JP2000511401A - Adapter molecules that target viral particles to cells - Google Patents

Abstract

  (57) [Summary] The present invention relates to molecular adapter molecules for targeting viral particles, especially retroviral particles, to cells. The adapter molecule comprises a first amino acid sequence having a binding affinity for a cell surface molecule and a second amino acid sequence having a binding affinity for a virus surface molecule. Adapter molecules are useful in gene therapy to target retroviral delivery vectors to specific cell types.

Description

DETAILED DESCRIPTION OF THE INVENTION       Adapter molecules that target viral particles to cells   The present invention relates to adapter molecules that facilitate binding of virus particles to cell surface molecules. I do. The present invention also provides a nucleic acid encoding an adapter molecule, expressing an adapter molecule. Cell lines, and the use of adapter molecules in gene therapy. Introduction   A common goal of gene therapy is the most commonly used nuisance ex viv o To make the gene deliverable in vivo, rather than in a method. Deliver in vivo In developing new technologies, targeting delivery systems to specific cell types It would be of considerable advantage if the gene could be delivered selectively. The present invention Achieves targeted gene delivery using retroviral vectors A new method for achieving this is described.   Recently, some good results in targeting retroviral vectors was gotten. Current retroviral vectors used for human applications Is an amphotropic envelope that infects a wide range of cell types from diverse species It is indiscriminate in that it carries loop proteins. It is used in the laboratory Carry an ecotropic envelope that only infects rodent cells In contrast to other major types of retroviral vectors. Effective in vivo To achieve targeted and selective gene transfer. Considerable effort has been expended in producing retroviral vectors to be I have. The main approach is to use EGF (Han et al., 1995 PNAS 92, 9747; Cosset et al., 1995 J Virol 69, 6314), EPO (Kasahara et al., 1994 Science 266, 1375), pepti containing RGD Do (Valsesia-Wittman et al., 1944 J. Virol 68, 4609), antibody fragments (Russell et al. 1993 Nucl. Acids Res. 21, 108), single chain antibodies (Tearina and Domburg, 1995 J. Virol 69, 2659, Somia et al., 1995 PNAS 92, 7570) Was to construct an envelope protein. In addition, target liver cells Chemical modifications of the envelope protein have been used for enveloping (Neda et al., 1). 991 J.C. Biol. Chem. 266, 14143), to bind viruses to host cells. Tin-streptavidin crosslinks have been used (Roux et al., 1989 PNAS 86, 9079). . Often, these "targeted" retroviruses have the appropriate cells Has been demonstrated to bind specifically, but in all cases Was significantly lower than that obtained with the amphotropic envelope. further Any chimeric envelope can be used to perform any gene transfer , Co-expression of unmodified envelope proteins, viral deglycosylation, Requires any of the cell treatments with endosomal modifiers (e.g., chloroquine) I do. Numerous technologies in this area have been published, but none of them are Teach how to efficiently target retroviruses to specific cell types Not in.   Features common to all of the above methods of targeting retroviral vectors Symptoms indicate that the SU component of the viral envelope protein is directed to a specific cell surface molecule. The SU component has been modified in some way to orient it. Embe This is because the structure and function of the rope protein is very sensitive to changes. That the modification of the envelope of a species does not lead to effective targeting gene transfer Is clear.   Therefore, improved delivery of genetic material from retroviral vectors to target cells Method is needed. The present invention aims to meet such needs. The present invention   In accordance with the present invention, targeting an allotropic receptor to a particular cell type, An alternative to using a modified allotrophic vector to deliver genes to these cells And state the new approach. This is because the envelope protein is Has the considerable advantage of causing binding and fusion events similar to those infecting vesicles You.   The present invention, in one embodiment, comprises a first having a binding affinity for a cell surface molecule. And a second amino acid having a binding affinity for a virus surface molecule Acid sequence, and the cell surface component by virus particles having the virus surface molecule. An adapter molecule is provided that mediates infection of cells expressing the offspring.   Said second amino acid sequence is preferably derived from a viral receptor and is most preferably Alternatively, it consists essentially of the extracellular portion of the virus receptor to which the virus particles bind.   The first amino acid sequence in the adapter molecule of the present invention is specific for a cell surface molecule. Any amino acid sequence having different affinities may be used. For example, antibodies or antibodies Derivatives, natural ligands or derivatives of natural ligands, or phage display (pha ge display). It can be a peptide. Examples of antibody derivatives suitable for use in adapter molecules include: A single chain antibody (scFv). scFv targets any specific cell surface molecule Can be used to Particularly useful scFvs are tumor specific cell surface Has binding affinity for native and tissue-specific cell surface proteins .   The first and second amino acid sequences described herein may be glycosylated or the like. It is understood that this may involve the usual modifications associated with proteins and peptides. Will be.   In the adapter molecule of the present invention, the first amino acid sequence is A second amino acid sequence at the amino terminus or at the first amino acid sequence It is reasonable that the sequence can be located at the amino terminus and the second amino acid sequence at the carboxy terminus. Will be understood.   The adapter molecule of the present invention mediates infection of cells with virus particles as described. Through. As used herein, the term "infection" refers to a virus particle. Attached to the vesicle surface, meaning that genetic material is introduced into cells from viral particles . Thus, the adapter molecule is involved in the process of transduction, ie, virus-mediated genetics. Can help with child introduction.   The first and second amino acid sequences may be covalent or Non-covalently bound. Adapter, if covalently bound The molecule can be produced from a single fusion gene or by chemical crosslinking. Meeting Are non-covalent, both components are streptavidin-biotin bridges, or Can be bound by other binding partners.   In another aspect, the invention encodes an adapter molecule described herein. Provide a nucleic acid. Whether intracellular or extracellular expression of the adapter molecule is desired Thus, a nucleic acid may or may not also include a secretory signal coding region. You. If a secretory signal coding region is present, it can be, for example, yeast, E. coli. coli, or yeast, E. coli, for expression and secretion in mammalian cells. Or it can be a mammalian signal.   The virus surface molecule to which the adapter molecule of the present invention binds is not required but is preferred. Or an unmodified homotrophic viral protein. Thus, the present invention provides Identify virus vectors with minimal modification to the virus envelope To cell surface molecules. However, one of the virus envelopes Parts (e.g., specific viral surface molecules of interest) Can be profitable.   In a further aspect, the invention includes a nucleic acid as described herein, wherein Genetically engineered to allow the expression of adapter molecules described in Provide the produced cells.   In the context of gene therapy, the adapter molecules described herein may Immediately prior to administration of the tropic vector particles, target cells can be administered either in vivo or in vitro. Administered to vesicles. One aspect of the invention also relates to the use of pharmaceuticals used in gene therapy. There is provided the use of an adapter molecule as described herein in the manufacture.   In the accompanying drawings:   FIG. 1 is a schematic diagram of a basic amino acid carrier in a membrane.   FIG. 2 shows the principle of the targeting molecule adapter of the present invention.   FIG. 3 illustrates the construction of one embodiment of the targeting adapter of the present invention. The following shows the scheme.   Receptors for all mouse allotropic viruses (F-MLV, R-MLV, and Mo-MLV) Mouse cationic amino acid carrier (Albritten et al., 1989 Cell 57, 659; Kim Et al., 1991 Nature 352, 725; Wang et al., 1991 Nature 352, 729). In this specification , Its receptor is referred to as the ecotropic retroviral receptor (ERR). This is yeast Deficiencies associated with arginine, histidine and choline carriers in It is a membrane protein that cannot be removed. By analogy with other carriers, and also by membrane protein Measurement programs (membrane protein prediction programs) Thus, putative regions that span the membrane in the amino acid sequence of the carrier can be identified. these Is schematically shown in FIG. MEMSAT program (Jones et al., 1994 Biochemistr y 33, 3038), we have determined that the coordination of the transmembrane region is 37-58, 65-84, 108 ~ 129,166 ~ 183,192 ~ 210,243 ~ 266,281 ~ 305,330 ~ 349,379 ~ 396,404 ~ 423, 487-510, 519-540, 553-572, and 582-599.   Analysis of specific mutants, and human and mouse cationic amino acid delivery Several studies, including body comparisons, have shown that viruses It has been suggested to interact with the three extracellular domains (210-243). This is Has the following amino acid sequence:   VKGSIKNWQLTEEDFGCNNNDTNVKYGEGGFMP [SEQ ID NO: 1]   Tyrosine and glutamic acid residues shown in bold are particularly relevant to viral infections. (Eiden et al., 1993 J. Virol. 67, 4056; Yoshimoto et al., 1993 J. Virol. 67, 1310; K avanaugh et al., 1994 JBC 269, 15445; Yoshimoto et al., 1993 J. Virol. 67, 1310; MacL eod et al., 1990 MCB 10, 3663). Herein, this region of the protein is ERRed Called 3.   In one particular embodiment of the invention, ERRed3 is ERRed3 specific cell surface Bound to a second molecule that is targeted to the molecule. The combined molecules are , Known as targeting adapters. This allows, for example, Specific targeting of the domain to a particular cell type is achieved. Once specific When bound to a cell type, the third domain is responsible for the reception of allotropic retroviral particles. Used as body. Thereby, the gene carried by the retrovirus vector Selective delivery to specific cell types is achieved. The above system is suitable for gene therapy In general.                                   Example   Example 1   In this first example, a single line directed to the cell surface marker CD4 Chain antibody (scFv) to ERRed3 and target the receptor fragment to CD4 + cells To run. This adapter molecule delivers the anti-HIV gene to cells susceptible to HIV. Useful to reach.   Techniques for generating scFvs are well established and are usually available from Pharmacia Biotech and Novagen. Available in kits sold by such companies. scFv code fragment a Generate OKT4 monoclonal antibody using Biotech kit Produced from RNA from the emerging hybridoma. Then this fragment Ligated to the coding sequence of ERRed3 using standard genetic engineering techniques. The latter The fragments are produced by PCR using the following primers:   To get a constrained loop similar to the real extracellular domain The fragment consists of amino acids 210-243 with additional cysteine residues located at each end. Code.   The structure of the fusion gene is as shown in FIG. 3a (ERR-3 = homotropic retrovirus). The third extracellular region of the Luz receptor; the site in parentheses is the site destroyed by ligation Is). The short coding sequence encoding the secretory signal sequence is then Added to the 5 'end of the sequence to produce the coding sequence shown in Figure 3b. Secretory signal Sequence can be obtained from any gene, such as tPA or immunoglobulin genes. (TPAss = tPA secretion signal sequence). In this example, the tPA sequence is used. , Which is added by ligation of oligonucleotides of the following sequence :  This allows the adapter to be secreted from mammalian cells and referred to as OKT4sc-ERRed3. A fragment (FIG. 3b) is generated. This fragment is then pCl- insert into a mammalian expression vector such as neo (Promega), and the resulting plasmid is pCln-ADAPT-OKT4 was shown. Next, this plasmid was transiently transfected into 293T cells. Used to transfect. 48 o'clock After a short time, the cell supernatant was collected. This supernatant contains the secretion adapter.   The supernatant sample was filter sterilized and 1 ml was placed in a dish containing HeLa-CD4 cells . In a control experiment, 1 ml of untransfected 293T cell supernatant was -Placed in a dish containing CD4 cells. The cells were exposed to the supernatant for 1 hour and then⁶ Aliquot 1 ml of the allotrophic retroviral vector preparation containing transducing particles / ml. One was added to both dishes. Uses HIT vector system (Soneoka et al., 1995 NAR 23, 628) When preparing retrovirus vector particles, the retrovirus vector particles It carries the vector genome HIT111 containing the E. coli lacZ gene. After 48 hours, remove the dishes from X-g The cells were stained with al and the blue cells receiving the vector were counted. Supernatant containing adapter In the absence of any blue cells were not seen. If an adapter is included, About 10^Four~Ten^FiveTransducer / ml vector was observed. This is because the adapter is CD4 + It indicates that it mediates gene transfer into cells. In further experiments, HeLa cells If cells were used, no blue cells were seen in the adapter supernatant and the adapter -Substantial blue cells if OKT4 antibody is added to HeLa-CD4 cells prior to treatment with supernatant Significant decrease was seen.   Adapter molecules are responsible for various gene fusions with subtly different junctions between scFv and ERRed3. Can be produced by a combination. Example 2   If the targeting molecule is not an antibody, but any other A method similar to that described in Example 1 was used, except that it was a ligand. Can be In this example, the initial VCAM was prepared using standard recombinant DNA procedures. Were fused to ERRed3. VCAM-ERRed3 Tan The protein is produced in a mammalian, yeast, or E. coli expression system, followed by OKT4svERRed3 Use the retroviral vector as described above for proteins and Target to cells. Example 3   The construction is similar to that described in Examples 1 and 2, except that the ERRed3 sequence Amino terminal component, and the OKT4scFv or VCAM1 domain is the carboxy terminal component. You.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 19/00 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 C07K 14 / 47 5/10 16/28 // C07K 14/47 C12N 5/00 A 16/28 A61K 37/02 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, C , CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT , UA, UG, US, UZ, VN, YU (72) Inventors Kingsman, Susan, Mary United Kingdom OEX 52 SEF Oxon, Islip, Middle Street, Graystones (no address)

Claims (1)

[Claims] 1. A first amino acid sequence having binding affinity for a cell surface molecule; A second amino acid sequence having a binding affinity for a surface molecule; Infection of Cells Expressing the Cell Surface Molecule by Virus Particles Having a Surface Molecule An adapter molecule that mediates 2. 2. The method according to claim 1, wherein the second amino acid sequence is derived from a virus receptor. Adapter molecule. 3. The second amino acid sequence is a virus receptor to which the virus particle binds; 3. The adapter molecule of claim 2, consisting essentially of an extracellular portion. 4. The first amino acid sequence is a single-chain antibody directed to the cell surface molecule. The adapter molecule according to any one of claims 1 to 3, wherein 5. The first amino acid sequence is derived from a natural binding ligand of the cell surface molecule. The adapter molecule according to any one of claims 1 to 3, wherein 6. The first and second amino acid sequences are covalently fused. 6. The adapter molecule according to any one of 1 to 5. 7. The adapter according to any one of claims 1 to 6, which is used for gene therapy. -Molecule. 8. A nucleic acid encoding the adapter molecule according to any one of claims 1 to 7. 9. 9. The nucleic acid according to claim 8, which also encodes a secretion signal. Ten. The adapter molecule according to any one of claims 1 to 6 can be expressed. A genetically engineered cell comprising the nucleic acid according to claim 8 or 9, which is capable of functioning. . 11． 8. The method according to claim 1, which is for the manufacture of a medicament for use in gene therapy. Use of an adapter molecule according to any one of the preceding claims.