScFvs and Fab' fragments (which have a similar molecular weight to scFv.mGM-CSF protein) have a half-life in plasma of 2.8 and 7.5 hours, respectively (Colcher D et al J Natl Cancer Inst 1990; 82: 1191-1197). An antibody-GM-CSF fusion protein has a half-life of approximately 30 hours (Dranoff G., et al. Proc. Natl. Acad. Sci USA 1993; 90: 3539-3543). Hence, it is likely that repeated administration would be required to maintain effective therapeutic levels. Using a recombinant adenovirus to direct expression of these proteins in vivo should allow low levels of expression to be maintained over a longer period of time. Levels of MFE-23. mGM-CSF exceeding 1000 ng/ml serum have been produced in mice. These levels induce fatal toxicity, causing leukocytosis, hepatosplenomegaly and pulmonary haemorrhage (Dranoff G et al Proc Natl Acad Sci U S A 1993; 90: 3539-3543) and in this experiment resulted in sacrifice of such mice due to toxicity. Lower doses of recombinant virus produce lower potentially therapeutic but non-toxic levels (< 25 ng/ml serum) of the protein. There are a number of strategies which could allow longer expression from each administration and effective repeated use of recombinant adenoviruses if that is required. One approach to prolong the expression of the fusion protein in vivo would be to use a retrovirus particularly a lentivirus. Lentiviruses have been used to achieve prolonged and substantial expression of proteins compared to adenovirus based systems (Kafric et al Nature Genetics 17 Nov. 97). Certainly, the requirement for repeated administration of adenovirus in therapy protocols has prompted studies on ways of reducing the host immune response to the adenovirus and hence extending the period of transgene expression. Immunomodulatory agents that have an antagonistic effect on the immune response have been studied (Lei D et al Hum. Gene Ther. 1996; 7: 2273-2279, Kay MA et al Nat. Genet. 1995; 11: 191-197). Second generation adenoviral vectors containing further deletions of adenoviral genes encoding structural proteins have also been constructed. This markedly increases the period of transgene expression and highlights a role for adenoviral protein expression in eliciting a host immune response (Engelhardt JF et al Proc Natl Acad Sci U S A 1994; 91: 6196-6200). The most extreme of these adenoviral vectors have all adenoviral coding sequences deleted (Kochanek S et al Proc Natl Acad Sci U S A 1996; 93: 5731-5736, Fisher KJ et al Virology 1996; 217: 11-22, Hardy S et al J Virol 1997; 71: 1842-1849). They require a helper virus for the production of adenoviral particles. The absence of adenoviral protein expression in infected cells in vivo increases the persistence of the adenovirus. Studies in animals tolerised to the transgene product have shown that expression from such vectors can last for >80 days post infection. In non-tolerised animals, expression was lost by 42 days (Chen HH et al Proc Natl Acad Sci U S A 1997; 94: 1645-1650). The use of these type of adenoviral vectors may allow us to achieve expression of scFv.GM-CSF proteins for a longer period in vivo. The level of protein produced may also be controlled by the amount of virus administered relative to body weight and also by the incorporation of gene regulatory sequences (Hu SX et al Cancer Res 1997; 57: 3339-3343). The recently described positive feedback tetracycline control system may be particularly effective when used in constructs in accordance with the invention (Agha-Mohammedi S & Hawkins RE Gene Therapy 1997 in press). These experiments show that is possible to express a scFv with therapeutic potential at levels which are biologically active and sustained for around 13 days. This approach has a number of theoretical and practical attractions and may be used in animals for anti-tumour activity. In the future it may be possible to extend this system as indicated above to allow repeated administration and also to target the expression to organs of interest. For example, physical means could be used to direct expression to the liver (a common site of colorectal cancer metastasis) or the peritoneum (a common site of ovarian cancer metastasis) to obtain higher levels of scFv fusion proteins where they are most needed. It may also be possible to target expression of the scFv fusion to specific tissues either using tissue specific promoters (Rothmann T et al Gene Ther 1996; 3: 919-926) in the constructs of the invention or by targeting gene delivery (Watkins SJ et al Gene Therapy 1997; 4: 1004-1012). An approach to targeting certain tissues would be to remove relevant cells from an organism infect them with a virus based construct in vitro in accordance with the invention and then to replace the cells in the organism for example using the technique of Murphy J E et al Human Gene Therapy 8: 1867 - 1979, November 1997. Indeed, supplies of infected cells could be maintained to treat a patient over a prolonged period. Further aspects of the invention and preferred embodiments will be apparent from the appended claims. The construction of scFv-murine GM-CSF constructs in accordance with the invention and their uses will now be described, by way of example only, with reference to the accompanying drawings Figures 1 to 12 in which: Fig. 1 is a schematic diagram of constructs in accordance with the invention; Fig. 2 illustrates the in vitro expression of constructs in accordance with the invention; Fig. 3 is a Western Blot of the results of the expression of various constructs in accordance with the invention; Fig. 4 illustrates antigen binding by fusion proteins in accordance with the invention; Fig. 5 illustrates the results of a cell proliferation assay for mGM-CSF bioactivity; and Fig. 6 illustrates the in vivo expression of fusion proteins in accordance with the invention in mice; FigJ: shows pAdlox.scFv-IL2 or HuFc(IgGl) generated from pAdlox.scFv-GM-CSF (Whittington et al 1998); Fig. 8 A illustrates the IL2 produced by HeLa cells when infected with recombinant adenovirus of the invention. Fig.8 (B) illustrates the specificity in ELISA against solid phase NIP-BS A and CEA (Calbiochem) of the 48 hour supernatant; Fig.9 illustrates the specificity in ELISA against solid phase NIP and CEA (Calbiochem) of the 48 hour supernatant; Fig. 10 illustrates the effects of administration of constructs in accordance with the invention on mice; Fig. 11 shows the sequence of scFV-mIL2 fusion constructs; and Fig. 12 shows the sequence of Hu-Fc(IGGl) construct inserted in the above vector.The murine EL2 in the above vector is replaced by the Notl-Xbal fragment encoding the hinge and Fc region of HU-IgGl as indicated below. 1. Construction and in vitro expression of the scFv.mGM-CSF gene fusion To determine the optimum cassette for eukaryotic expression of the scFv. mGM-CSF fusion proteins, various vectors were constructed containing different combinations of promoter sequence and leader sequence with scFv.mGM-CSF DNA (Fig. 1). Fig. 1 is a restriction map showing the components of the scFv.mGM-CSF expression cassette. The Hind m/Xbal fragment containing the OM leader sequence, scFv and mGM-CSF DNA was cloned into the multiple cloning site of pAdlox for constructing the recombinant adenoviruses. The CMV promoter is already present within pAdlox (L = th OM or VH1 leader sequence). 2. Antibodies and scFv-mGM-CSF expression cassette In particular, two scFv antibody fragments are used. One, scFv B1.8, is specific for the haptens 4-hydroxy-3-nitrophenyl acetyl/4-hydroxy-3-nitro-5-iodo-phenylacetyl (NP/NIP) and is derived from a hybridoma (Hawkins RE & Winter G Cell Eur J Immunol.1992; 22: 867-870). The other, MFE-23, is derived from a phage library and binds to human carcinoembryonic antigen (CEA), which is highly expressed on many tumour types including lung, colorectal and ovarian cancers (Chester KA et al Lancet 1994; 343: 455-456). To construct a scFv.mGM-CSF expression cassette, the Hindm/Pst I fragment containing the VH1 leader sequence in the eukaryotic expression vector pVACl (Stevenson FK et al Immunol Rev 1995; 145; 211-228) was replaced with the oncostatin-M leader sequence (OM) (Malik N et al Mol Cell Biol 1989; 9: 2847-2853). The engineered signal sequence contains an optimal Kozak start sequence (ACCATGG) (Kozak M Cell 1986; 44: 283-292) and a Cla I site was introduced at 69 bp to allow for future manipulations. The mGM-CSF DNA was generated by PCR from mGM-CSF cDNA using oligonucleotides to introduce Not I and Xba I restriction sites, to allow cloning into pVACl, and replace the mGM-CSF signal sequence with a short linker peptide (Ala Ala Ala Gly) to allow fusion to the C-terminus of scFv proteins. The individual scFv DNA's were inserted into the vectors as Pst I/Not I restriction fragments. Vectors to allow comparison of expression using the RSV promoter versus the CMV promoter were generated by excision of the RSV promoter on a Bgl II/Hind HI fragment and addition of the CMV promoter from pCEP4 (Invitrogen) as a Ace I Hind HI fragment. An Xba I site present in this fragment was deleted to leave a unique Xba I site within pVACl. Different constructs were tested to determine optimal expression by transient transfection into 79 IT or HeLa cells and assay of mGM-CSF in the media after 72 hours using an ELISA kit (Endogen, MA, USA) The promoters studied were derived from the Rous sarcoma virus (RSV) and cytomegalo virus (CMV). The leader sequences were VH1 from the murine antibody heavy chain (Stevenson FK et al Immunol Rev 1995; 145; 211-228) and the OM signal sequence from Oncostatin M (Malik N et al Mol Cell Biol 1989; 9: 2847-2853). The expression of the B1.8.mGM-CSF fusion cloned into these vectors was studied in 791T(ATCC:CRL-7798) and HeLa(ATCC:CCL-2) cells. Two cell lines were used to allow for natural differences in the level of expression and protein secretion. In all experiments, cell lines were cultured in DMEM (Sigma) supplemented with 10% FCS, lOOU/ml streptomycin, lOOU/ml penicillin and 2mM L-glutamine at 37°C/5% CO 2 . The B 1.8. mGM-CSF protein was produced from all the different expression vectors but the level of expression and secretion was higher using the CMV promoter in conjunction with the OM leader sequence in both 79 IT and HeLa cells (data not shown). The OM leader sequence produced higher levels of B 1.8. mGM-CSF secretion than the VH1 leader with both the RSV and CMV promoters(data not shown). Hence the combination of CMV promoter and OM leader sequence was used for subsequent experiments involving the expression of scFv.mGM-CSF proteins in eukaryotic cells. Overall this resulted in 4-fold and 9-fold higher expression than our original vector (pVACl) in HeLa and 79 IT cells, respectively. 3. Generation of recombinant adenoviruses ThescFvs, B1.85 and MFE-23 6, descrobed above, which bind to NIP (4-hydroxy-3-nitro-5-iodo-phenylacetyl) and CEA (carcinoembryonic antigen) respectively were used. The B1.8.mGM-CSF and C23.mGM-CSF gene fusions were subcloned into the Hind HI and Xba 1 sites of plasmid pAdlox to yield plasmids pAdlox.B1.8.mGM-CSF and pAdlox.MFE-23.mGM-CSF. The plasmids were individually co-transfected into CRE8 cells with adenovirus Y5 DNA. The Y5 adenovirus, pAdlox plasmid and CRE8 cell line were generously provided by Dr. S Hardy, Somatix (Hardy S et al J Virol 1997; 71: 1842-1849). The Y5 adenovirus is replication deficient due to deletions in the viral El and E3 genes. The packaging site is directly flanked by loxP sites so that the packaging site is deleted by recombination when the virus is cultured in CRE8 cells, a derivative of 293 cells containing the Cre recombinase gene. Hence there is negative selection for growth of the Y5 virus in CRE8 cells. Growth is restored by recombination with the loxP site in pAdlox, which contains a packaging site (Hardy S et al J Virol 1997; 71: 1842-1849). The CRE8 cells promoted recombination between the loxP site in ρAdlox.B1.8.mGM-CSF or pAdlox.MFE-23.mGM-CSF and Y5 virus. Cytopathic effects on the CRE8 cells were observed at 10 days post transfection. Control CRE8 cells transfected with either of the pAdlox plasmids or Y5 DNA did not exhibit any cytopathic effects. The recombinant viruses were passaged twice in CRE8 cells to reduce contamination by Y5 virus. Digestion of viral DNA, isolated from cells at each passage, with BsaBI confirmed the presence of the recombinant viruses Y.B1.8.mGM-CSF and Y.MFE-23. mGM-CSF. The titre of virus present after the second passage was approximately 10 10 /ml cell lysate. 4. Expression of the scFv.mGM-CSF proteins in vitro The YB1.8.mGM-CSF and YMFE-23. mGM-CSF viruses were used to infect HeLa cells. These were cultured to 70% confluence and, after removal of the culture medium, approximately 10 6 cells were infected in triplicate with viral lysate at a multiplicity of infection (moi) of 1000 in a low volume of cell medium. After incubation for 1 hour at 37°C/5% CO 2 , the viral lysate was removed and the cells washed three times in an excess of culture medium before addition of an appropriate volume of fresh medium (5m./10 6 cells) and further incubation at 37°C/5% Co 2 . Culture supernatant was removed at appropriate time points for analysis by ELISA and Western blot. Media samples were collected at time points up to 48 hours. The samples were assayed for mGM-CSF by ELISA and also examined by Western blot. To assay for NIP binding MicroTest HI Flexible assay plates (Falcon) were coated overnight at 40°C with NIP.BSA conjugate (20mg/ml) in PBS (Hawkins RE & Winter G Cell Eur J Immunol.1992; 22: 867-870). After blocking for 1 hour with PBS/2%BSA and washing with PBS, culture supernatant diluted in PBS/2%BSA was added to the appropriate wells and incubated for 1 hour at room temperature. After washing three times, bound scFv.mGM-CSF protein was detected using the biotinylated anti-mGM-CSF antibody, Streptavidin-POD conjugate and BM Blue POD substrate according to the protocol in the mGM-CSF ELISA mini-kit (Endogen, MA, USA) To assay for binding to CEA on the surface of cells, aliquots of LS 174T cells (2 x 10 5 ), washed with PBS/2% BSA, were incubated with culture supernatants diluted 50:50 in PBS/2% BSA for 1 hour at 40C. Bound scFv.mGM-CSF protein was detected using rabbit anti-mGM-CSF (Sigma, USA) (1/100 in PBS/2%BSA for 30 minutes at 40°C) and FTTC conjugated goat anti-rabbit Ig (Sigma, USA)(1/160 in PBS/2%BSA for 30 minutes at 40°C). Between incubations cells were washed three times in PBS/2%BSA. Data was acquired using a Becton Dickinson FACScan and LYSIS II software and analysed using WinMDI 2.4 . At t=0 there was no detectable scFv.mGM-CSF protein in any of the media samples, confirming that scFv.mGM-CSF present in the viral lysate had been removed by washing after infection of the HeLa cells. B1.8.mGM-CSF and MFE-23. mGM-CSF were detected in the culture supernatants from 4 hours post infection (Figure 2). Specifically, in the work presented in Fig. 2, a Western blot of the samples from the infection confirmed the data obtained by ELISA. The ability of Y.MFE-23.mGM-CSF to direct the expression of the scFv fusion protein in a variety cells was tested in two murine and five human cell lines. MFE-23. mGM-CSF was produced in all those tested. There was only a minor difference in the level of secretion of MFE-23.mGM-CSF in HeLa compared to HeLa-CEA (HeLa expressing surface CEA following transfection with full length human CEA). The lower efficiency of adenoviral infection of murine cells resulted in lower levels of expression than obtained in human cell lines (data not shown). A lOOng sample of each scFv.mGM-CSF protein in culture supernatant (as determined by ELISA) was examined by SDS PAGE and Western blot in comparison to 10 ng of recombinant mGM-CSF, expressed in Escerichia coli (Sigma, Missouri, USA), to check for any specific proteolytic cleavage in the fusion proteins (Figure 3). Specifically in the work presented in Fig. 3 100 ng MFE-23. mGm-CSF (Lane A), lOOng B1.8.mGm-CSF (Lane B) and lOng recombinant mGM-CSF, expressed in E. coli, (Lane C) were electrophoresed through a 7.5% SDS PAGE gel and transferred to 4.5: nitrocellulose. The proteins were detected using rabbit anti-mGM-CSF according to the methods. The molecular weight markers are Rainbow™ coloured protein markers (Amersham, UK). Protein bands of the correct size (46 kDa) were observed for the scFv.mGM-CSF fusion proteins. There were no other bands representing possible specific proteolytic cleavage products of the fusion protein. As the band of recombinant mGM-CSF is clearly visible, it suggests that any proteolytic products would represent a small percentage («10%) of the protein sample. 5. Bioactivity of the scFv-mGM-CSF proteins Antigen binding activity of B 1.8 and MFE-23 scFvs as fusion proteins with mGM-CSF proteins was tested by ELISA and FACS analysis, respectively. Samples of culture supernatant from HeLa cells infected with Y.B1.8.mGM-CSF or Y.MFE-23.mGM-CSF were taken immediately post infection and again after 24 hours incubation. Samples were tested by ELISA against NIP antigen (Figure 4A). Specifically, in the experiments reflected in Fig. 4A, wells of a 96 well microtitre assay plate were coated with 20μg ml NIP.BSA conjugate. Uncoated wells were used as a negative control. Wells were incubated with culture supernatants containing either MFE-23.mGm-CSF or B1.8.mGM-GSF. Bound protein was detected using the biotinylated anti-mGM-CSF antibody from the MGM-CSF ELISA kit (Endogen, MA, USA) as described in the Methods. All samples were assayed in triplicate. The B1.8.mGM-CSF protein binds to the NIP antigen while, as expected, the MFE-23. mGM-CSF protein does not. The B1.8.mGM-CSF and MFE-23. mGM-CSF proteins, in culture supernatants taken 24 hours post infection, were tested by FACS analysis against LS 174T cells. LS 174T, a human colon carcinoma cell line(ATCC:CL-188), expresses high levels of CEA. Specifically, 2 x 10 5 LS 175T cells, a colon carcinoma line expressing high levels of CEA, incubated with culture supernatants of either MFE-23.mGM-CSF or B1.8.mGM-CSF. Bound fusion protein was detected with rabbit anti-mGM-CSF (Sigma, UK) and FTTC-conjugated goat anti-rabbit Ig (Sigma, UK). The data was acquired using a Becton Dickinson FACScan and LYSIS II software and analysed using WinMDI 2.4. The FACS analysis (illustrated in Fig. 4B) shows that MFE-23.mGM-CSF protein binds to LS 174T cells while B1.8.mGM-CSF does not. The ability of mGM-CSF within the scFv.mGM-CSF proteins to promote cell proliferation was demonstrated by a bioassay using FDC-P1 cells. FDC-P1 (ATCC:CRL-12103) is a murine myeloid cell line dependent on IL-3 or mGM-CSF for growth. The B1.8.mGM-CSF and MFE-23. mGM-CSF proteins were compared to recombinant mGM-CSF (Sigma, Missouri, USA), expressed in E. coli. HeLa cell culture medium was used as a negative control. Samples between O.OOlng and 50ng were incubated with FDC-P1 cells for 48 hours and proliferation measured by 3 H-thymidine incorporation as shown in Fig. 5. In the experiment the results of which are shown in Fig. 5, aliquots of 5000 FDC-P1 cells, a mGM-CSF-dependent cell line, were incubated with between 50ng to O.OOlng of each of the following: MFE-23. mGM-CSF, B1.8.mGM-CSF and mGM-CSF, which was prepared commercially from protein expressed bacteria, was the positive control. Culture medium was taken from HeLa cells was the negative control. All samples were set up in triplicate. After 48 hours incubation, tritiated thymidine (l:Ci/sample) was added for a further 8 hours incubation. Proliferation was measured using a scintillation counter and is expressed as the number of counts/20 seconds of incorporated thymidine. There was no incorporation by the negative control. The EC50 is the concentration of mGM-CSF that elicits a 50% increase in cell growth and hence thymidine incorporation. The B1.8.mGM-CSF and MFE-23. mGM-CSF proteins appear to have similar bioactivity to recombinant mGM-CSF, as determined by FDC-Pl proliferation. The EC50 of the scFv.mGM-CSF proteins, which is the effective concentration of growth factor that elicits a 50% increase in cell growth in a cell based bioassay, is approximately 0.5 ng/ml. Hence both the scFv and mGM-CSF domains of the fusion proteins are bioactive in vitro. 6. Expression of MFE-23.mGM-CSF in vivo To assess the expression of MFE-23.mGM-CSF protein in vivo, C57B1 6 mice were injected intravenously with RMFE-23.mGM-CSF adenovirus purified by CsCl. Mice were injected with between 100ml and 1ml of adenovirus at a titre of approximately 10 12 pfu/ml in a total volume of lOOμl. Sera samples, prior and post infection, were tested by ELISA using the ELISA kit (Endogen for mGM-CSF) (Figure 6). Mice receiving 100ml of virus produced high levels of MFE-23. mGM-CSF (> 1000 ng/ml serum) and were sacrificed after 4 days due to systemic toxicity caused by GM-CSF. The remaining mice injected with 1-lOml of virus produced lower levels of MFE-23.mGM-CSF (~ 2-25 ng/ml serum). Protein expression was detected four days post infection; with levels of expression falling to 0-3 ng/ml serum at 13 days post infection. No MFE-23.mGM-CSF could be detected by 26 days post infection. Serum samples from Mouse 3 were tested for anti-adenoviral antibodies by ELISA using Y.MFE-23. mGM-CSF coated assay plates. 16 Anti-adenoviral antibodies were not present prior to adenoviral infection but could be detected in sera samples from 4 days post-infection (data not shown). Mice No. 1 and 2 were culled at 4 days due to symptoms of systemic toxicity caused by high levels of mGM-CSF. No mGM-CSF could be detected in the remaining mice at 26 days. The DNA encoding the OM leader sequence and scFv.mGM-CSF proteins, MFE-23. mGM-CSF and B 1.8. mGM-CSF, in the eukaryotic expression vector pVACl were excised by digestion with HindHI and Xbal. The fragments were subcloned. The recombinant viruses were then constructed by co-transfection of Sfil digested pAdlox.MFE-23. mGM-CSF or pAdlox.B 1.8. mGM-CSF with Y5 DNA into CRE8 cells, according to the method described, to yield adenoviruses YMFE-23. mGM-CSF and YB1.8.mGM-CSF (Hardy S et al J Virol 1997; 71: 1842-1849). The recombinant viruses were passaged twice in CRE8 cells to reduce contamination with Y5 adenovirus. CRE8 cell lysates were prepared by three rounds of freeze-thawing. The viral titre was approximately 10 9 -10 10 /ml cell lysate. Recombinant adenoviral particles were purified on CsCl gradients and titred by comparison to a control virus in cytopathic effect assays, using standard methods. 7. SDS PAGE and Western Blots To assess the size of the expressed proteins, and to detect possible degradation, Western blots were used (Towbin, H. et al, Proc. Natl. Acad. Sci. 1979, 76: 4350-4354). Samples were electrophoresed through a 7.5% SDS polyacylamide gel under denaturing conditions using a Mini-Protean II cell (BIORAD, USA). Proteins were transferred to nitrocellulose using the Semi-Phor (Hoefer, USA) semi-dry transfer system. Recombinant mGM-CSF and scFv.mGM-CSF proteins were detected using rabbit anti-mGM-CSF (Sigma, USA)(1/1000 in PBS/0.1% Tween-100), HRP conjugated goat anti-rabbit Ig (Sigma, USA)(l/2000 in PBS/0.1% Tween-100) and then detected using a chemiluminescent detection system according to the protocol supplied with the ECL Western blotting kit (Amersham, UK). Cell Proliferation Assay mGM-CSF bioactivity was measured using the mGM-CSF/IL-3 dependent haemopoetic precusor line FDC-Pl (Dexter T. M et al J. Exp. Med (1980 152: 1036-47). FDC-Pl cells were cultured in DMEM/10%FCS/10% WEHI-3 CM at 37°C in 5% CO 2 . The cells were harvested, washed three times in PBS, counted and resuspended at a concentration of 10 5 cells/ml in DMEM/10% FCS. Recombinant mGM-CSF and culture supernatant containing MFE-23. mGM-CSF or B1.8.mGM-CSF were serially diluted in a final volume of 50ml DMEM/10% FCS in 96-well flat bottomed tissue culture microwell plates. A 50ml aliquot (5000 cells) of FDC-Pl cells was then added to each well. All wells were set up in triplicate. Wells containing no growth factor (HeLa cell culture supernatant) were used as a negative control. Plates were incubated for 48 hours at 37°C in 5%CO 2 . Proliferation was measured by incorporation of tritiated thymidine 3 H-TdR (lmCi/well). After 8 hours incubation, cells were harvested onto absorbent glass fibre paper and the radioactivity determined using a scintillation counter. 9. Mouse studies C57B1/6 mice, bred under specific pathogen-free conditions and maintained according to Home Office approved institutional protocols, were injected in the tail vein with different amounts (total volume lOOμl) of pure ΨC23.mGM-CSF diluted in lOmM Tris ρH7.5,lmM MgC12,135mM NaCl,10% glycerol. Blood samples were collected at different time points and the sera assayed for the presence of mGM-CSF using the mouse GM-CSF ELISA kit (Endogen, MA, USA). The following results provides further examples of scFv-fusions that may be delivered for in vivo expression using appropriate viral vectors. The fusions are scFv-IL2 and scFv-HuFc(IgGl). The scFv's used are derived from Bl-8 a hybridoma cell line which expresses an antibody with anti-NIP activity and MFE-23 a scFv made from a phage display library (Whittington et al, 1998). 10. Preparation of ScFv-IL2 fusion In order to undertake model mouse experiments the scFv was linked to murine IL2. The IL2 gene was subcloned by PCR from a vector (pVAC4) containing the mouse IL2 gene which was previously cloned from the spleen of a C57BL/6 mouse. The PCR primers incorporate Notl and Xbal sites in the 5' and 3' primers respectively. The PCR fragment encoding IL.2 was cloned into pAdlox.scFv.GM-CSF as a Notl -Xba fragment to produce pAdlox. scFv-IL2. The clones were sequenced and a clone with the correct sequence selected from further use. Both B1-8-IL.2 and MFE-23-IL2 fusions were made. For details of sequence see Fig 11. A recombinant adenovirus was then made as previously described by cotransfecting Cre8 cells with pAdlox. scFv-IL2 and Ψ5 viral DNA. After loxP driven recombination and amplification by 3 rounds of lysis and re-infection of Cre8 cells a large viral stock was produced and purified by density gradient purification. The virus was then characterised and the expressed protein assessed after infection of HeLa cells at an MO1 of 1. This revealed high level expression of IL2 fusion with retention of the specificity of the scFv portion (Figure 8A & B). The data illustrate high levels of expression of IL2 (comparable to GM-CSF (Whittington et al 1998)) and retained specificity. Figure 8 (A) illustrates the levels of IL2 produced by HeLa cells when infected with recombinant adenovirus at an MO1 of 1. The IL2 was measured using an IL2 specific ELISA (Endogen). Fig.8 (B) illustrates the specificity in ELISA against solid phase NIP-BSA and CEA (Calbiochem) of the 48 hour supernatant. The assay uses solid phase antigen with the scFv-IL2 fusion being detected with biotinylated anti-mIL2 antibody. Streptavidin-POD conjugate and BM Blud POD substrate according to the protocol in the mIL2 ELISA mini-kit (Endogen, Cambridge, MA, USA). 11. ScFv-HuFc(IgGl) This construct was prepared for both Bl-8 and MFE-23 using methods similar to those described above. The HuFv(IgGl) was PCR cloned using specific primers with Notl and Xball ends from cDNA derived from human peripheral blood lymphocytes. After cloning into pAdlox.scFv-HuFc(IgGl) clones were DNA sequenced and a clone with the correct sequence identified (Fig.12). Recombinant adenoviruses were then prepared for both scFv's as described above. They were again characterised and used to infect HeLa cells at an MO1 of 1. the supernatant was collected at various timepoints and assayed for binding to antigen and presence of the Fc using plates coated with antigen and an anti-human Fc-HRP conjugate (Sigma) (Figure 9). These results indicate that significant amounts of both recombinant antibodies are clearly produced and retain the appropriate specificity. Figure 9 illustrates the specificity in ELISA against solid phase NIP and CEA (Calbiochem) of the 48 hour supernatant. The assay uses solid phase antigen with the scFv-Fc fusion being detected with anti-human Fc-HRP (Sigma) and BM Blue POD substrate. Purified Ad5.Bl-8-Fc was also used to inject Balb/c mice. The serum was taken at various timepoints and assayed for anti-NIP activity and anti-Hu-Fc activity in an ELISA using NIP coated plates and detecting with an anti-murine-Fc-HRP and BM Blue POD substrate (Figure 10). In the experiment depicted in Fig. 10, 10 9 Pfu Ad5-Bl-8-Fc were injected into the tail vein of three Balb/c mice at day 0 and serum samples taken from the tail veins at days 0, 3, 7, 14, 21, 28. The samples were assays for anti-NIP activity and to assess the immune response to the (human)Fc region by ELISA against NIP-BSA and Human IgGl (Sigma) - results represent mean of three mice; assays at 1:100 serum dilution for anti-NIP assay and 1:1000 dilution for anti-Fc assay. As Fig 10 illustrates, the anti-NIP activity initially rises significantly from day 3 to day 7. This is likely to be as a result of accumulation of the scFv-Fc in the serum due to its large size (lOOkDa). After that the mouse response to the human Fc region is clear and is (presumably responsible for the rapid decline in serum levels (more rapid than with scFv-GM-CSF (Whittington et al. 1998) - which is smaller (45kDa)). This would not be apparent if the Hu-Fc construct was used in a person where the scFv-Fc would be anticipated to increase further for a significantly longer time.

Claims: Claims 1. A method of producing a fusion protein comprising an antibody portion and a further biologically active portion, the method comprising forming a virus-based construct including a DNA sequence encoding the fusion protein and infecting cells or an organism with the thus constructed virus-based construct whereby the fusion protein is expressed in vitro in the cells, or in vivo in the organism. 2. A method according to claim 1 in which the virus based construct is based on an adenovirus, retrovirus, adeno-associated virus, herpes simplex virus, or a synthetic virus based particle. 3. A method according to claim 2 in which the adenovirus lacks at least some native sequence. 4. A method according to claim 3 in which the adenovirus lacks all adenovirus sequences. 5. A method according to claim 4 in which the cells or organism are supplied with a helper virus. 6. A method according to claim 2, 3 or 4 in which the adenovirus is ψ5. 7. A method according to claim 2, and in which the virus based construct is based on an retrovirus, in which the retrovirus is a lentivirus. 8. A method according to any preceding claim, and in which the the fusion protein is expressed in vivo, in which the organism expresses therapeutic levels of the fusion protein. 9. A method according to claim 5 in which the therapeutic levels of the fusion protein are from about 1 ng ml "1 to about lOμg ml "1 serum. 10. A method according to claim 9 in which the therapeutic levels are about 1 to 100 ng per ml. 11. A method according to claim 8, 9 or 10 in which the levels of the fusion protein produced are sufficient to produce a therapeutic effect. 12. A method according to claim 11 in which the therapeutic effect is selected from reduction of tumour size, reduction of active agent, reduction of autoimmune disease, reduction of graft v host disease, systemic immune response. 13. A method according to any preceding claim in which the antibody portion comprises a single chain Fv portion. 14. A method according to any preceding claim in which the antibody portion of the protein is selected from or derived from fragments of IgGl, IgG2, IgG3, IgG4 or IgGE. 15. A method according to any preceding claim in which the further biologically active portion is selected from or derived from cytokines, interferons, enzymes, toxins, cell surface ligands for cellular receptors and F v derived portions of antibodies. 16. A method according to any claim 15 in which the cytokine is selected from GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15. 17. A method according to claim 15 in which the interferon is interferon α or interferon γ. 18. A method according to claim 15 in which the enzyme is carboxypeptidase or β-glucorinidase. 19. A method according to claim 15 in which the toxin is Pseudomonas exotoxin, endotoxin, or ricin. 20. A method according to claim 15 and in which the further biological portion is an enzyme, in which a pro-drug which is converted to an active form by the action of the enzyme. 21. An in vivo method according to any preceding claim in which the organism is a mammal. 22. A method according to claim 21 in which the mammal is selected from cattle,, equids, rodents, and humans. 23. A fusion protein comprising a functional antibody portion and a functional further biologically active portion. 24. A fusion protein according to claim 23 in which the further functional biologically active portion comprises at least a portion of GM-CSF or IL-2. 25. A fusion protein consisting of scFv-IL2 or scFv-HuFc(IgGl). 26. An virus-based construct comprising a DNA sequence arranged to express a fusion protein according to claim 23, 24, or 25. 27. An adenovirus based construct according to claim 26 in which the virus-based portion is derived from a Ψ5 adenovirus. 28. A method of expressing a fusion protein according to claim 23, 24 or 25 in vitro, the method comprising contacting cells with an virus-based construct including a DNA sequence encoding the fusion protein and infecting cells with the thus-constructed virus-based construct whereby the fusion protein is expressed in the cells. 29. A method according to claim 28 in which the cells are removed from an organism prior to infection with the virus based construct and are later replaced in the organism whereby the fusion protein is expressed in the organism. 30. A method of providing therapeutically useful levels of an fusion protein in an organism, the method comprising infecting the organism with an virus-based construct according to claim 26 or 27. 31. A method according to claim 29 or 30 in which the organism is a mammal. 32. A method according to claim 31 in which the mammal is selected from cattle, equids, rodents and humans. 33. A method or construct or fusion protein according to any preceding claim in which the antibody portion is obtained from a phage display library. 34. Cells infected with a virus based construct or containing a fusion protein construct according to any preceding claim.

Inventors: HAWKINS ROBERT EDWARD, WHITTINGTON HAYLEY ANN, HAWKINS, ROBERT, EDWARD, WHITTINGTON, HAYLEY, ANN
Applicants: HAWKINS ROBERT EDWARD, UNIV BRISTOL, WHITTINGTON HAYLEY ANN, HAWKINS, ROBERT, EDWARD, UNIVERSITY OF BRISTOL, WHITTINGTON, HAYLEY, ANN
Application year: 1999
Main classification: C07K16
IPC codes: A61K38, A61K48, C07K14, C07K16