JPH07502753A - Transvascular and intracellular transport of lipidated proteins - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Transvascular and intracellular transport of lipidated proteins Technical field of the invention The present invention provides methods for targeting proteins, such as antibodies, to subcellular compartments within eukaryotic cells. Method: Compositions consisting of proteins, modified proteins for use in human therapy Provided is a method for increasing the uptake of a protein by an organ: and a method for producing a modified protein. It is something. The modified proteins of the invention contain an attached lipid moiety and include one or more the acyl group of the carbohydrate or the side chain of the carbohydrate by various covalent chemical reactions provided is bound to proteins through

Background of the invention Numerous naturally occurring or modified proteins are available as diagnostic agents for use in humans and livestock. and/or as a therapeutic agent. However, proteins are generally Transport across the membrane is negligible and is usually carried across the cell membrane to intracellular compartments. I just can't reach it.

Thus, for example, transcription factors, intracellular enzymes and cytoarchitectonic structural proteins. Is it possible to generate antibodies against purified intracellular proteins such as If such antibodies do not rupture the cell membrane, they will enter the complete cell and target the antigen within the cell. It is only possible to combine them.

The emergence of monoclonal antibody technology in the mid-1970s heralded a new era in medicine. It was done. Initially, researchers and clinicians need to determine which antigens can bind to a given antigen site. Homogeneous antibodies with various immunological effector functions can be obtained in near-infinite quantities. Ta. These proteins are known as ``monoclonal antibodies'', but for example very promising for eliminating harmful cells, microbial pathogens and viruses in living organisms. It was thought that Includes antigens present within intracellular compartments in intact cells. Specific monochromes with binding specificity for almost all desired antigenic epitopes The method of "generating local antibodies" is a pharmaceutical "magic bullet" (no side effects). It promised an abundance of ``harmless drugs''.

Unfortunately, adequate treatments based on monoclonal antibodies and polyclonal antisera are lacking. The development of therapeutic products is hampered by the numerous drawbacks inherent in the chemistry of naturally occurring antibodies. thwarted. First, immunoglobulins cannot cross the plasma membrane and are generally It is only internalized as a result of efficient endocytic mechanisms. , antibodies generally do not effectively reach intracellular locations. Second, antibodies are generally It cannot pass through vascular membranes (e.g. endothelial basement membranes), and these membranes prevents efficient uptake of antibodies into the body and interstitial spaces. Therefore, capillary a specific biologically active immunoglobulin molecule that passes through a gate and enters an intracellular location If we had an effective way to obtain this, we could develop treatments for many important diseases. It will be possible. For example, the life cycle of retroviruses such as old V involves intracellular replication. A specific monoclonal that is necessary and reactive with the virus-encoded protein. Antibodies can easily reach the intracellular locations where retrovirus replication occurs. If possible, there are several essential factors for the production of infectious pylons from infected cells. The polypeptide encoded by the virus will likely be inhibited or blocked.

Deliver immunoliposomes or various molecules contained in liposomes to target cells , manufactured as a delivery system to potential targets. immunoliposomes utilizes immunoglobulins as targeting reagents, and acylated immunoglobulins are , tethered in the lipid bilayer of liposomes, and acylated in immunoliposomes. Targeting specific cell types with foreign antigens captured by globulins Directing liposomes (Connor and Huang, J., Ce1l Biol.

Volume 101, page 582, 1985; Huang L, Biochemist ry, vol. 24, p. 29, 1985 HBabbitt et al., Biochemi stry, vol. 23, p. 392o, 1984; Connor et al.; Proc. , Natl, Acad, Sci, (U, S, A,), vol. 81, 171 5, 1984; 11uang et al. 1.1. Biol, Chem, 25 Volume 8, page 14034, 1983 H5hen et al., Biochim, Biphy s, Acta, vol. 689, p. 31, 1982;) Iuang et al., Bio chim.

Biophys, Acta, volume 716, page +40, 1982; Huang g et al., J. Immunol.

MethodS, vol. 46, p. 141, 1981: and Huang et al., J. Biol.

Chem, vol. 255, p. 8015, 1980). Immune liposomes are generally , conjugation of liposomal bilayers by cross-linking agents such as N-hydroxysuccinimide. Immunoglobulins linked to sil substituents and thus immobilized in the liposomal lipid bilayer Contains Therefore, this cross-linked immunoglobulin is attached to liposomes. specific external antigens by binding to external cellular antigens. Helps target liposomes to cell types. This kind of method It helps to target liposomes to specific cell types, but immunoliposomes Somes are particularly useful as pharmaceutical transport vehicles to transport proteins to intracellular locations. There are a number of significant drawbacks that limit it.

To make it easier for proteins to cross the capillary barrier and enter cells Attempts have been made to modify proteins (European Patent No. 0329185) ). However, no completely satisfactory method has yet been reported in the art. anti Non-specific cationization of proteins such as the body on)’ to increase transvascular and intracellular transport of proteins. A method has been reported (US patent application Ser. No. 07/693.872). deer Current methods of producing cationized immunoglobulins are Compare the binding affinity of immunoglobulins to a given epitope (approximately 9006) compared to non-cationized immunoglobulin suitable for . Generally, a cationization reaction creates an amino acid in the polypeptide sequence of an immunoglobulin. Carboxylate of spartate and glutamate residues with putrescine or A carbodiimide bond of a diamine such as hexanediamine is formed. first class These chemical modifications with amino acids probably improve the secondary and secondary properties of immunoglobulins. The tertiary structure is thought to be destroyed, and the loss of binding affinity has not been adequately explained. It's clear. These methods also target proteins located within the variable region of the immunoglobulin chain. It cationizes the tamate and aspartate residues to some extent, thus increasing the binding affinity. There is a significant loss of tee and/or specificity.

Chemical modification of small molecules has also been proposed as a way to increase the transport of small bioactive compounds. It is being proposed. Felgner (PCT Patent Application Publication No. WO91/17242 ) is the production of lipid complexes consisting of lipid vesicles and biologically active substances contained within them. Discloses its composition. Also, Felgner et al. (PCT Patent Application Publication No. W091/16 No. 024) to facilitate the transport of small bioactive molecules in plants and animals. Discloses cationic parasitic compounds that are claimed to be useful. liposome and polycationic nucleic acids have been proposed as a method for delivering polynucleotides into cells. ing. Because liposomes often exhibit a narrow spectrum of cell specificity, DN When A is coated on the outside of liposomes, its DNA becomes sensitive to cellular nucleases. It often becomes sexual. A new polycationic lipospermine compound has a wide range of cellular Indicate the range: Behr et al., Proc, Natl, Acad, Sci, USA.

86, p. 6982, 1989), and DNA is coated with these compounds. It will be done. In addition, a combination of neutral and cationic lipids can be used for transfection of animal cells. It has been proposed as a sorption method (Rose et al., BioTechniques , vol. 10, p. 520, 1991).

Other methods of facilitating drug transport, particularly across the blood-brain barrier, Attention or lysis of hydrophilic drugs Pharmacology-based methods have been utilized, including conversion to sex drugs. most of the latency In the cation method, hydroxyl groups, carboxyl groups and primary amines of pharmaceuticals are blocking the -i group and making the drug more lipid-soluble, so that it easily crosses the blood-brain barrier. and transport it. Pardridge and Schimmel U.S. Pat. No. 4.902 ．． No. 505 deals with receptor-mediated transcytosis. discloses chimeric peptides that facilitate transport by.

Therefore, specific proteins such as antibodies can pass through the capillary barrier and Methods to facilitate intracellular transport, as well as intracellular proteins and monochrome Human and vertebrate diseases are amenable to treatment with targeted reagents such as natural antibodies. Used to treat disease 1. %6 Pharmaceutical compositions of such immunoglobulins are known in the art. It is widely requested.

Summary of the invention Immunoglobulin primary polybebutide sequences using relatively nonspecific coupling chemistry A method that increases the transport of antibodies into cells by attaching cationic substituents to The methods of the prior art are based on the secondary, tertiary and/or quaternary structure of the protein. (found) that these structural changes are cationized. This is clearly responsible for the loss of binding affinity observed with .

To overcome this, the present invention proposes a method for adding protein to proteins such as immunoglobulins. substituents that substantially destroy the biological activity (e.g., antigen binding) of the protein. A method of linking to the carbohydrate side chains of the protein, generally by co-existence bonding, such that It provides:

The present invention generally involves lipidating the carbohydrate moieties of glycoproteins or glycopeptides. The present invention provides a method for producing a lipidated protein. Generally, the present invention This method generally involves covalently bonding a lipid to the carbohydrate portion of a protein. Therefore, it is used to attach lipids such as riboamine to polypeptides, and this The carbohydrate moiety is generally chemically oxidized and then reacted with riboamine. to produce lipidated proteins. The resulting lipidated protein is generally increased ability to cross the blood vessel barrier and reach parenchymal cells in various organs; It has advantageous pharmacokinetic properties such as increased ability to reach internal compartments. present invention In one embodiment, proteins specific for transcription factors (e.g. Fos, Ju When proteins such as n, AP-1,0CT-1゜NP-AT) are lipidated, , the intracellular localization of lipidated proteins is promoted.

The present invention also provides for effective transport in vivo through the capillary barrier, and for mammals. Provides a method for producing lipidated antibodies that can be internalized into cells It is something to do. This method of the invention provides a method for reducing carbohydrate substituents on immunoglobulins. Chemically link at least one lipid substituent (e.g. riboamine) to a carbohydrate. Lipidized immunoglobulin (carbohydrate-1i) Those who manufacture 1ipidized immunoglobulin) With respect to the method, the lipidated immunoglobulin produced can be localized intracellularly. Book In another embodiment of the invention, at least one lipid substituent (e.g. riboamine) Covalently linked to the non-carbohydrate portion of a protein or polypeptide (e.g. A An amide bond with a carboxyl substituent on the side chain of sp or Glu residue or Cys by the formation of 1,000-onythyl bonds with residues). In addition, unilateral fatty acids have amine substituents on their side chains. can be linked to Arg or Lys residues by.

Similarly, lipid substituents can be covalently attached to peptidomimetics. Pepti Peptide analogues are typically pharmaceutically available as non-peptide drugs with properties similar to the template peptide. Used in industry. These types of non-peptide compounds are called “peptide analogues” ” (“peptide mimetics” or -peptide mimet ics-) (Fauchere, J., Adv.

Drug Res, vol. 15, p. 29, 1986; Veber and Fre Idinger, TlN5゜392, 1985: and Evans et al., J. , Med, Chem, vol. 30, p. 1229, 1987. Furthermore, these The literature is hereby incorporated by reference), which is usually used for computer-based molecular modeling. Therefore, it is manufactured. Peptides that are structurally similar to peptides used to treat Analogs can be used to produce equivalent therapeutic and prophylactic effects. generally peptide analogs are paradigm polypeptides (i.e., have biological or pharmacological activity). is similar in structure to polypeptides known in the art and further described below. -CH2NH-, -C)I, S-, -C Il, -CH, -+ -CH=CH- (cis and trans), -COCH ,-, -CH(0)()C1,- and -CIl, So- have one or more peptide bonds optionally replaced with bonds that are (“Ch. emistry and biochemistry of Am1no Ac 1ds.

Peptides, and Proteins″B, edited by Weinstein Collection, Marcel Dekker, = New York, 267 pages (1983) 5 patula, A, F, paper; 5 patula.

A, F, ``Pepti'' in Vega Data (1983), Volume 1, No. 3 de Backbone Modifications” (Overview) +Mo rley, J, S, Trends Pharm Sci 463-468 Page, 1980 (overview); Hudson, D., et al., Int. J. Pept. 　Prot　Res.

Volume 14, pages 177-185, 1979 (Ctt2NH, CHzC112); 5patola.

A, F, et al., Life Sci, vol. 38, pp. 1243-1249, 1986 Year(-CH,-3);t(ann, M, M,, J, Chem Soc P arkin Trans i pp. 307-314, 1982 (-CIl-C1 1-, cis and trans) + AImquist, R, G, et al., J Med Chem, vol. 23, pp. 1392-1398, 1980 (-COCtlz- ); Jennings-White, C. et al., Tetrahedron Lett, Volume 23, Page 2533, 1982 (-COCH,-); 5z Elke, M. et al., European Patent Application No. 45665 (1982), CA : 97 : 39405 (1982) (-C1l(011)CJ(,- );1lolladay.

M, W, et al., Tetrahedron Lett, vol. 24, 4401-44 04 page, 1983 (-C(OH)CH2); and! Iruby, V.J. , Life Sci, Volume 31, pp. 189-+99, 1982 (-CH2- 5-). Each of these documents is incorporated by reference in this application]. Particularly preferred non-peptide The bonds are -CH,NH-. Such peptide analogs can be used to implement polypeptides. There are important advantages over other aspects, such as more economical production, greater chemical safety qualitative, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered properties. It has the advantages of isomerism (eg, high spectrum biological activity) and reduced antigenicity. Pe Lipidation of putido analogs is based on quantitative structure-activity data and/or molecular modeling. one or more acyl linkages in non-interfering positions of the peptide analog, as predicted by the to covalently bond directly or through a spacer (e.g. amide group) Therefore, this is usually done. Such non-interfering positions are generally where the peptide analog binds. It is a location that does not come into direct contact with the macromolecular substance (for example, receptor) that produces a therapeutic effect. Be The lipidation reaction of the peptide analogues is used to achieve the desired biological or pharmacological activity of the peptide analogues. shall not substantially impede sexual activity.

The present invention also provides lipidated antibodies that can cross vascular membranes and enter intracellular compartments. In particular, the virus coats gene production, which is an essential component of the virus life cycle. intracellular immunotherapeutic targets such as proteins (e.g. HIV-I Tat protein), Physically active intracellular antigens (e.g. Cgos, c-src, c-myc, Cancer genes such as c-lck (p56), c-fyn (p59) and c-abl genes, proteins) and/or transmembrane or extracellular antigens (e.g. IL-2 receptor, PDGF receptor, EGF receptor, NGF receptor, Gl+ receptor lipids that bind to the body or polypeptide hormone receptors (such as TNF receptors) The present invention relates to therapeutic and diagnostic compositions comprising lipidated proteins, such as lipidated antibodies.

Other proteins that can be targeted by lipidated antibodies include However, the following ones, namely c-ras p21. c-her-2 tan Activates protein, c-rat, various G proteins and/or G proteins proteins (GAP), transcription factors such as NF-AT, calcinin urin and cis-trans prolyl isomerase. Anti-lipidation of the present invention The body is a radio contrast agent or a diagnostic reagent, such as a magnetic resonance imaging component, to a specific location within the body, e.g. Organs, tissues, body compartments, cell types, neoplasms or other anatomical structures (e.g. It can be used to localize to pathological lesions (e.g., pathological lesions). Moreover, the lipid of the present invention Antibodies can be used to bind therapeutic agents such as chemotherapeutic drugs, radiosensitizers, radiosensitizers, etc. radionuclides, which can be used to localize drugs such as antibiotics to specific locations within the body. Wear. Alternatively, the lipidated antibodies of the present invention may be intravesicular target antigen, transmembrane or membrane-binding antigen targets (e.g. γ -glutamyl transpeptidase, c-ras' p21. rasGAP ), or extracellular antigen targets (i.e., β-aminoproteins in the brains of Alzheimer's patients). treatment by neutralizing (i.e. binding and inactivating) It can be used to The lipidated antibody of the present invention can pass through the blood-brain barrier and common to immunoglobulins circulating in the fluid or lymphatic system It can react with extracellular antigen targets that cannot. In addition, the anti-lipidation agent of the present invention The body processes viral envelope glycoproteins, lipids, or proto-oncogene proteins. Transcripts such as protein kinase regions (c-src, c-abl) Reacts with intracellular proteins on smembrane proteins to form an infectious envelope inhibit the production or kinase activity of the infected virus, respectively.

detailed description ” Unless otherwise specified, all technical and scientific terms used in this application are It has the same meaning as commonly understood by those skilled in the technical field to which it belongs. Ru. Methods and materials similar or equivalent to those described in this application may be used to carry out the invention. Preferred methods and materials that can be used for testing are described below. Book For purposes of the invention, the following terms are defined as follows.

As used in this application, the 20 common amino acids and their abbreviations follow conventional convention. (Immunology-A 5 synthesis, 2nd edition, E, S .

Golub and Tori, edited by R.Gren, 5inauer As5ociate S Corporation, Sunderland, Massachusetts, USA, 1991; ).

As used in this application, the term "equivalent to" refers to a polynucleotide sequence or a reference. homologous (i.e., strictly homologous to all or part of the polynucleotide sequence) have no evolutionary relationship and are identical), or the polypeptide sequence is identical to the reference polypeptide. means identical to the peptide sequence. In contrast, “complementary to” As the term is used in this application, complementary sequences of a reference polynucleotide sequence Homologous in whole or in part. For example, Nucleotid The code sequence “TATAC” corresponds to the reference sequence “TATAC” and the reference sequence “GT Complementary to "ATA".

As used in this application, the terms "substantially similar" or "substantially identical" refer to polypeptides. Characteristics of a polypeptide sequence or a nucleic acid sequence, such that the polypeptide sequence is compared to a reference sequence. that the sequence is at least 50% identical and that the nucleic acid sequence is at least 50% identical to the reference sequence; This means that at least 7o96 sequences are identical. Percent sequence identity Rates are calculated excluding small deletions or additions totaling less than 2596 of the reference sequence. Ru. The reference sequence is a constant domain, such as the constant domain of a constant immunoglobulin gene. The reference sequence may be a subset of a larger sequence or may be a small number in the case of polynucleotides. at least 18 nucleotides in length, and for polypeptides at least is also 6 amino residues long.

The term "naturally occurring", when applied to an object and used in this application, means that the object has a natural appearance. It means that it can be attached. For example, present in living organisms (including viruses) can be isolated from natural sources and intentionally modified by humans or in the laboratory. A polypeptide or polynucleotide sequence that is not present is a naturally occurring sequence. Heaven Lipotans that are found naturally and can be isolated from living organisms that have not been processed by humans. Proteins (e.g. naturally occurring isoprenylated or myristylated proteins) The material is a naturally produced seaweed paste material.

A “glycosylation site” is an amino acid that eukaryotic cells recognize as a place where sugar residues are linked. means a amino acid residue. Amino acids linked to carbohydrates such as oligosaccharides are one Generally asparagine (N-linked), serine (〇-linked) and threonine (〇-linked) is the residue of The specific site of linkage is herein defined as the “glycosylation site sequence” It is generally signaled by an amino acid sequence called . For N-linked glycosylation The sequence of the glycosylation site is -Asn-X-3er or -Asn-X-Thr - (where X is any normal amino acid other than proline). 〇-Linked glyco The predominant glycosylation site sequence for lylation is -(Thr or 5er )-X-X-Pro- (where X is any conventional amino acid). Group The recognition sequence for lycosaminoglycans (a particular type of sulfated sugar) is -3er-Gl y-X-GIY- (where X is any conventional amino acid). term" N-bond' and "〇-bond" refer to the bonding site between the sugar molecule and the amino acid residue. means a chemical group that is useful. N-linked sugars are linked through an amino group; 〇-linked sugars is bonded through a hydroxyl group. However, all glycosyl glycosides in proteins The glycosylation site is not necessarily glycosylated, and some proteins The substance is secreted in both glycosylated and non-glycosylated forms. , while for other proteins, the sequence of one glycosylation site is sufficient for glycosylation. contains other glycosylation site sequences that are glycosylated but not glycosylated. Ru. Therefore, all glycosylation site sequences present in a polypeptide are Of course, it is not the glycosylation site that actually connects the sugar residue. Early during biosynthesis In the glycosylation reaction, a “core carbohydrate” or “core oligosaccharide” is inserted ( Proteins, 5 structures and Mo1ecularPr inciples, 1984, edited by Creighton, W.F. References from Eman and Company, New York, USA are incorporated into this application. shall be incorporated herein by reference).

As used in this application, the term “glycosylating cell” refers to cells capable of oxidation, in particular N-linked cells containing at least one mannose residue. "0 = core oligosaccharide" and/or 0 = linking sugar expressed in said cell. at least one glycoprotein of at least one polypeptide, especially a secreted protein It is a eukaryotic cell that can add to the dylation site sequence. Therefore, glycosylated cells are Reaction of binding of sugar residues to glycosylation site sequences in proteins or polypeptides the cell actually has at least one enzymatic activity that catalyzes the reaction. Glycosylation of two expressed polypeptides. For example, but not limited to, Mammalian cells are generally glycosylated cells. others such as insect cells and yeast eukaryotic cells are glycosylated cells.

The term “antibody” as used in this application refers to the immunoglobulin superfamily A protein consisting of one or more polypeptides substantially encoded by a gene of (e.g. T, 1lonjo, P, W, Alt and T, H, Ra edited by bbitts “1mmunoglobulin Genes”, 1989 , Academic Press, San Diego, California, USA. A, F. Williams and A. N. Barcla, pp. 361-387. Y's paper “The Immunoglobulin Gene Superfam ily”, this document is incorporated into this application). For example, However, antibodies contain some or all of the heavy chains and some or all of the light chains. contain only some or all of the key ingredients. be recognized Immunoglobulin genes include λ, α, γ (IgG+, [gG2.IGz, I gG<).

δ, ε, and μ constant region genes, as well as a vast number of immunoglobulin variable region genes I have a child. Full-length immunoglobulin “light chain” (approximately 25 kd or 214 atoms) Ni+, terminal variable region gene (approximately 110 amino acids) and C0 It is coated with a constant region gene of 0II-terminus or λ. full-length immunograph Robulin “heavy chain” (approximately 50 kd or 446 amino acids) is similarly Variable gene (approximately 116 amino acids) and one of the remaining constant region genes above For example, it is encoded by the γ gene (encoding approximately 330 amino acids).

Antibodies include, but are not limited to, the following: immunoglobulin fragments ( For example, Fab, F(ab)2. Fv), single-linked immunoglobulins, texture immunoglobulins, humanized antibodies, primates Primatized antibodies and various light chain-heavy chain combinations be. The antibody can be applied to glycosylated cells (e.g., human lymphocytes), ibritoma cells, etc. cells, yeast, etc.) or in non-glycosylated cells (e.g. E. coli i)] or synthesized by chemical methods or polynucleotides. It can be produced in an in vitro translation system that directly translates using a template.

As used herein, the term "lipidated antibody" refers to immunoglobulin one or more carbohydrate moieties linked at the conjugation site (e.g. glycyldio Kutadecylamine, dilauroyl phosphatidylethanolamine or dioc (by covalent bonding of boamines like tadecylamidoglycylspermicin) It represents an antibody modified by lipid derivatization. Generally, lipid substituents such as riboamine is co-linked to naturally occurring glycosylation sites through naturally occurring carbohydrate moieties. . However, changes in the sequence of glycosylation sites (generally coded for immunoglobulin chains) (by site-directed mutagenesis of polynucleotides to be coded) and/or altered pattern of glycosylation (e.g. glycosylated cells other than lymphocytes or Expression of polynucleotides encoding immunoglobulins in lymphocytes of other species immunoglobulin can be produced. Lipid substituents linking to one or more naturally occurring or non-naturally occurring carbohydrate portions of the chain. I can do it. Antibodies can be produced by direct polypeptide synthesis or in non-glycosylated cells (e.g. For example, when produced by biosynthesis using a phage display library), Generally, carbohydrate substituents are attached by chemical or enzymatic modification followed by (or lipidation of carbohydrates and then preparing immunoglobulins) (may be attached to phosphorus).

As used herein, the term "lipidated protein" generally refers to carbohydrate moieties. Proteins modified by binding lipids (e.g. riboamine) through (including multimeric proteins, glycoproteins and polypeptides of various sizes) ) means. Lipidated proteins are produced by derivatizing proteins The resulting lipidated protein is a protein bound to naturally occurring lipids. and lipoproteins. Biologically active proteins (e.g. For example, enzymes, receptors, transcription factors), the lipidation reaction reduces biological activity. must not be significantly destroyed (e.g. at least about 15% of the native biological activity % must be conserved in the lipidated protein). lipidated peptide The analog has at least about 25 to 9 of the pharmacological activity of the corresponding non-lipidated peptide analog. Must hold 596.

``The term alkinodi means a fully saturated aliphatic group that is cyclic, branched, or means a straight chain group. Examples of alkyl groups include methyl, ethyl, cyclo Propyl, cyclopropylmethyl, 5eC-butyl, heptyl and dodecyl be. All of the above alkyl groups are unsubstituted or have one or more non-interfering positions. It may be substituted with a substituent, and non-interfering substituents include, for example, halogen; C, -C.

Ryorukoxy; C, -C, acyloxy: formyl: alkylene dioxy; benzene phenyl or benzyl (respectively halogen, C, -C, alkoxy or or optionally substituted with 1 to 3 substituents selected from C, -C, and acyloxy. There are) The term "non-disturbing" refers to Characteristics of substituents that do not adversely affect any reaction. 2 or more Al When kill groups are present in a given molecule, each independently, unless otherwise specified. selected from “alkyl”.

The term "alkylene" refers to a fully saturated binary containing only carbon and hydrogen. refers to a valent radical, and can be a branched or straight-chain radical. This term is Examples include methylene, ethylene, n-propylene, t-butylene, i-benzene. There are radicals such as n-hebutylene, n-hebutylene, etc. All of the above groups are unsubstituted. or -1 or more non-interfering substituents, the non-interfering substituents Examples of the group include halogen; C, -C, alkoxy: C, -C, acylskine; Formyl: Argylenedioxy: Benzyloxy: Phenyl or Hensyl ( each selected from halogen, C, -C, alkoxy or C, -C, acyloxy. optionally substituted with 1 to 3 substituents). The use of “non-disturbing” The term refers to substituents that do not adversely affect the reactions carried out in accordance with the process of the invention. Show characteristics. If more than one alkylene group is present in a given molecule, especially Unless otherwise specified, each is independently selected from "alkylene".

The term Arinobi, represented by Ar, includes a monomer having 6 to 20 carbon atoms. Contains cyclic or fused carbocyclic aromatic groups. Examples of aryl groups include Then there are phenyl and naphthyl. These groups may be substituted with one or more non-interfering substituents. and the non-interfering substituents are, for example, lower alkyl; lower alkenyl; Lukinyl-lower lucoxo; lower alkylthio; lower alkylsulfinyl; low alkylsulfonyl: dialkylamine; halogen; hydroxy: dimethyl; Phenyloxy, benzyl: device substituents consisting of benzoyl and nitro , optionally substituted with additional non-interfering substituents.

The term "amino" refers to the group -NH.

The term "alkylcarbonyl' refers to the group -(CIIRI)-CO-, in which R 5 means α-position). R1 is hydrogen, alkyl or amino group . Preferably R1 is an amino group.

Desirable fruit 7il! iDeaf explanation According to the present invention, proteins such as antibodies can be chemically modified to improve gearpilary-related A novel method for facilitating passage through the gate and introduction into cells is provided. Generally these The method includes at least one non-interfering lipid substituent (e.g. glycyl dioctadecyl Min, glycyldioctadecylamine, glycyldioctadecylamine, dilau Roylphosphatidylethanolamine and glycyldioctadecageno ylamide) on a reactive site of a protein molecule (e.g. carbon oxidized with periodic acid). This method consists of covalently bonding to a hydrate moiety). Various non-interfering lipid substitutions lipidated proteins, such as the lipidated antibodies of the present invention, by attaching groups to proteins. can be manufactured. For example, without limitation, the following examples of lipids: riboamine, Ribopolyamines and fatty acids (e.g. stearic acid, oleic acid, etc.) are important A lipidated protein can be obtained by conjugating it to a protein.

In general, lipids are carbohydrates that are bound to proteins (e.g., carbohydrates in glycoproteins). covalently bonded to the chemical side chain). Against naturally occurring carbohydrate side chain boamino Although the novel glycosylation site is preferably used to perform Encodings obtained by genetically manipulating coding polynucleotides Glycosylated polypeptides are produced by expressing polynucleotides in glycosylated cells. Polypeptides can be made by manufacturing peptides.

Glycosylated proteins are lipidated and lipidated proteins pass through blood vessels. Can promote transport, organ uptake and intracellular localization including intranuclear localization . Glycosylated polypeptides, such as antibodies, are commonly treated with oxidizing agents (e.g. periodic acid). chemically oxidized by carboxyl and/or aldehyde side groups generates and then reacts with riboamine to bind riboamine to the protein. Generates a covalent bond (generating an amide or imide, respectively). carbohydrates in general The side chain oxidation reaction involves at least one reactive carboxyl or aldehyde group. It is a partial oxidation reaction that produces some molecules that are oxidized and other molecules that are unoxidized or fully oxidized or something like that.

However, in order to be lipidated by reaction with riboamine, the glycoprotein must be acidified. to form at least one side aldehyde group capable of reacting with the riboamine. However, lipidated proteins can also be made by attaching them to carboxyl side groups. You can make a crispy texture.

The side group carboxyl or aldehyde group of the oxidized glycoprotein is It has a carbonyl carbon derived from oligosaccharides and is added directly or directly to the protein. through a pacer (e.g. the unoxidized portion of an N-linked or -linked carbohydrate side chain). It is a carboxyl group or an aldehyde group that is covalently bonded. N-bond and Beauty. - The carbohydrate chain of bonds is incompletely oxidized and has multiple generates a number of reactive aldehyde and carboxyl groups and then reacts with riboamine. It is preferable to comply. For example, branched (mannose).

one or more complex N-linked molecules having a (β-N-acetylgelcosamino) core Glycoproteins with oligosaccharides can be prepared using a suitable oxidizing agent, generally acid limited by periodate. It is most commonly partially oxidized in a chemical reaction. N-acetylglucosamine (NA G), mannose, galactose, fucose (6-dioxygalactose), N-acetylneuraminic acid (sialic acid), glucose, N-acetyl lumulamine acid, N-acetylgalactosamine, xylose, or a combination of these monosaccharide units The bound oligosaccharide containing the oligosaccharide is oxidized and then reacts with riboamine to form a lipid. Proteins, more specifically carbohydrate-bound lipidated proteins (carb ohydrate-1inked l1pidized proteins) generate. Monosaccharide units other than those specifically listed as examples (including non-naturally occurring monosaccharides) Glycoproteins containing linked oligosaccharides with It can be covalently bonded to proteins to produce lipidated protein proteins.

Riboamines have at least one acyl group and at least one free amine (i.e. (i.e., primary or secondary amine). The present invention also provides oxidation at least one substituent that can be substituted by reaction with a carbohydrate It can also be carried out using riboamine, which has a tertiary amine with Conceivable.

An example of a riboamine with a primary amine is shown in FIG. For example, according to the present invention, sugar Lipids are produced by reacting proteins with linear riboamine represented by the following formula. It is possible to produce modified proteins.

i.e. Formula: Nl, -R-(CL), -CO.

[Wherein, R is di-substituted alkyl (alkylene), preferably methylene (-C11 ,-); I, 4-disubstituted cyclohexyl; di-substituted aryl (arylene), Preferably 1,4-disubstituted phenyl (phenylene); formula: -(CHR,)- Amide group represented by CO-NH- (in the formula, R3 is hydrogen or an amino group): alkyl carbonyl, preferably α-amino substituted alkylcarbonyl; Ester, preferably a group represented by the formula CI+, -0-PO, -0- It is riboamine. In the formula, n is generally an integer of 1 to 50, preferably is about 5-30, more preferably about 10-25 and most usually about 15-20 is an integer. In general, n is determined and selected by engineers according to the guidelines below. do. That is, if the molecule to be lipidated is large (i.e. larger than about 10 kD) n is small in order to increase the hydrophobicity of the resulting lipidated protein. At least about 8 to 12 or more are preferred.

Also, if the molecule to be lipidated is small (e.g. oligopebutyl), n is generally It is within the range of 2 to 18, or larger if you want to further increase the hydrophobicity of the lipidated molecule. to save.

The invention can also be practiced using branched riboamines.

Examples of the riboan include those represented by the following formula.

[In the formula, R' is trisubstituted alkyl, preferably -CH, -C1+< or l , 2.4-trisubstituted cyclohexyl; trisubstituted aryl, preferably I, 2.4 -trisubstituted phenyl, formula -(CHRI) -Co-N < (wherein R5 can also be hydrogen or an amino group); an amide group represented by the formula CHR, -NH-CI+< (formula R2 is hydrogen or an imino group represented by an amino group or the formula: CtlzN imino group represented by <; or phosphoric acid diester, preferably formula: -C)l, -CL -o -PO, -0-CI.

CH(CO2-)! It is a riboamine represented by the following group. In addition, m in the formula and n are independently selected and are generally integers from 1 to 50, preferably from about 5 to 30. , more preferably an integer from about 10 to 25 and most usually from about 15 to 20. one In general, n is determined and selected by an engineer according to the following guidelines. In other words, fat If the molecules being purified are large (i.e. proteins larger than about 10 kD), the m and/or at least about 8 to 12 or more is preferred. Also, if the molecules to be lipidated are small (e.g. oligonucleotides), n is generally within the range 2 to 18, but lipidated molecules is increased when an increase in the hydrophobicity of is desired.

Almost all glycoproteins can be oxidized to carbohydrates according to the method of the present invention. It can be lipidated by reacting the side chain with a polyamino acid. Figure 2 schematically depicts a glycosylated antibody and a carbohydrate-conjugated lipidated antibody of the present invention, respectively. Shown below. Non-glycosylated proteins can be linked through appropriate cross-linkers. conjugated to lipids (e.g. by carbodiimide coupling chemistry) .

According to the present invention, specific for a given intracellular epitope due to strong affinity. Novel lipidated antibodies are provided that can bind to. These antibodies are loaded into intracellular compartments. easily accessible and at least approximately] XIO'M-', preferably l XIO'M-' to IXIO''M-', more preferably at least about IX It has a binding affinity of IO'M-' or higher. of the present invention Lipidated antibodies generally attach to naturally occurring carbohydrate side chains on the donor immunoglobulin chain. has a linked lipid substituent, and the substituent is an intracellular transmembrane or have formed antibodies specifically reactive with extracellular epitopes. carbohydrates are exempt Since it is located in the FC part of epiglobulin, this carbohydrate residue can be used for lipidation reaction. Therefore, even if the antibody is chemically modified, the affinity of the antibody for the antigen is substantially lost. (Rodwell et al., Proc. Na1l, Acad. Sci, (Ll, S, A), vol. 83, p. 2632, 1986). Fat of the present invention A modified antibody generally retains sufficient affinity for its antigen and its binding activity can be determined by any of a number of antibody-antigen binding assays known in the art. Can be easily measured. These antibodies can be produced economically in large quantities. It is used, for example, in treating various human diseases in various ways.

It constitutes the basic structural unit of one form of immunoglobulin or antibody. This form is a tetramer, consisting of two identical pairs of immunoglobulin chains, each pair containing one light Has a chain and one heavyweight. In each pair, both the light chain and heavy chain variable regions Involved in antigen binding, and the constant region is involved in antibody effector function. antibody In addition, immunoglobulins exist in a variety of other forms. For example, Fv, F ab and (Fab')2 and Ninomiya hybrid antibodies, fusion proteins (e.g. bacteriophage display libraries) and other forms! 1! ( For example, Lanzavecchia et al., Eur, 1. Immunol, No. 17, p. 105, 1987) and single chain forms (e.g. t(usto n et al., Proc, Natl.

Acad, Sci, U, S, A, vol. 85, pp. 5879-5883, 19 1988 and Bird et al., 5science, vol. 242, pp. 423-426, 1 988) (Generally, 1lood et al., “Immunology-1 2nd edition 1984, Benjamin Co., New York, USA: and) lunkal. lli tier and) food, Nature, vol. 323, 15-16 p. 1986).

Antibodies can be used in glycosylated cells (e.g., human lymphocytes, hybridoma cells, yeast, etc.). ) or in non-glycosylated cells (e.g. E. coli), or by chemical Synthesized by biological methods or directly translated using a polynucleotide template It can be manufactured by an outside translation system. Hybridoma cell lines and immunoglobulins One origin of polynucleotides encoding Cυ1ture Collection (Rockville, Maryland, USA) It is in. Methods for expressing heterologous proteins in recombinant hosts, polypeptide chemistry Synthesis and in vitro translation are well known in the art and are described in the following references: It is clearly stated. Namely, Maniatis et al., Molecular Clo ning: A Laboratory Manual (1989) 2nd edition , New York City, Cold Spring Harbor, United States; Berger and and Kimmel.

Methods in Enzymology, Volume 152, Guide to Mo1ecular Cloning Techniques (1987) , Academic Press Inc., San Francisco, California, USA. ・Diego; Merrifield, J, Am, Chem, Soc, , vol. 91, p. 501, 1969; Chaiken, 1. M,, CRCC rlt, Rev, Biochem.

Volume 11, page 255, 1981 Hkaiser et al., 5science, volume 243 , p. 187, 1989; Merrifield, B., 5science, Volume 232, page 342, 1986; Kent.

S, B, H, Ann, Rev, Biochem, Volume 57, Page 957 , 1988; and Offford, R.E., Sem1 synthetic Proteins, 1980, Wiley Publishing Co., Ltd. . Note that these documents are incorporated by reference in this application. Produced within non-glycosylated cells The antibodies to be used are conjugated to lipids using trifunctional cross-linkers or, preferably, translated. After translation, glycosylation occurs in glycosylation systems such as purified canine pancreatic microsomes. (Mueckler and Lodish, Ce1l, 44 Vol. 629, 1986; and Waller, P., Meth, E. nzymol, vol. 96, p. 84, 1983. These documents are incorporated by reference in this application. ). Alternatively, a combination of polynucleotides that encode antibodies Screened prokaryotes such as antibody fragment display libraries isolated from an expression library and then expressed in glycosylated cells to induce glycosylation. It is also possible to produce antibodies that contain antibodies. By these methods, naturally occurring and/or can obtain glycosylated antibodies with non-naturally occurring glycosylation patterns. Ru. Such glycosylated antibodies can be lipidated by the method of the present invention. Can be done.

Glycosylation of immunoglobulins improves immunoglobulin effector functions, may have a significant impact on structural stability and secretion rate from antibody-producing cells. has been reported (Leatherbarrow et al., Mo1. Immuno 22, p. 407, 1985). Carbohydrates involved in these properties The group is generally linked to the constant (C) region of the antibody. For example, the CH2 domain Glycosylation of IgG at position paragine 297 is associated with either 117G or complement-dependent cell lysis. It is necessary to obtain 1 q of sufficient Yotto to activate the classical pathway of the reaction (TaO and and Morrison, J. Immunol.

143, p. 2595, 1989). Asparagine 402 in C113 domain Glycosylation of IgM at the position ensures proper lateralization and cytolytic activity of the antibody. (Muraoka and Shulman, J., immuno l.

142, p. 695, 1989). The C,1 and C,3 domains of IgA antibodies If glycosylation sites such as positions 162 and 119 are removed, intracellular degradation occurs (1), At least 90% of secretion is inhibited (Talor and Wall, Mol.

Ce11. Biol, vol. 8, p. 4197, 1988).

Glycosylation in the variable region (part 7) of immunoglobulins has also been observed. So x and 1load are (Proc, Natl, Acad, Sci, US A, vol. 66, p. 975, 1970), approximately 2096 human antibodies are 7-part glycosylated. It was reported that it was zyllized. ■ Glycosylation of the region is caused by N-linkages in the heptad sequence. The combined glycolylation signal Asn-Xaa-3er/Thr may occur accidentally. It is believed that this occurs due to Not recognized in the art.

Therefore, lipidation cannot be performed on antibodies with naturally occurring glycosylation patterns. It's generally preferable. When creating glyphsylation sites in antibodies, novel glycosyl It is preferable to introduce the conjugation site into the framework region of the constant region or the variable region, These regions may be less likely to adversely affect the antigen-binding activity of the antibody. . It is generally best to place new glycosylation sites in antibodies in the constant region. Delicious.

Alternatively, only a portion of the secondary antibody structure and lipid substituents (e.g. Polypeptide flags with carbohydrate side chains that can be derivatized with fragments of which can be produced by one or more immunoglobulins. It has binding activity (eg, antigen-binding activity). These polypeptide fragments The intact antibody is subjected to proteolytic cleavage by methods known in the art. or expression vectors containing sequences encoding immunoglobulin proteins. For example, to create a Fab fragment CH, the position after the region To make a (Fab’)2 fragment, place the site after the hinge region. It can be created by inducing a specific sudden displacement. A single chain antibody is ■, and V. can be produced by linking them with a DNA linker ( (See Huston et al., supra and Bird et al., supra). Many similar remains Genes related to immunoglobulins, which consist of genes, contain distinct functional regions, each with These genes have novel properties because they have one or more different biological activities. can be fused to functional regions derived from other genes. Immunoglycerides used in the present invention The nucleic acid sequence that produces Robulin can ultimately express the desired antibody and different types of polynucleotides (e.g. genomic or cDNA or RN) , synthetic oligonucleotides, etc.) and components (e.g. V, J, D, and region C) and in a variety of different ways. Appropriate composition Although the current production method is to link the sequence and genome sequence, arrays are also available (European Patent Publication No. 0239400 and Rei chmann et al., Najure, vol. 332, pp. 323-327, 1988. (see).

A DNA sequence encoding an immunoglobulin and/or an immunoglobulin chain is For example, hybridoma clones that can be produced by methods known in the art. Thus (Kohler and Milstein, Eur.

, 1. Immunol, vol. 6, p. 511, 1976; this document is incorporated herein by reference. ), or any number of origins (“ATCC Catalog f　Ce1lLines　and　llybridomas'', America n Type Culture Co11ection, Maryland, USA Rockville, D.C.; this document is hereby incorporated by reference). Ru. DNA sequences encoding immunoglobulin chains are known in the art and described below. can be obtained by conventional cloning methods described in various publications. for example Maniatis et al., Molecular CIoning: A Labor atory Manual 2nd edition, 1989, Cole, New York, USA Spring Harbor and 3erger and Kimmel, Meth ods in Enzymology, Volume 152, Guide to Mole cular Cloning Techniques, 1987, Academ ic Press Inc., San Diego, Caliphate, United States However, these are incorporated by reference in this application.

As mentioned above, the DNA sequence is operably linked to an expression control sequence (all (i.e., after the expression control sequences have been arranged to ensure their functionality), the host generally It is expressed in cells that contain polypeptides. These expression vectors can be delivered as episomes or It can generally be replicated within the host organism as an essential part of the host's chromosomal DNA.

Usually, the expression vector contains a selectable marker, such as tetracycline resistance or G4. 18- To detect cells containing resistance and transformed with the desired DNA sequence (See, eg, US Pat. No. 4,704,362).

E. coli is a particularly useful prokaryotic host for cloning the [lNA sequences of the present invention]. one of. Other suitable microbial hosts for use include bacilli, e.g. For example, Bacillus subtilis and other Bacteria of Enterobacteriaceae, e.g. Salmonella IrE, Serrati a) Bacteria of the genus Pseudomonas and various Pseudomonas species be. In these prokaryotic hosts, expression control sequences that are compatible with the host cell (e.g. Expression vectors can be made that generally contain a starting point). Additionally, a large number of There are various known promoters, such as the lactose promoter system and the tryptopyl promoter system. trp promoter system, β-galactosidase promoter system, or Examples include the promoter system derived from phage λ. These promoters can be any together with operator sequences to generally control expression and initiate transcription and translation. It has a sequence of liposome binding sites for completion. protein e.g. Antibodies are expressed in non-glycosylated cells, but post-translationally, they are expressed within the glycosylated system. (Mueckler and Lodish, supra) Reference: This document is hereby incorporated by reference in this application).

Other microorganisms such as yeast can also be used for expression. Genus Satucharomyces ( Saccharomyces) microorganisms are preferred host glycosylated cells. Yes, promoter containing 3-phosphoglycerate kinase or other glycolytic enzymes and expression control sequences such as replication initiation sites, termination sequences, etc., as desired. Have the appropriate vector.

In addition to microorganisms, cultures of mammalian tissue cells also express polypeptides of the invention. (Winnacker, “From Genes o C1ones”, VCt (Publishers, USA, New York State of New York, 1987). Eukaryotic cells are currently the preferred microorganisms.

a large number of suitable host cells capable of secreting intact immunoglobulins. This is because systems have been developed in the art. These cells include CHO cells. cell lines, various CO3 cell lines, HeLa cells, preferably myeloma cell lines, and and transformed B cells or hybridomas. The expression base of these cells Vectors include, for example, origins of replication, promoters, enhancers (Queen et al., I mmunol, Rev.

89, pp. 49-68, 1986), as well as liposome binding sites, RNA spaces. Necessities such as price sites, polyadenylation sites and transcription terminator sequences It has expression control sequences such as processing information sites. Favorable expression control Sequences include immunoglobulin gene, SV40, adenovirus, cytomegalovirus This is a promoter derived from the virus, bovine papilloma virus, etc.

Important NA upper segments (e.g. heavy chain and light chain encoding sequences and expression control sequences) A vector containing the vector can be introduced into a host cell by known methods. Naoko These methods vary depending on the type of cell host. For example, calcium chloride trans The fection method is usually used for prokaryotic cells, whereas calcium phosphate treatment Physical or electroboration methods are used for other cell hosts (general , Maniatis et al., Molecular Cloning: A La laboratory Manual, Cold Spring t(arbor Press, 1982).

Whole antibodies of the invention, dimers thereof, individual light and heavy chains or other immunoglobulin forms Once expressed, ammonium sulfate precipitation, affinity columns, columns by standard methods in the art, including chromatography, gel electrophoresis, etc. (Generally, R, 5 copies, "Protein") Purification”, Springer-Verlag, 1982 (see New York, USA). For pharmaceutical use, an average of at least about 90 to 9596 Substantially pure immunoglobulin is preferred and is 98-99% or so. Homogeneity greater than or equal to this is most preferred. partially or homogeneously purified as desired If so, the polypeptide may be used therapeutically (including in vitro) or in assays, It is used to develop and implement immunofluorescence staining methods (generally 1mmunolog ical Methods.

Volumes ■ and ■, Lefkovits and Pernism Collection, Academic Press, New York, New York, USA, 1979 and 19 (see 1981).

The methods of the invention may include whole immunoglobulins or binding fragments thereof, e.g. Fab) is preferably used. Generally, lipidated antibodies are human IgM or IgM. The antibody is of the M isotype, but if desired, constant regions of other mammals may be used. I can do it. IgA, IgG, IgM, IgE.

Lipidated antibodies of the IgD class can be produced. The lipidated antibody of the present invention is human, Murine, bovine, equine, porcine or non-human primate antibodies are preferred, but also human. Alternatively, murine antibodies are more preferred. The present invention provides methods for producing various lipidated antibodies. These antibodies include, but are not limited to, chimeric antibodies, suitable for humans. adapted antibodies, primate adapted antibodies, Fv fragments, toxin-antibody conjugates , isotope-antibody conjugates and imaging agent-antibody conjugates. There is a union. For in vivo imaging, lipidated antibodies must be appropriately labeled with a diagnostic label. The drug is administered to the patient and its location is then measured multiple times following administration. diagnostic report (e.g. T0n, other radioligands, radioactive contrast agents or radioactive Various methods of labeling antibodies with opaque dyes are known in the art.

Proteins other than immunoglobulins and oligopeptides (i.e., peptidyl-linked polypeptides having from 2 to about 50 amino acid residues linked together) are included in the present invention. It can be lipidated depending on the method.

Naturally occurring glycoproteins (e.g. gamma-glutamyl transpeptidase, thrombocytopenia) Modulin, glucose transporter protein) is lipidated through carbohydrate binding Although virtually all polypeptides are preferred substrates for Covalently attached to the amino acid side chain by a cross-linking agent (e.g. N-hydroxysuccinimide) Lipidization can be achieved by In another embodiment of the invention, less At least one lipid substituent (e.g. riboamine) can be added to proteins or polypeptides. (e.g., Asp or Glu residues). Amide bond with side chain carboxyl substituent or thioester with Cys residue (by generating bonds). In addition, fatty acids have Arg or Lys residues in their side chains. It can be attached via an amine substituent. Transvascular and intracellular transport Examples of non-glycosylated proteins that are lipidated to promote Is there any of the following proteins: c(as, c-myc, c-src, NF- There are AT and HMG COA reductases. Physiological farnesylation Native proteins that undergo oxidation, geranylgeranilation, and valmitylation. Naturally produced lipoproteins, such as lipid protein, are natural products, so in this application, they are referred to as “lipidated proteins.” Do not define it as "quality".

The lipidated antibody of the present invention and its pharmaceutical composition can be administered parenterally, i.e., subcutaneously, intramuscularly or intramuscularly. It is particularly useful for intravenous or intravenous administration. Compositions of the invention for parenteral administration typically contain The immunoglobulins of the present invention may be dissolved in an acceptable carrier, preferably an aqueous carrier. The cocktail is made up of solutions. Although a variety of aqueous carriers can be used, e.g. Examples include water, buffered water, 0.4% saline, and 0.3% glycine. these Solutions are sterile and generally free of particulate matter. These compositions are usually Sterilize using known sterilization methods. The compositions of the invention provide appropriate physiological conditions. may contain pharmaceutically acceptable auxiliary substances necessary for pH adjustment. Reagents that buffer and modify toxicity, such as sodium acetate and sodium chloride. um, potassium chloride, calcium chloride, sodium lactate, human albumin, etc. Can be mentioned. The concentration of antibody in these formulations can vary over a wide range. vinegar i.e., from less than about 0.5%, usually about 1%, or at least about 1% by weight. , up to 15 or 20% by weight, and for the particular administration selected. Depending on the mode, the selection is primarily based on fluid volume, viscosity, etc.

Thus, a typical pharmaceutical composition for injection consists of 1 ml of sterile buffered water and a lipidated immunoglobulin. It can be prepared by containing 1 to 1 Omg of Robulin.

A typical composition for intravenous infusion contains 250 ml of sterile Ringer's solution and 150 mg of antibody. It can be prepared by having The actual manufacturing method of the composition for parenteral administration is known and obvious to those skilled in the art, eg Remington 's Pharmaceutical 5science, 15th edition, MackP Publishing Company, 1980, Pennsylvania, USA , described in detail by Easton. This publication is incorporated by reference into this application. To be.

The lipidated proteins and antibodies of the invention are frozen or lyophilized for storage and then It may be reconstituted with a suitable carrier before use. This method is used for normal immunoglobulin freeze-drying and recycling methods known in the art. You can use the construction method. Freeze-drying and reconstitution to varying degrees of activity For example, in the case of normal immunoglobulins, IgM antibodies are IgG antibodies. (there tends to be a greater loss of activity), usage levels must be adjusted to compensate. Those skilled in the art are well aware that this must be the case.

Compositions containing lipidated proteins (e.g. antibodies) of the invention or cocktails thereof can be administered for prophylactic and/or therapeutic treatment. In therapeutic applications, The composition cures or at least partially inhibits the disease and its complications Administer to the patient in sufficient amount to The appropriate amount to accomplish this Effective dose for this purpose is determined by the severity of the infection and the severity of the disease. Depending on the general state of a person's own immune system, generally about 1 to about 200 mg of antibody /- single dose, and 5 mg to 25 mg/- single dose are more commonly utilized.

The substances of the invention are generally used to treat serious disease conditions, i.e. life-threatening or life-threatening conditions. It must be noted that this is used when there is a possibility that .

For prophylactic use, compositions containing the immunoglobulins of the invention or cocktails thereof is administered to patients who are not yet diseased to strengthen their resistance.

Such an amount is defined as a "prophylactically effective dose." For this application, the exact amount is Again, it depends on the patient's health status and general level of immunity, but generally 0.1 ~25 mg/-doses.

Single or multiple administrations of the compositions of the invention may be administered at a rate of administration selected by the treating physician. Performed with bells and patterns. In any case, the pharmaceutical formulation of the present invention provides effective treatment for patients. of the lipidated protein and/or lipidated protein of the invention in an amount sufficient to treat You have to donate your body.

For diagnostic purposes, the lipidated antibodies of the invention may be labeled or unlabeled. . The unlabeled antibody may include another labeled antibody (second antibody) that is reactive with the lipidated antibody of the present invention, e.g. For example, using a combination of antibodies specific for the constant regions of human immunoglobulins. It comes like that. Alternatively, lipidated antibodies may be directly labeled. Many types of signs e.g. radionuclides, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors , ligands (especially hubtens), radioactive contrast agents, metal chelating agents, etc. Ru. Many types of diagnostic mapping methods are available and known to those skilled in the art. It is. For example, without limitation, antibodies that bind to tumor antigens (e.g., anti-CEA antibodies) body) is lipidated, conjugated to a radioactive contrast agent or a magnetic mapping substance, injected into a human patient and then detected to locate the tumor or metastatic lesion It will be done.

The lipidated immunoglobulin of the present invention can be used for diagnosis and therapy. For example, limit Although not specified, the lipidated immunoglobulin of the present invention may be used to treat cancer, autoimmunity or viral infections. It can be used for infectious diseases. When treating cancer, the antibodies of the invention generally It is known to those skilled in the art that c gene products are preferred in certain cancer cells. binds to antigens expressed in The lipidated antibodies of the invention preferably contain pathogenic molecules. Mutant proteins such as the c-ras oncogene product with sequences (e.g. neoplastic sequences) Proteins (e.g. proteins in which Set at position 12 of p2+”’ is substituted) binding to a mutant protein with a substitution at position 12,13,59 or 61 of the Ru. When treating autoimmune diseases, the antibodies of the invention generally target intraactivated T cells. Important regulatory proteins mainly expressed in Binds many other intracellular proteins known to those skilled in the art (e.g. Fun damental Immunology, 2nd edition, edited by W. E. Pau1, See Raven Press, New York, New York, USA. Naoko (This document is hereby incorporated by reference).

When treating viral infections, the antibodies of the invention will generally be used to treat viral infections. proteins expressed within cells, such as polymers coated by various viruses. V-I Tat and many others known to those skilled in the art. (e.g., Virology, 2nd edition, B, N , edited by Fields et al., 1990, RavenPress, USA, New See York State, New York. This document is incorporated by reference in this application).

The kit may also be used to protect against or detect cellular activity. for the presence of intracellular proteins in selected cells or for the diagnosis of a disease. Lipidated antibodies can also be supplied for use. Therefore, the composition of the subject of the invention can be used alone or in combination with additional antibodies specific for the desired cell type. It is usually provided in lyophilized form in a container. The lipidated antibody of the present invention can also be labeled. Tris, phosphate, Buffers such as carbonates, stabilizers, biocides, inert proteins such as serum Rubumin, etc., and put it in the kit along with a set of usage taxes. Generally speaking These substances are present in an amount less than about 5% by weight relative to the amount of active antibody and are typically A total amount of at least about 0.0C11% by weight is present. Also It may be desirable to include inert fillers or excipients to dilute the active ingredient. There are many cases where it is true. and in this case, the excipient is present in an amount of about 1 to 99% by weight of the total composition. It exists in A second antibody capable of binding to the lipidated antibody of the present invention is used in the assay. If so, the second antibody is usually placed in a separate vial. This second antibody is generally labeled and are formulated in a manner similar to that of the antibodies described above, and are generally themselves lipid-free. Qualified.

The lipidated antibodies of the present invention are also useful for improved diagnostic and protein purification methods. suitable for For example, many intracellular proteins are unstable (e.g. have half-lives short or susceptible to proteolytic reactions) or prone to aggregation (e.g. β-amylo is difficult to purify and/or detect for diagnosis. . Lipidated antibodies can penetrate living cells and bind to specific intracellular target antigens. Can be done. This antibody-antigen binding stabilizes the target antigen and Enzymes involved in the degradation of target antigens (e.g. proteases, ubiquitin conjugating enzymes, glycans) cosidases) are blocked, facilitating detection and/or purification of the target antigen.

In one variation of the invention, a lipidated antibody that specifically binds an intracellular target antigen is Live cells containing intracellular target antigens and under physiological conditions (e.g. cell culture conditions, cell conditions) and then incubate the appropriate attachment organ (e.g., for about 10 minutes to several hours). Incubate. The lipidated antibody of the present invention specifically binds to a target antigen and Either generates an antibody complex, or this complex is more susceptible to degradation and/or Not susceptible to agglomeration. The cells are then fixed and permeabilized to absorb the lipidated antibody-bound target. An antigen-antibody complex consisting of a target antigen is usually a target antigen that specifically binds to the lipidated antibody. Detected by a secondary antibody. An example of a preferred label to be attached to the secondary antibody is F LTC, rhodamine, horseradish peroxidase conjugate, alkaline phosphata -ase conjugate, β-galactosidase conjugate, biotinyl moiety, radioisotope, etc. What is it? In some embodiments, the secondary antibody is lipidated, immobilized and/or Eliminate the permeabilization step and replace non-specific staining with thorough washing of cell samples. except. It is also possible to directly use a lipidated and labeled primary antibody and omit the secondary antibody. Both are possible. Labeled protein A can also be used to detect secondary (lipidated) antibodies. It can be used in place of a secondary antibody.

Lipidated antibodies can also be used to bind to specific intracellular target antigens to determine the biochemical properties of the target antigen. It can be used to perform intracellular treatments such as altering the

For example, heteromultimeric proteins (e.g. transcription factors, G proteins) or homogeneous Multi-subunit proteins such as dimeric proteins (e.g. polymerized tubulin) biochemical activities that require interactions between molecules (e.g. GTPase activity) This intermolecular interaction with one or more subunits by lipidated antibodies that specifically bind and prevent functional interaction of its subunits. The first one is blocked. For example, the AP-1 transcription factor (F), which binds to Jun and is active during transcription. os/, Jun heterodimer) necessary to generate a portion of Fos (e.g. The lipidated anti-Fos antibody that binds to the functional AP-1 [■) to inhibit AP-1-mediated gene transcription. Also, for example, lipidated anti-ras Antibodies are used to detect the ras antibiotics necessary for proper signal transduction function. Auxiliary proteins such as GTP/GDP-binding sites, i.e. GAP The activity of intracellular ras in living cells is increased by binding to the part of ras that captures Modify.

1:J, The examples below are provided for illustrative purposes only and do not limit the invention. It's not a thing.

Rchr et al., Proc, Natl, Acad, Sci, (tl, s, A,) , 86 @, p. 6982, 1989. glinoldioctade by coupling a syn residue to dioctadecylamine. Got Silat. Note that the documents listed in -I- are incorporated by reference in this application. To〉Jiroki Cycarbonyl-glycyl-p-nitrophenol (1 equivalent) and triethylamine (1,1 eq.) in CH2Cl2 for 5 h, followed by H7 and lO%Pd/ c-containing CHtCLt/EtOII was added and reacted for 1 hour.

Bovine IgG (Sigma) 2mg, 1.5ml -1-Oppendorff Dissolved in 400ml of 300mM NatfCo in a vial. new tone Add 5 μm of the prepared Na1O4 solution (42 mg/ml in water) and The bottle was wrapped in aluminum foil and gently shaken at room temperature for 90 minutes. obtained The reaction medium was transferred to a PD-10 column (P Fraction 1) was injected into the column (Harmacia), and then the column was divided into 500 μm portions. fractions were eluted. Fraction No. 7 (both fractions between 3 ml and 3.5 ml) is Approximately 1.6 mg of bovine [[(G Was.

A DMSO solution of glycyldioctadecylamide was prepared (5 mg of the above lipid was added to D ml of MSOI and stirred vigorously for several minutes). Under these conditions the above lipids was not sufficiently dissolved. Carefully remove 50 μl of this solution to remove any undissolved lipids. ), and the amount obtained as described above in the Esoventorf High Refill bottle. The mixture was added to 7350 μl in both aliquots. Place the vial in aluminum foil. The mixture was gently shaken for 20 hours at room temperature.

Then 100 μl of a solution of NaBH+ (10 mg/ml in water) was added. 1 o'clock After a while, 40 μm of a solution of ethanolamine (15 μl in 1 ml water) was added.

After a further 1 hour, the reaction mixture was adjusted to 100 mM t (EPES buffer p++8 ．． The sample was injected onto a PD-10 column that had been equilibrated for 5 minutes. Both components containing lipidated 1gG ( between 3 ml and 3.5 ml) were collected and stored above water.

Labeling with 14c-acetic anhydride Using “C-acetic anhydride (500 μCi, Amersham) in Hensen. (10x lO'cpm/uI), 5 μm each of this solution was applied twice, Containing lipidated 1gG in Eppendorf vials after 10 minute intervals. 500 μl was added to both aliquots. This reaction was performed on ice. Unmodified I [(G Solution (800 μg in roomM tlEPEs pt18.5) 500 u I was treated similarly.

After 30 minutes, the vial was heated to 20-25°C and the Ig labeled with "C" Free “C-acetate” was extracted using a PD-10 column equilibrated with PBS. Separated from. The incorporated radioactivity is approximately 10X IO’cpm for 500μg There was.

Organ uptake testing Male Swiss Alpino mice (20 g body weight) were used. +40-labeled lipidated 1g G or “c” labeled control 1gG-containing PBS looμI (approximately 400,000 dpm) was injected into the tail vein for intravenous administration. After 30 minutes or 3 hours , kill a mouse, collect its blood in a test tube containing EDTA, and remove its brain (cerebellum). and brainstem), pancreas, one kidney and one liver were excised. these organs Homogenized in 117.41 ml of 10mM Tris buffer 1) and obtained Homocine) Transfer 500 μl each into a Beckman scintillation counter. It was counted by The protein concentration in these homogenates was determined by the Platford assay. It was measured by the standard method (Coomassie Blue). The blood was centrifuged and 20 μl of plasma was collected. Both parts were counted. Table I lists 14 (intakes) of the brain, liver, pancreas, and kidneys. The ratio obtained by dividing the radioactivity of 1 μg of protein by the radioactivity of 1 μl of plasma (data (expressed in μl/μg protein).

Table■ Brain 0.20±0.03 0.32+0008 0.94±0.05 1.75 +, 0.09 Kidney 1.46±0.31 9.50±2.19 1.17±0. 04 3.54+0.22 Liver 1.26±0.26 4.95±0.82 0 ．． 66±0.04 7.59±0.27 tile 1.08+0.27 4.54+ 0.76 1.13 ± 0.03 12.0 + 2.15 Control group for 30 minutes Four and six mice were used for each 3-hour period, respectively. Lipidated 1gG was administered. Each group used 6 mice at both time points. data is average The value is ±sem.

A monoclonal antibody that specifically captures the old V-1 Tat protein was used as described above. Lipidation was carried out according to the method described in Example 1. That is, the carbohydrates of the antibody are Oxidation with urate followed by covalent attachment of glycyldioctadecyl amide to release carbonate The lipidated antibody was generated and the product was dissolved in PBS from the final PD-10 column. I let go.

Sup Tl cells were placed in a 24-well plate (100.000 cells/ml , in 2 ml of modified RPMI 1640 culture medium). Cells are grown under one of the following conditions: Cultured. i.e. for the first 5 days of the experiment, (1) two controls without additional treatments) , (2) in the presence of added native anti-Tat antibody (15 μg/ml), or (3) Cultured in the presence of lipidated anti-Tat antibody (11.7 μg/ml). day-day At the end of the eye, old V-1111B was added to two wells of control cells and native anti-Ta. Contains cells treated with t antibody or lipidated anti-Tat antibody. culture. Live cells were counted daily. Approximately 500 untreated old V-infected cells ．． 000 cells/ml, and then the number increases after about 8 days. began to decline due to the cytotoxic effects of Uninfected cells have approximately 1.000.000 cells. cells/ml. Even if infected cells were treated with native anti-Tat antibody, Cells were not protected from the cytotoxic effects of the virus. On the other hand, anti-lipidation The Tat antibody protects infected cells from the cytotoxic effects of the IIV-1 virus. Fully defended. This protection is at least approximately It lasted 5 days. The results are shown in Figure 3.

In another experiment, SupTl cells were cultured and maintained as described in the previous example. cultured without treatment or infection, or infected with old V-IIIB without treatment. infection by treatment with native anti-Tat antibody (lμg/ml), or The cells were infected with lipidated anti-Tat antibody (1 μg/ml). The latter three above In the conditions, virus was added at the end of day 1 of culture. Under the latter two conditions Native or lipidated antibodies were present from day 1 to day 7.

The data shown in table Although it has almost no effect against viral cell damage, lipidated antibodies can protect cells in culture from viral cytotoxicity. showed significant protection against harmful effects and significantly decreased reverse transcriptase activity. ing. The latter data above suggest that lipidated antibodies may inhibit the replication of old V-1 within cells. This suggests that it was possible to do so.

Table■ ) Viable cell number and reverse transcriptase activity in Sup Tl cells infected with 11V-1 Effect of lipidated anti-Tat antibody on gender untreated, uninfected 1.15 1.28 1 ．． 52 1.63 1,69 1.76 1.72 Untreated, infected 1. +2 1 ．． 2 1.18 1. +2 0.92 0.64 0.51 Untreated, uninfected 2 2122+! Untreated, infected 3 2 4 175 264 337 367 infected with HIV-1 before adding the supernatant containing IIV-1 virus. Anti-Ta in native or lipidated form starting from day 1 until day 10 post-infection. t antibody or rsCD4 (all proteins at 1 μg/ml). used). The cell number and reverse transcriptase (RT) activity in the culture medium were determined on the second day after infection. I started taking measurements every day. By day 10, the native anti-Tat antibody increased the number of cells or R Although the lipidated anti-Tat antibody had no significant effect on T activity, the untreated It increased cell viability by approximately 70% compared to cells infected in situ, and increased RT activity. The degree was reduced. Culture was continued for 30 hours without further addition of antibody. lipid The effect of the transformed anti-Tat antibody lasted for 3 days, which indicates that the transformed anti-Tat antibody Accumulates intracellularly in high enough amounts to sustain protection against viral infection/replication It shows that it was done. Cell viability and l'lT of lipidated 1-Tat antibody The magnitude of the effect on activity was that observed for the same dose of rsCD4. was very similar.

Even if the concentration of lipidated anti-Tat antibody was increased to 10 μg/ml, the RT activity was still higher than that. It did not decrease.

Example 3 Performance of lipidated anti-Tat antibody to inhibit Tat transcriptional activity of HIV-I LTRI The 11eLa cell line was stably transfected with a polynucleotide expressing CD4. ]・Shita. The memplan receptor mediates IIIV-1 infection and is an operable receptor. containing a reporter construct having long terminal repeats (LTRs) of IIIV-1 in combination; Drive transcription of bound reporter gene (chloramphenifur acetylate Transferase: CAT). These cells (HLCD 4-CAT) are Susceptible to HIV-1 infection that produces functional Tat protein. and newly synthesized The generated Tat binds to the old VI LTR and transcribes the bound CAT gene.

Therefore, the magnitude of CAT expression depends on the extent of 111V-1 infection and intracellular T. The activity of at protein is 5' proportional.

Cultured l1ela cells (3XI06 in DMEM containing 1096 fetal bovine serum) cells/ml) at the same concentration (1 or IO μg/ml) of various antibodies (native or or lipidated form) or soluble recombinant CD4 (rscCD4) The supernatant of a culture of cells containing 111. (100 μm) was added. Cells were harvested 24 hours later and CAT expression was tlo (1984). Each experiment was performed four times and at four different times. went. Figure 4 shows that lipidated anti-Tat antibody significantly inhibits CAT activity (approximately 75%). , while native (unlipidated) anti-Tat antibody, lipidated anti-gl)120 antibody or r It shows that sCD4 was much less effective at inhibiting CAT activity.

These data demonstrate that lipidated anti-Tat antibodies or their intracellular targets, i.e. Tat, are significantly and capture the LTR/reporter gene construct as a transcriptional activator. It has been shown that the target activity can be inhibited.

Furthermore, data showing the route of entry of lipidated anti-Tat antibodies into 1leLa cells are It has been reported that La cells do not undergo phagocytosis, so the transport mechanism is endogenous. This shows that it is probably not necessary to create a system.

Glycyldioctadecylamide is described by Behr et al., Proc. Natl., Ac. ad.

Sci, (U, S, A,), vol. 86, p. 6982, 1989. We decided to combine the glycine residue with dioctadecylamine in a method that Therefore, it is obtained. The above documents are incorporated by reference into this application. pencil oxyca Luponyl-glycyl-p-nitrophenol (Ik) and triethylamine ( 1,1 eq.) in Cl12CI2 for 5 hours, followed by H, and lO%Pd/ C12C12/E1.011 was added and reacted for 1 hour.

Anti-human c-myc Ig Glycolylated canine immunoglobulin that specifically binds human c-myc protein under specific conditions (Evan et al., Mo1.Ce11.Biol, vol. 5). 3610 pages, 1985, this document is incorporated by reference in this application'), 10% Hybridoma cell line MYC in RPM 1640 containing 0% bovine fetal serum CT9- B7.3 (ATCC CRL 1725), MYCCTl4-G 11.3 (ATCC CRL +727) and MYCI-9E 10.2 ( ATCCCRL 1729) was prepared and secreted by culturing separately. Monoclonal antibodies are collected and purified by conventional methods known in the art.

Approximately 2 mg of each purified monoclonal antibody was added to a 1.5 ml Eppendorf vial. Dissolve in 300mM Na1lC0,400μm in a rub bottle. freshly prepared Add 50 μm of Na1Ot solution (42 mg/ml in water) and pour into the vial. Wrap in aluminum foil and shake gently for 90 minutes at room temperature. Then that The reaction medium of was added to a PD-10 column (P Fraction 1) was injected into a 500 μm column Elute in fractions. less when measured using the Platford protein assay Both portions, each containing approximately 500 μg of IgG, are collected.

Prepare a DMSO solution of glycyldioctadecylamide (add the above lipid 5mHB to Add to ml of DMSOl and stir vigorously for several minutes). Under these conditions, the above fat quality is not fully dissolved. Carefully remove 50 μm of this solution and Add to 350 μl of the purified IgG fraction obtained as above in the Hyal bottle. . Wrap the vial in aluminum foil and let the mixture stand at room temperature for 20 hours. Shake gently.

100 uI of a solution of NaRH, (10 mg/ml in water) are then added. 1 After an hour, add 40 μl of a solution of ethanolamine (15 μm in 1 ml water). . After an additional hour, the reaction mixture was transferred to a PD-10 column pre-equilibrated with PBS. Inject into. Both volumes containing lipidated dog anti-human-myc IgG (3 m 1 and 3.5 mL) and store on ice.

Anti-tlMG CoA reductase Ig intracellular enzyme 11MG CoA reductor Glycosylated immunoglobulins that specifically bind to n et al. 1.1. Biol, Chem, Volume 258, Page 8450, 1783 (This document is incorporated by reference in this application). 4. ! Contains 5g/l glucose, 5% horse serum and 2.5% fetal bovine serum Hypuritoma cell line A9 (ATCC CRI, 1811) in DMEM Monoclonal antibodies are produced by culturing them separately, and the secreted monoclonal antibodies are collected. It is then purified by conventional methods known in the art.

Approximately 2 mg of each purified monoclonal antibody was placed in a 1.5 ml Eppendorf vial. Dissolve 300mM Nat(C(L) in 400μl of freshly prepared N Add 50μI of a1Ot solution (42mg/ml in water) and remove the vial. Wrap in aluminum foil and shake gently at room temperature for 90 minutes.

The reaction medium was transferred to a PD-10 column (pre-equilibrated with 10mM Na2COz). The column was then injected into a 50 Gμm column (Pharmacia). Elute in two fractions. as measured using the Platford Protein Assay. Both aliquots containing approximately 500 μg of IgG are collected.

Prepare a DMSO solution of glycyldioctadecylamide. Add 3 mg of the above lipid to C. Add to 1 ml of DMSO and stir vigorously for several minutes). Under these conditions, the above fat quality is not fully dissolved. Carefully remove 50 μm of this solution and Add to 350 μm of purified LgG fraction obtained as above in a vial. . Wrap the vial in aluminum foil and let the mixture stand at room temperature for 20 hours. Shake gently.

A solution of NaB)L (10 mg/ml in water) +00 uI is then added. 1 After an hour, add 40 μm of a solution of ethanolamine (15 μl in 1 ml water) . After an additional hour, the reaction mixture was transferred to a PD-10 column pre-equilibrated in PBS. inject into the tank. Lipidated anti-t (MG CoA reductase IgG-containing fraction ( between 3 and 3.5 ml) and stored on ice.

Example 5 Production of lipidated immunoglobulins reactive with transmembrane protein Behr et al., Proc, Natl, Acad, Sci, (U, S, A,), Glycine according to the method described in Vol. 86, p. 6982, 1989. Glycyl diotadecyl by linking the An amide is obtained. The above documents are incorporated by reference into this application. Penzyloxy p-nitrophenol (1 equivalent) and triethylamine ( 1,1 eq.) in CILCI2 for 5 hours, followed by H, and 1006Pd/ c-containing CI+ and CI2/Ej011 are added and reacted for 1 hour.

Anti-ras Ig Glycosylated canine immunoglobulin that specifically binds to ras oncogene protein The products are produced by culturing them separately as follows. Sunaocchi 4.5g/j? glucose , 2mM L-glutamine, 1mM sodium pyruvate, non-essential amino acids, I XBME vitamins, 0. ]mM hypoxanthine, 0.032mM thymidine, D containing 0.05 mg/ml gentamicin and 10o6 Uno fetal serum. Hybridoma cell line 142-24E5 (ATCC 1188679 ; U.S. Pat. No. 5.015.517 and U.S. Pat. No. 5.030.565. these ), and +96L-tamino, IIT and 1 Iscove's DMEM containing fetal serum 006 IEM) under certain conditions (U.S. Pat. No. 4,820,631, which is incorporated herein by reference). ) culturing hybridoma cells MX (ATCCHB 9158), The monoclonal antibodies secreted from these hybridoma lines were collected and It is purified by conventional methods known in the field.

Approximately 2 mg of each purified monoclonal antibody was added to a 1.5 ml Eppendorf vial. Dissolve in 300mM NatlCo 3400μm in a rub bottle. freshly prepared Add 50μl of Na102 solution (42mg/ml in ice) and pour into the vial. Wrap in aluminum foil and shake gently at room temperature for 90 minutes.

The reaction medium was then transferred to a PD-10 column (pre-equilibrated with 10mM Na2COz). Inject fraction 1) into a column (Pharmacia), then load the column with 500 μl each. Elute in fractions. at least as measured using the Platford Protein Assay Both aliquots containing approximately 500 μg of IgG are collected.

Prepare a DMSO solution of glycyldioctadecylamide (5 mg of the above lipid was added to D into 1 ml of MSO and stir vigorously for several minutes). Under these conditions, the above fat quality is not fully dissolved. Carefully remove 50 μm of this solution and place it in an Eppendorf tube. Add to both 350 μm portions of the purified 1 gG obtained as above in the vial. Add. Wrap the vial in aluminum foil and let the mixture sit at room temperature. Shake gently for 20 hours.

NaB11. 100 ul of a solution (10 mg/ml in I+20) of do. After 1 hour, add 40 μl of a solution of ethanolamine (15 μl in 1 ml water). Added. After an additional hour, the reaction mixture was pre-equilibrated with PBS using PD-IO. Inject into column. Both volumes containing lipidated dog anti-ras IgG (3 ml and 3 ml) ．． 5 ml) and store on ice.

The hybridoma cell line mentioned in the above example is American Type Cul. From ture Co11ection (Rockville, Maryland, USA) Available (ATCCCe1l Lines and Hybrid omas (1992) 7th edition, this document is incorporated herein by reference).

Enzyme γ-glutamyl transpeptidase (GGT: EC2,3,2,2 ) is the decomposition reaction of γ-glutamyl compound with glutathione. by hydrolyzing the mill moiety or transferring the moiety to a suitable receptor. It is a widely distributed enzyme that catalyzes

GGT is a heterodimeric glycoprotein that is glycosylated and released into the mature enzyme. It is synthesized as a precursor protein that is cleaved into two subunits. GGT is It is anchored to the cell membrane through the N-terminal part of its heavy subunit. of the above enzymes The active site is located in the extracellular part of the molecule, which is heavily glycosylated. .

GGT was administered to rat kidneys according to methods previously reported in the art. separately purified from human liver cancer cells cultured with liver (Barouki et al., J. Biol, Chem, vol. 259, p. 7970, 1984; Curt Hoys and lIaghey, Enzyme, vol. 24, p. 383, 197 9 years; Matsuda et al., J.

Biochem, vol. 93, p. 1427, 1983; Taniguchi et al., J. Natl, Cancernst, vol. 75, p. 841, 19 85; Tate and Meister, Methods Enzymol, 113, p. 400, 1985: and Toya et al., Ann, N.Y.

Acad, Sci, vol. 417, p. 86, 1983. These documents are ).

Approximately 1 mg each of purified rat and human GGT preparations in 1.5 ml Dissolve in 400 μl of 300 mM NatlCo in a Ruf vial.

Add 50 μl of freshly prepared Na1 (L solution (42 mg/ml in water)) Then wrap the vial in aluminum foil and gently incubate at room temperature for 60 minutes. Shake. The reaction medium was then mixed with P pre-equilibrated with 10mM Na2COz. Fraction 1) was injected onto a D-IQ column (Pharmacia), then the column is eluted in 500 μm fractions. Using the Platford Protein Assay Measure and collect fractions containing at least about 100 μg of GGT.

Prepare a DMSO solution of glycyldioctadecylamide (5 mg of the above lipid was added to D ml of MSOI and stir vigorously for several minutes). Under these conditions, the above Lipids are not fully dissolved. Carefully remove 50 μm of this solution and Added to 350 μm of the purified GGT fraction obtained as above in a microvial. Add. Wrap the vial in aluminum foil and store the mixture at room temperature for 2 hours. Shake gently for 0 hours.

Next, 100 μl of a solution of NaB11a (10 mg/ml in water) is added. 1 After an hour, add 40 μm of a solution of ethanolamine (15 μm in 1 ml water) . After an additional hour, the reaction mixture was transferred to a PD-10 column pre-equilibrated with PBS. Inject into. Contains lipidated human and rat GGT (between 3 ml and 3.5 ml). Collect both parts and store on ice.

The obtained human and rat lipidated GGT fractions contain γ-glutamyl transpeptidyl Dase activity was determined using conventional assays (Tate and Meister, 1983, supra). literature; this literature is hereby incorporated by reference), and lipidated human G The specific activities of GT and lipidated rat GGT are measured.

Lipidated human and rat GGT was purified using 1251 iodine using chloramine T. Approximately 50 μg of radioiolated lipids were radiolabeled by GGT is administered to rats by intraperitoneal injection. After 24 hours, kill the rats. Tissue samples were then taken and subjected to autoradiography to detect each lipidated GGT. Determine the pattern of localization of seeds within organs.

Lipidated antibodies are able to localize intracellularly within living cells and capture intracellular targets. To demonstrate that anti-actin antibodies were lipidated and cultured Swiss 3T3 fibroblasts The ability to penetrate cells and bind to the cytoskeletal protein actin was evaluated. Not yet A denatured anti-actin antibody (unexplored) was used as a control.

I would like to use the following method to prepare protein A-purified heron anti-actin polyclonal antibody. and converted into lipid. Riboamine glycyldioctadecylamide, periodate acid Covalently attached to the carbohydrate moiety of anti-actin antibodies by sodium borohydride reduction Combined. Antibodies are about 0.2 to 1. Concentration of Omg/ml, 300mM Dissolved in Na1lCO*0.81. Freshly made Na1O4 aqueous solution (42m g/ml) 50μm and then the incubation vial. It was wrapped in aluminum foil and gently shaken at room temperature for 90 minutes. reaction mixture Next, a PD-10 column (Phar Macia, New Chassis, USA), 10mM Purified by elution with NatCOz. Glinoldioctadecylamide benzene solution (10 mg/ml) 50111 and both parts containing the antibody (eg A2.

The obtained The reaction mixture was incubated for 20 hours at room temperature with gentle shaking. new Add 100 μm of a specially prepared aqueous solution of NaBIl (IOrng/ml). , incubated for 1 hour at room temperature, followed by a solution of ethanolamine (in 1 ml of water) 50 μm of 15 μm of ethanolamine dissolved in the solution was added. Further at room temperature After 1 hour, the generated lipidated antibodies were transferred to PD-10 cells equilibrated with phosphate buffered saline. Purified by ram chromatography.

El, ISA certification Lipidated anti-actin antibodies and lipidated anti-rat antibodies (described above) are native (non-lipidated) ) for anti-actin or anti-Tat antibodies against specific antigens. rated Nity. Even if the anti-actin antibody or anti-Tat antibody is lipidated, its residual The affinity of these antibodies for their respective antigens compared to denatured (non-lipidated) antibodies I can't ignore the tee (I didn't miss it).

Intracellular immunological staining Lipidated anti-actin antibodies can capture intracellular actin in living cells. To demonstrate that lipidated anti-actin antibodies or native anti-actin antibodies were 3T3 cells for 1 hour, followed by thorough washing to remove residual anti-actin antibodies. Removed. The cells are then fixed and permeabilized. ize), anti-actin antibody was detected with a fluorescently labeled secondary antibody. unaltered (Non-lipidated) In cells preincubated with anti-actin antibody, there is a specific Although no differential staining could be detected, pre-ink staining along with lipidated anti-actin antibody Specific actin staining (e.g. stained actin cells) ) was clearly recognized. Lipidated anti-actin antibody used before fixation The staining pattern observed for fixation and permeabilization was Then, using a native (non-lipidated) anti-actin antibody incubated with cells. It was similar to what was seen. These data indicate that lipidated anti-actin antibodies We were able to reach and capture intracellular actin, and the labeled secondary demonstrated that it was recognized and captured by the antibody, which indicates that the lipidated anti-acid This shows that the Chin antibody is functional and substantially complete.

Although the invention has been described in detail by way of example for purposes of clarity of understanding, certain modifications may be made. It is obvious that modifications and modifications may be practiced within the scope of the claims.

Brief description of the drawing FIG. 1 shows structural formulas representing various gluepoamino compounds that can be used in the present invention. right The column exemplifies branched-chain riboamines, and the column on the left exemplifies straight-chain riboamines. .

Figure 2 shows (1) an immunoglobulin tetramer (two light chains linked to two heavy chains); (2) Schematic illustration of the glycosylated antibody of the present invention, and (2) the binding to the carbohydrate of the present invention. Therefore, it is a schematic illustration of lipidated immunoglobulin. For example, but not limited to , branched riboamine substituents are partially oxidized carbohydrates of immunoglobulin tetramers. It is shown that it is attached to the side chain of the compound. The side chains of such carbohydrates are C Located in the M, V, CL and/or VL regions.

Figure 3 shows the in vitro survival of cells infected with old V-1 versus native (i.e. (nonlipidated) anti-Tat immunoglobulin has no effect, whereas lipidated Figure 2 shows the beneficial effects of anti-Tat immunoglobulin.

Figure 4 shows that the lipidated anti-Tat antibody significantly inhibits CAT activity (about 7596 inhibition). On the other hand, native (non-lipidated) anti-Tat antibody, lipidated anti-gp120 antibody or rsC D4 has significantly lower efficacy in inhibiting CAT activity in 1LcD4-CATm cells. Indicates that the

FIG.i Engraving (no changes to the content) Glycosylated antibodies FIG　2゜ Engraving (no changes to the content) Oral control, non-infected FIG. 3B.

Engraving (no changes to the content) 3: Lipidated anti-GP120 antibody 4: Soluble fat CD4 FIG. Procedural amendment (formality) October day of Heisei et al.

Claims (1)

[Claims] 1. Lipid substituents are covalently attached to proteins to produce lipidated proteins. Let: Collecting the produced lipidated protein; How to modify mechanical properties. 2. 2. The method of claim 1, wherein the lipid substituent is a lipoamine. 3. The joining step is further oxidizing carbohydrates of glycosylated polypeptides to produce oxidized glycoproteins; then The oxidized glycoprotein is reacted with lipoamine under appropriate reaction conditions to form a lipidated protein. Generate protein; 3. The method of claim 2, comprising the steps of: 4. Lipoamine has the following formula: NH2-R-(CH2)n-CH3 [Wherein, R is di-substituted alkyl (alkylene); 1,4-disubstituted cyclohexyl; Di-substituted aryl (arylene); formula: -(CHR1)-CO-NH- (in the formula R1 amide group represented by hydrogen or amino group); alkylcarbonyl, and selected from the group consisting of phosphoric acid diesters; n is a linear chain represented by 1 to 50; The method according to claim 2, wherein the poamine is a poamine. 5. Lipoamine formula: NH2-R'-(CH2)n-cm2 (CH2)m CH3 [In the formula, R' is trisubstituted alkyl: trisubstituted aryl: formula -(CHR1)-C Amido group represented by O-N< (wherein R1 is hydrogen or an amino group): formula -CH Imino group represented by R2-NH-CH< (wherein R2 is hydrogen or an amino group) or an imino group represented by the formula -CH2-N<; or a phosphoric acid diester; m is 1 ~50; n is 1 to 50; and m and n are independently selected] 3. The method according to claim 2, wherein the lipoamine is lipoamine. 6. The method according to claim 5, wherein the branched-chain lipoamine is glycyldioctadecylamide. Law. 7. 2. The method according to claim 1, wherein the protein is a naturally occurring glycoprotein. 8. proteins encoded by immunoglobulin superfamily genes 2. The method according to claim 1. 9. Immunoglobulin superfamily genes encode μ or γ heavy chains The method according to claim 8. 10. 8. The method according to claim 7, wherein the naturally occurring glycoprotein is an antibody. H. 11. The method according to claim 10, wherein the antibody is a monoclonal antibody. 12. at least one lipore in which a lipidated protein is attached to a carbohydrate side chain The method according to claim 1, which contains a min residue. 13. Contacting cells with lipidated antibodies that specifically bind intracellular proteins A method of binding an intracellular protein to an antibody intracellularly, comprising: 14. The lipidated antibody has at least 14. The method of claim 13, containing one lipoamine residue. 15. 15. The method according to claim 14, wherein the lipoamine is glycyldioctadecylamide. . 16. 14. The method of claim 13, wherein the lipidated antibody is administered in vivo to a non-human animal. 17. The ratio of lipidated antibodies that have the same amino acid sequence and the same glycosylation pattern 17. The method of claim 16, wherein the antibody is taken up by the organ in greater amounts than comparable naturally occurring antibodies. . 18. Also contains a therapeutically effective dose of lipidated protein to treat a disease. or a composition used for prevention. 19. 19. The composition of claim 18, wherein the lipidated protein is an antibody. 20. 20. The composition of claim 19, wherein the antibody binds to an intracellular protein. 21. Claim 20 wherein the intracellular protein is a protein encoded by a virus. Compositions as described. 22. The protein encoded by the virus is the Tat protein encoded by HIV-1. 21. The composition according to claim 20, which is a protein. 23. A prophylactic composition containing a lipidated antibody effective for prophylaxis. 24. A composition containing a lipidated antibody conjugated to a diagnostic reporter. 25. 19. The composition of claim 18, comprising a carbohydrate-bound lipidated protein. 26. The carbohydrate-bound lipidated protein is a carbohydrate-bound lipidated immunoglobulin. 26. The composition according to claim 25. 27. Pharmaceutically acceptable carbohydrate-bound lipidated immunoglobulin and excipients Compositions to be contained. 28. administering to the patient a lipidated antibody containing a diagnostic reporter; Detects lesions where diagnostic reporters are preferentially localized; lesions consisting of steps How to diagnose the condition.