TARGETED DELIVERY OF VIRUS VECTOR TO MAMMALIAN CELLS

Abstract: A complex between a virus the cell binding receptor of which has been inactivated and an antibody, said antibody having the ability to interact with a specific antigen on the surface of a mammalian cell different from the cellular structure which would otherwise mediate binding of the virus to the cell surface and which antigen has the ability to mediate entrance of a virus vector or infectious virus into mammalian cells.

Description: TARGERED DELIVERY OF VIRUS VECTOR TO MAMMALIAN CELLS The present invention relates to a complex between virus the cell binding receptor of which has been inacti vated and an antibody. The invention is applicable to si tuations where it is desireable to introduce viruses int selected mammalian cells for medicinal purposes. Viruses enter mammalian cells following binding be¬ tween a viral receptor and a chemical structure, or li- gand, in the cell membrane. This initial binding is a first and necessary step leading to the entry of virus into the cell and the subsequent transcription of the viral genome and replication of the virus within the cel Rather detailed knowledge about the structure and speci¬ ficity of many viral receptors is at hand. For example, the case of influenza virus the specificity of the virus receptor is analogous to the specificity of antibodies against certain tumor antigens, in that the influenza virus hemagglutinin binds to NeuAc2-3Gal-R, which is pre¬ sent on the same structures as many human tumor associat antigens. The main object of the present invention is to pro¬ vide techniques enabling introduction of viruses into mam malian cells for providing desired biological action in such cells. Another object of the invention is to enable virus vectors or infectious viruses to be directed to specific cells in a living animal body. Yet another purpose is to provide techniques making it possible for viruses to selectively enter specific cells in a living animal body. For these and other purposes the invention provides for a complex between a virus and an antigen-binding substance selected from antibodies, fragments of anti¬ bodies and antigen-binding peptides, the cell binding receptor of said virus being inactivated. Said substance has the ability to interact with a specific antigen on the surface of a mammalian cell which antigen is different from the cellular structure which would otherwise mediate binding of the virus to the cell surface. Furthermore, said substance has the ability to mediate entrance of a virus vector or infectious virus into the cell. The substance can either be a monoclonal antibody or a fragment of a whole antibody. The substance can be bound to the virus in different ways, such as by chemical conjugation, by bridging the substance to the virus by an immunochemical reagent, or by using a bifunctional substance binding to the virus as well as to a cellular antigen. The substance may also be expressed on the virus sur¬ face envelope following cloning into the virus genome of gene(s) for whole antibodies or fragments thereof. The inactivation of the viral cell receptor in accor¬ dance with the present invention can take place by chemi¬ cal means, by a specific antibody or by gene technological manipulation of the viral genome. Thus, the gene for the viral cell receptor or parts thereof can be replaced with antibody genes , The complex according to the present invention is in¬ tended for medicinal use. Thus, the complex can be used as a vector to introduce genes into cells or organs for spe¬ cific therapeutical purposes. The complex may also be used to express viral antigens on the cell surface for such purposes or it can be used to cause an infection in cells carrying the substance against which the antibody is reac¬ tive. By using an antibody which binds to a cellular mem¬ brane antigen which can be internalized into the cell, virus can bind to the cell via an antibody-antigen reac¬ tion and then penetrate into the cell and replicate caus¬ ing a viral infection. Furthermore, since antigens are known which are more or less unique for certain types of cells, virus can be selectively directed to enter and in- feet specific cells in the body. Examples of such antige are some cell-differentiation antigens, such as CD19 on B cells, and certain tumor-associated antigens on carcinoma cells. The invention will therefore make it possible to di¬ rect virus vectors or infectious viruses to specific cell in the body with the possibility of introducing functiona genes into selected cells or organs in vivo or to cause a viral infection in certain cells. The following table shows examples of viral receptor and their cellular ligands. Receptors such as these are possible candidates for use within the present invention. TABLE 1 Some viral receptors and their cellular ligands. Virus Cellular ligand Distribution Viral recepto Epstein- CD21 B cells gp350/220 Barr Influen- Sialyloglycolipid Many cell Hemagglutinin za A or Sialyloglyco- types protein containing NeuAc2-3Gal-R Para- Sialic acid-con- Many cell HN, H and G myxovirus taining carbohyd- types prot trates Retrovirus Protein Many cell SU protein types When virus replicates in the cell certain viral pro¬ teins are degraded to peptides. These peptides are bound to so called MHC antigens within the cell and are there¬ after transported to the cell membrane where the peptide- MHC complex serve as target for a very important defense system, i.e. virus specific cytotoxic T cells, which actu ally kill the virus infected cell. Certain virus, e.g. retroviruses, are integrated into the host cell genome in the form of provirus which may thereafter be replicated. By "genetic engineering" methods retroviruses can be mani¬ pulated so that they no longer replicate but can be used as vectors to introduce new genes into mammalian cells * . The use of retroviral vectors offers a practical means for gene therapy in humans. However, at the present time the use of retroviral vectors are largely restricted to cells of hemopoietic origin. There are several technical routes along which the cell-binding receptor of a virus can be replaced with an antibody. Some principal ways are listed below and illu¬ strated in the appended drawings, viz. figures 1 to 3. 1. The viral receptor can be blocked with an antibody which may in turn be conjugated with an antibody of the desired specificity. 2. The viral receptor can be inactivated by chemical- or gene technological methods and the replacing antibody can then be chemically coupled directly to the virus par¬ ticle. 3. The gene for the viral receptor can be replaced with the genes for the replacing antibody. The possibilities inherent in this invention are many. One area is gene therapy in humans where retroviral vectors have already been used to introduce genes into human cells for therapeutical purposes. The present inven¬ tion may be expected to increase the efficiency of this approach as well as making possible new areas of use, such as the deliberate introduction of genes into tumor cells. Another area is immunotherapy of tumor diseases where the invention migt be used for gene therapy or to intro¬ duce virus into tumor cells with the intent of expressing viral antigens at the cell surface converting the cells into possible targets for the virus-specific cytotoxic T cells. The invention will be further illustrated in the fo lowing by specific examples which are not construed to r strict the scope of the invention otherwise than defined by the appended claims. EXAMPLE 1 The following examples demonstrate that the binding specificity of a virus can be blocked and replaced with that of an antibody. In the examples the binding and in¬ fection of Influenza virus A/PR/8 to human colon carcino cells Colo 205 is used. The cell binding of influenza vi rus is depending on a specific binding molecule, Hemaggl tinin (HA), which is present in the viral envelope, and which binds to NeuAc2-3Gal-R, present on many mammalian cells. In this example the following strategy was employed 1. The binding of A/PR/8 to the target cell was blocked by means of the monoclonal antibody HK-PEG-1 a- gainst influenza HA (Koprowski H, Gerhard W and Croce CM Production of antibodies against influenza virus by soma tic cell hybrids between mouse myeloma and primed spleen cells. Proc.Natl.Acad.Sci USA, Vol. 74, pp 2985-2988, 1977). The cell line producing this antibody is obtainab from the American Type Culture Collection, 12301 Parklaw Drive, Rockville, Maryland 20852, USA. 2. A new binding specificity was conferred on the virus by another antibody which had been chemically con¬ jugated to the HK-PEG-1 antibody used to block the natur cell binding of the virus. The antibody OKT9 (US Patent 4,364,934) which reacts with the transferrin receptor on proliferating mammalian cells was used for this purpose. An antibody against the transferrin receptor was used be cause the transferrin receptor is rapidly and constituti nally internalized by the cells along with an antibody which has been reacted with the receptor (Schwartz AL: Cell Biology of Intracellular Protein Trafficking, Ann.Rev.Immunol., Vol 8, pp 195-229, 1990). The cell lin producing the OKT9 antibody is obtainable from the Ameri¬ can Type Culture Collection. The expected dual effect on virus binding of the an¬ tibody conjugate used is schematically shown in Fig. 4 of the drawings. EXAMPLE 2 Influenza A/PR/8 was obtained from Statens Bakterio- logiska Laboratorium, S-10521 Stockholm, Sweden. HK-PEG-1 and OKT9 antibody Hybridoma cells were obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Mary¬ land 20852, USA. Hybridoma cells were grown in Iscove's medium supplemented with 10% fetal calf serum. Antibody production was performed in dialysis tubings (Sjδgren- Jansson E and Jeansson S: Large-scale Production of Mono¬ clonal Antibodies in Dialysis Tubin, J.Immunol.Meth., Vol 84, pp 359-364, 1985) and antibodies were purified by af¬ finity chromatography on a Protein A Sepharose CL-4B (Pharmacia, Uppsala, Sweden)(Ey PL, Prowse SJ and Jenkin CR: Isolation of pure IgGl, IgG 2 and Ig ?h immunoglobu- lins from mouse serum using Protein A-Sepharose. Immuno- chemistry Vol 15, pp 429-436, 1978). Antibody conjugates: Antibody conjugates were prepared using the hetero- bifunctional reagent N-succinimidyl (2 pyridyldithio)- propionate, SPDP (Pharmacia, Uppsala, Sweden) (Carlsson J, Drevin H and Axen R: Protein thiolation and reversible protein protein conjugation. N-succinimidyl 3(2 pyridyldi¬ thio)propionate - a new heterobifunctional reagent. Biochem.J., Vol 173, pp 723-737, 1978). Conjugates were purified from unreacted antibody by gel filtration on Superdex 200 (Pharmacia, Uppsala, Sweden). Titration of virus by Hemagglutination: Hemagglutination of human blood group 0 red blood cells was performed in 96-well trays using standard me¬ thods (Mahy BWJ, ed: Virology, a practical approach. IRL Press, Oxford, 1985). Colo 205 colon carcinoma cells were obtained from t American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA, and maintained in Iscove' medium with 10% fetal calf serum. Results Inhibition of viral binding by HK-PEG-1 antibody and HK- PEG-1 conjugates. Inhibition of viral binding to its cellular ligand was measured by the hemagglutination method. Virus, 800 HAU (HemAgglutinating Units)/ml, was incubated for lh at room temperature with antibody at different concentratio and then allowed to agglutinate human red blood cells. H PEG-1 antibody caused complete inhibition of agglutinati at 7μg/ml or more wheras the corresponding figure for HK- PEG-1 conjugated to 0KT9 was 10 μg/ml. Ability of HK-PEG-1 conjugates to mediate viral infection HK-PEG-1 or HK-PEG-1 conjugated to 0KT9 was incubate with 100 HAU of A/PR/8 virus for 1 hour at room tempera¬ ture in order to block the HA molecules in the viral en¬ velope. Thereafter the virus preparations were incubated . together with 2x10 Colo 205 cells in 1 ml PBS for 1 hour at 37°. The cells were washed three times with PBS by c n tr.ifugation, suspended in 1 ml of complete medium and in¬ cubated at 37° for 24 hours. Presence of virus in the me¬ dium was assessed by hemagglutination. The results are shown in Table 2 below. It is evident that the conjugatio of 0KT9 to HK-PEG-1 allows the virus to infect the cells by giving the virus a new binding site in the cell mem¬ brane, thereby overcoming the inhibition caused by the HK .PEG-1 antibody alone. The decreasing infectivity with increasing antibody concentration is probably an effect of formation of large virus-antibody complexes since such complexes are less effectively taken up into the cell. TABLE 2 Production of virus as measured by hemagglutination by Colo 205 cells infected with Influenza A/PR/8 pretreated with different antibodies. Antibody Concentration (μg/ml HA-titer None 1/16 Negative (<l/2) Negative (<l/2) Negative (<l/2) 1/8 1/4 1/2

Claims: CLAIMS 1. A complex between a virus the cell binding re¬ ceptor of which has been inactivated and an antigen- binding substance selected from antibodies, fragments o antibodies and antigen-binding peptides, said substance having the ability to interact with a specific antigen the surface of a mammalian cell different from the cellular structure which would otherwise mediate bindin of the virus to the cell surface and which antigen has ability to mediate entrance of a virus vector or infectious irus into mammalia, cells. 2. The complex according to claim 1, wherein the s stance is a monoclonal antibo ττ . 3. The complex according to claim 1, wherein the s stance is a fragment of a whole antibody, whether prepa by chemical means or by expression of antibody genes or DNA sequences containing information derived from antib genes. 4. The complex according to claim 1, wherein the s stance is an antigen-binding peptide, whether derived f an antibody sequence or not. 5. The complex according to any preceding claim, wherein the substance is bound to the virus by chemical conjugation. 6. The complex according to any preceding claim, wherein the substance is bridged to the virus by an immu nochemical reagent, such as an anti-species antibody, avidin or streptavidin. 7. The complex according to any preceding claim, wherein the substance is a bifunctional antibody binding to a virus as well as to a cellular antigen. 8. The complex according to any of claims 1 to 4, wherein antibody binding site(s) is (are) expressed on t viral surface following cloning of antibody gene(s) or D sequence(s) coding for antibody binding structure(s) into the viral genome. 9. The complex according to any of claims 1 to 4, wherein the substance is reactive with a cellular antigen capable of mediating entry of a virus into a cell. 10. The complex according to any preceding claim,- wherein the viral cell receptor has been inactivated by chemical means, by a specific antibody or by gene techno¬ logical manipulation of the viral genome. 11. The complex according to any of claims 1 to 9, wherein the gene for the viral cell receptor or parts thereof has been replaced with antibody genes. 12. The complex according to any preceding claim for medicinal use. 13. The complex according to claim 12 for use as a vector to introduce genes into cells or organs for thera¬ peutical purposes. 14. The complex according to claim 13 for use to ex¬ press viral antigens on the cell surface for therapeutical purposes. 15. The complex according to claim 13 for use for therapeutical purposes to cause an infection in cells carrying the antigen against which the substance is re¬ active.

Inventors: ENERBAECK SVEN, LINDHOLM LEIF, STRANNEGAARD OERJAN, ENERBAECK, SVEN, LINDHOLM, LEIF, STRANNEGAARD, OERJAN
Applicants: THERA GENE HB
Application year: 1992
Main classification: A61K48
IPC codes: A61K39, A61K48, A61P31, C07K16, C07K19, C12N15, C12P21